Exploration of Structure-Activity Relationship of Aromatic Aldehydes Bearing Pyridinylmethoxy-Methyl Esters as Novel Antisickling Agents

Article abstract of DOI:10.1021/acs.jmedchem.0c01287

Aromatic aldehydes elicit their antisickling effects primarily by increasing the affinity of hemoglobin (Hb) for oxygen (O2). However, challenges related to weak potency and poor pharmacokinetic properties have hampered their development to treat sickle cell disease (SCD). Herein, we report our efforts to enhance the pharmacological profile of our previously reported compounds. These compounds showed enhanced effects on Hb modification, Hb-O2 affinity, and sickling inhibition, with sustained pharmacological effects in vitro. Importantly, some compounds exhibited unusually high antisickling activity despite moderate effects on the Hb-O2 affinity, which we attribute to an O2-independent antisickling activity, in addition to the O2-dependent activity. Structural studies are consistent with our hypothesis, which revealed the compounds interacting strongly with the polymer-stabilizing αF-helix could potentially weaken the polymer. In vivo studies with wild-type mice demonstrated significant pharmacologic effects. Our structure-based efforts have identified promising leads to be developed as novel therapeutic agents for SCD.

Full text of DOI:10.1021/acs.jmedchem.0c01287

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Article
Exploration of Structure−Activity Relationship of Aromatic
Aldehydes Bearing Pyridinylmethoxy-Methyl Esters as Novel
Antisickling Agents
Piyusha P. Pagare, Mohini S. Ghatge, Qiukan Chen, Faik N. Musayev, Jurgen Venitz,
Osheiza Abdulmalik, Yan Zhang,* and Martin K. Safo*
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ABSTRACT: Aromatic aldehydes elicit their antisickling effects primarily by increasing the affinity of hemoglobin (Hb) for oxygen
(O2). However, challenges related to weak potency and poor pharmacokinetic properties have hampered their development to treat
sickle cell disease (SCD). Herein, we report our efforts to enhance the pharmacological profile of our previously reported
compounds. These compounds showed enhanced effects on Hb modification, Hb-O2 affinity, and sickling inhibition, with sustained
pharmacological effects in vitro. Importantly, some compounds exhibited unusually high antisickling activity despite moderate effects
on the Hb-O2 affinity, which we attribute to an O2-independent antisickling activity, in addition to the O2-dependent activity.
Structural studies are consistent with our hypothesis, which revealed the compounds interacting strongly with the polymer-stabilizing
αF-helix could potentially weaken the polymer. In vivo studies with wild-type mice demonstrated significant pharmacologic effects.
Our structure-based efforts have identified promising leads to be developed as novel therapeutic agents for SCD.
vascular nitric oxide (NO) bioavailability, painful crises, multi-
organ damage, morbidity, and mortality.9−11
INTRODUCTION
■
Sickle cell disease (SCD) is an inherited genetic disorder that
is caused by a mutation at the sixth position of the β-chain of
normal hemoglobin (HbA), which results in substitution of a
polar glutamic acid (Glu) residue for a hydrophobic valine
(Val) to form sickle Hb (HbS).1 Under hypoxic conditions,
the concentration of the low affinity deoxygenated HbS
(deoxyHbS) increases, which polymerizes into long, rigid, and
insoluble fibers with concomitant sickling of red blood cells
(RBCs). The polymerization process is initiated by a primary
interaction between β2Val6 from one deoxyHbS molecule and
a hydrophobic acceptor pocket in the region of β1Ala70,
β1Phe85, and β1Leu88 of another deoxyHbS molecule.2−4
The polymerization process is further worsened because of
high levels of 2,3-diphosphoglycerate present in sickle RBCs,
which is an adaptive process to increase tissue oxygenation in
SCD patients.5−8 The hypoxia-induced RBC sickling leads to
multiple secondary pathophysiological events, for example,
adhesion of RBCs to tissue endothelium, oxidative stress/
damage, hemolysis of RBCs, hemolytic anemia, inflammation,
vaso-occlusion, impaired microvascular blood flow, decreased
Several secondary contacts between the deoxyHbS mole-
cules also play important roles in stabilizing the HbS polymers,
as demonstrated by a number of naturally occurring mutations
that disrupt these polymer-stabilizing contacts and in turn
reduce HbS polymerization and RBC sickling. For example,
αAsn78 → Lys (Hb Stanleyville) leads to increase in the
solubility of deoxyHbS heterotetramers, mitigating the severity
of SCD.12−14
Approximately, 100,000 Americans have SCD, and the
Centers for Disease Control and Prevention (CDC) has
declared this disease as a major public health concern.15 Four
drugs have currently been approved for treating SCD. These
Received: July 23, 2020
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Figure 1. Structures of 5-HMF, vanillin, INN-298, INN-312, SAJ-310, TD-7, and GBT-440.
include hydroxyurea (HU),16,17 which has been used for about
two decades. There is reported lack of response to HU in up to
30% of patients due to a complex variety of reasons.18 L-
Glutamine (Endari), an antioxidant, was approved in 2017 by
the US FDA and expected to reduce painful crises and adverse
events;19 however, the European regulatory body recommen-
ded against approval of the medication because of limited
evidence of efficacy in phase III trials.20 GBT-440 (Aka
Voxelotor or Oxbryta),21 (Figure 1), and Crizanlizumab22
were approved in 2019. The latter is a monoclonal antibody
targeting P-selectin, to reduce the frequency of painful vaso-
occlusive crises. As intracellular polymerization of deoxyHbS
remains the principal cause of the pathophysiology-associated
SCD, GBT-440, which directly prevents hypoxia-induced RBC
sickling by increasing Hb affinity for oxygen, offers immense
therapeutic potential.23 Although, in the clinic, GBT-440
showed proof-of-principle disease-modifying potential by
increasing Hb levels and reducing hemolysis in patients,
there was no evidence of efficacy against vaso-occlusion. Thus,
there is a need for novel aromatic aldehydes for treating SCD
with better clinical outcomes.
INN33 or SAJ39 series and then to TD35 series of compounds
(Figure 1), significantly improved antisickling activities have
been observed. Among these, the INN and SAJ compounds are
pyridyl derivatives of vanillin, while the TD compounds
represent further modifications of the INN compounds (with a
hydroxymethyl group on the pyridine ring) rationalized to
stereospecifically inhibit deoxyHbS polymer formation by
perturbing the αF-helix and contribute to the antisickling
activity of the compounds through the O2-independent
antisickling mechanism. At every stage, co-crystallization of
these compounds in complex with Hb revealed insights into
their binding and guided our future structure-based mod-
ifications. These compounds, however, still underwent
significant metabolism leading to suboptimal pharmacokinetics
properties, for example, short duration of action and low
bioavailability, mainly because of the highly susceptible
aldehyde pharmacophore. Nonetheless, the development of
vanillin,40 5HMF,38,41 and GBT-44042 demonstrated that these
allosteric modulators of Hb carry no significant toxicity even at
high doses and thus established aromatic aldehydes as a
scaffold to design potential therapeutics for SCD.
Prior to GBT-440, several aromatic aldehydes were studied
as allosteric effectors of hemoglobin (AEHs) for their
antisickling activities.24,25 Specifically, these compounds form
Schiff-base adducts with the Val1 amines of both α-subunits of
Hb increase Hb-O2 affinity and ameliorate the primary
pathophysiology of SCD, that is, RBC sickling. Several
challenges relating to the application of aromatic aldehydes
for treating SCD became apparent during the early years of
their development. These include nonspecific binding to
plasma proteins and susceptibility of the active aldehyde
moiety to rapid oxidative metabolism by several enzymes, for
example, aldehyde oxidase (AO) and aldehyde dehydrogenases
(ALDHs),26−32 that compromise the effectiveness of these
compounds dramatically. Another major challenge is the
difficulty in designing pharmaceutically applicable agents that
are capable of sustained modification of the large amounts of
intracellular Hb (∼5 mmol) in the body. These problems led
to termination of clinical trials of vanillin and 5-HMF
(Aes103)29 for SCD. GBT-440 appeared to have overcome
some of these problems. While the long-term benefits of GBT-
440 are still unclear, the need remains critical for additional
investigations into strategies to further enhance this class of
compounds. In our continuous efforts to develop aromatic
aldehydes for SCD, we have adopted a structure-based
approach to modify the antisickling agent, vanillin, resulting
in several generations of potent, allosteric, and covalent-
binding antisickling derivatives.33−39 From vanillin to the
In this current work, we aimed to design a series of aromatic
aldehydes that potently improve the antisickling activity of
aromatic aldehydes while addressing some of the challenges
associated with the rapid metabolism of the aldehyde
pharmacophore.
RESULTS AND DISCUSSION
■
Molecular Design. Two molecules of vanillin bind to the
α-cleft of Hb in a symmetry-related fashion, forming Schiff-
base interactions with the N-terminal amines of the two α-
subunits. Our previous studies identified several vanillin
derivatives, for example, SAJ-310,39 INN-312,34 and INN-
29834 (Figure 1) with improved in vitro antisickling potency
compared to vanillin.35 Some of these compounds, for
example, INN-312 with the pyridinylmethoxy substitution
ortho to the aldehyde group, showed a second novel O2-
independent antisickling activity, although weak, which was
attributed to weak hydrophobic interactions with a surface-
located αF-helix. Based on structural studies, we subsequently
modified the SAJ and INN series of compounds to generate
another series of compounds termed TD, by incorporating the
hydroxymethyl group on the pyridine ring. The rationale was
to increase protein interactions with the α-cleft residues to
potently increase Hb oxygen affinity (O2-dependent antisick-
ling activity), as well as strongly interact with the surface-
located αF-helix to directly weaken polymer interactions (O2-
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independent antisickling activity). The most promising
compound, TD-7, however undergoes significant metabolism
leading to suboptimal pharmacokinetics properties, for
example, short duration of pharmacologic action and low
bioavailability. In addition, the co-crystal structure of TD-7
with Hb revealed that the hydroxymethyl group did not make
interactions with the αF-helix as anticipated.35 Therefore, we
decided to pursue further structure-based modifications to
design better pharmacological agents.
In the current study, we structurally modified TD-7 by first
replacing the hydroxymethyl group on the pyridine ring with a
methyl ester group, as well as varying its position on the
pyridine ring while maintaining the meta-methoxy on the
benzaldehyde ring (Table 1, compounds 1−7). Introduction of
lization and/or increase interactions with Hb to improve both
the O2-independent and O2-dependent antisickling activities.
Next, to reduce oxidative metabolism of the aldehyde moiety,
we introduced an ortho-hydroxy group on the benzaldehyde
ring (Table 1, compounds 8−14). As previously observed, the
ortho-hydroxy is expected to form an intramolecular hydrogen-
bond interaction with the aldehyde moiety to reduce
enzymatic metabolism.43,44
In summary, two series of compounds, one with the 5-
methoxy group (1−7) and second with the 2-hydroxy group
(8−14) on the aromatic aldehyde ring, were synthesized, as
shown in Table 1. Each series contained 2- and 3-pyridine-
substituted benzaldehydes with the methyl esters at different
positions on the pyridine ring. Because of unavailability of
reagents as well as limitations in the synthetic feasibility, the 4-
substituted pyridine derivatives were not included.
Table 1. Structures of Target Compounds
Chemistry. All target compounds in Table 1 were
synthesized in two steps. The first step was a radical-catalyzed
Wohl−Ziegler bromination reaction of a methyl-substituted
pyridylmethyl ester to give bromomethyl-substituted pyridyl-
methyl ester. The starting materials used for these reactions
were commercially available methyl-substituted methyl esters
of nicotinic acid and picolinic acid. N-Bromosuccinimide
(NBS) was used as the brominating agent, while 2,2′-azobis(2-
methylpropionitrile) (AIBN) was applied as the radical
initiator. The bromomethyl pyridine intermediates obtained
were then used to form an ether linkage between the phenolic
oxygen of either 2-hydroxy-5-methoxybenzaldehyde or 2,6-
dihydroxybenzaldehyde via Williamson’s synthesis using the
previously reported procedure (Scheme 1).35,39 The synthesis
of TD-7, as a positive control, has been reported.33,39 The
compounds were obtained in reasonable yields (75−90%),
fully characterized, and then investigated for binding
interactions with Hb, as well as in vitro and in vivo
pharmacokinetics and/or pharmacodynamics (PDs) studies
using normal and sickled blood or wild-type mice.
Compounds Potently Increase Hb Affinity for Oxy-
gen. Aromatic aldehydes are known to bind to Hb
allosterically and modify it, either to increase its O2 affinity
[stabilizing the relaxed (R) state] or increase O2 delivery
[stabilizing the tense (T) state].25,45−50 Increasing the Hb-O2
affinity is the primary mechanism by which these compounds
elicit antisickling effects as the R-state Hb is resistant to
polymerization. Thus, oxygen equilibrium curve (OEC)
studies were first conducted using normal human whole
blood to determine the effects of the compounds on Hb’s
affinity for oxygen.
the methyl ester group was expected to lead to closer
interactions with the αF-helix and increase polymer destabi-
Scheme 1. Synthetic Route for Target Compounds
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Figure 2. Concentration-dependent shift in the Hb-O2 affinity of (a) m-methoxy-substituted and, (b) o-hydroxy-substituted compounds in normal
whole blood after 1.5 h incubation.
Figure 3. Time-dependent shift in the Hb-O2 affinity of (a) m-methoxy-substituted and, (b) o-hydroxy-substituted compounds in normal whole
blood at a concentration of 2 mM.
Dose-dependent studies were carried out using 0.5, 1, and 2
mM concentrations of the compounds and normal whole
blood (hematocrit adjusted to 30%) at 37 °C as previously
reported.33 The concentration of Hb in blood (∼5.0 mmol)
warrants the use of such high concentrations of the tested
compounds. Whole blood comprises of plasma proteins,
enzymes, and RBC cell membrane, which may play an
interfering role on the activity of the compounds as compared
to purified Hb. Thus, whole blood was used in this study to
obtain results more representative of the native system.
As shown in Figure 2, the compounds increased Hb affinity
for O2 in normal whole blood to varying degrees. The low or
insignificant activity shown by compounds 1 and 8 was likely
because of their low solubility in most solvents, including
dimethyl sulfoxide (DMSO). Thus, compounds 1 and 8 were
not studied further. The low activity of compounds 3, 5, 10,
and 12 compared to TD-7 could be due to the low intrinsic
affinity for Hb or due to plasma protein binding and/or
difficulty in RBC membrane permeation. It could also be due
to significant metabolism by enzymes in the blood and/or
RBCs that are known to play deteriorating roles in the
bioavailability and potency of antisickling aromatic alde-
hydes.26,29,30,41 In comparison, compounds 2, 6, 7, 9, 11,
and 13 showed significant increase in the Hb-O2 affinity.
Vanillin, at 2 mM concentration, only increased Hb O2 affinity
by 21%,33 whereas TD-7 showed almost similar potency as
most of the best compounds. These observations confirmed
that the structural modifications have conserved the potent
Hb-O2 affinity in vitro activity previously observed in TD-7, the
parent lead.
function,26−30 resulting in most of these compounds having
poor oral bioavailability, which has stymied their development.
Therefore, one of the major objectives of this work was to
identify compounds that may be less susceptible to the
oxidative metabolism of the aldehyde pharmacophore. For this
reason, time-dependent Hb-O2 affinity studies were conducted
with 2 mM concentrations of the compounds with human
blood (hematocrit adjusted to 30%) at 37 °C for 24 h using
TD-7 as control. Although, oxidative metabolism of aldehydes
occurs by several enzymes in various tissues including AO,31,32
it is primarily by ALDH in RBCs;26−28 therefore, whole blood
serves as an appropriate matrix for the measure of oxidative
metabolism in vitro. Among the meta-methoxy derivatives,
compound 2 showed optimal activity between 1 and 4 h which
then decreased to about 70% of its activity by 8 h and
sustained through the 24 h. Compounds 3 and 5 showed
maximum potency at 8 h followed by a gradual decrease over
the course of 24 h to about 70 and 60% of its original activity,
respectively. Interestingly, three of the meta-methoxy deriva-
tives, including compounds 4, 6, and 7 appear to be stable
against oxidative metabolism in whole blood. Compound 4
shows a very slow onset and reached its highest potency at 24
h. Such a slow onset may not be optimal because other
metabolizing enzymes, such as in the liver, could deactivate this
compound before reaching therapeutic potency. Compounds 6
and 7, on the other hand, have quick onset and showed a
sustained effect during the 24 h experiment (Figure 3a). In
comparison, TD-7, similar to some of the meta-methoxy
derivatives, also after reaching a maximal effect at 1 h gradually
decreased in potency, and at 24 h, showed a loss of almost 45%
of its activity. Clearly, the meta-methoxy-substituted derivatives
show varying oxidative metabolism in whole blood and/or
As pointed out above, aromatic aldehydes are highly
susceptible to oxidative metabolism of the active aldehyde
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Figure 4. Inhibition of sickling by (a) m-methoxy-substituted and, (b) o-hydroxy-substituted compounds in SS RBCs.
Figure 5. Concentration-dependent Hb modification (adduct formation) by (a) m-methoxy-substituted and, (b) o-hydroxy-substituted compounds
in SS RBCs.
RBC, probably because of differences in their affinity to the
metabolizing enzymes, ALDH. What is not clear is whether,
when administered in vivo, compounds 4, 6, and 7 would still
maintain their resistance to oxidative metabolism of the
aldehyde.
compounds for their O2-dependent antisickling properties. We
incubated the compounds at 0.5, 1.0, and 2.0 mM
concentrations with blood from individuals with homozygous
SS (hematocrit 20%), under hypoxic conditions (2.5% O2/
97.5% N2) at 37 °C for 2 h. The concentration-dependent
antisickling effect was observed for most of the tested
compounds (Figure 4). Although compounds 4, 12, and 14
showed dose-dependent sickling inhibition, their antisickling
effects were significantly weaker when compared to the other
compounds. At a lower concentration of 0.5 mM, compounds
2, 5, 9, and 13 showed more than 35% sickling inhibition as
compared to only 26% by TD-7. At 1.0 mM concentration,
compounds 5, 6, 9, and 13 showed more than 70% inhibition
as compared to 47% by TD-7, with compounds 5 and 13
almost exhibiting 100% sickling inhibition (Figure 4a,b). At 2.0
mM concentration, several of the tested compounds showed
more than 90% sickling inhibition.
This observation clearly indicates that the structural
modifications have led to improvement in increasing the
antisickling potencies of the compounds when compared to
TD-7. In comparison, GBT-440 at 0.5 and 1.0 mM decreased
the O2-dependent sickling by 56% and 98%, respectively,
suggesting that at lower concentration, GBT440 shows better
potency. This enhanced potent activity of GBT-440 has been
suggested to be because of reduced binding stoichiometry, that
is, it binds Hb at a 1:1 ratio52 while our compounds bind Hb at
a 2:1 ratio.
Unlike the meta-methoxy-substituted compounds (Figure
3a), all the ortho-hydroxy-substituted compounds (Figure 3b)
showed sustained action over the duration of the experiment,
suggesting all compounds are resistant to oxidative metabolism
in whole blood. Interestingly, compounds 10, 11, and 12
showed relatively slow onset of action compared to the other
compounds, which we speculate to be due to slow RBC
membrane permeation. As previously reported, formation of
intramolecular hydrogen bonding between the aldehyde
carbonyl group and the ortho-hydroxy group acts as a
protection to prevent undue metabolism of the aldehyde.43,51
It is clear from the forgoing that not only did we maintain the
Hb-O2 potencies of these compounds but also achieved one of
our primary objectives of increasing the metabolic stability of
the aldehyde moiety.
The compounds have a major effect of Hb cooperativity as
the shape of the OEC changed from sigmoidal to hyperbolic
with addition of the compound (Supporting Information S1),
as a result of the stabilization of the R-state Hb and restraining
the cross talk between the Hb subunits that is important for
the R → T transition.24,25
Compounds Potently Increase In Vitro Antisickling,
Hb-Modification, and Hb-O2 Affinity Activities with
Sickle RBCs. Because the pathophysiology of SCD, including
RBC sickling, is driven by hypoxia-induced polymerization of
deoxyHbS, we further characterized these high-O2 affinity
The remarkable enhanced antisickling activity of the
compounds compared to TD-7 led us to investigate whether
such an improvement is solely the consequence of their ability
to bind Hb and modify it to the higher O2 affinity form.
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Figure 6. Concentration-dependent shift in Hb-O2 affinity by (a) m-methoxy-substituted and, (b) o-hydroxy-substituted compounds in SS RBCs.
Figure 7. Linear correlation between increase in the Hb-O2 affinity, antisickling, and Hb-modification properties for 0.5 mM (blue), 1 mM (green),
and 2 mM (gray) concentrations of compounds.
Aliquot samples from the above antisickling study were
hemolyzed and utilized for dose-dependent Hb-O2 affinity
and Hb modification studies. As observed with normal whole
blood (Figure 2), the compounds increased Hb affinity for
oxygen and Hb modification in dose-dependent fashions
(Figure 5). The modification effect by the compounds were in
most instances similar or better than observed with TD-7. For
example, compounds 7, 9, 10, 11, 13, and 14 showed almost
complete modification of HbS at 2.0 mM concentration
compared to the ∼80% by TD-7. At 1.0 mM concentration,
compounds 5, 6, 7, 9, and 14 modified HbS similar to TD-7
(∼60%), while compounds 10, 11, and 13 showed more than
80% HbS modification at the same concentration (Figure 5).
In few instances, such as with compounds 2, 3, 4, and 12, TD-
7 showed better a Hb modification effect. Interestingly, while
compounds 4 and 12 expectedly showed weaker antisickling
effects than TD-7, compounds 2 and 3 appear to show
comparable or stronger antisickling effects when compared to
TD-7. In comparison, GBT-440 as expected from its potent
antisickling activity showed very high Hb modification to the
high affinity adduct form of 55% (0.5 mM) and 100% (1.0
mM).
Of most interest is that despite the fact that most of these
compounds, with the exception of 4, 12, and 14, show better
antisickling effects than TD-7, they conversely increase Hb
affinity for oxygen to a lesser degree when compared to TD-7
(Figures 4 and 6). Similar to TD-7, GBT-440’s high
antisickling activity (56% and 98% at 0.5 and 1.0 mM) also
corresponded to very high P50 shifts of 41 and 80%,
respectively. The unusual weak correlation between the
antisickling and P50 shift of our newer derivative compounds
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Table 2. Sickling Inhibition, Hb-O2 Affinity, and Hb-Modification Studies Using SS RBCs with the Seven Compounds
% sickling inhibition
1 mM
% Hb-O2 affinity
% Hb-modification
compd
0.5 mM
2 mM
0.5 mM
1 mM
2 mM
0.5 mM
1 mM
2 mM
2
3
5
6
9
10
13
41.8 0.7
17.5 1.1
37.6 0.9
29.1 1.2
43.3 4.1
7.6 1.1
71.7 0.9
48.2 1.2
97.7 1.5
81.6 1.8
70.6 4.1
54.2 0.1
90.7 11
99 2.6
88 5.5
5.9 0.7
4.8 2.1
4.4 0.9
9.9 0.7
12.2 2.1
5.4 1.4
16.3 12
12.8 1.9
20.1 1.3
13.9 1.5
19.5 0.8
18.8 2.1
29.2 1.1
44.2 1.6
21.3 2.1
37.1 2.5
25.4 1.1
32.3 0.7
39.6 1.3
48.6 0.4
60.1 2.1
32.1 0.5
20.9 1.5
30.2 0.5
36.1 1.1
46 3.2
49.9 1.6
41.7 1.8
62.3 1.9
64 1.3
64.4 2.7
81.1 0.6
67.5 2.2
63.8 3.2
79.2 0.6
81.3 1.4
90.8 1.2
100 0.7
100 1.1
99.1 1.1
96.5 0.7
98.8 1.8
93.2 4.4
98.9 2.1
29.2 1.6
47.8 4.6
39.5 12
92.5
5
Figure 8. Time-dependent recovery of parent compounds 9 and 10 at 0.5 and 1 mM; (a) representative chromatograms of 1 mM compound 9 and
(b) compound 10 at 0, 4, 8, and 24 h.
as discussed below is likely because of a second mechanism of
O2-independent antisickling activity.
between antisickling activity and P50 shift (Figure 7). The
weak correlation between the PD parameters of the
compounds presented here clearly suggests additional unique
mechanism of antisickling action that is particularly independ-
ent of Hb oxygen affinity. Compounds 2, 3, 5, 6, 9, 10, and 13
demonstrated the most potent antisickling effects despite only
marginally increasing Hb affinity for oxygen (Figure 7, Table
2). For example, while compound 9 inhibits sickling and
increases Hb-O2 affinity by 43 and 12% at 0.5 mM (71 and
19% at 1.0 mM), respectively, GBT-440 on the other hand
inhibits sickling and increases Hb oxygen affinity by 56 and
41%, at 0.5 mM (100 and 80% at 1 mM), respectively. This
substantial antisickling activity, but at a relatively much lower
Hb-O2 affinity increase by some of our compounds, we
The pharmacologic properties of aromatic aldehydes,
including their ability to modify Hb, increase Hb affinity for
oxygen, and inhibit hypoxia-induced RBC sickling are known
to be highly correlated. For example 5-HMF, when incubated
with sickle blood results in perfect direct correlations (r2 = 1)
between any two of the biological parameters, that is, Hb
adduct, P50 shift, or sickling inhibition.41 With the newer
derivative compounds, even though we observed a relatively
good correlation between the Hb adduct and P50 shift (r2 =
0.8−0.9) (Figure 7), it is weaker than reported for 5-HMF41 or
GBT-440. Even more so, the correlation between antisickling
activity and Hb modification are weaker and especially
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propose, is because of the polymer destabilization effect that
potentially led to an O2-independent antisickling activity
contribution, which is currently under further investigation.
Stability Studies of Compounds Using Whole Blood
Show Limited Hydrolysis of the Parent Methyl Ester.
The parent compounds reported here contain the methylester
moiety, which could potentially hydrolyze into their corre-
sponding acids and affect the PK or PD properties. We
therefore studied representative compounds, 9 and 10, for
their stability in buffer and whole blood using reversed-phase
high-performance liquid chromatography (HPLC). The
compounds at 0.5 and 1 mM concentrations were incubated
in normal whole blood (hematocrit adjusted to 30%) at 37 °C,
and aliquots obtained at 0, 4, 8, and 24 h were subjected to
deproteinization and extraction of the compounds in organic
solvent (acetonitrile). The purified extracts were subjected to
HPLC analyses to detect and quantify the amount of the
parent compound present, as well as any metabolized
derivatives. The acid analogue of compound 10 was used as
a control, which as expected eluted before the parent
compound (Figure 8). Both parent compounds showed
formation of the carboxylic acid analogue, but at very low
levels (Figure 8). Compound 10 demonstrated a time-
dependent hydrolysis of the methyl ester to the acid analogue,
with 89 4.4% (0.5 mM) and 83 1.7% (1 mM) of parent
compound 10 retained at 24 h (Figure 8a,c). Interestingly,
compound 9 did not show any observable metabolism until 24
h, with 81 1.4% (0.5 mM) and 78 2.4% (1 mM) of the
parent compound 9 being retained (Figure 8a,b). Thus, during
the course of 24 h, only approximately 20% of compounds 9
and 10 metabolized into the carboxylic acid form, indicating
that the antisickling activity of both compounds (and in
general all compounds) which were conducted within 3 h of
experimentation is likely exclusively due to the parent methyl
esters.
determined by the molecular replacement method with the
Phenix program53 using the native R2-state crystal structure
(PDB ID 1BBB) as a search model. Structure refinement was
carried out using both Phenix and CNS,54 and model
correction was carried out using COOT.55 The crystal
structures of COHb-6 (PDB ID 6XDT) and COHb-9 (PDB
ID 6XE7) complexes were refined to 1.9 and 2.0 Å,
respectively. The overall tetrameric structures of both
complexes were indistinguishable from 1BBB (RMSD 0.3 Å),
and their detailed crystallographic data are summarized in
Table 3.
Table 3. Crystallographic Data and Refinement Statistics for
COHb-6 and COHb-9 Complexes
compound
6 (PDB ID6XDT)
Data Collection Statistics
P212121
9 (PDB ID6XE7)
space group
P212121
unit-cell a, b, c (c5) 63.68, 82.98, 105.03
62.64, 82.76, 104.77
resolution (c5)
unique reflections
redundancy
29.32−1.90 (1.97−1.90) 29.29−2.00 (2.07−2.00)
40,600 (3764)
37,109 (3683)
4.27
4.04
completeness (%)
average I/σ(I)
Rmerge (%)
92.5 (86.9)
13.4 (3.3)
5.5 (37.3)
98.9 (99.7)
8.5 (2.3)
7.9 (42.8)
a
Refinement Statistics
resolution (c5)
no. of reflections
Rwork (%)
30.00−1.90
40,573
18.81
30.00−2.00
37,580
19.94
b
Rfree (%)
23.91
25.14
rmsd bonds (c5)
rmsd angles (deg)
0.006
0.97
0.006
0.83
Dihedral Angles
most favored (%)
allowed (%)
98.00
1.78
98.00
1.96
Average B (c52)/Atoms
X-ray Crystallography Shows Compounds Bind as
Predicted and Provide Atomic Basis for Their Potent
Antisickling Activities. To verify our molecular design,
which was intended toward these compounds interacting with
the αF-helix of Hb, as well as explain the unusual functional/
biological effects of the compounds, we selected two
compounds, one each from the meta-methoxy-substituted
and ortho-hydroxy-substituted benzaldehyde series, namely,
compounds 6 and 9 respectively, for co-crystallization studies
with CO-liganded Hb (COHb) in the R2-state conformation.
COHb was used for the crystallographic study because the
structures of O2-Hb and COHb complexes are very similar,
while the latter is comparatively easy to purify, more stable,
and easier to work with. Also, normal Hb (HbA) instead of
HbS was used for the crystallographic study because the
quaternary structures as well as the size and geometry of the α-
cleft binding site are similar in the two Hb structures.4,52
Freshly made solution of the compounds was added to
deoxygenated Hb (30 mg/mL protein) at an Hb tetramer-
compound ratio of 1:10. The complex mixture was saturated
with CO and allowed to incubate for 2 h to form the COHb-
compound complex. Co-crystallization experiments were
carried out under low salt precipitant conditions (10−20%
PEG6000, 100 mM HEPES buffer, pH 7.4), where cherry red
needle crystals, isomorphous to previous aromatic aldehyde
Hb-R2 state complex crystals, appeared in 1−3 days and were
used to collect X-ray diffraction data.35,39 The crystal structures
of the COHb complexes with compounds 6 and 9 were
all atoms
protein
ligand
water
a
32.00
31.50
29.50
36.90
34.90
34.40
35.00
39.00
b
Rmerge = ΣhklΣi|Ii(hkl) − ⟨I(hkl)⟩|/ΣhklΣiIi(hkl). Rfree was calculated
from 5% randomly selected reflection for cross-validation. All other
measured reflections were used during refinement.
As expected, compounds 6 and 9 bind in a symmetry-related
fashion, forming Schiff base interactions between the aldehyde
group of the compounds with the αVal1 nitrogens (∼1.5 c5) of
both α1- and α2-subunits at the α-cleft of Hb. Also, as
anticipated, the ortho-substituted pyridinylmethoxy-methyl
ester groups directed upward toward the surface of the α-
cleft and close to the αF-helix (Figures 9 and 10). Because
both molecules in their respective structures bind in a
symmetrical fashion, detailed interaction analyses with Hb
will be focused on α2Val1 binding, which appears to be more
ordered than the α1Val1 binding, especially with compound 9.
The benzaldehyde ring of both compounds 6 and 9
superimposed closely, making both intra- and inter-subunit
hydrophobic interactions with α2Ala130, α2Ser131,
α2Thr134, and α1Thr134 (Figures 9−11). The pyridine
rings of the two bound molecules formed extensive face-to-face
π−π stacking interactions (3.4−3.8 c5) (Figures 9 and 10).
These interactions serve to stabilize the R-state and increase
the oxygen affinity of Hb, as reported previously.25
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Figure 9. Crystal structure of Hb in the R2 conformation in complex with two molecules of compound 9 bound at the α-cleft (a) overall crystal
structure of Hb in the R2 conformation in the complex with two molecules of compound 9 in the central water cavity. (b) Close-up view of
compound 9 bound at the α-cleft of Hb. (c) Close-up view of compound 9 showing interactions with the residues of the αF-helix. (Hb is shown as
cartoon with α1 subunit in green, α2 in yellow, β1 in pink, and β2 in gray; hemes are shown as sticks; compound 9 is shown in orange sticks).
Figure 10. Crystal structure of Hb in the R2 conformation in complex with two molecules of compound 6 bound at the α-cleft (a) overall crystal
structure of Hb in the R2 conformation in complex with two molecules of compound 6 in the central water cavity. (b) Closeup view of compound
6 bound at the α-cleft of Hb. (c) Closeup view of compound 6 showing interactions with the residues of the αF-helix. (Hb is shown as cartoon with
α1 subunit in green, α2 in yellow, β1 in pink, and β2 in gray; hemes are shown as sticks; compound 6 is shown in cyan sticks; water molecules are
shown as red spheres).
Interestingly, the ortho-hydroxy group of compound 9 makes
both direct and water-mediated hydrogen bonding interactions
with the αVal1 nitrogen. This interaction may help increase the
binding affinity of compound 9 with the protein (Figure 3).
The pyridinylmethoxy-methyl ester group in compound 9,
which is directed toward the surface of the α-cleft, is involved
in a hydrogen bonding interaction with the backbone nitrogen
of α2Met76 of the αF-helix (3.5 c5) (Figure 9), which is
missing in TD-7 binding.35 In addition, the pyridinylmethoxy-
methyl ester group is also involved in hydrophobic interactions
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Figure 11. Structural comparison of compounds 6 and 9 bound at the α-cleft of Hb. (a) superposition of compounds 6 and 9 molecules showing
interactions with residues from αF-helix. (b) 90° rotated view of (a). (Hb is shown as cartoon with α1 subunit in green and α2 in yellow;
compounds 6 and 9 are shown in cyan and orange sticks, respectively).
with α2Val73, α2Asp74, and α2Pro77 (2.7−4.0 Å) residues,
which in TD-7 is relatively weaker (3.5−4.3 Å).35 These αF-
helix interactions, we believe lead to perturbation of the helix
to destabilize the polymer, explaining the unusually high
antisickling effect (through O2-independent mechanism) at the
relatively low P50 shift of this compound. As noted above,
GBT-440 also binds to the α-cleft of the R2-state Hb to make
Schiff-base interaction with the αVal1 amine. Interestingly,
unlike our compounds and previously studied aromatic
aldehydes that bind two molecules, one each to α1-subunit
and α2-subunit, GBT-440 only binds one molecule at the α-
cleft because of the isopropyl−pyrazole substituent that
sterically prevents two molecules from binding.52 Nonetheless,
unlike compound 9, GBT-440 does not make any close contact
with the αF-helix, and thus, we do not expect this molecule to
have any significant O2-independent antisickling activity,
consistent with our comparative study that show strong
correlation between its P50 shift and antisickling activity, and
also as reported in the literature.52
Similar to compound 9, compound 6 also binds with the
pyridinylmethoxy-methyl ester group orientated toward the
αF-helix. Nonetheless, the ester group forms stronger intra-
subunit hydrophobic interactions with α2Pro77 (3.1 c5)
compared to compound 9 (3.5 c5) (Figures 10 and 11). In
addition, the methoxy groups of the ester form several water-
mediated hydrogen bonding interactions with the backbone
atoms of α2Val73, α2Asp75, and α2Met76 of the αF-helix
(Figure 10). Similar to compound 9, compound 6 interactions
with the αF-helix likely lead to perturbation of the αF-helix,
consistent with this compound unusually potent antisickling
effect when compared to the relatively small effect on the Hb
oxygen affinity.
cation-exchange HPLC to determine the levels of drug-
modified Hb (adducts) and to conduct OEC studies to
determine the degree of P50 shift or Hb-O2 affinity.
All compounds showed significant in vivo modification of
intracellular Hb with increasing levels from 1 h to the 6 h
experimental period (Figure 12). Mice treated with compound
Figure 12. Time-dependent modification of Hb in wild-type mice (n
= 6) after 150 mg/kg IP administration of test compounds.
9 demonstrated the highest levels of modified Hb (16.9
1.0% at 6 h), followed by compound 10 (9.6 1.9%). Only
1.5 0.3% modified Hb was observed in mice treated with the
control TD-7. Corresponding changes were observed in
oxygen affinity at the measured time points of 3 and 6 h
(Figure 13). Mice treated with compound 9 showed the
Compounds Showed Improved In Vivo PD Effects.
Through systematic SAR studies, we successfully identified
several compounds that showed significantly enhanced
pharmacologic activity in vitro compared to the previous
precursor TD-7. Several of these compounds showed sustained
action over 24 h. Among the most potent compounds
identified, compounds 9 and 10 were selected for further in
vivo PD studies using wild-type mice (C57BL/6). The mice
received a single IP dose of 150 mg/kg (a total of 6 mice).
Blood samples were obtained prior to treatment (0), and 1, 3,
and 6 h post treatment, via submandibular bleeding and
subjected to hemolysis using standard methods. Clarified
lysates, free of cell debris and RBC ghosts, were analyzed by
Figure 13. Time-dependent Hb-O2 affinity shifts in the wild-type
mice (n = 6) after 150 mg/kg IP administration of test compounds.
highest levels of shifts (14.5% 2.2 at 6 h). Compound 10
also increased Hb-O2 affinity, although relatively lower by 5.5%
1.9. In contrast, only approximately 1% 0.2 shift in the
Hb-O2 affinity was observed in the mice treated with TD-7.
These preliminary in vivo studies demonstrate improvement in
the pharmacologic profile of our aromatic aldehyde class of
allosteric effectors of Hb.
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reaction was cooled to room temperature, and the solvent evaporated.
The mixture was then extracted using dichloromethane and water
followed by washing the organic layer with brine. The organic layer
was dried over sodium sulfate, filtered, evaporated, and the crude
product was purified using column chromatography.
CONCLUSIONS
■
Intracellular polymerization of deoxygenated sickle hemoglo-
bin remains the principal cause of the pathophysiology
associated with SCD. Naturally occurring and synthetic
AEHs have been investigated as potential therapeutic agents
for the treatment of SCD.33,35,36,39−41,52 Even though this class
of compounds showed significant potency in vitro, several of
them lack good PK properties and therefore are not feasible to
treat a chronic disease that requires significant levels of Hb
modification for efficacy. These drawbacks have severely
hampered the clinical development and utility of aromatic
aldehydes to treat SCD. For example, our group studied 5-
HMF; however, because of metabolic instability of the
aldehyde that translated to poor oral bioavailability, the
phase II clinical study was terminated.24,56 In previous reports,
our group has demonstrated a systematic and rational
structure-based approach to develop several aromatic alde-
hydes that exhibited excellent antisickling activity. In the
current report, we modified the TD compounds to overcome
the disadvantages associated with potency and oxidative
metabolism by derivatizing the methyl alcohol on the pyridines
to a methyl ester as well as protecting the aromatic aldehyde
with an ortho-hydroxy group. These compounds exhibited
several novel properties that are missing from the previous
generation compounds. They showed sustained pharmacologic
activity in vitro, as well as both sustained and significantly
improved activity in vivo. Remarkably, some of the compounds
potentially exhibited additional mechanism of action inde-
pendent of the primary O2-dependent antisickling effect.
Structural studies of compounds 6 and 9 complexes provided
plausible explanations for these enhanced antisickling activities
that involves perturbing the αF-helix leading to O2-
independent antisickling activity. These novel results are
encouraging and provide guidance for the further development
of potent antisickling lead compounds. Compounds 9, 10, and
13 are currently undergoing formulation for detailed in vivo PD
studies.
Methyl 5-(bromomethyl)nicotinate (vi) and methyl 2-
(bromomethyl)benzoate (vii) were purchased from Sigma-Aldrich
(St. Louis, MO).
Methyl 6-(Bromomethyl)nicotinate (i).57 The title compound was
prepared following the general procedure as a white solid in 66%
1
yield. H NMR (400 MHz, DMSO-d6): δ 9.16 (d, J = 1.56 Hz, 1H),
8.30 (dd, J = 8.12, 2.2 Hz, 1H), 7.53 (dd, J = 8.12, 0.48 Hz, 1H), 4.58
(s, 2H), 3.96 (s, 3H). MS (ESI) m/z: 229.98 [M + H]+.
Methyl 2-(Bromomethyl)nicotinate (ii).58 The title compound was
prepared following the general procedure as an orange solid in 65%
1
yield. H NMR (400 MHz, DMSO-d6): δ 8.71 (dd, J = 4.8, 1.76 Hz,
1H), 8.28 (dd, J = 7.92, 1.76 Hz, 1H), 7.33 (m, 1H), 5.04 (s, 2H),
3.98 (s, 3H). MS (ESI) m/z: 229.98 [M + H]+.
Methyl 6-(Bromomethyl)picolinate (iii).59 The title compound
was prepared following the general procedure as a white solid in 66%
1
yield. H NMR (400 MHz, DMSO-d6): δ 8.05 (d, J = 7.68 Hz, 1H),
7.99 (t, J = 7.72 Hz, 1H), 7.77 (d, J = 7.68 Hz, 1H), 4.67 (s, 2H), 3.98
(s, 3H). MS (ESI) m/z: 229.98 [M + H]+.
Methyl 2-(Bromomethyl)isonicotinate (iv).60 The title compound
was prepared following the general procedure as a dark blue solid in
1
66% yield. H NMR (400 MHz, DMSO-d6): δ 8.77 (dd, J = 5, 0.64
Hz, 1H), 8.02 (m, 1H), 7.77 (dd, J = 5, 1.56 Hz, 1H), 4.81 (s, 2H),
3.91 (s, 3H). MS (ESI) m/z: 229.98 [M + H]+.
Methyl 5-(Bromomethyl)picolinate (v).61 The title compound was
prepared following the general procedure as a white solid in 66%
1
yield. H NMR (400 MHz, DMSO-d6): δ 8.78 (t, J = 1.48 Hz, 1H),
8.06 (d, J = 1.36 Hz, 2H), 4.82 (s, 2H), 3.89 (s, 3H). MS (ESI) m/z:
229.98 [M + H]+.
General Procedure for Synthesis of Compounds 1−14. A
mixture of 2-hydroxy-5-methoxybenzaldehyde or 2,6-dihydroxyben-
zaldehyde (1 equiv) and corresponding bromo-pyridinylmethoxy-
methyl ester (1 equiv) was dissolved in anhydrous N,N-
dimethylformamide (DMF). Anhydrous potassium carbonate
(K2CO3) (1.2 equiv) was added to this mixture, and the reaction
was stirred at room temperature for 8−10 h. The solvent was then
evaporated, and the reaction mixture extracted with ethyl acetate and
water. The organic layer was dried over sodium sulfate, filtered,
solvent evaporated, and the crude product was purified using column
chromatography.
EXPERIMENTAL SECTION
■
Methyl 6-((2-Formyl-4-methoxyphenoxy)methyl)nicotinate (1).
The title compound was prepared following the general procedure as
a white solid in 82% yield. IR (Diamond, cm−1): 2922, 2867, 1721,
1669, 1620, 1585, 1499, 1446, 1391, 1370, 1286, 1224, 1174, 1141,
Chemistry. All reagents used in the synthesis and functional assays
were purchased from Sigma-Aldrich (St. Louis, MO) or Thermo
Fisher Scientific (Waltham, MA) and utilized without additional
1
purification. H NMR and 13C NMR spectra were obtained on a
1
1121; H NMR (400 MHz, DMSO-d6): δ 10.49 (s, 1H), 9.08 (m,
Bruker 400 MHz spectrometer, and tetramethylsilane was used as an
internal standard. Peak positions are given in parts per million (δ).
Column chromatography was performed on silica gel (grade 60 mesh;
Bodman Industries, Aston, PA). Routine thin-layer chromatography
was performed on silica gel GHIF plates (250 μm, 2.5 × 10 cm;
Analtech Inc., Newark, DE). MS spectra were obtained from a
PerkinElmer Flexar UHPLC with an AxION 2 time-of-flight mass
spectrometer, and the molecular weight of the compounds was within
0.005% of calculated values. Infrared spectra were obtained on
Thermo Nicolet iS10 FT-IR. Purity of the compounds was
determined by HPLC using Varian Microsorb 100-5 C18 column
(250 × 4.6 mm), using Prostar 325 UV−vis (210 nm) as the detector.
The HPLC parameters used were injection volume = 15 μL, sample
concentration = 3 mM, mobile phase = 60MeCN−40H2O, flow rate =
1 mL/min.
1H), 8.36 (dd, J = 8.16, 2.16 Hz, 1H), 7.80 (d, J = 8.12 Hz, 1H), 7.25
(m, 3H), 5.42 (s, 2H), 3.91 (s, 3H), 3.77 (s, 3H); 13C NMR (100
MHz, DMSO-d6): δ 188.94, 165.00, 161.04, 154.62, 153.62, 149.59,
137.82, 125.03, 124.77, 122.85, 121.31, 115.97, 110.78, 71.02, 55.59,
52.38. Mass spectrum m/z: found 324.07 [M + Na]+; calcd, 324.08
[M + Na]+. The purity of the compound was checked by HPLC and
was found to be 96% pure.
Methyl 2-((2-Formyl-4-methoxyphenoxy)methyl)nicotinate (2).
The title compound was prepared following the general procedure as
a pale yellow solid in 82% yield. IR (Diamond, cm−1): 2920, 1719,
1684, 1667, 1622, 1583, 1535, 1492, 1445, 1404, 1372, 1276, 1215,
1
1168, 1142; H NMR (400 MHz, DMSO-d6): δ 10.25 (s, 1H), 8.74
(dd, J = 4.76, 1.48 Hz, 1H), 8.23 (dd, J = 7.8, 1.44 Hz, 1H), 7.55 (m,
1H), 7.2 (m, 3H), 5.56 (s, 2H), 3.78 (s, 3H), 3.75 (s, 3H); 13C NMR
(100 MHz, DMSO-d6): δ 188.73, 166.14, 155.39, 155.29, 153.46,
151.55, 138.47, 126.17, 124.86, 123.66, 122.98, 116.13, 110.06, 70.98,
55.61, 52.54. Mass spectrum m/z: found 302.11 [M + H]+, 324.09 [M
+ Na]+; calcd, 302.10 [M + H]+, 324.08 [M + Na]+. The purity of the
compound was checked by HPLC and was found to be 98% pure.
Methyl 6-((2-Formyl-4-methoxyphenoxy)methyl)picolinate (3).
The title compound was prepared following the general procedure as
The compounds were synthesized according to the general
procedures for similar compounds.33,35,39
General Procedure for the Synthesis of Compounds i−vii. A
mixture of the methyl-substituted pyridylmethyl ester (1 equiv) and
α,α′-azoisobutyronitrile (AIBN) (10%) was dissolved in carbon-
tetrachloride (CCl4). The solution was heated at 48 °C, and NBS (1.1
equiv) solution in CCl4 was added dropwise and stirred for 5 h. The
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a white solid in 82% yield. IR (Diamond, cm−1): 2953, 2851, 1720,
1682, 1618, 1585, 1494, 1396, 1369, 1296, 1222, 1169, 1141; H
309.06 [M + Na]+. The purity of the compound was checked by
HPLC and was found to be 99% pure.
1
Methyl 2-((2-Formyl-3-hydroxyphenoxy)methyl)nicotinate (9).
The title compound was prepared following the general procedure
as a pale yellow solid in 58% yield. IR (Diamond, cm−1): 2956, 1713,
1618, 1637, 1571, 1459, 1435, 1396, 1370, 1287, 1238, 1170, 1141;
1H NMR (400 MHz, DMSO-d6): δ 11.67 (s, 1H), 10.19 (s, 1H), 8.75
(dd, J = 4.8, 1.68 Hz, 1H), 8.25 (dd, J = 7.84, 1.68 Hz, 1H), 7.55 (m,
2H), 6.66 (d, J = 8.2 Hz, 1H), 6.53 (d, J = 8.4 Hz, 1H), 5.58 (s, 2H),
3.79 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 193.68, 166.15,
162.26, 161.44, 155.02, 151.73, 138.75, 138.31, 126.02, 123.60,
110.66, 109.49, 103.51, 70.59, 52.50. Mass spectrum m/z: found
288.09 [M + H]+, 310.07 [M + Na]+; calcd, 288.08 [M + H]+, 309.06
[M + Na]+. The purity of the compound was checked by HPLC and
was found to be 99% pure.
NMR (400 MHz, DMSO-d6): δ 10.47 (s, 1H), 8.03 (m, 2H), 7.87 (d,
J = 7.32 Hz, 1H), 7.26 (m, 3H), 5.38 (s, 2H), 3.90 (s, 3H), 3.76 (s,
3H); 13C NMR (100 MHz, DMSO-d6): δ 189.03, 164.99, 156.95,
154.68, 153.59, 146.99, 138.57, 125.05, 125.01, 123.95, 123.84,
115.99, 110.76, 71.08, 55.59, 52.39. Mass spectrum m/z: found
302.10 [M + H]+, 324.08 [M + Na]+; calcd, 302.10 [M + H]+, 324.08
[M + Na]+. The purity of the compound was checked by HPLC and
was found to be 98% pure.
Methyl 2-((2-Formyl-4-methoxyphenoxy)methyl)isonicotinate
(4). The title compound was prepared following the general procedure
as a white solid in 82% yield. IR (Diamond, cm−1): 2923, 1729, 1686,
1
1608, 1566, 1450, 1382, 1292, 1276, 1216, 1190, 1171; H NMR
(400 MHz, DMSO-d6): δ 10.43 (s, 1H), 8.81 (dd, J = 5, 0.56 Hz,
1H), 8.02 (s, J = 1.44 Hz, 1H), 7.80 (dd, J = 5.04, 1.56 Hz, 1H), 7.24
(m, 3H), 5.41 (s, 2H), 3.91 (s, 3H), 3.76 (s, 3H); 13C NMR (100
MHz, DMSO-d6): δ 188.81, 165.02, 157.80, 154.78, 153.63, 150.44,
137.79, 125.09, 122.92, 121.82, 120.33, 116.25, 110.73, 71.27, 55.58,
52.81. Mass spectrum m/z: found 302.10 [M + H]+, 324.08 [M +
Na]+; calcd, 302.10 [M + H]+, 324.08 [M + Na]+. The purity of the
compound was checked by HPLC and was found to be 98% pure.
Methyl 3-((2-Formyl-4-methoxyphenoxy)methyl)picolinate (5).
The title compound was prepared following the general procedure as
a pale yellow solid in 80% yield. IR (Diamond, cm−1): 2897, 1712,
1671, 1607, 1567, 1490, 1445, 1398, 1365, 1275, 1232, 1165, 1139,
1106; 1H NMR (400 MHz, DMSO-d6): δ 10.35 (s, 1H), 8.65 (dd, J =
4.6, 1.28 Hz, 1H), 8.2 (dd, J = 7.84, 0.76 Hz, 1H), 7.66 (dd, J = 7.88,
4.68 Hz, 1H), 7.24 (m, 3H), 5.49 (s, 2H), 3.83 (s, 3H), 3.77 (s, 3H);
13C NMR (100 MHz, DMSO-d6): δ 188.81, 166.12, 154.66, 153.55,
148.49, 146.69, 136.89, 133.04, 126.43, 124.89, 122.96, 115.79,
110.68, 67.44, 55.56, 52.34. Mass spectrum m/z: found 324.08 [M +
Na]+; calcd, 324.08 [M + Na]+. The purity of the compound was
checked by HPLC and was found to be 98% pure.
Methyl 5-((2-Formyl-4-methoxyphenoxy)methylpicolinate) (6).
The title compound was prepared following the general procedure as
a pale yellow solid in 80% yield. IR (Diamond, cm−1): 2962, 2864,
2767, 1719, 1684, 1673, 1588, 1489, 1458, 1395, 1355, 1274, 1218,
1198, 1150, 1127; 1H NMR (400 MHz, DMSO-d6): δ 10.40 (s, 1H),
8.84 (d, J = 1.36 Hz, 1H), 8.10 (m, 2H), 7.28 (m, 2H), 7.2 (d, J =
2.92 Hz, 1H), 5.39 (s, 2H), 3.89 (s, 3H), 3.76 (s, 3H); 13C NMR
(100 MHz, DMSO-d6): δ 188.77, 154.89, 154.57, 148.62, 135.93,
135.67, 135.93, 135.67, 125.92, 125.17, 123.28, 114.92, 111.30, 68.52,
55.90, 52.97. Mass spectrum m/z: found 302.10 [M + H]+, 324.09 [M
+ Na]+; calcd, 302.10 [M + H]+, 324.08 [M + Na]+. The purity of the
compound was checked by HPLC and was found to be 98% pure.
Methyl 5-((2-Formyl-4-methoxyphenoxy)methyl)nicotinate (7).
The title compound was prepared following the general procedure as
a white solid in 82% yield. IR (Diamond, cm−1): 2969, 1718, 1670,
1602, 1572, 1498, 1435, 1405, 1382, 1279, 1217, 1185, 1160, 1119;
1H NMR (400 MHz, DMSO-d6): δ 10.37 (s, 1H), 9.06 (d, J = 2 Hz,
1H), 8.97 (d, J = 2.04 Hz, 1H), 8.42 (t, J = 2.04 Hz, 1H), 7.29 (m,
2H), 7.2 (d, J = 3.08 Hz, 1H), 5.38 (s, 2H), 3.9 (s, 3H), 3.77 (s, 3H);
13C NMR (100 MHz, DMSO-d6): δ 188.87, 165.04, 154.64, 153.64,
152.90, 149.49, 135.99, 132.63, 125.44, 125.13, 122.88, 116.21,
110.71, 67.83, 55.58, 52.49. Mass spectrum m/z: found 324.08 [M +
Na]+; calcd, 324.08 [M + Na]+. The purity of the compound was
checked by HPLC and was found to be 99% pure.
Methyl 6-((2-Formyl-3-hydroxyphenoxy)methyl)picolinate (10).
The title compound was prepared following the general procedure as
a pale yellow solid in 60% yield. IR (Diamond, cm−1): 3065, 3010,
2956, 2890, 1738, 1697, 1640, 1614, 1583, 1462, 1454, 1400, 1358,
1
1297, 1242, 1190, 1171, 1150; H NMR (400 MHz, DMSO-d6): δ
11.74 (s, 1H), 10.44 (s, 1H), 8.04 (m, 2H), 7.91 (dd, J = 7.48, 1.12
Hz, 1H), 7.52 (t, J = 8.4 Hz, 1H), 6.7 (d, J = 8 Hz, 1H), 6.56 (d, J =
8.4 Hz, 1H), 5.39 (s, 2H), 3.90 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): δ 193.79, 164.99, 162.43, 160.69, 160.62, 149.59, 138.65,
137.84, 124.81, 121.26, 110.79, 109.81, 103.42, 70.49, 52.41. Mass
spectrum m/z: found 288.09 [M + H]+, 310.07 [M + Na]+; calcd,
288.08 [M + H]+, 309.06 [M + Na]+. The purity of the compound
was checked by HPLC and was found to be 99% pure.
Methyl 2-((2-Formyl-3-hydroxyphenoxy)methyl)isonicotinate
(11). The title compound was prepared following the general
procedure as a pale yellow solid in 58% yield. IR (Diamond, cm−1):
2961, 1733, 1716, 1639, 1620, 1577, 1460, 1441, 1373, 1342, 1293,
1235, 1215, 1174; 1H NMR (400 MHz, DMSO-d6): δ 11.68 (s, 1H),
10.39 (s, 1H), 8.81 (dd, J = 5, 0.6 Hz, 1H), 8.03 (d, J = 0.56 Hz, 1H),
7.81 (dd, J = 5, 1.56 Hz 1H), 7.51 (t, J = 8.4 Hz, 1H), 5.41 (s, 2H),
3.91 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 193.51, 165.02,
162.37, 160.80, 157.43, 150.43, 138.66, 137.84, 121.89, 120.38,
110.83, 109.74, 103.56, 70.69, 52.83. Mass spectrum m/z: found
288.09 [M + H]+, 310.07 [M + Na]+; calcd, 288.08 [M + H]+, 309.06
[M + Na]+. The purity of the compound was checked by HPLC and
was found to be 99% pure.
Methyl 3-((2-Formyl-3-hydroxyphenoxy)methyl)picolinate (12).
The title compound was prepared following the general procedure as
a pale yellow solid in 54% yield. IR (Diamond, cm−1): 3092, 2923,
2851, 1774, 1712, 1632, 1599, 1566, 1515, 1478, 1366, 1276, 1231,
1180, 1136, 1072; 1H NMR (400 MHz, DMSO-d6): δ 11.72 (s, 1H),
10.32 (s, 1H), 8.65 (dd, J = 4.64, 1.52 Hz, 1H), 8.22 (m, 1H), 7.66
(dd, J = 7.88, 4.64 Hz, 1H), 7.54 (t, J = 8.4 Hz, 1H), 6.66 (d, J = 8
Hz, 1H), 6.57 (d, J = 8.4 Hz, 1H), 5.51 (s, 2H), 3.84 (s, 3H), 13C
NMR (100 MHz, DMSO-d6): δ 193.59, 166.09, 162.46, 160.68,
148.53, 146.54, 138.72, 136.83, 132.78, 126.44, 110.74, 109.79,
103.26, 67.04, 52.35. Mass spectrum m/z: found 310.08 [M + Na]+;
calcd, 309.06 [M + Na]+. The purity of the compound was checked
by HPLC and was found to be 99% pure.
Methyl 5-((2-Formyl-3-hydroxyphenoxy)methyl)picolinate (13).
The title compound was prepared following the general procedure as
a white solid in 54% yield. IR (Diamond, cm−1): 2954, 1731, 1679,
1644, 1618, 1574, 1458, 1440, 1392, 1357, 1289, 1249, 1177, 1144,
1
1122; H NMR (400 MHz, DMSO-d6): δ 11.74 (s, 1H), 10.37 (s,
Methyl 6-((2-Formyl-3-hydroxyphenoxy)methyl)nicotinate (8).
The title compound was prepared following the general procedure
as a white solid in 58% yield. IR (Diamond, cm−1): 3299, 2959, 1726,
1H), 8.86 (s, 1H), 8.12 (m, 2H), 7.54 (t, J = 8.4 Hz, 1H), 6.72 (d, J =
8.32 Hz, 1H), 6.56 (d, J = 8.4 Hz, 1H), 5.40 (s, 2H), 3.89 (s, 3H);
13C NMR (100 MHz, DMSO-d6): δ 193.83, 162.42, 160.69, 148.72,
147.01, 138.64, 136.32, 135.91, 135.78, 124.64, 109.79, 103.37, 67.31,
52.39. Mass spectrum m/z: found 310.08 [M + Na]+; calcd, 310.08
[M + Na]+. The purity of the compound was checked by HPLC and
was found to be 99% pure.
Methyl 5-((2-Formyl-3-hydroxyphenoxy)methyl)nicotinate (14).
The title compound was prepared following the general procedure as
a white solid in 54% yield. IR (Diamond, cm−1): 2907, 1719, 1643,
1617, 1584, 1460, 1429, 1397, 1370, 1292, 1246, 1179, 1152, 1121;1H
1
1694, 1620, 1458, 1437, 1382, 1343, 1285, 1233, 1172, 1122; H
NMR (400 MHz, DMSO-d6): δ 11.73 (s, 1H), 10.45 (s, 1H), 9.09 (s,
1H), 8.34 (d, J = 8.12, 1H), 7.81 (d, J = 8.28 Hz, 1H), 7.51 (t, J = 8.4
Hz, 1H), 6.67 (d, J = 8.52 Hz, 1H), 6.56 (d, J = 8.36 Hz) 5.41 (s,
2H), 3.90 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 193.85,
165.39, 163.86, 160.84, 160.41, 150.66, 138.40, 138.12, 125.47,
120.52, 111.05, 110.86, 102.24, 70.91, 52.47. Mass spectrum m/z:
found 288.08 [M + H]+, 310.08 [M + Na]+; calcd, 288.08 [M + H]+,
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NMR (400 MHz, DMSO-d6): δ 11.72 (s, 1H), 10.33 (s, 1H), 9.05 (d,
J = 1.8 Hz, 1H), 8.99 (d, J = 1.76 Hz, 1H), 8.43 (t, J = 2 Hz, 1H),
7.55 (t, J = 8.2 Hz, 1H), 6.73 (d, J = 8.2 Hz, 1H), 6.56 (d, J = 8.44 Hz,
1H), 5.39 (s, 2H), 3.91 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ
193.74, 176.82, 165.04, 162.40, 160.72, 152.87, 149.53, 138.63,
135.99, 132.32, 110.80, 109.76, 103.42, 67.28, 52.49. Mass spectrum
m/z: found 288.08 [M + H]+; calcd, 288.08 [M + H]+. The purity of
the compound was checked by HPLC and was found to be 99% pure.
Blood. Normal whole blood was collected from adult donors at the
Virginia Commonwealth University after informed consent, in
accordance with regulations of the IRB for Protection of Human
Subjects. Hb was purified from discarded normal blood samples using
standard procedures.62 Leftover blood samples from individuals with
homozygous SS were obtained and utilized, based on an approved
IRB protocol at the Children’s Hospital of Philadelphia, with
informed consent.
Oxygen Equillibrium Curve Studies. The OEC study to
determine the effect of the compounds on Hb affinity for oxygen was
conducted following the standard procedure.34,41 Whole blood (30%
hematocrit) was incubated in the absence or presence of compounds
(0.5, 1.0, and 2.0 mM) solubilized in DMSO. Following, the blood-
compound samples were incubated in an IL 237 tonometer
(Instrumentation Laboratories, Inc., Lexington, MA) for about 7
min at 37 °C against a gas mixture containing O2 concentrations of
0.804, 2.935, and 5.528% and allowed to equilibrate at O2 tensions of
6, 20, and 40 mmHg. After equilibration, the sample was removed via
syringe and aspirated into a ABL 700 series table top automated blood
gas analyzer (Radiometer America, Inc., Westlake, OH) to determine
total hemoglobin (tHb), hematocrit (Hct), pH, pCO2, partial pressure
of oxygen (pO2), and the Hb-O2 saturation (sO2) values. The
measured values of pO2 and sO2 at each oxygen saturation level were
then subjected to nonlinear regression analysis using the software
Scientist (Micromath, Salt Lake City UT) with the following equation
Finally, for the Hb adduct formation studies, the above-clarified
lysates were subjected to cation-exchange HPLC (Hitachi D-7000
Series, Hitachi Instruments, Inc., San Jose, CA), using a weak cation-
exchange column (Poly CAT A: 30 mm × 4.6 mm, Poly LC, Inc.,
Columbia, MD). A commercial standard consisting of approximately
equal amounts of composite HbF, HbA, HbS, and HbC (Helena
Laboratories, Beaumont, TX) was utilized as reference isotypes. The
areas of new peaks, representing HbS adducts, were obtained,
calculated as percentage fractions of total Hb area and reported as
levels of modified Hb.
Stability Studies of Compounds Using Whole Blood. Normal
whole blood (hematocrit 30%) was incubated in the absence and
presence of 0.5 and 1 mM of compounds 9 and 10. Aliquots were
removed at 0, 4, 8, and 24 h of incubation, and deproteinization was
carried out using 0.1 M formic acid in acetonitrile (HPLC grade) at
60−65 °C for 30 min. Following this, the samples were centrifuged at
12,000 rpm for 20 min to obtain a clear supernatant which was
sequentially filtered through 0.43 and 0.22 μM filters to further clean
the samples for HPLC analysis. Compounds 9, 10, and the
corresponding carboxylic acid derivative of compound 10 were used
as the positive controls, while blood in the absence of any compound
was used as the negative control. HPLC analysis was carried out at
261 nm wavelength using the Phenomenex Luna C18 5μ 100A 150 ×
4.6 mm column with a gradient consisting 0.1% formic acid in water
(Buffer A) and 0.1% formic acid in acetonitrile (Buffer B) and a flow
rate of 1.5 mL/min. Elution times for compounds and their
metabolites were approximately 9.6 and 11.8 min, respectively.
X-ray Crystallography. Co-crystals of liganded Hb with
compounds 6 and 9 were obtained following similar reported
procedures.35,39 In short, freshly made solution of compound in
DMSO was added to deoxygenated (deoxy) Hb (30 mg/mL protein)
at an Hb tetramer-compound ratio of 1:10. Following, the complex
mixture was saturated with carbon monoxide and allowed to incubate
for 2 h to form the COHb-compound complex. Sodium
cyanoborohydride (NaBH3CN) was then added to this mixture to
reduce the Schiff-base adduct formed between the protein and
compound to the corresponding irreversible alkylamine covalent
bond. The resulting solution was crystallized using 10−20%
PEG6000, 100 mM HEPES buffer, and pH 7.4 using the batch
method as previously published. Single-cherry red needle crystals were
formed in 1−3 days and were used to collect X-ray diffraction data at
100 K using Rigaku MicroMax 007HF X-ray Generator, Eiger R 4M
Detector, and Oxford Cobra Cryo-system (The Woodlands, TX). The
crystals were first cryoprotected with 80 μL mother liquor mixed with
62 μL of 50% PEG6000. The data set was processed with the CrysAlis
PRO 2015 program (Rigaku Oxford Diffraction Ltd. Yarnton,
Oxfordshire, England) and the CCP4 suite of programs.63 The
crystal structures of the COHb-compound complexes were
determined by a molecular replacement method with the Phenix
program,53,64 using the native R2-state crystal structure (PDB ID
1BBB) as a search model. The structures were refined using both
Phenix and CNS, while model building and correction were carried
out using COOT.54,55
pO2NmmHg
sO2 % = 100 ×
P5N0(mmHg) + pO2N(mmHg)
This equation was used to calculate P50 and Hill coefficient (N)
values. ΔP50 (%) was determined as
ΔP (%) = 100 × (P in the absence of compound
50
50
− P in the presence of compound)
50
/(P in the absence of compound)
50
For concentration-dependent OEC studies, 0.5, 1.0, and 2 mM
concentrations of each compound to be tested were used, whereas for
time-dependent OEC studies, 2 mM of each compound was used.
Aliquots of each sample was tested at 1, 2, 4, 8, 12, and 24 h to obtain
the P50 and ΔP50 values.
Hemoglobin Modification, Oxygen Equilibrium, and Anti-
sickling Studies Using Human Sickle Blood. Blood suspensions
from subjects with homozygous SCD (hematocrit 20%) were
incubated under air in the absence or presence of compounds (0.5,
1, and 2 mM) at 37 °C for 1 h to ensure that binding has attained
equilibrium. Following, the suspensions were incubated under hypoxic
conditions (certified gas mix of 2.5% O2/97.5% N2) at 37 °C for 2 h.
Aliquot samples were fixed with 2% glutaraldehyde solution without
exposure to air and then subjected to microscopic morphological
analysis of bright field images (at 40× magnification) of single layer
cells on an Olympus BX40 microscope fitted with an Infinity 2 camera
(Olympus) and the coupled Image Capture software. The residual
samples were washed in phosphate-buffer saline and hemolyzed in
hypotonic lysis buffer for subsequent analyses.38,41
For oxygen equilibrium studies, approximately, 100 μL aliquot
samples from the above-clarified lysate were added to 4 mL of 0.1 M
potassium phosphate buffer, pH 7.0, in cuvettes and subjected to
hemoximetry analysis using the Hemox Analyzer (TCS Scientific
Corp., New Hope, PA) to assess P50 shifts. Degree of Hb-O2 affinity
shift (ΔP50) was expressed as percentage fractions of control DMSO-
treated samples.
In Vivo Pharmacologic Effect of Compounds in Wild-Type
Mice. We tested in vivo pharmacologic effects of compounds 9 and 10
(and TD-7 as control) in C57BL/6 mice. All animal experiments were
performed according to the institutional ethical guidelines on animal
care and approved by the Institute Animal Care and Use Committee
at the Children’s Hospital of Philadelphia (approval number: IAC 19-
000113). Equal numbers of male (n = 3) and female (n = 3) mice
were used for the study. The animals were treated with a single
intraperitoneal (IP) (150 mg/kg) dose of the compounds. Blood
samples were obtained prior to treatment (0) and 1, 3, and 6 h post-
treatment, via submandibular bleeding, and subjected to hemolysis
using standard methods. Clarified lysates, free of cell debris, and RBC
ghosts were analyzed by cation-exchange HPLC to determine the
levels of drug-modified hemoglobin (adducts) and utilized to conduct
oxygen equilibrium studies to determine degrees of shift in P50 values
using methodologies described earlier for in vitro studies on human
samples.
M
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Article
Statistical Analyses. All functional and biological assays
evaluating antisickling properties, Hb modification, and oxygen
affinity changes were conducted in three biological replicates. Results
are reported as mean values with standard deviations, from triplicate
analyses.
Author Contributions
M.K.S. and Y.Z. conceived and oversaw the project and
finalized the manuscript. P.P. drafted the manuscript. P.P.
conducted the chemical synthesis. P.P. and M.S.G. conducted
the in vitro studies with normal human blood. P.P. and F.N.M.
conducted the crystallography studies. O.A. designed, con-
ducted, and analyzed in vitro and in vivo biochemical assays
and edited the manuscript. Q.C. conducted in vitro antisickling
and in vivo studies.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01287.
■
sı
Notes
Molecular formula strings (CSV)
The authors declare the following competing financial
interest(s): Virginia Commonwealth University has filed a
patent related to compounds 1-14.
OEC of final compounds; final 2Fo − Fc electron density
map of COHb-6 and COHb-9 complexes; representa-
tive plot for cation-exchange HPLC patterns of modified
Hb; spectral data of final compounds (1H and 13C
graphs); and purity data of final compounds (HPLC
graphs) (PDF)
ACKNOWLEDGMENTS
■
This work was supported by NIH/NIMHD grant MD009124
(M.K.S.). Structural biology resources were provided by NIH
Shared Instrumentation Grant S10OD021756 (M.K.S.) and
Virginia General Assembly Higher Education Equipment Trust
Fund (HEETF) to Virginia Commonwealth University
(M.K.S.).
Accession Codes
The atomic coordinate and structure factor files have been
submitted to the Protein Data Bank under accession codes
6XDT and 6XE7 for the complex structures of compounds 6
and 9, respectively. Authors will release the atomic coordinates
and experimental data upon article publication.
ABBREVIATIONS
■
AUTHOR INFORMATION
Corresponding Authors
Hb, hemoglobin; SCD, sickle cell disease; HbS, sickle
hemoglobin; RBC, red blood cell; HU, hydroxyurea; AEH,
allosteric effector of hemoglobin; ALDH, aldehyde dehydro-
genase; OEC, oxygen equilibrium curve; SS, homozygous
sickle cell trait; HbA, normal hemoglobin; pO2, partial pressure
of oxygen; pCO2, partial pressure of carbon dioxide; sO2,
oxygen-saturation; Hct, hematocrit; tHb, total hemoglobin; P50,
partial pressure of oxygen at which 50% of hemoglobin is
saturated with oxygen
■
Yan Zhang − Department of Medicinal Chemistry and The
Institute for Structural Biology, Drug Discovery, and
Development, School of Pharmacy, Virginia Commonwealth
University, Richmond, Virginia 23298, United States;
orcid.org/0000-0001-8934-7016; Email: yzhang2@
vcu.edu
Martin K. Safo − Department of Medicinal Chemistry and The
Institute for Structural Biology, Drug Discovery, and
Development, School of Pharmacy, Virginia Commonwealth
University, Richmond, Virginia 23298, United States;
orcid.org/0000-0002-9536-1743; Email: msafo@
vcu.edu
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