An investigation of structure‐activity relationships of azolylacryloyl derivatives yielded potent and long‐acting hemoglobin modulators for reversing erythrocyte sickling

Article abstract of DOI:10.3390/biom10111508

Aromatic aldehydes that bind to sickle hemoglobin (HbS) to increase the protein oxygen affinity and/or directly inhibit HbS polymer formation to prevent the pathological hypoxia‐induced HbS polymerization and the subsequent erythrocyte sickling have for s

Full text of DOI:10.3390/biom10111508

biomolecules
Article
An Investigation of Structure-Activity Relationships
of Azolylacryloyl Derivatives Yielded Potent
and Long-Acting Hemoglobin Modulators for
Reversing Erythrocyte Sickling
Abdelsattar M. Omar 1,2,
*
, Osheiza Abdulmalik 3 , Mohini S. Ghatge 4, Yosra A. Muhammad 1,
Steven D. Paredes 4, Moustafa E. El-Araby 1 and Martin K. Safo 4,
*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Alsulaymanyah,
Jeddah 21589, Saudi Arabia; ymuhammad@kau.edu.sa (Y.A.M.); madaoud@kau.edu.sa (M.E.E.-A.)
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA;
abdulmalik@email.chop.edu
Department of Medicinal Chemistry, School of Pharmacy and Institute for Structural Biology, Drug
Discovery and Development, Virginia Commonwealth University, Richmond, VA 23219, USA;
msghatge@vcu.edu (M.S.G.); sdpar97@gmail.com (S.D.P.)
2
3
4
*
Correspondence: Omar: asmansour@kau.edu.sa (A.M.O.); msafo@vcu.edu (M.K.S.);
Tel.: +966-567-68-14-66 (A.M.O.); +1-804-828-7291 (M.K.S.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 15 September 2020; Accepted: 30 October 2020; Published: 2 November 2020
Abstract: Aromatic aldehydes that bind to sickle hemoglobin (HbS) to increase the protein oxygen
affinity and/or directly inhibit HbS polymer formation to prevent the pathological hypoxia-induced
HbS polymerization and the subsequent erythrocyte sickling have for several years been studied for the
treatment of sickle cell disease (SCD). With the exception of Voxelotor, which was recently approved
by the U.S. Food and Drug Administration (FDA) to treat the disease, several other promising
antisickling aromatic aldehydes have not fared well in the clinic because of metabolic instability of
the aldehyde moiety, which is critical for the pharmacologic activity of these compounds. Over the
years, our group has rationally developed analogs of aromatic aldehydes that incorporate a stable
Michael addition reactive center that we hypothesized would form covalent interactions with Hb to
increase the protein affinity for oxygen and prevent erythrocyte sickling. Although, these compounds
have proven to be metabolically stable, unfortunately they showed weak to no antisickling activity.
In this study, through additional targeted modifications of our lead Michael addition compounds, we
have discovered other novel antisickling agents. These compounds, designated MMA, bind to the
α
-globin and/or β-globin to increase Hb affinity for oxygen and concomitantly inhibit erythrocyte
sickling with significantly enhanced and sustained pharmacologic activities in vitro.
Keywords: hemoglobin; sickle cell disease; Michael addition; antisickling; oxygen equilibrium
1. Introduction
Sickle cell disease (SCD) is a genetic hematologic disorder affecting over 100,000 people in the
U.S. and several millions worldwide [
residue Glu6 with a hydrophobic Val6 residue, changing normal Hb (HbA) into sickle Hb (HbS).
When deoxygenated or under hypoxic conditions, the pathogenic Val6 residue on one HbS tetramer
1,2]. The disease occurs as a result of a replacement of a polar
β
β
β
makes a hydrophobic interaction with an adjacent HbS tetramer, polymerizing into long and rigid
fibers, which is further stabilized by several intermolecular interactions between the HbS tetramers,
Biomolecules 2020, 10, 1508; doi:10.3390/biom10111508
www.mdpi.com/journal/biomolecules

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causing sickling of red blood cells (RBCs) [
3–
8]. The rigid RBCs lead to several interrelated secondary
pathophysiological events, including but not limited to, impaired blood flow, hemolysis, painful
vaso-occlusion (VOC), adhesion of RBCs to tissue endothelium, oxidative stress, decreased vascular
nitric oxide (NO) bioavailability, inflammation, and eventually chronic endothelial and organ damage
that ultimately leads to poor quality of life and decreased life expectancy [1,2,9–11].
Currently, there are four approved therapies for SCD. The first is hydroxyurea (HU), which
works by inducing fetal Hb (HbF) production to inhibit HbS polymerization [12]. A reported lack of
response in about 30% of patients, partly due to poor compliance, tend to limit its use [13
L-glutamine (Endari), which reduces oxidative stress was approved to treat SCD [15]. However, it has
limited efficacy [16 17]. In 2019, two other drugs, Crizanlizumab (Adakveo)[18] and Voxelotor [19
,14]. In 2017,
,
]
(GBT-440 or Oxbryta) were approved. The former is a monoclonal antibody that targets P-selectin to
reduce the frequency of painful vaso-occlusive crises [18]. Voxelotor, an aromatic aldehyde binds to
Hb and prevents HbS polymerization by increasing Hb oxygen (O2) affinity [19–22].
Hb functions in equilibrium between the unliganded or deoxygenated (Deoxy) tense (T) state,
which exhibits low affinity for ligand, and an ensemble of oxygenated relaxed states (collectively
referred to as R state) that exhibit high affinity for oxygen [23–25]. A plot of partial pressure and
Hb oxygen saturation gives a sigmoidal oxygen equilibrium curve (OEC). The degree of shift in the
OEC is reported as a decrease (left-shift) or increase (right-shift) in P50 (oxygen tension at 50% Hb
O2 saturation). Since only deoxygenated T-state HbS polymerizes, allosteric modulation of Hb with
compounds that destabilize the T-state and/or stabilize the R-state to increase Hb O2 affinity could
prevent the hypoxia-induced HbS polymerization and sickling of RBC [23–33].
Several compounds, including aromatic aldehydes, e.g., Voxelotor, 5-HMF, vanillin, and their
derivatives [20–33], thiols [34,35], and Michael addition compounds [36,37], are known to covalently
bind to Hb and pharmacologically increase its oxygen affinity and/or directly destabilize the polymer to
prevent RBC sickling. Aromatic aldehydes form Schiff base covalent interactions with the N-terminal
α
Val1nitrogen at the
α
-cleft of Hb, while thiols and Michael addition compounds, in most part
Cys93 of Hb. Aromatic aldehydes have been the most studied class of
react covalently with
β
compounds, with Voxelotor approved for the treatment of SCD [19]. Nonetheless, unlike Michael
addition compounds, most aromatic aldehydes undergo significant and rapid metabolic oxidation
of the aldehyde moiety [38–40], resulting in poor pharmacokinetic (PK) properties, hampering their
development into viable therapeutic agents.
To overcome the limitation of aromatic aldehydes susceptibility to oxidative metabolism, our group
has over the years embarked on the development of Michael addition compounds [36
these compounds as expected demonstrated metabolic stability, they showed weaker antisickling
potencies compared to the aromatic aldehydes [36 37]. In this report, we have further refined the
,37]. Although,
,
structures of these earlier Michael addition compounds, resulting in some of the compounds exhibiting
a significant improvement in their pharmacologic properties.
2. Materials and Methods
2.1. Study Approvals
At Virginia Commonwealth University (VCU), normal whole blood was collected from adult
donors (>18 years) after informed consent, in accordance with regulations of the IRB for Protection of
Human Subjects (IRB #HM1) by the Institutional Review Board at VCU. At the Children’s Hospital of
Philadelphia (CHOP), leftover blood samples from individuals with homozygous SS who had not been
recently transfused, were obtained and utilized based on an approved IRB protocol (IRB# 11-008151)
by the Institutional Review Board at CHOP, with informed consent. All experimental protocols and
methods were performed in accordance with institutional (VCU and CHOP) regulations.

Biomolecules 2020, 10, 1508
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2.2. Chemistry
Melting point was performed using OptiMelt Automated Melting Point System Digital Image;
Processing Technology SRS, Stanford Research Systems (Sunnyvale, CA, USA). NMR spectroscopy
was recorded on Bruker AVANCE III 400 (Bruker, Fällanden, Switzerland). LCMS spectroscopy was
performed on Agilent Technologies 1260 Infinity LC/MSD system with DAD
\
ELSD Alltech 3300
and Agilent; LC MSD G6120B mass-spectrometer (Santa Clara, CA, USA). High-resolution mass
\
spectroscopy (HRMS) was performed by separation and mass spectrometric detection techniques
and were performed with an Infinity 1260 UHPLC system (Agilent Technologies, Waldbronn,
Germany) coupled to a 6224 Accurate Mass TOF LC/MS system (Agilent Technologies, Singapore).
Chromatographic separation was achieved using Agilent Zorbax C18 column (Agilent Technologies,
Santa Clara, CA, USA) (100mm
formic acid in water and mobile phase B consisted of 0.1% formic in acetonitrile. Injection volume
×
2.1mm, 1.9 µm particle size). Mobile phase A consisted of 0.1%
of the◦samples solutions was 1
µL. Separation was performed at a constant flow rate of 0.4ml/min
at 40 C. A linear gradient started at 5% mobile phase B and ramped to 95% in 6.5 min, flushed
1.5 min at 95% B. The column was re-equilibrated for 2 min with 5% mobile phase A. Positive ion mass
spectra of the column eluate were recorded in the range of m/z 100–1500 at a measuring frequency of
9500 transients/s and a detection frequency of 4GHz. The Agilent ion source was operated using the
following conditions: pressure of nebulizing gas (N2) was 40 psi; temperature and flow rate of drying
gas (N2) was 320 ◦C and 6 L/min, respectively; The capillary voltage was set to 4 kV, the fragmentor
potential to 120V and the skimmer potential to 75 V. Solvents and chemicals were HPLC grade. Water
was purified by Millipore Water Purification System. All the chemicals, reagents and solvents had
been purified and/or dried in according to well-known literature methods; vendors’ names: UORSY
and Enamine (Kiev, Ukraine).
2.2.1. Synthesis of the MMA-100 Series of Compounds
Preparation of (E)-3-(1H-imidazol-2-yl)-1-(5-methoxypyridin-2-yl)prop-2-en-1-one (MMA-101)
To a solution of 5-methoxypicolinic acid (1a) (0.68 g, 3.58 mmol) in DCM (20 mL) was added
1,1’-carbonyldiimidazole (0.58 g, 3.58 mol) at 30 ◦C. The reaction mixture was stirred at this temperature
for 3 h, whereupon N,O-dimethylhydroxylamine hydrochloride (0.349 g, 3.58 mmol) was added,
and the solution stirred for 2 hr. The reaction mixture was poured into water (20 mL), and the
organic layer separated, washed with brine (30 mL), dried over anhydrous sodium sulfate, and filtered.
The filtrate was concentrated under reduced pressure to give N,5-dimethoxy-N-methylpicolinamide
(2a) (0.632 g, 90% yield).
Methylmagnesium bromide (3.2 M solution in 2-MethylTHF, 1.5 mL) was added via syringe
to a solution of N,5-dimethoxy-N-methylpicolinamide (2a) (632 mg, 3.22 mmol) in THF (12 mL)
under argon atmosphere at 0 ◦C. The resulting mixture was warmed to RT and left to stir for 10h.
The resulting mixture was quenched with aq NH4Cl (8mL) at 0 ◦C and extracted with EtOAc (2
×
15 mL).
Organic layers were combined, washed with brine (10 mL), dried over Na2SO4 and evaporated to give
1-(5-methoxypyridin-2-yl)ethan-1-one (3a) (0.432 g, 87% yield) for use without further purification.
The solution of NaOH (0.336 g, 8.4 mmol) in EtOH was added to the solution of
1-(5-methoxypyridin-2-yl)ethan-1-one (3a) (0.423 g, 2.8 mmol) and 1H-imidazole-2-carbaldehyde
(0.268 g, 2.8 mmol) in EtOH (15 mL) under argon at rt. The reaction mixture was stirred for 24h. After
this time 10% aqueous HCl was added at 0 ◦C to reaction mixture (for establish pH = 4.5) and then
concentrated under reduced pressure. The resulting residue was dissolved in aq NaHCO3 and filtered
to give MMA-101 (0.45 g, 70% yield). Melting point: 190–192 ◦C.1H-NMR (400 MHz, DMSO-d6)
(d, J = 2.9 Hz, 1H), 8.31 (d, J = 16.1 Hz, 1H), 8.11 (d, J = 8.7 Hz, 1H), 7.63 – 7.52 (m, 2H), 7.28 (s, 2H),
δ 8.44
3.94 (s, 3H).13C-NMR (126 MHz, Chloroform-d)
δ 187.68, 158.84, 146.86, 144.20, 137.54, 131.78, 124.72,
121.31, 120.90, 56.54. LC-MS (ESI), RT = 0.775 min, m/z 230.20 [M + H]+; HRMS (ESI), RT = 3.583 min,
m/z 230.0934 [M + H]+, formula C12H11N3O2.

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Preparation of (E)-3-(1H-imidazol-2-yl)-1-(5-methoxypyridin-3-yl)prop-2-en-1-one (MMA-102)
The compound was synthesized from (1b) following the procedure in MMA-101 synthesis,
and gave (0.193g, 65% yield) MMA-102 with the following characterization profile. Melting point:
185–192 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
12.82 (s, 1H), 8.92 (d, J = 2.5 Hz, 1H), 8.28 (dd, J = 8.7,
2.5 Hz, 1H), 7.84 (d, J = 15.6 Hz, 1H), 7.50 (d, J = 15.6 Hz, 1H), 7.32 (s, 2H), 6.99 (d, J = 8.7 Hz, 1H), 3.96
(s, 3H);13C-NMR (126 MHz, DMSO-d6)
δ 186.93, 166.65, 149.51, 143.87, 139.28, 132.16, 127.84, 121.47,
111.49, 54.44. LC-MS (ESI), RT = 0.806 min, m/z 230.0 [M + H]+; HRMS (ESI), RT = 0.55 min, m/z
230.0927 [M + H]+, formula C12H11N3O2.
Preparation of (E)-N-(4-(3-(1H-imidazol-2-yl)acryloyl)phenyl)methanesulfonamide (MMA-103)
NaOH (412 mg, 10.3 mmol) in aqueous solution (6 mL) was added drop-wise to a solution of
N-(4-acetylphenyl)methanesulfonamide (3c) (730 mg, 3.43 mmol) and 1H-imidazole-2-carbaldehyde
(330 mg, 3.43 mmol) in ethanol (8 mL) while stirring. The resulting reaction mixture was stirred at 60 ◦C
for 7h. The ethanol was evaporated, followed by addition of water, and the mixture was neutralized
by citric acid to pH ~7. The product was extracted with DCM, washed two times with water, and
dried over Na2SO4. After evaporation of DCM, the product was purified by HPLC to give MMA-103
(150 mg, 15% yield). Melting point: 216–218 ◦C 1H-NMR (400 MHz, DMSO-d6)
(s, 1H), 8.04 (d, J = 8.6 Hz, 2H), 7.85 (d, J = 15.6 Hz, 1H), 7.48 (d, J = 15.6 Hz, 1H), 7.35 (d, J = 8.6 Hz,
δ: 12.82 (s, 1H), 10.41
2H), 7.31 (s, 2H), 3.14 (s, 3H).13C-NMR (126 MHz, DMSO-d6)
δ: 187.67, 143.96, 143.58, 132.63, 131.94,
130.51, 130.51 121.57, 118.18, 40.5. LC-MS (ESI), RT = 0.735 min, m/z 292.0 [M + H]+.
Preparation of (E)-3-(1H-imidazol-2-yl)-1-(4-(trifluoromethoxy)phenyl)prop-2-en-1-one (MMA-104)
(720mg, 3.54 mmol) of the ketone (3d) was reacted at room temperature for 7h following the same
procedure used in MMA-103, and gave (150 mg, 15% yield) product MMA-104 with the following
characterization profile. Melting point: 150–156.5 ◦C.1H-NMR (400 MHz, DMSO-d6)
δ
12.89 (s, 1H),
8.17 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 15.7 Hz, 1H), 7.58 (d, J = 8.3 Hz, 2H), 7.51 (d, J = 15.6 Hz, 1H), 7.33
(s, 2H).13C-NMR (126 MHz, DMSO-d6)
δ 188.05, 151.97, 143.79, 136.81, 132.90 (2C), 131.13, 131.12, 131.0
(2C), 130.9, 121.34(2C). LC-MS (ESI), RT = 0.938 min, m/z 283.0 [M + H]+; HRMS (ESI), RT = 2.946 min,
m/z 283.0696 [M + H]+, formula C13H9F3N2O2.
Preparation of (E)-5-(3-(1H-imidazol-2-yl)acryloyl)-2-methoxybenzonitrile (MMA-105)
1-(4-methoxyphenyl)ethan-1-one(6)(13.01 g, 0.086 mol, 1eq.) was added to solution of octane
bis(tetrafluoroborate) (F-TEDA) (30 g, 0.086 mol, 1 eq.) and I2 (11 g, 0.043 mol, 0.5eq.) in 100 mL
CH3CN, and the mixture stirred at room temperature for 16 h. The mixture was quenched with aqueous
NaOH-5%, the organic layer separated, and the aqueous layer was extracted with EtOAc (3 × 30 mL).
The organic layers were combined, washed with water (2 × 20 mL) and brine (2 × 15 mL), dried over
Na2SO4, and concentrated in vacuum to give 8.53 g of 1-(3-iodo-4-methoxyphenyl)ethan-1-one (7)
(yield = 61.3%). CuCN(4 g, 0.0465 mol, 1.5eq.) was added to solution of (7) (8.5 g, 0.0309 mol, 1 eq.) in
10 mL DMF, and the mixture stirred at 130 ◦C for 16 h. The reaction mixture was quenched with aqueous
HCl-10%, the organic layer separated, and the aqueous layer was extracted with EtOAc (3 × 20 mL).
The organic layers were combined, washed with water (2
×
10 mL) and brine (10 mL), dried over
Na2SO4, and concentrated in vacuum to give (3.5 g, 65% yield) of 5-acetyl-2-methoxybenzonitrile (3e).
Subsequently, (3e) was reacted with the aldehyde (1 eq) and NaOH (1.3 eq.) following the
procedure used in MMA-101. Water was poured to the reaction mixture, extracted with DCM
(
3 × 10 mL). The organic layers were combined, washed with water (3 × 10 mL), dried over Na2SO4,
and concentrated in vacuum to give(yield = 61.3%) of the crude product. Purification by HPLC (15–25%
water-ACN, flow: 40 mL/min (loading pump 5 mL/min ACN)) yielded (100 mg, 2%) of MMA-105,
which had the following characterization profile. Melting point: 177–178.9 ◦C. 1H-NMR (400 MHz,
DMSO-d6)
δ 12.89 (s, 1H), 8.17 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 15.7 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.30

Biomolecules 2020, 10, 1508
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(d, J = 15.6 Hz, 1H), 7.33 (s, 2H), 3.96 (s, 3H); LC-MS (ESI), RT = 0.840 min, m/z 254.2 [M + H]+; HRMS
(ESI), RT = 0.745 min, m/z 254.0931 [M + H]+, formula C14H11N3O2.
Preparation of (E)-3-(1H-imidazol-2-yl)-1-(6-((tetrahydrofuran-3-yl)oxy)pyridin-3-yl)prop-2-en-1-one
(MMA-106)
Compound1-(6-methoxypyridin-3-yl)ethan-1-one (3b) (0.432 g, 2.86 mmol) was dissolved in
hydrogen bromide solution 33 wt.% in acetic acid and was refluxed for 48 hr. The reaction mixture was
cooled and product filtered to give1-(6-hydroxypyridin-3-yl)ethan-1-one (4b) (0.305 g, 78% yield).
To a solution of 1-(6-hydroxypyridin-3-yl)ethan-1-one (4b) (0.305 g, 2.23 mmol) in dry DMF
(10 mL) was added Cs2CO3 (1.44 g, 4.46 mmol) and the reaction mixture stirred at RT for 30 min.
Following, tetrahydrofuran-3-yl methanesulfonate (0.37 g, 2.23 mmol) was added at RT, and the reaction
mixture was heated at 60 ◦C overnight. The resulting mixture concentrated under reduced pressure.
The resulting residue taken up in water (30 mL) and extracted with EtOAc (3
anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude was purified
×
15 mL), dried over
by HPLC to give 1-(6-((tetrahydrofuran-3-yl)oxy)pyridin-3-yl)ethan-1-one (5b) (0.184 g, 41% yield).
(
5b) was reacted with the aldehyde and NaOH following the procedure for MMA-101, to
give the product MMA-106 (0.126 g, 50% yield) after HPLC purification, which had the following
characterization profile. Melting point 20 ◦C; 1H-NMR (400 MHz, DMSO-d6)
9.11 (t, J = 2.8 Hz, 1H),
δ
8.79 (d, J = 15.0 Hz, 1H), 8.37 (dd, J = 8.7, 2.5 Hz, 1H), 7.78 (s, 2H), 7.57 (dd, J = 15.6, 3.5 Hz, 1H), 7.01
(d, J = 8.5 Hz, 1H), 5.71 – 5.57 (m, 1H), 4.02 – 3.70 (m, 4H), 2.36 – 2.16 (m, 1H), 2.10 – 1.96 (m, 1H);
13C-NMR (151 MHz, DMSO-d6)
δ 185.98, 166.06, 150.31, 141.51, 139.63, 128.78, 127.21, 124.76, 122.57,
112.36, 77.32, 72.97, 66.82, 32.96. LC-MS (ESI), RT = 1.788 min, m/z 286.2 [M + H]+
Preparation of (E)-3-(1H-imidazol-2-yl)-1-(5-((tetrahydrofuran-3-yl)oxy)pyridin-2-yl)prop-2-en-1-one
(MMA-107)
Compound MMA-107 was synthesized from (3a) following the procedure used for MMA-106, and
gave (0.189 g, 50% yield) product with the following characterization profile. Melting point: 149–150 ◦C.
1H-NMR (400 MHz, DMSO-d6)
δ
12.93 (s, 1H), 8.44 (d, J = 2.8 Hz, 1H), 8.31 (d, J = 16.1 Hz, 1H), 8.10 (d,
J = 8.8 Hz, 1H), 7.60 (dd, J = 8.8, 3.0 Hz, 1H), 7.56 (d, J = 16.2 Hz, 1H), 7.29 (s, 2H), 5.27 (t, J = 5.3 Hz,
1H), 4.10 – 3.71 (m, 4H), 2.37–2.20 (m, 1H), 2.10–1.93 (m, 1H); 13C-NMR (151 MHz, DMSO-d6)
187.60,
δ
156.87, 146.80, 144.03, 138.33, 131.62, 124.65, 122.51, 120.93, 72.55, 66.88, 32.73; LC-MS (ESI), RT = 0.757
min, m/z 286.2 [M + H]+; HRMS (ESI), RT = 1.494 min, m/z 286.1185 [M + H]+, formula C15H15N3O3.
2.2.2. Synthesis of the MMA-200 Series of Compounds
Preparation of 1-(4-methoxyphenyl)prop-2-en-1-one (MMA-202)
To a suspension of powdered aluminum chloride (0.723 g, 5.42 mmol) in dry DCM (40 mL) at
0 ◦C was added slowly 3-chloropropionyl chloride(0.455 mL, 4.74 mmol) and anisole (8a) (0.491 mL,
4.52 mmol) under cooling with water. The orange solution was stirred at room temperature for 3 h
and left standing for about 12h. The dark orange solution was poured onto ice (20 g), the organic
layer was separated, and the aqueous layer extracted two times with CH2Cl2. The collected organic
phases were washed with water (2
×
20 mL), several times with aqueous NaOH (2%, 50 mL) until the
organic phase did not turn back to yellow, and then twice with water (10–15 mL). The organic phase
was dried over MgSO4 and the solvent removed under reduced pressure. Purification by gradient
column chromatography [SiO2, CH2Cl2/petroleum ether (40/60), 20:1 to CH2Cl2] gave 0.923 g (97%
yield) of 3-chloro-1-(4-methoxyphenyl)propan-1-one (9a) as a colorless solid.
TEA (0.83 mL) was added to a solution of compound 3-chloro-1-(4-methoxyphenyl)propan-1-one
(0.923 g, 4.64 mmol) (9a) in 20 mL CH3CN, stirred and refluxed for 1 hr. Then the reaction mixture was
cooled, concentrated under vacuum, followed by addition of 50 mL H2O and extraction with DCM
(3
×
10 mL). The organic layers were combined, washed with water (2
×
5 mL), dried over Na2SO4,

Biomolecules 2020, 10, 1508
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filtrated and the solvent removed under reduced pressure at RT. Purification by gradient column
chromatography [SiO2, CH2Cl2/petroleum ether (40/60), 20:1 to CH2Cl2] gave 0.562 g of MMA-202
(78% yield). Melting point 20 ◦C; 1H-NMR (400 MHz, Chloroform-d)
δ
7.93 (d, J = 8.8 Hz, 2H), 7.14 (dd,
J = 17.0, 10.5 Hz, 1H), 6.93 (d, J = 8.8 Hz, 2H), 6.39 (dt, J = 17.1, 1.5 Hz, 1H), 5.84 (dt, J = 10.5, 1.5 Hz,
1H), 3.84 (s, 3H); 13C-NMR (126 MHz, Chloroform-d)
δ 189.21, 163.55, 132.14, 131.02, 130.21, 129.22,
113.85, 55.48; LC-MS (ESI), RT = 1.162 min, m/z 163.00 [M +H]+; HRMS (ESI), RT = 5.487 min, m/z
163.0752 [M + H]+, formula C10H10O2.
Preparation of 1-(4-methoxyphenyl)prop-2-yn-1-one (MMA-204)
To a solution of 4-methoxybenzoic acid (11) (200 mg, 1.31 mmol) dissolved in dry DCM (5 mL),
1,1’-carbonyldiimidazole (212.42 mg, 1.31 mmol) was added proportionally. Reaction mixture was
proceeded by ultrasound (US) for 20 min. Next, N,O-dimethylhydroxylamine hydrochloride (193 mg,
1.97 mmol) and TEA (199.5 mg, 1.97 mmol) were added, and the reaction mixture was proceeded by
US for 5h. The mixture was washed with water and purified by column chromatography (Eluent
EtOAc\Hexane) to give N,4-dimethoxy-N-methylbenzamide (12) (189 mg, 73.8% yield).
Trimethylsilylacetylene (89 mg, 0.902 mmol) was dissolved in THF (5 mL) and cooled to 0 ◦C. To this
solution was added n-BuLi as a 2.5%solution in hexane (0.4 mL, 0.902 mmol), dropwise. After 20 min,
Weinreb amide (12) (160 mg, 0.820mmol) was added dropwise via syringe, and the reaction was allowed
to slowly warm to 25 ◦C over 2 h. The reaction was diluted with Et2O, poured into saturated aqueous
citric acid solution, and the aqueous phase was extracted twice by EtOAc. Organic phases were dried
with Na2SO4, filtered, and solvent was evaporated. The residue was purified by flash chromatography
(Eluent EtOAc
70.3% yield).
\Hexane) to give 1-(4-methoxyphenyl)-3-(trimethylsilyl)prop-2-yn-1-one (13) (134mg,
1-(4-methoxyphenyl)-3-(trimethylsilyl)prop-2-yn-1-one (13) (250 mg, 1.08 mmol) was dissolved in
THF (5 mL) and cooled to 0 ◦C, and potassium hydrofluoride (51 mg, 0.646 mmol) in 2 mL of water
was then added. Reaction mixture was stirred for 5h, and the reaction was allowed to slowly warm to
25 ◦C over 2h. The reaction was diluted with CH2Cl2, the mixture was poured into saturated aqueous
hydrochloric acid solution, and the aqueous phase was extracted twice by CH2Cl2. Organic phases
were dried with Na2SO4, decanted, and solvent was evaporated. The residue was purified by flash
chromatography (Eluent EtOAc
\
Hexane) to give MMA-204 (114 mg, 66% yield). Melting point 86 ◦C;
8.10 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 3.86 (s, 3H), 3.36
1H-NMR (400 MHz, Chloroform-d)
δ
(s, 1H).13C-NMR (126 MHz, Chloroform-d)
δ 175.94, 164.79, 132.15, 131.16, 129.60, 113.96, 80.41, 80.04,
55.63; HRMS (ESI), RT = 5.542 min, m/z 161.0597 [M + H]+, formula C10H8O2.
Preparation of 1-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)prop-2-en-1-one (MMA-205)
Phenol (8b) (3.65 g, 0.039 mol, 1 eq.) was dissolved in CH2Cl2 (20 mL) and cooled to 0 ◦C. AlCl3
(5.5 g, 0.0403 mol, 1.03 eq.) was added in small portions, keeping internal temperature below 5 ◦C.
The reaction mixture was stirred for 10 minutes, followed by dropwise addition of a solution of
3-chloropropanoyl chloride (5 g, 0.0397 mol, 1.01 eq.) in CH2Cl2 (10 mL), and with stirring overnight
at 0 ◦C. The reaction mixture was quenched with H2O (30 mL), the layers separated, and the water
phase was extracted with CH2Cl2 (2
×
20 mL). The combined organic layers was washed with H2O
(2 × 10 mL) and brine (2 ×
10 mL), dried over Na2SO4 and evaporated. The obtained crude product
was purified by column chromatography to give 3-chloro-1-(4-hydroxyphenyl)propan-1-one as orange
oil (9b) (0.5 g, 7% yield).
To a precooled (0 ◦C) solution of (9b) (0.5 g, 2.7 mmol) in dihydropyran (5 mL), catalytical amount
of TsOH*H2O was added and the mixture stirred overnight. Evaporation of the solvent gave the crude
product of 3-chloro-1-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)propan-1-one (10) (1 g, 0.0017mol,
about 45% yield), which was used in the next step without purification.
◦
To the solution of (10) (crude 1 g, ~1.7mmol) in CH2Cl2 (10 mL) at 0 C, a solution of TEA
(1 g, 10mmol, 4–5eq.) in CH2Cl2 (10 mL) was added in a dropwise, followed by stirring overnight.

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Evaporation of the solvent and purification of the crude residue resulted in MMA-205 (120 mg,
0.5 mmol, 25% yield); 1H-NMR (500 MHz, Chloroform-d)
7.95 (d, J = 8.7 Hz, 2H), 7.17 (dd, J = 17.1,
δ
10.6 Hz, 1H), 7.12 (d, J = 8.7 Hz, 1H), 6.43 (d, J = 17.0 Hz, 1H), 5.88 (d, J = 10.5 Hz, 1H), 5.53 (s, 1H), 3.91
– 3.80 (m, 1H), 3.66 – 3.59 (m, 1H), 2.07 – 1.95 (m, 1H), 1.90 (dd, J = 7.1, 3.5 Hz, 2H), 1.80 – 1.47 (m, 3H);
13C-NMR (126 MHz, Chloroform-d)
δ 189.4 (s), 161.1 (s), 132.2 (s), 130.9 (s, J = 7.1 Hz), 129.3 (s), 116.1
(s), 96.1 (s), 62.1 (s), 30.1 (s), 25.1 (s), 18.5 (s); LC-MS (ESI), RT = 3.061 min, m/z 149.2 [M -C5H9O +H]+;
HRMS (ESI), RT = 1.085 min, m/z 149.06174 [M -C5H9O +H]+, formula C9H8O2.
2.3. In Vitro Time-Dependent Hb Oxygen Equilibrium Studies Using Normal Whole Blood
To determine the metabolic stability of the compounds in whole blood, the MMA compounds
were used to conduct time-dependent studies on Hb oxygen equilibrium using normal whole blood
as previously described [30]. Briefly, blood samples (hematocrit 30%) in the presence of 2 mM
concentration of compounds were incubated at 37 ◦C for 24 h with shaking (at 140 rpm). At 1.5, 3,
6, 8, 12, and/or 24h time intervals, aliquots of this mixture were removed and further incubated in
TM8000 Thin film tonometer (Meon Medical Solutions) to equilibrate at oxygen tensions 6, 20, and
40 mmHg for approximately 10 minutes at 37 ◦C. The samples were then aspirated into an ABL 800
Automated Blood Gas Analyzer (Radiometer) to determine the pH, partial pressure of CO2 (pCO2),
partial pressure of oxygen (pO2), and Hb oxygen saturation values (SO2). The measured values of pO2
(mmHg) and SO2 were then subjected to a non-linear regression analysis using the program Scientist
(Micromath, Salt Lake City, UT) to estimate P50 as previously reported [30]. The observed P50 shifts
values in %P50 shifts were plotted as function of time [hrs].
2.4. In Vitro Hemoglobin Modification, Oxygen Equilibrium and Antisickling Studies Using Human
homozygous Sickle cell (SS) Blood
The MMA series of compounds (MMA-101, MMA-102, MMA-103, MMA-104, MMA-105,
MMA-106, MMA-107, MMA-202, MMA-204, and MMA-205) and the positive control vanillin were
studied for their abilities to inhibit hypoxia-induced RBC sickling (RBC morphology study) and
increase Hb oxygen as previously published [28–33]. Briefly, homozygous SS blood (hematocrit: 20%)
suspensions were incubated under air in the absence or presence of 2 and 5 mM concentration of test
compounds at 37 ◦C for 1h. Following, the suspensions were incubated under hypoxic condition (2.5%
oxygen) at 37 ◦C for 2h. Aliquot samples were fixed with 2% glutaraldehyde solution without exposure
to air, and then subjected to microscopic morphological analysis. The residual samples were washed in
phosphate-buffered saline, and hemolyzed in hypotonic lysis buffer for the Hb oxygen equilibrium
experiment. For the study, approximately 100 µL aliquot samples from the lysate were added to 4 mL
of 0.1M potassium phosphate buffer, pH 7.0, in a cuvette and subjected to hemoximetry analysis using
Hemox™ Analyzer (TCS Scientific Corp.) to assess P50 shifts and Hill coefficient values (n50).
2.5. Reverse-Phase HPLC Studies to Determine which Hb Subunit Interacts with the Compounds
To determine whether the MMA compounds bind to the α- and/or β-globin chains to effect
the Hb oxygen binding property, hemolysates from the above antisickling studies with MMA-202,
MMA-204, and MMA-205 (2 mM) were subjected to reversed-phase (RP) HPLC on a Hitachi D-7000
HSM Series, using a Jupiter 5
a gradient from 20% to 60% acetonitrile in 0.3% trifluoroacetic acid in 15 min, with UV detection at
215 nm as previously published [28].
µ
m C-4 50
×
4.6 mm column, (Phenomenex®, Torrence, CA) and
2.6. Reactivity of MMA Compounds toward Csyteine Residues of Hb
The accessible sulfhydryl groups in Hb, and their reactivity with the MMA compounds, MMA-102,
MMA-202 and MMA-204, were determined by following the exchange reaction of the thiols in Hb and
DTNB (Ellman’s reagent) at 412 nm (
an aqueous solution of Hb (50 M in PBS) was mixed with MMA-102, MMA-202, MMA-204, or the
ε
= 14,150 M−1cm−1) as previously reported by us [36]. Briefly,
µ

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positive control Ethacrynic acid (ECA) at 2 mM final concentration in a final volume of 500
µL. ECA
is known to form covalent interaction with βCys93 of Hb [41]. The mixture was incubated at room
temperature for 3 h, with shaking at 100 rpm, and then transferred ◦to a microfiltration centrifugal
tube (MWCO 10 kDa) and centrifuged at 7000 rpm for 30 min at 4 C to separate Hb from excess
reagents. Hb was washed with PBS and centrifuged again to a final volume of 100
25 L of each Hb solution was added to 0.1 M potassium phosphate buffer (475 L) at pH 8.0 and
incubated at 25 ◦C for 1 h (non-DTNB control samples). Another set of 25
L Hb solution was added to
phosphate buffer (465 L) and 10
L of DTNB (10 mM in buffer) and incubated at 25 ◦C for 1h. Before
µL. Following,
µ
µ
µ
µ
µ
centrifuging the non-DTNB control samples, the absorbance of each sample was noted at 576 nm to
determine the concentration of Hb in each sample. Both sets of tubes were centrifuged using different
centrifugal filters (7000 rpm, 20 min, 4 ◦C) to collect the yellow filtrate (2-nitro-5-thiobenzoate), which
was quantified by measuring absorbance at 412 nm.
3. Results and Discussion
3.1. Rationale for Design of MMA Compounds
Aromatic aldehydes, the most studied antisickling agents, form Schiff base through a reversible
chemical reaction with the N-terminal
and increase the protein affinity for oxygen [20
metabolism by aldehyde dehydrogenase (ALDH), particularly in the liver and RBCs [38
α
Val1 amine of the
33]. Nonetheless, their susceptibility to oxidative
40] leading to
α-subunit to stabilize the R-state HbS
–
–
poor PK properties, have greatly hampered their development [30,33,42]. The chronic nature of SCD,
as well as the large amount of Hb that needs to be modified to reach therapeutic dose, clearly requires
long acting antisickling agent. To overcome the aldehyde moiety limitation, we rationally developed a
new class of antisickling agents (imidazolylacryloyl derivatives; Figure 1) that incorporated a stable
Michael addition reactive center that we hypothesized would form covalent interaction with the Hb
β-subunit residue βCys93 to increase Hb oxygen affinity [36,37]. Although, these compounds were
metabolically stable, they showed weak or no antisickling activity. Interestingly, crystallographic
study of a representative compounds, KAUS-15 and KAUS-12, showed that these compounds instead
of binding to
cavity of deoxygenated Hb to make covalent interaction with the N-terminal
-subunits [36]. Thus, unlike aromatic aldehyde that bind to liganded Hb and stabilize the R-state
βCys93, because of their carboxylate moiety, were being directed to the central water
α
Val1 amines of the two
α
Hb to increase the protein affinity for oxygen, binding of the KAUS compounds to deoxygenated
Hb ultimately led to no significant effect on Hb affinity for oxygen, and in some instances even
decreased Hb affinity for oxygen. Accordingly, we reported a second generation of KAUS compounds
by removing the acidic group (azolylacryloyl derivatives; Figure 1), expecting the novel compounds to
direct and bind to
βCys93. Expectedly, these compounds, as exemplified by KAUS-33 and KAUS-38,
bind covalently to βCys93 [37].
Figure 1. Earlier Hb modulators based on Michael Addition scaffold.
KAUS-33 and KAUS-38 showed promising antisickling activities with long durations of action;
however, they have limited solubility, and their potency was significantly less than aromatic
aldehydes [37]. This potentially would require larger doses in order to achieve sufficient therapeutic
effects. Since KAUS-33 and KAUS-38 were considered viable leads, we decided to refine and optimize

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these azolylacryloyl molecules to increase their biological activity while still maintaining the Michael
addition reactive center for their metabolic stability (Figure 2).
Figure 2. Design of new Michael Addition compounds as hemoglobin modifiers.
In the first modification (Figure 2), compounds MMA-101, MMA-102, MMA-106, and MMA-107
(imidazyl Michael addition compounds) have the benzene ring replaced with a pyridine ring to increase
solubility and RBC distribution, as well as reactivity of the Michael addition moiety. Similar observations
have been reported for aromatic aldehydes, where substitution of benzene with pyridine considerably
increased the compounds reactivity and RBC distribution [20]. MMA-106 and MMA-107 are pyridyl
derivatives of KAUS-33, where the acid-labile 2-tetrahydropyranyl moiety has been replaced with the
3-tetrahydrfuranyl, a smaller, polar but more stable group. The compounds MMA-103, MMA-104 and
MMA-105, also imidazyl Michael addition compounds contain electron-withdrawing groups that are
expected to decrease the electron density on the benzene ring (c.f. KAUS-33 and KAUS-38) aiming to
investigate this electronic factor on the binding potency. In addition, the sulfonamide analogue MMA-103
is also able to form several hydrogen bonding as both acceptor and donor within the active site.
In another modification exercise to increase potency, we made a radical change to the scaffold with
the aim of decreasing possible steric hindrance at the βCys93 reactive site. For covalent interaction
to form between KAUS molecules and
has to move closer to the SH group of
β
Cys93, the imidazole moiety of the Michael addition group
Cys93, while at the same time avoiding steric contact with
β
the surrounding binding site residues. As indicated by manually docking the KAUS compounds to
the binding site, such potential steric interactions would ensue unless there is a re-arrangement of
the binding site residues, which could have contributed to the marginal potency of these compounds.
Therefore, three subset of compounds, namely MMA-202, MMA-204, and MMA-205, without the
imidazolyl group (non-imidazyl Michael addition compounds) were designed and synthesized to test
this hypothesis.

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3.2. Chemical Synthesis
The MMA-100 series of compounds were prepared as outlined in Scheme 1, and detailed syntheses
are described in the experimental section. Briefly, the compounds were synthesized by converting the
methoxypicolinic acids (1a,1b) to the corresponding ketones (3a,3b) via Weinreb amide intermediate
(2a,2b). The ketones were condensed with the aldehyde, 1H-imidazole-2-carbaldehyde, to form the
Michael addition products MMA-101 and MMA-102. MMA-103 and MMA-104 were synthesized by
reacting the corresponding ketones (3c,3d) with the aldehyde, 1H-imidazole-2-carbaldehyde under the
same reaction conditions. For MMA-106 and MMA-107, the methoxy group of the ketones (3a,3b) was
converted to the hydroxyl group of the ketones (4a,4b) to make the corresponding tetrahydrofuran-3-oxy
group in (5a,5b), which were then reacted with the aldehyde to form the final products MMA-106
and MMA-107. The 5-acetyl-2-methoxybenzonitrile (3e) was synthesized by iodination followed
by cyanation of methyl 4-methoxyphenyl ketone (6), which was condensed with the aldehyde to
form MMA-105.
Scheme 1. Synthetic scheme of MMA-100 series. (
N,O-dimethylhydroxylamineHCl, 2 h. (ii) CH3MgBr, THF, Ar, 0 ◦C
2-carbaldehyde, NaOH, EtOH, Ar, RT (60 ◦C for MMA-103). (iv) HBr/AcOH, reflux, 48 h. (
i
) (1) 1,10-carbonyldiimidazole, DCM, 30 ◦C, 3 h; (2)
RT, overnight. (iii) 1H-imidazole-
) (1) Cs2CO3,
60 ◦C, overnight. (vi) I2, F-TEDA,
→
v
dry DMF, RT, 30 min; (2) tetrahydrofuran-3-yl methanesulfonate, RT
→
CH3CN, RT, 16 h. (vii) CuCN, 130 ◦C, 16 h. The yields ranged from 2% to 70%.
The MMA-200 series of compounds were prepared as outlined in Scheme 2, and detailed syntheses
are described in the experimental section. MMA-202 and MMA-205 synthesis started with acylation
of anisole and phenol (8a and 8b) under Lewis acid catalysis to give the ketonic intermediates (9a
and 9b). The phenol (9b) was converted to 2-tetrahydropyranyl ether (10) by the reaction with
3,4-dihydro-2H-pyran (DHP) under a Brønsted acid catalysis. The 3-chloropropionyl ketones (9a
and 10) were converted to the enone Michael addition final products MMA-202 and MMA-205 via

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treithylamine-initiated
β-elimination. For the alkyne MMA-204, 4-methoxybenzoic acid (11) was
coupled with N,O-dimethylhydroxylamineHCl to form the Weinreb amide (12). The amide was reacted
with trimethylsilylacetylene forming (13), followed by a fluoride-induced elimination of the protective
silyl group to produce the ynone final product MMA-204. The yields of the reactions described above
ranged from 15% to 70%.
Scheme 2. Synthetic scheme for MMA-200 series. (
(0 ◦C overnight for 9b). (ii) TEA, CH3CN, reflux, 1 h. (iii) TsOH. H2O, dihydropyran, 0 ◦C, overnight.
iv) TEA, DCM, 0 ◦C, overnight. ( ) (1) 1,10-carbonyldiimidazole, dry DCM, ultrasound, 20 min; (2)
i
) AlCl3, dry DCM, 0 ◦C
→
RT 3 h then 12 h standing
(
v
N,O-dimethylhydroxylamine HCl, TEA, ultrasound, 5 h. (vi) trimethylsilylacetylene in THF, n-BuLi in
hexane, 0 ◦C
to 70%.
→
25 ◦C, 2 h. (vii) KHF2, THF, 0 ◦C for 5 h, then 25 ◦C for 2 h. The yields ranged from 15%
3.3. MMA Compounds Demonstrated Sustained Pharmacologic Effects In Vitro
As noted above, aromatic aldehydes suffer from oxidative metabolism by aldehyde dehydrogenase,
aldehyde oxidase in the liver, blood and other tissues that leads to short half-life and suboptimal
bioavailability [28
in vitro, vanillin, 5-HMF, and several other aromatic aldehydes showed significant metabolism of the
aldehyde [29 33], resulting in shortening of their allosteric and antisickling activities [32 33], which
contrasts with Michael addition compounds that showed longer duration of action [36 37]. Thus,
,30,42,43]. In a recent study, we also showed that when incubated in whole blood
–
,
,
one of our main objectives for developing these compounds was to improve on the pharmacokinetic
properties of aromatic aldehydes by replacing the aldehyde moiety with a more metabolically stable
Michael addition reactive center, that is expected to increase the duration of action of these compounds.
This was tested by incubating 2 mM of the MMA compounds with fresh normal adult whole blood
(Hct of ~30%) at 37 ◦C as a surrogate measure of systemic metabolism, and following their effect on Hb
oxygen binding affinity. At defined time points (1.5, 3, 6, 8, 12, or 24 h), aliquot samples were drawn,
subsequently analyzed for their P50-shifts relative to the initial P50 value, using three-point tonometry,
and the results presented in Figure 3.

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Figure 3. Time-dependent P50-shift of HbA in normal blood (30% hematocrit) incubated with 2 mM
test compound. ( ) Effect of MMA-100 series of compounds on Hb affinity for oxygen. ( ) Effect of
MMA-200 series of compounds on Hb affinity for oxygen.
A
B
The results are shown in Figures 4 and 5, and in the Table 1. The MMA-200 series of compounds
showed the most potent antisickling activities (Figure 4A; Table 1). MMA-202, with the highest
biological effect, inhibited sickling by 50% and 67% at 2mM and 5mM, respectively. MMA-204, the
second most potent compound inhibited sickling by 42% and 63%, and lastly MMA-205 with 32% and
52% inhibitory activities, respectively. These values compare with sickling inhibition of 16% and 53%
by vanillin at 2 mM and 5mM, respectively, suggesting significant improvement in the antisickling
activities of the MMA-200 series of compounds especially at the lower dose of 2 mM. The slightly
antisickling potency of MMA-202 over MMA-204 may suggest that the propenoyl reactive center has
a slight advantage over the propynoyl reactive center since the two compounds are structurally similar.
It also appears that the more bulky THP substituent of MMA-205 is not optimal for biological activity
since MMA-202 and MMA-204 with smaller methyl substituent showed better antisickling activity.

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Figure 4. Concentration-dependent SS cells (20% hematocrit) sickling and oxygen equilibrium (P50
shift in vitro by MMA-200 series of compounds. Cyan and red bars denote compound concentrations
of 2 mM and 5 mM, respectively. ( ) Sickling inhibition of SS cells under hypoxia for 2 h, reported
as mean values from three replicates, and calculated by normalizing to untreated control samples as
previously reported [28]. ( ) Degree of shift in P50. % P50 values were calculated by normalizing to
untreated control samples as shown in Table 1. Hemolysates from the antisickling assay were used
for the oxygen equilibrium study. ( ) Representative oxygen equilibrium curves of lysates from the
)
A
B
∆
C
MMA-202, MMA-204, and MMA-205 antisickling study.
Figure 5. Cont.

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Figure 5. Concentration-dependent SS cells (20% hematocrit) sickling inhibition and oxygen
equilibrium (P50) shift in vitro byMMA-100 series of compounds. Cyan and red bars denote compound
concentrations of 2 mM and 5 mM, respectively. (
for 2 h, reported as mean values from two replicates, and calculated by normalizing to untreated
control samples as previously reported.[28] ( ) Degree of shift in P50. % P50 values were calculated by
A) Sickling inhibition of SS cells under hypoxia
B
∆
normalizing to untreated control samples. Hemolysates from the antisickling assay were used for the
oxygen equilibrium study.
Table 1. Oxygen equilibria and antisickling studies on MMA-200 series of compounds using human
sickle blood a.
∆P50 (%) c
Sickling Inhibition (%) b
Compound
2.0 mM
5.0 mM
2.0 mM
5.0 mM
MMA-202
MMA-204
MMA-205
Vanillin
MMA-101
MMA-102
MMA-103
MMA-104
MMA-105
MMA-106
MMA-107
Vanillin
49.5 ± 14.8
41.5 ± 15.3
31.9 ± 19.3
15.9 ± 5.5
21.3 ± 3.7
33.2 ± 8.9
20.9 ± 2.9
10.4 ± 2.3
11.7 ± 9.6
35.3 ± 13.7
1.3 ± 3.7
66.8 ± 6.6
63.2 ± 19.4
51.5 ± 12.4
53.0 ± 3.9
25.9 ± 8.8
27.1 ± 2.1
14.5 ± 5.5
12.4 ± 5.4
13.5 ± 2.8
33.5 ± 10.0
9.1 ± 2.9
19.7 ± 2.2
19.7 ± 4.0
10.9 ± 4.8
11.5
43.1 ± 9.1
32.9 ± 3.7
28.5 ± 2.8
26.5
17.4
7.6
2.0
3.5
0.4
12.0
3.0
11.5
22
12.3
6.9
8.4
0.5
21.2
13.2
26.5
15.9 ± 5.5
53.0 ± 3.9
a
All studies were conducted with SS cells suspensions (20% hematocrit) incubated with 2 and 5 mM of each test
compound. The final concentration of DMSO was < 2% in all samples, including in control samples. b % Sickled cells,
reported as mean values from 3 replicates for the MMA-200 series of compounds and 2 replicates for the MMA-100
c
series of compounds. %P50 is the oxygen pressure at which the hemolysates are 50% saturated with oxygen.
∆
P50(%) was mean values from 3 replicates for the MMA-200 series of compounds, and single measurements for the
MMA-100 series of compounds.
The six tested MMA-100 series of compounds, including MMA-101, MMA-102, MMA-104,
MMA-105, MMA-106, and MMA-107 achieved maximum P50 shifts of 12–18%, with MMA-102 and
MMA-104 showing the most shift (Figure 3A). Nonetheless, MMA-102 and MMA-104 show significantly
different activity profiles. While MMA-104 showed almost no P50 shift at 24 hrs from its high activity
of 18% at 1.5h, the rest of the compounds appear to show sustained activity, and in fact, three of the
compounds, MMA-101, MMA-102, and MMA-106 showed increasing activity even at 24 hrs (P50 shifts
of 15%, 18%, and 15%, respectively). With the exception of MMA-105, the rest of the compounds

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appear to show relatively fast onset, although their activities seem to decline at the 3 h time point and
then started going up again. (Figure 3A).
Interestingly, the non-imidazyl MMA-200 compounds showed significantly more potent activity
(P50 shifts of 24–31%) than the imidazyl MMA-100 compounds (P50 shifts of 12–18%), but with delayed
activity with maximum P50 shifts at ~6h, which persisted until 8 h, followed by a decrease to the 24 h
time (Figure 3B). MMA-202 showed the highest P50 shift (31%), followed by MMA-204 (27%), and
lastly MMA-205 (24%) (Figure 3B), indicating that the bulkier tetrahydro-2H-pyran-2-yl (THP) group
had no positive impact on the overall binding and/or Hb oxygen affinity. Interestingly, propynoyl
MMA-204 showed the fastest onset with a P50 shift of 20% at 3h, with the propenoyl MMA-202 and
MMA-205 showing corresponding shifts of 17% and 11%, respectively.
It is clear from the above study that the MMA-200 series of compounds are more potent than the
MMA-100 series of compounds; however, the latter seem to show better-sustained activity in whole
blood. We speculate that the MMA-100 compounds with non-terminal propenone reactive center are
less susceptible to metabolism compared to the MMA-200 compounds with terminal propenoyl or
propynoyl reactive centers. Nonetheless, all the MMA series of compounds appear to show better
metabolic stability or longer duration effect than typical aromatic aldehydes, at least in whole blood,
the latter are known to lose their activity after 2–4 hours [29,32,33].
Unlike the MMA-200 series of compounds, the MMA-100 series of compounds, for the most part
showed significantly lower antisickling activities. Interestingly, even though MMA-102 and MMA-106
show relatively high activities, they were not dose-dependent (Figure 5A). The antisickling results
show the same trend as the above oxygen affinity results, where the MMA-200 series of compounds
with terminal propenoyl or propynoyl reactive centers are more biologically active than the MMA-100
series of compounds with the non-terminal 2-substituted propenone reactive center.
Using aliquots from the same incubation sample from the above antisickling study, we tested
the compounds effect on Hb affinity for oxygen at 2 mM and/or 5mM, and the results are shown
in Figure 4B,C, Figure 5B and Table 1. The high antisickling activity observed for the MMA-200
series of compounds are reflected in the significant increase in Hb oxygen affinity, which also follow
a similar trend as the antisickling results. Figure 4C presents representative oxygen equilibrium
curves of lysates from the MMA-202, MMA-204, and MMA-205 antisickling study that clearly show
dose-dependent effect. At 2mM and 5mM, MMA-202 increased Hb oxygen affinity by 20% and
43%, respectively, followed by MMA-204 with 20% and 33%, and lastly MMA-250 with 11% and
29%, respectively (Figure 4B). These values compare with 12% and 28% by vanillin, respectively,
suggesting significant improvement in the allosteric activity of these compounds, especially MMA-202
and MMA-204. As previously reported for allosteric effectors of Hb (T-state or R-state Hb stabilizers),
the three MMA-200 series of compounds showed major effect on Hb cooperativity [30]. The shape of
the OEC changed from sigmoidal to hyperbolic with increasing compound concentration (Figure 4C).
At 5 mM concentration, the Hill coefficient decreased from 2.86
±
0.7 (unliganded) to 1.96
±
0.08
(MMA-202), 1.98 0.08 (MMA-204), 2.07 0.04 (MMA-205), and 2.02
±
±
±
0.07 for the control vanillin.
The reduced cooperativity is expected because of the apparent weakening of interdimer interactions in
the T-state and strengthening of the interdimer interactions in the R-state with compound binding.
As expected from their relatively weak to no antisickling activities, the MMA-100 series of
compounds did not show any significant effect on Hb affinity for oxygen with SS blood (Figure 5B).
Clearly, the MMA-200 series of compounds are potently superior to the MMA-100 series of compounds.
Likely, the MMA-200 compounds with terminal reactive center encounter less steric hindrance to
the
βCys93 binding site, while the MMA-100 compounds because of the non-terminal 2-substituted
propenone reactive center (as part of a bridge) require significant re-arrangement of the
βCys93
binding pocket to allow for effective compound interaction, resulting in reduced binding affinity for
the latter compounds.
Comparatively, the lead compounds, KAUS-33 and KAUS-38 (at 2 mM concentration) inhibited
sickling by 7% and 9%, and increased Hb oxygen affinity by 15% and 12%, respectively [37]. Likewise,

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the aromatic aldehyde, 5-HMF (at 2 mM concentration), which recently underwent phase I/II clinical
studies for the treatment of SCD but failed due to poor PK properties (as a result of metabolically unstable
aldehyde) inhibited SS sickling and increase Hb oxygen affinity by 25% and 33%, respectively [28]. These
observations suggest a successful outcome of our targeted structural modifications, especially with
MMA-202 and MMA-204. An interesting observation worth pointing out is that despite the relatively
low P50 shifts (20%) at 2 mM concentration, MMA-202 and MMA-204 demonstrated substantial
antisickling effects of 50% and 42%, respectively. Note that this compares with 25% sickling inhibition
and 33% P50 shift by 5-HMF, and 16% sickling inhibition and 12% P50 shift b.
3.4. MMA Compounds React and Modify Hb α-Globin and/or β-Globin
The antisickling activity of aromatic aldehydes is dependent on Schiff base interaction between
the aldehyde moiety and the Hb
thiols, it is dependent on disulfide bond formation between the thiol sulfur and Hb
atoms at the surface of the protein [34 35]. Michael addition compounds, like the thiols, also form
covalent interaction between their -unsaturated carbon and the sulfur atom of Cys93 to affect their
antisickling activities [36 37]. Interestingly, some Michael addition compounds, e.g., KAUS-12 and
KAUS-15 as mentioned above are known to also bind to Val1 of Hb at the -cleft in addition to
their Cys93 binding, the former only observed with deoxygenated Hb, which incidentally led to
α
Val1 amines (Schiff base adduct) at the
α
-cleft [20
–
33]. while for
βCys93 sulfur
,
β
β
,
α
α
β
stabilization of the T-state Hb to decrease the protein affinity for oxygen [36]. We investigated which
Hb subunits are modified by our test compounds, by performing reverse phase HPLC analyses study
on the MMA-200 series of compounds, MMA-202, MMA-204, and MMA-205 (2 mM), and the results
are shown in Figure 6. The top chromatogram (untreated control sample) shows
and -globin peaks. The -globin represents ~7% of the -like chains. The second chromatogram
shows modification of both -globin (~27%) by MMA-202, followed by the
chromatogram for MMA-205, with ~20% modified -globin peak, which co-elutes with the -globin
peak (26.4%
10% modified
exclusively with the
is forming covalent interactions with the
other Hb modifiers, binding of the MMA-200 series of compounds at the
disruption of the T-state stabilization salt-bridge interaction between His146 and
allosteric equilibrium to the R-state to increase the oxygen affinity of the protein [34
binding site is located on the surface of the protein, and interaction with Cys93 is also expected to
contribute to the antisickling activity by weakening the interactions between adjacent HbS molecules in
the polymer. Of the three compounds, MMA-202 uniquely also binds to the -globin, and most likely
at the -cleft to make covalent interaction with Val1 amine as observed for aromatic aldehydes and
β-globin, α-globin
γ
γ
β
β-globin (~39%) and
α
β
γ
β
+
γ
). Finally, a similar pattern is seen with MMA-204, but with only approximately
-globin (~17% ). It is clear that while MMA-204 and MMA-205 appear to bind
-chain, MMA-202 binds to both the -chain and -chain. Most likely, MMA-202
Val1 amine at the -cleft, as well as the Cys93 sulfur. Like
Cys93 site should lead to
Asp93, shifting the
35]. The Cys93
β
β + γ
β
α
β
α
α
β
β
β
β
,
β
β
α
α
α
the Michael addition compounds KAUS-12 and KAUS-15. However, unlike, KAUS-12 and KAUS-15
that bind to the T-state Hb and concomitantly decrease the protein affinity for oxygen, we expect
MMA-202 to bind to the R-state Hb and contribute to the observed biological activities, consistent with
its potent antisickling effect.

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Figure 6. Reverse-phase HPLC of 0 mM (control) and 2 mM of MMA-202, MMA-204, and MMA-205
after incubation with intact SS cells. Identities of modified peaks were assigned based on the balance
between α and β-like (β and γ globin) chains.
3.5. MMA Compounds React with Free βCys93 of Hb
Our expectation was that the MMA compounds will bind to
activity, and although the RP-HPLC study suggests binding of the compound to the
does not identify Cys93 as the interacting amino acid. To determine whether Cys93 is indeed the
-subunit residue involved in the interaction with these molecules, we studied the reactivity of the
βCys93 to effect their antisickling
β-subunit, it
β
β
β
MMA compounds, MMA-102, MMA-202 and MMA-204 (at 2 mM concentration) with the accessible
sulfhydryl groups in Hb that was quantified by the disulfide exchange reaction of the cysteine thiols
and DTNB (Ellman’s Reagent) at 412 nm. There are six cysteine residues in Hb, including a pair each
of
crystallography, and DTNB assay [34
Cys93 because it is the only cysteine that is solvent exposed for reaction [36
solution-based sulfhydryl assay identified only two accessible thiols, out of the six present in Hb that
are likely the two Cys93 residues. As expected from their biological potency, the MMA-200 series of
compounds, as exemplified by MMA-202 and MMA-204 were the most reactive, showing only about
10% freely available SH of Cys93 (i.e., forming ~90% adduct with Cys93) (Figure 7), whereas the
least potent MMA-102 showed only 10% adduct formation. Ethacrynic acid (ECA), which has been
studied for its antisickling activity [36 37 41], is known to form covalent adduct with Cys93 showed
70% adduct with Cys93 (Figure 7). It is clear from this study that the biological activities of the MMA
βCys93,
α
Cys104, and
β
Cys112, however as previously reported using mass spectroscopy, X-ray
37 44 45], compounds preferentially form covalent adduct with
37 44]. Consistently, the
–
,
,
β
,
,
β
β
β
,
,
β
β

Biomolecules 2020, 10, 1508
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compounds are in part due to their reaction with
the non-polymer forming R-state HbS.
βCys93 that leads to an increasing concentration of
100
80
60
40
20
0
Control
ECA
MMA 102 MMA 202 MMA 204
Figure 7. Percentage free SH available on Hb after 3h incubation with MMA compounds.
4. Conclusions
Even though aromatic aldehydes remain promising as antisickling agents, their therapeutic
development into clinically useful drugs have been hampered by poor PK properties due in most part
to the metabolically unstable aldehyde moiety, with the notable exception of Voxelotor. Considering
the fact that Voxelotor is currently the first and only approved molecule in the class of Hb modifiers,
the need remains to develop additional candidates. We have developed novel compounds with
Michael addition reactive centers that not only showed superior potency over several known aromatic
aldehydes, but most importantly, these compounds show improved in vitro metabolic profiles. It is
expected that these properties will translate into sustained and improved pharmacologic activities
in vivo.
Author Contributions: A.M.O., O.A., M.K.S., and M.E.E.-A. contributed to study conception. Y.A.M., M.E.E.-A.,
and A.M.O. synthesized the compound under study. O.A., M.K.S., M.S.G., and S.D.P. collected and performed
biochemical experiments. A.M.O., O.A., M.K.S., and M.E.E.-A. analyzed and interpreted data. A.M.O, O.A.,
M.K.S., and M.E.E.-A. wrote the manuscript. All authors provided final approval of the manuscript. All authors
have read and agreed to the published version of the manuscript.
Funding: This project was funded by the National Plan for Science, Technology, and Innovation (MAARIFAH),
King Abdulaziz City for Science and Technology, the Kingdom of Saudi Arabia; Award number 14-BIO80-03.
Acknowledgments: The authors acknowledge with thanks Science and Technology Unit, King Abdulaziz
University, for technical support.
Conflicts of Interest: The authors declare no competing interests.

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