Identification of a novel class of covalent modifiers of hemoglobin as potential antisickling agents

Organic &

Biomolecular Chemistry

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Identiﬁcation of a novel class of covalent modiﬁers

of hemoglobin as potential antisickling agents†

Cite this: Org. Biomol. Chem., 2015,

13, 6353

A. M. Omar,*^a,bM. A. Mahran,^cM. S. Ghatge,^dN. Chowdhury,^dF. H. A. Bamane,^e

M. E. El-Araby,^a,fO. Abdulmalik^gand M. K. Safo*^d

Aromatic aldehydes and ethacrynic acid (ECA) exhibit antipolymerization properties that are beneﬁcial for

sickle cell disease therapy. Based on the ECA pharmacophore and its atomic interaction with hemoglobin,

we designed and synthesized several compounds – designated as KAUS (imidazolylacryloyl derivatives) –

that we hypothesized would bind covalently to βCys93 of hemoglobin and inhibit sickling. The com-

pounds surprisingly showed weak allosteric and antisickling properties. X-ray studies of hemoglobin in

complex with representative KAUS compounds revealed an unanticipated mode of Michael addition

between the β-unsaturated carbon and the N-terminal αVal1 nitrogen at the α-cleft of hemoglobin, with

no observable interaction with βCys93. Interestingly, the compounds exhibited almost no reactivity with

the free amino acids, ^L-Val, L-His and L-Lys, but showed some reactivity with both glutathione and L-Cys.

Our ﬁndings provide a molecular level explanation for the compounds biological activities and an impor-

tant framework for targeted modiﬁcations that would yield novel potent antisickling agents.

Received 23rd February 2015,

Accepted 16th April 2015

DOI: 10.1039/c5ob00367a

www.rsc.org/obc

aﬃnity for ligands, and the liganded or oxygenated relaxed (R)

state, which exhibits high aﬃnity for ligands.^4–6Unless noted

Introduction

Sickle hemoglobin (Hb S), under low oxygen tension and/or otherwise, the R-state is used to represent the ensemble of

when deoxygenated, polymerizes into rigid and insoluble relaxed Hb states that include the classical R-state, R2-state,

fibres that cause the primary pathophysiology associated with R3-state, RR2-state, RR3-state, etc.^7,8The crystal structure of

sickle cell disease (SCD), leading to several secondary patho- Hb, either in the T- or R-state quaternary conformation, is

logical eﬀects, including, but not limited to, adhesion of red made up of two αβ heterodimers (α1β1 and α2β2) arranged

blood cells (RBCs) to tissue endothelium, oxidative stress, around a 2-fold axis of symmetry to form a central water cavity

hemolysis of RBCs, decreased vascular nitric oxide bioavail- that is accessed via an α-cleft or a β-cleft. Allosteric eﬀectors,

ability, inflammation, vaso-occlusion, impaired microvascular both endogenous and synthetic, are known to bind to the

blood flow, and painful crises.^1–3

central water cavity, the α-cleft, β-cleft, or the surface of the

Hemoglobin (Hb) functions in equilibrium between the protein, and modulate the allosteric properties of Hb, leading

unliganded or deoxygenated tense (T) state, which exhibits low to a further increase or decrease in its aﬃnity for oxygen.^4,7–14

For example, the natural allosteric eﬀector, 2,3-diphospho-

glycerate (2,3-DPG), binds to the β-cleft of Hb, and preferen-

^aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz

tially stabilizes the T-state relative to the R-state and produces

University, Alsulaymanyah, Jeddah 21589, Saudi Arabia.

a low-aﬃnity Hb.⁹Interestingly, sickle RBCs have a signifi-

E-mail: asmansour@kau.edu.sa

cantly reduced aﬃnity for oxygen compared to normal RBCs,

presumably as a result of an increased intracellular concen-

tration of 2,3-DPG in the erythrocytes, which compensates for

the lower hematocrit, leading to both increased and premature

release of oxygen and concomitant RBC sickling.^15,16This has

led to a rational approach to treat the disease by shifting the

Hb oxygen equilibrium curve (OEC) to the left (i.e., stabilizing

the R-state and/or destabilizing the T-state), producing a high-

aﬃnity Hb.^{7,8,10,11,17–20}Unlike non-covalent binders of Hb,

covalent binders have proven to be potential antisickling

agents by increasing the oxygen aﬃnity of Hb. Several aromatic

aldehydes are known to have this allosteric property by

^bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University,

Cairo 11884, Egypt

^cDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria

University, Alexandria, Egypt

^dDepartment of Medicinal Chemistry, School of Pharmacy and Institute for

Structural Biology and Drug Discovery, Virginia Commonwealth University,

Richmond, VA 23219, USA. E-mail: msafo@vcu.edu

^eDepartment of Biochemistry, Faculty of Medicine, King Abdulaziz University,

Alsulaymanyah, Jeddah 21589, Saudi Arabia

^fDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Helwan

University, Cairo 11790, Egypt

^gDivision of Hematology, The Children’s Hospital of Philadelphia, PA 19104, USA

†Electronic supplementary information (ESI) available. See DOI: 10.1039/

c5ob00367a

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forming

a

Schiﬀ-base interaction in

a

symmetry-related than aromatic aldehydes, necessitating lower and less frequent

fashion with the two N-terminal αVal1 nitrogens at the α-cleft doses. Biological analysis, including OEC and sickle RBC mor-

of liganded Hb (in the R2-state conformation), and through phological antisickling studies, showed the compounds to

several inter-subunit mediated hydrogen-bond and/or hydro- exhibit weak or no allosteric and antisickling activities when

phobic interactions cross-link the two α-subunits to stabilize compared to ECA. Structural study of Hb in complex with two

the R-state Hb.^{7,8,10,11,17–20}Of note is that eﬀectors preferen- of the compounds, KAUS-12 and KAUS-15, provides molecular

tially bind to the α-cleft of liganded Hb in the R2-state confor- insight into the unexpected biological eﬀect, and oﬀers impor-

mation because the α-cleft of the other relaxed states of Hb, tant direction and guidance for structure-based redesign of

including the classical R-state, is sterically crowded.^7,18It is the compounds to yield potent left-shifting and antisickling

notable that, unlike liganded Hb or oxygenated Hb (oxyHb), agents.

binding to unliganded Hb or deoxygenated Hb (deoxyHb)

appears to inhibit non-specific chloride binding and/or break

inter-subunit hydrogen-bond interactions, leading to T-state

Results

Design

destabilization.^7,18,21,225-Hydroxymethyl-2-furfural (5-HMF,

aka Aes-103) is one such left-shifting allosteric eﬀector that

Ethacrynic acid increases Hb aﬃnity for oxygen, and expect-

edly shows antisickling properties.^37,38In a reported crystallo-

graphic study of ECA complexed to deoxyHb, it was shown that

the β-unsaturated carbon of the eﬀector reacted covalently

with the sulfur atom of βCys93 (Michael addition reaction;

Scheme 1), while the rest of the molecule makes hydrogen-

bond and/or hydrophobic interactions with αPro44, βAsp94,

βHis97, and αThr41.³⁸The interaction at the βCys93 binding

site destabilizes the T-state by abolishing a salt-bridge inter-

action between βHis146 and βAsp94 in deoxyHb, shifting the

allosteric equilibrium to the R-state and increasing the protein

aﬃnity for oxygen. Binding of ECA to the surface-located

βCys93 was also suggested to prevent stereospecific interaction

between Hb S molecules, providing a secondary antisickling

eﬀect.³⁸Alkyl isothiocynates,²³and quite recently thiols,^10,24

have also been shown to covalently bind to βCys93, acting in a

similar mechanistic manner as ECA to increase Hb aﬃnity for

oxygen. Using ECA as a scaﬀold, we rationally designed and

synthesized nine imidazolylacryloyl derivatives with varying

chloro-substitutions on the phenyl ring, as well as varying

carboxylate arm lengths (Fig. 1). These compounds have

similar a Michael acceptor chemotype α,β-unsaturated ketone

as ECA, and it was expected that the β-unsaturated carbon

would react in a similar covalent manner to ECA, i.e., under-

going a Michael addition reaction with the sulfur of βCys93.

The replacement of the ethyl group attached to the β-unsatu-

rated carbon in ECA with an imidazole in the KAUS molecules

binds to both R-state and T-state Hb, and has been shown to

prevent Hb S polymerization and erythrocyte sickling.^10,17,18

5-HMF is currently in phase II clinical studies for the treat-

ment of SCD. A potential problem for the use of aromatic alde-

hydes for SCD therapy is their poor pharmacokinetic

properties (due to metabolic instability of the aldehyde func-

tion as a result of aldehyde dehydrogenase metabolism) which

may necessitate large amounts and frequent dosing to reach

therapeutic level.¹⁰This has prompted several studies to find

alternate covalent modifiers of Hb, such as isothiocynates and

thiols, to treat the disease.^23,24

Eﬀectors that right-shift the OEC to produce low oxygen-

aﬃnity Hb have also been studied for treating ischemia-related

cardiovascular diseases, where more oxygen is needed to heal

tissue or organs, and also as radiation enhancers in the radio-

therapy of hypoxic tumors.^25–28Right-shifters encompass both

covalent binders, such as aromatic aldehydes,^29,30and non-

covalent binders, e.g. aromatic propionates.^{12,13,31–34}Structural

studies of these eﬀectors show that the compounds stabilize

the T-state through inter-subunit or cross-linking

interactions.^{12,13,31–34}Some of these right-shifters have also

been shown to bind to R-state Hb and destabilize this

conformation.^34–36From the foregoing, it is clear that Hb

eﬀectors can bind both T-state and R-state Hb; however, the

direction of the OEC shift is dependent on which Hb state is

preferentially stabilized and/or destabilized.

Ethacrynic acid (ECA), used as a diuretic, also increases the

oxygen aﬃnity of Hb and/or causes stereospecific inhibition of

polymer formation with a concomitant antisickling eﬀect.^37,38

In contrast to aromatic aldehydes, the antisickling activity of

ECA is through interaction with the surface-located Hb

residue, βCys93 via a Michael addition reaction. Unfortunately,

the diuretic activity of ECA precludes its use as an oral thera-

peutic agent for the treatment of SCD. The current study was

initiated with the principle of forming a similar covalent

adduct between imidazolylacryloyl derivatives (designated as

KAUS molecules) and βCys93 that not only would increase the

oxygen aﬃnity of Hb, but do so with greater antisickling

potency than ECA, as well as with reduced or no diuretic

eﬀect. We also posited that these compounds, with a reactive

Scheme 1 Schematic representation of the Michael addition reaction

between the β-unsaturated carbon of ethacrynic acid (ECA) and the

α,β-unsaturated ketone, would be more metabolically stable βCys93 sulfur of deoxyHb.

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A modified synthetic route (Scheme 3) was used for the pro-

pionic acid derivatives 1b, 1e and 1h, since the alkylation of

the hydroxyl group using ethyl-3-bromopropionate was not suc-

cessful. The protected aldehyde, 2-(2-bromoethyl)-1,3-dioxo-

lane was used for alkylation as a precursor for the acid. The

acid intermediate 8 was obtained by deprotecting the cyclic

acetal using aq. HCl, followed by oxidation using oxone.⁴⁵The

final products (1a–1h and 1i) were purified using silica gel

column chromatography to give purities >95% as indicated by

HPLC (UV detection) and/or LC-MS.

Fig. 1 Synthesized imidazoloylacryloyl derivatives.

Oxygen equilibrium curve (OEC) studies

was also anticipated to increase the reactivity of the β-carbon

and hence accelerate the Michael addition reaction. It was also

envisaged that the hydrophobic chlorobenzene ring, imidazole

ring and/or the varying carboxylate moiety would make various

hydrogen-bond/hydrophobic interactions with the βCys93

binding pocket residues, including αLys40, αThr41, αSer49,

αPro44, βGlu90, βAsp94, βHis97, and the C-terminal residue

βHis146, which would confer binding stability to these com-

pounds, and hence a greater destabilization eﬀect on the

T-state, leading to greater increase in Hb oxygen aﬃnity when

compared to ECA.

Compounds that increase the oxygen aﬃnity of Hb are

expected to shift the OEC to the left, and the degree of shift is

reported as an increase or decrease in P₅₀(the oxygen tension

at 50% Hb O₂saturation), while the degree of allosteric charac-

ter is indicated by the slope of the oxygen binding curve (n₅₀).

The eﬀect of the nine KAUS molecules on Hb aﬃnity for

oxygen was determined using normal human blood and multi-

point tonometry as previously reported,¹⁸and the results are

shown in Table 1. At 2 mM concentration, the compounds

showed very little or no left-shifting eﬀect on the OEC

(0.0–2.8 mmHg) compared to the 5.7 mmHg left-shift by ECA.

One compound, KAUS-10, seems to right-shift the OEC by

2.1 mmHg. There appears to be some correlation in the OEC

shift and the varying carboxylate arm length in the three

classes of compounds, where the phenoxybutyrates (with a

longer carboxylate arm) seem to give the greatest shift to the

left, while the phenoxyacetates (with a shorter carboxylate arm)

show the smallest shift, and in some instances even right-

shifts the OEC. There is no apparent correlation between the

allosteric activity of the compounds and the varying chloro-

substitutions on the phenyl ring.

Chemistry

For the phenoxyacetic acid and phenoxybutyric acid derivatives

(1a, 1c, 1d, 1f, 1g and 1i), the synthetic sequence in Scheme 2

was followed, where the alkylation step was carried out using

the corresponding bromo-ethyl esters.^39,40The ester intermedi-

ate 3 was hydrolyzed to obtain the corresponding acid inter-

mediate 4 that was used for the condensation step. The

condensation of methylketone intermediate 4 with trityl-

protected imidazole-3-carbaldehyde 5⁴¹was accomplished

under catalysis by sodium hydroxide to give the protected imi-

dazolylacryloyl final precursors 6.^42,43The target phenoxyacetic

Antisickling studies

and phenoxybutyric acid derivatives were obtained after Compounds that increase the oxygen aﬃnity of Hb are

removal of the trityl protecting group under standard acidic expected to inhibit RBC sickling.^7,8,11,18Selected compounds,

conditions.⁴⁴

including KAUS-12, KAUS-13, KAUS-15, KAUS-16, and

Scheme 2 Synthetic scheme for compounds 1a, 1c, 1d, 1f, 1g and 1i.

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Scheme 3 Synthetic scheme for compounds 1b, 1e and 1h.

first reacted with deoxyHb or carbonmonoxy Hb (COHb), fol-

lowed by crystallization using high-salt conditions as pre-

viously described.^11,18,22,46Unlike aromatic aldehydes, which

co-crystallize with deoxyHb to give both T- and R2-state crys-

tals,^11,18we only observed T-state crystals during the co-crystal-

lization experiment with deoxyHb. The T-state crystals are

isomorphous with the native deoxyHb crystal (PDB code

2DN2), necessitating the use of 2DN2 as the starting model for

the refinement of both complex structures. The two structures

refined to 1.7 Å and 1.9 Å, respectively, and the structural stat-

istics are summarized in Table 2. The refined KAUS-12 and

KAUS-15 complex structures are deposited in the PDB with the

ID codes 4ROL and 4ROM, respectively.

The co-crystallization experiment of KAUS-15 or KAUS-12

with COHb resulted in the formation of classical R-state crys-

tals that diﬀracted to 2.0 and 2.8 Å, respectively. The iso-

morphous R-state native COHb structure (1LJW) was used as

the starting model to refine the complexes. Unlike the

deoxyHb complex structures, which showed apparent binding

of the KAUS compounds (see below), none of the COHb struc-

tures showed bound compound. Since the liganded structures

did not have any bound eﬀector, and were also determined to

be indistinguishable from the native 1LJW structure, the

refinements were terminated at R_factor/R_freeof 23.5/26.4 and

26.4/33.5, respectively.

Table 1 The eﬀect of compounds on Hb aﬃnity for oxygen using

normal whole blood^a

Ave. dev.

b

c

d

Comp Name

R₁R₂

N

Mean P₅₀from mean ΔP₅₀n₅₀

Ctr

1a

1b

1c

1d

1e

1f

DMSO^e

ECA

KAUS-10 Cl Cl

KAUS-11 Cl Cl

KAUS-12 Cl Cl

KAUS-13

KAUS-14

KAUS-15

KAUS-16 Cl

KAUS-17 Cl

KAUS-18 Cl

38.3

32.7

40.4

38.1

36.9

37.4

38.2

35.6

38.3

37.8

37.4

0.0

0.2

0.0 2.3

−5.7 1.9

2.1 2.2

−0.2 2.2

−1.4 1.9

−1.0 2.4

−0.1 2.1

−2.8 1.9

0.0 2.3

1

2

3

1

2

3

1

2

3

0.23

0.47

0.14

0.05

0.3

0.55

0.74

0.15

0.42

H

Cl

H

1g

1h

1i

−0.5 2.3

−0.9 2.1

b

^aThe results are the means of 2 measurements. P₅₀is the oxygen

pressure at which normal RBCs (22% hematocrit) are 50% saturated

with oxygen. ^cΔP₅₀is P₅₀of compound-treated cells – P₅₀of control.

^dn₅₀is the Hill coeﬃcient at 50% saturation with oxygen. ^eThe final

concentration of DMSO was about 2% in all samples, including the

control.

KAUS-17, at 2 mM concentration were tested for their abilities

to prevent RBC sickling under hypoxic conditions.^11,18As

expected from the weak or no left-shifting activities, the com-

pounds showed very little to no observable antisickling eﬀect,

with even KAUS-16 and KAUS-17 promoting slight RBC sick-

ling (Fig. 2). In comparison, the control ECA inhibited about

18% RBC sickling.

KAUS compounds bind at the α-cleft of deoxygenated Hb with

the amine of the N-terminal αVal1

We assumed, based on the ECA interaction with Hb, that the

primary site of reaction of the KAUS molecules would be at the

βCys93 binding site; however, the crystallographic studies with

KAUS-12 and KAUS-15 showed no apparent binding of either

compound to βCys93 in the classical R-state or T-state struc-

tures. Instead, we observed that the compounds were bound at

Co-crystallization of deoxygenated Hb or carbonmonoxy Hb

with KAUS-12 or KAUS-15

X-ray crystallography was used to determine the binding site of the α-cleft of the T-state structures (Fig. 3A and B). There was

KAUS-12 and KAUS-15 in both deoxygenated Hb and carbon- no apparent binding of the compounds at the α-cleft of the

monxy Hb (COHb) to explain the unexpected biological activi- classical R-state structures, which we attribute to steric crowd-

ties of the imidazolylacryloyl derivatives. The compounds were ing at this binding pocket.^7,18

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Fig. 2 Morphology of SS cells after incubation with various compounds (2 mM) or without compound under 4% oxygen at 37 °C for 5 h. (A) In the

absence of a test compound, most of the cells underwent sickling (85%). (B) At 2 mM, most of the compounds, including KAUS-15 depicted here,

showed very little or no observable antisickling eﬀect. (C) At 2 mM, KAUS-16 promoted sickling of the cells. (D) At 2 mM, ECA inhibited sickling of

the cells by ∼18%.

Table 2 Crystallographic data and reﬁnement statistics for deoxyHb in complex with KAUS-12 and KAUS-15; values in parentheses refer to the out-

ermost resolution bin

Data collection statistics

KAUS-12

KAUS-15

Space group

P2₁

Cell dimensions (Å)

Resolution (Å)

No. of measured reflections

Unique reflections

Redundancy

a = 61.64, b = 76.57, c = 53.20; β = 98.02

33.59–1.70 (1.76–1.70)

159 264

52 644 (5225)

3.03 (3.03)

a = 61.98, b = 78.92, c = 53.26; β = 98.44

33.75–1.90 (1.97–1.90)

134 725

37 044 (3482)

3.64 (3.80)

I/σI

8.4 (4.4)

97.7 (96.8)

7.4 (19.3)

22.7 (5.0)

92.5 (87.8)

3.4 (16.3)

Completeness (%)

R_merge^a(%)

Structure refinement

Resolution limit (Å)

No. of reflections

R_work(%)

33.59–1.70 (1.78–1.70)

52 489 (6138)

18.2 (27.0)

33.18–1.90 (1.99–1.90)

37 014 (4143)

21.3 (30.6)

R_free^b(%)

20.6 (30.1)

24.8 (34.2)

R.m.s.d. standard geometry

Bond lengths (Å)

Bond angles (°)

Dihedral angles (%)

Most favored regions

Allowed regions

Average B-factors (Å²)

All atoms

Protein alone

Water

KAUS

Heme

0.008

1.5

0.008

1.4

93.2

6.8

93.0

7.0

16.0

13.5

29.7

34.9

10.3

29.7

28.1

39.6

51.4

23.8

^aR_mege= ∑_hkl∑_i/I_hkli− <I_hkli>/∑_hkl∑_i< I_hkli>. ^bR_freewas calculated with 5% excluded reflection from the refinement.

The deoxyHb complex structures of KAUS-12 and KAUS-15,

Although the compounds bind to T-state Hb, they are

and the native deoxyHb 2DN2 structure, are quite similar to characterized by weak and highly disordered electron densities

each other, with a root mean square deviation (rmsd) of (Fig. 3C–F). There are several reported cases where bound left-

∼0.4 Å, indicating no significant change in the overall T-state shifting eﬀectors at the α-cleft of deoxyHb show weak and dis-

quaternary conformation upon eﬀector binding. Similarly, ordered density even though these compounds bind covalently

there are no apparent diﬀerences in the heme environments, to the protein.^11,18This is in contrast to right-shifting

the interdimer interfaces, or the inter-subunit hydrogen-bond/ eﬀectors, especially very potent ones, which are normally well-

salt bridge interactions that are unique to T-state Hb. Thus, ordered as a result of extensive inter-subunit mediated inter-

the overall quaternary and tertiary structures are very similar actions with the protein.¹²Since the bound compound den-

among the three T-state structures.

sities are weak, their refined positions may not be totally

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α2Lys127, α2Ala130, α1Thr137, α1Ser138, α1Tyr140, α1Arg141

and β2Trp37. The butyrate, which is directed toward the

middle of the central water cavity, could also make direct and/

or water-mediated inter-subunit and intra-subunit hydrogen-

bond/salt-bridge interactions with the surrounding residues,

α1Lys99, α2Lys99, α1Pro95, α1Arg141, and α1Asp126. Similar

interactions as discussed above for the α2Val1 bound molecule

are also observed for the symmetry-related α1Val1 bound

molecule.

From the foregoing, it is apparent that the symmetry-

related molecules of KAUS-12 or KAUS-15 bind to the α-cleft of

deoxyHb, making possible hydrogen-bond/hydrophobic

mediated interactions that tie the two α-subunits together to

stabilize the T-state Hb. These cross-link interactions are remi-

niscent of monoaldehyde-monocarboxylate, monoaldehyde-

biscarboxylate, and bisaldehyde-biscarboxylate molecules that

also bind to deoxyHb and confer a similar stabilization eﬀect

on T-state Hb.^29,30The above observations are in contrast with

yet another class of aromatic aldehydes (monoaldehydes), e.g.

5-HMF, that bind to both deoxyHb and liganded Hb (in the

R2-state form), but while these compounds confer stability to

the R-state Hb through mediated inter-subunit interactions,

binding to deoxyHb destabilizes the T-state, and as a result the

allosteric equilibrium is shifted to the R-state Hb.^7,11,18

Reactivity of KAUS toward glutathione (GSH) and free amino

acids, ^L-Cys, L-Val, L-His and L-Lys

Although the structural studies suggest specific binding of the

compounds at the α-cleft of Hb, nevertheless, due to their

highly reactive Michael acceptor moiety, there is a likelihood

of non-specific binding to a number of nucleophiles, including

Fig. 3 Binding of KAUS-12 and KAUS-15 at the α-cleft of deoxyHb. (A)

Symmetry-related bound KAUS-12 (spheres) at the α-cleft of deoxyHb

(ribbons). (B) Symmetry-related bound KAUS-15 at the α-cleft of GSH and other amino acids on proteins. We therefore investi-

deoxyHb. (C) Diﬀerence electron density (F_o− F_c) map of the bound

KAUS-12 (before KAUS-12 was built into the model) contoured at 2.2σ.

(D) Final 2F_o− F_cmap of the bound KAUS-12 contoured at 0.7σ. (E)

Diﬀerence electron density (F_o− F_c) map of the bound KAUS-15 (before

gated possible GSH, ^L-Cys, L-Val, L-His or L-Lys conjugates with

KAUS-12, KAUS-15 or ECA using LC-MS or UPLC-MS analysis.

The LC-MS analysis of the reaction between KAUS-12 and

GSH after 1 h showed only traces (1.6%) of the adduct ion,

which increased to 9.7% after 3 h (Fig. S1A,† Table 3). A

similar result was observed with KAUS-15, which showed

10.6% and 18.6% of the adduct ion at 1 h and 3 h, respectively

(Fig. S1B,† Table 3). In contrast to the KAUS compounds, ECA

reacted strongly with GSH, disappearing completely after

60 minutes of reaction, and only its GSH adduct was observed

in the mass (Fig. S1C†).

The UPLC-MS analysis of the reactions between the com-

pounds (KAUS-12, KAUS-15 and ECA) and the four amino

acids, ^L-Cys, L-Val, L-His and L-Lys (which were incubated for

3 hours) showed only ^L-Cys reacting in any significant manner

with the three compounds (Table 4; Fig. S2–S5†). ECA was the

most reactive toward ^L-Cys (∼96%), followed by KAUS-15

(∼57%), while KAUS-12 showed the least reactivity (∼32%).

Interestingly, the KAUS compounds were unreactive toward

L-Val, L-His and L-Lys (Table 4; Fig. S2–S5†) even though struc-

tural studies suggest adduct formation with the N-terminal

valine of the protein. Similar observations have been reported

with aromatic aldehydes, where the Schiﬀ base equilibrium

constants between the aldehyde and the amine of the N-term-

KAUS-15 was built into the model) contoured at 2.2σ. (F) Final 2F_o− F_c

map of the bound KAUS-15 contoured at 0.7σ.

unambiguous, and therefore only general conclusions for com-

pound–protein interactions will be made.

For each T-state complex, we identified two-symmetry-

related diﬀerence electron densities close to α1Val1 and

α2Val1 (Fig. 3C–F). At each binding site, the αVal1 nitrogen

makes a covalent interaction with the β-unsaturated carbon

through a Michael addition reaction (Fig. 4A), which is ascer-

tained by an overlapping electron density of the two interact-

ing atoms (Fig. 3C–F). With a covalent interaction with one of

the αVal1 nitrogens (such as α2Val1), the imidazole ring of

KAUS-12 or KAUS-15 is in a position to make either direct or

water-mediated intra-subunit hydrogen-bond interaction with

the α2Ser131 and α2Thr134 hydroxyl groups (Fig. 4B and C).

The α,β-unsaturated ketone and the chlorophenyl ring moi-

eties could also make both intra-subunit and inter-subunit

hydrophobic interactions with the surrounding residues,

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Fig. 4 Interactions between the protein and KAUS-12 or KAUS-15. (A) Schematic representation of the Michael addition reaction between the αVal1

nitrogen of Hb and the β-unsaturated carbon of the KAUS molecule. (B) Possible interactions between the protein and the KAUS-12 molecule at the

α2Val1 binding site. For clarity, not all surrounding residues are shown. (C) Schematic representation of the possible interactions between KAUS-12

and deoxyHb at the α2Val1 binding site. Note that atomic distances are not given because of the uncertainty in the atomic positions of the bound

compound as a result of the weak density.

covalent interaction with another surface located Hb residue,

βHis117;³⁸however, it only showed very weak reactivity with

free ^L-His (3%). Similar to the KAUS compounds, ECA was

unreactive toward ^L-Val and L-Lys. The apparent reactivity of

the KAUS compounds, as well as aromatic aldehydes and ECA,

with the protein amino acids but not with the corresponding

free amino acids could be due to the fact that the protein

oﬀers stable binding environments for these compounds that

contribute to the stability of the covalent interactions.

Table 3 LC-MS measurements of relative ratios of KAUS-12, KAUS-15,

and ECA and their adduct products with GSH

Reaction

time (min) Peak

RT^a

(min) m/z

Max.

Ratio of Cpd/

adduct (%)

Area

KAUS-12

60

Compound 3.2

Adduct 2.4

Compound 3.1

Adduct 2.4

368.9 223 754 007 98.4

454.3 3 682 173 1.6

368.9 136 800 833 90.3

454.2 14 624 755 9.7

180

KAUS-15

60

Compound 2.9

Adduct 2.4

Compound 2.9

Adduct 2.3

334.9 151 292 991 89.4

642.2 17 918 962 10.6

334.9 156 535 264 81.4

642.2 35 710 465 18.6

Reactivity of KAUS-15 towards βCys93 of Hb

180

Both solution and structural studies suggest ECA forms a

covalent interaction with βCys93 of Hb,^37,38consistent with the

strong reactivity of this compound with free ^L-Cys. Our struc-

tural studies on the other hand show no adduct formation

^aRT = Retention time.

inal Val1 of Hb are 3- to 5-fold greater than typical Schiﬀ base with βCys93, even though the compounds do react with free

equilibrium constants with free amino acids or small molecule cysteine, although more weakly than ECA. We therefore con-

amines.²⁰We note that ECA has been reported to form a ducted a solution-based sulfhydryl assay with Hb incubated

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Table 4 UPLC-MS measurements of relative ratios of KAUS-12, KAUS-15, and ECA and their adduct products with diﬀerent amino acids

RT^a

(min)

Ratio of

Cpd/adduct

Amino Acid

Cysteine

Compound

Ethacrynic acid

KAUS-12

Peak

Max. m/z

Area

Compound

Adduct

Compound

Adduct

Compound

Adduct

4.43

2.63

2.45

1.79

2.12

1.34

302.7

423.7

368.7

489.7

334.8

455.8

898

3.7

23 733

19 901

9559

14 150

18 915

96.3

67.5

32.5

42.8

57.2

KAUS-15

Lysine

Ethacrynic acid

KAUS-12

Compound

Adduct

Compound

Adduct

Compound

Adduct

4.42

N/A

2.46

N/A

2.13

N/A

302.7

N/A

368.8

N/A

334.9

N/A

11 524

N/A

21 520

N/A

22 750

N/A

100

N/A

100

N/A

100

N/A

KAUS-15

Valine

Ethacrynic acid

KAUS-12

Compound

Adduct

Compound

Adduct

Compound

Adduct

4.42

N/A

2.46

N/A

2.15

N/A

302.8

N/A

368.8

N/A

334.9

N/A

13 128

N/A

31 545

N/A

23 461

N/A

100

N/A

100

N/A

100

N/A

KAUS-15

Histidine

Ethacrynic acid

KAUS-12

Compound

Adduct

Compound

Adduct

Compound

Adduct

4.42

1.71

2.46

N/A

2.16

N/A

302.8

457.9

368.8

N/A

334.8

N/A

12 934

382

29 432

N/A

17 020

N/A

97.1

2.9

100

N/A

100

N/A

KAUS-15

^aRT = Retention time.

with KAUS-15 (which showed greater reactivity with free the presence of ECA, almost 100% of the βCys93 thiols were no

cysteine than KAUS-12) and 5,5′-dithiobis-(2-nitrobenzoic acid) longer detectable, indicating complete reaction of ECA with

(DTNB) to determine the KAUS reactivity with βCys93. First, we the βCys93 thiols. In the presence of KAUS-15, about 78% of

measured the accessible thiol content in Hb, which was found the thiol groups were available to react with DTNB, suggesting

to be close to two, consistent with previous reports that a weak interaction between KAUS-15 and βCys93, perhaps

suggest that out of the six thiols present in Hb, only the two explaining why the KAUS compounds were not observed

βCys93 are solvent accessible and reactive.⁴⁷As expected, in crystallographically at the βCys93 binding site. It should be

noted that several compounds, including thiols and alkyl

isothiocynates, have been shown crystallographically to form a

covalent interaction with βCys93 of Hb.^10,23,24

Cytotoxicity of KAUS

We determined the potential toxicity of KAUS-12, KAUS-15,

and ECA by testing against the C-166 mouse fibroblast cell line

using the cell viability sulforhodamine B (SRB) assay,⁴⁶follow-

ing compound incubation in a concentration range from

0.01 μM to 100 μM over a 72 h period. While ECA contributes

to cell toxicity with an IC₅₀of 22 μM, the two tested com-

pounds were much less toxic (Fig. 5). When extrapolated from

the data, the IC₅₀values of both KAUS-12 and KAUS-15 were

greater than 1.6 mM.

Discussion

Fig. 5 Toxicity of compounds in the C-166 mouse ﬁbroblast cell line,

assessed using the SRB assay. Results are shown for KAUS-12 (squares)

One therapeutic strategy for SCD is to develop allosteric

eﬀectors of Hb that stabilize the relaxed state Hb and/or desta-

and KAUS-15 (triangles), in comparison to ECA (circles). The error on

each data point is the standard deviation of three measurements.

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bilize the tense state Hb to increase the concentration of the inter-subunit interactions; the latter interactions, which

high-oxygen-aﬃnity Hb species that do not polymerize.^7,10cross-link the two α-subunits, are expected to further

Finding such Hb allosteric molecules has been a challenge, stabilize the T-state Hb and decrease the protein aﬃnity

due in part to the diﬃculty of designing pharmaceutically for oxygen.

useful agents capable of modifying the large amounts of intra-

While βCys93 is buried in deoxygenated Hb, it is accessible

cellular Hb (∼5 mmol L⁻¹). However, this fact is alleviated by in liganded Hb and able to react with electrophiles.^10,24A thiol

the knowledge that therapeutic eﬃcacy could be obtained with containing compound was recently shown crystallographically

50% or less modification of Hb S, as evidenced by patients to form a covalent adduct with βCys93 in classical R-state Hb

who are heterozygous for SCD and are asymptomatic, as well and R3-state Hb.²⁴However, as noted above, we did not

as by the fact that patients that produce about 20% fetal Hb observe any KAUS compound at the βCys93 binding site in the

(Hb F) only have the mild form of the disease.^48–50Nonethe- classical R-state structure, even though our solution studies

less, it would still require significant amounts of compounds suggest possible binding of the compounds with this amino

to treat the disease, underscoring the realization that any acid, albeit weak. This weak binding may explain why the com-

potential antisickling agent would have to be highly specific pounds were undetected in our liganded crystal structure.

for Hb, as well as exhibit a long and sustained eﬀect. One class Unlike the T-state structure, which showed the KAUS com-

of allosteric eﬀectors, aromatic aldehydes, have become attrac- pound bound to the N-terminal αVal1 nitrogen, we did not

tive since they form a covalent, albeit transient, interaction observe such binding in the classical R-state structure,

with the α-subunit N-terminal αVal1 nitrogens of Hb, which consistent with the sterically hindered α-cleft of R-state Hb

leads to significant reduction of Hb S polymerization due to precluding binding at this pocket.^7,18Instead, eﬀectors that

the ability of the compounds to increase Hb S oxygen bind to the α-cleft of liganded Hb are known to bind to the R2-

aﬃnity.^{10,11,17–19}Unfortunately, the metabolic instability of state form^7,11,18Although we have not been able to obtain

aromatic aldehydes, as a result of aldehyde dehydrogenase liganded Hb crystals in the R2-state, we speculate that the

oxidation of the aldehyde functional group to the corres- KAUS compounds like aromatic aldehydes also bind to the

ponding inactive acid analog, makes the use of these α-cleft of R2-state Hb.

compounds to treat a large amount of Hb problematic. We

The fact that in general we do not observe a right-shifting

now report a new class of Hb covalent binding eﬀectors, eﬀect suggests that the interaction of the compounds with

imidazolylacryloyl derivatives, that were designed based on liganded Hb leads to stabilization of the R-state, as observed

the ethacrynic acid pharmacophore to form

a covalent for several potent left-shifting antisickling aromatic aldehydes

adduct with the surface-located amino acid βCys93. We that are known to bind to both R-state Hb and T-state

posited that this interaction should increase the protein Hb.^7,11,18Nonetheless, these aromatic aldehydes, unlike the

oxygen aﬃnity and/or stereospecifically prevent polymer con- KAUS molecules, do not add to the T-state stabilization,

tacts. It was also anticipated that the metabolically stable func- explaining their potent left-shifting properties.^7,11,18We also

tional group and the covalent interaction with Hb would lead note that while aromatic aldehydes bind to liganded Hb with

to both lower compound doses and a longer sustained thera- strong and well-resolved electron density, like the KAUS com-

peutic action.

pounds, their location in deoxyHb is characterized by very

Nine compounds, named KAUS, were successfully syn- weak and broken densities.^7,11,18

thesized (Fig. 1; Table 1; Schemes 2 and 3) and tested for their

The nine imidazolylacryloyl derivatives diﬀer by having

eﬀect on Hb oxygen binding properties, as well as their anti- varying chloro-substitutions on the phenyl ring and/or varying

sickling properties. Unexpectedly, the compounds showed no carboxylate arm lengths. Although subtle, the compounds with

or only weak biological eﬀects compared to ECA (Fig. 2; the longest and most flexible carboxylate arm length, i.e. the

Table 1). Some of the compounds even showed the opposite phenoxybutyrates (e.g. KAUS-12 and KAUS-15), appear to shift

pharmacological eﬀect by decreasing Hb aﬃnity for oxygen the OEC the most to the left, while the shortest and least flex-

and/or promoting cell sickling. To understand the unexpected ible, i.e. the phenoxyacetates (e.g. KAUS-10), shift the OEC the

behavior of these compounds on an atomic level, two of the least to the left or even shift it to the right. αLys99, which is

compounds KAUS-12 and KAUS-15 were co-crystallized with quite flexible, can easily move to interact with the varying

liganded Hb (COHb) and unliganded Hb (deoxyHb) and the chain-length carboxylates, and these interactions as noted

structures determined.

above are expected to stabilize the T-state. Most likely, the flex-

Contrary to our design expectation that the KAUS molecules ible phenoxybutyrates will make weaker inter-subunit inter-

would form a covalent adduct with βCys93 (Scheme 1), the actions with αLys99, and thus confer less constraint on the

structural studies with both deoxyHb and COHb showed no T-state, while the shorter chain phenoxyacetates will make a

observable binding at βCys93. Rather, the studies tighter interaction with αLys99 and confer more stability to the

with deoxyHb showed KAUS-12 and KAUS-15 to bind T-state, consistent with the trend in the allosteric properties.

covalently in a symmetry-related fashion at the α-cleft of the From the foregoing, the carboxylates may be responsible for

Hb via a Michael addition reaction that involved the β-unsatu- most of the inter-subunit interactions that stabilize the T-state,

rated carbon and the αVal1 nitrogens of Hb (Fig. 3 and 4). and removing them would presumably generate novel covalent

Notably, the two compounds make both intra-subunit and eﬀectors that would still bind at the α-cleft. However, instead

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of stabilizing the T-state they would rather destabilize it, shift-

ing the allosteric equilibrium to the R-state and exhibit a

Conclusion

potent antisickling eﬀect. Indeed, left-shifters, which co- Pharmacological stabilization of R-state Hb and/or destabiliza-

valently bind at the α-cleft, do not have carboxylates, while the tion of T-state Hb, which leads to an increase in Hb aﬃnity for

opposite is true for right-shifters.^{7,8,10,18,29,30}

oxygen, oﬀers a therapeutic strategy to treat SCD. We have

The question we pose is why do the KAUS compounds form designed a new class of Hb covalent modifiers, imidazolyl-

a covalent adduct with the N-terminal αVal1 amine, while ECA acryloyl derivatives, that were envisaged to increase Hb oxygen

(also with a β-unsaturated carbon) preferentially forms a aﬃnity and concomitantly prevent red blood cell sickling.

covalent adduct with the βCys93 thiol? First, the weakly In vitro analyses showed the compounds to exhibit very weak if

nucleophilic αVal1 amino group, which may have been unreac- any left-shifting and/or antisickling activities. Crystallographic

tive to the alkylacryloyl β-unsaturated carbon of ECA, has now studies of the compounds complexed to deoxyHb reveal an

become susceptible to electrophilic attack by the highly reac- unexpected result that showed the compounds binding not on

tive imidazolylacryloyl (arylacryloyl) β-unsaturated carbon. the surface of the protein as predicted but rather inside the

Consistent with when an electrophile (e.g. isothiocyanate or central water cavity, which stabilizes the T-state Hb and – para-

aldehyde) is directly attached to an aromatic ring (as observed doxically – explains their suboptimal activity. The observed

in aromatic aldehydes or aryl isothiocyanates), these com- mode of binding appears to be due to the increased reactivity

pounds are known to preferentially bind to the N-terminal of the β-unsaturated carbon and the highly specialized α-cleft

amines of the α-chain of Hb.^{11,18,23,29,30}On the other hand, if binding pocket that attracts and maximizes interactions with

the electrophile is attached to an alkyl group, as observed in the KAUS molecules, as well as provides stabilization to the

ethacrynic acid, alkyl isothiocyanates or alkyl aldehydes, the covalent adduct. Nonetheless, we are encouraged by the fact

compound does not bind to the α-cleft, with ethacrynic acid that these compounds still bind in a covalent manner, which

and alkyl isothiocyanates known rather to react with was one of our objectives, providing the framework for specific

βCys93.^23,37,38Second, and very importantly, the α-cleft seems modification of the KAUS molecules to develop potent anti-

to oﬀer a highly specialized complementary binding pocket for sickling agents.

diﬀerent classes of Hb eﬀectors that include aromatic alde-

hydes^{7,10,11,17,18,29,30}and aryl isothiocynates.²³This appears to

be true for the newly discovered imidazolylacryloyls that are

also attracted to the topologically complementary α-cleft

instead of the βCys93 binding site, with the protein binding

Experimental section

Materials and chemistry

environment and interactions serving to stabilize the Ethacrynic acid was purchased from Santa Cruz Biotechnology.

covalent adduct between the β-unsaturated carbon and the Glutathione (GSH), ^L-cysteine, L-valine, L-histidine, L-lysine,

αVal1 nitrogen. The absence of such a protein stabilization 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and sulforhod-

eﬀect may in part explain the non-reactivity of the compound amine were purchased from Sigma-Aldrich. Reagents and sol-

towards free valine.

vents for chemical synthesis were purchased from Sigma-

We note that even though diﬀerent classes of compounds Aldrich (USA), Alfa Aeser (UK) or Acros Organics (Belgium) as

bind at the same α-cleft, they exhibit diﬀerent allosteric beha- ACS-reagent grade and used without further purification.

viors, with the direction and magnitude of the allosteric shift Anhydrous solvents were prepared according to standard

depending on preferential stabilization of one state (T or R) methods. Solvents for LC-MS or UPLC-MS were purchased

over the other.^7,8,29,30Thus, the slight left-shift with some of from the same vendors mentioned above and used without

the compounds, e.g. KAUS-12 and KAUS-15, may mean that further purification. RPMI-1640, fetal bovine serum and other

the stabilization eﬀect on the R-state (most likely binding to cell culture materials were purchased from Lonza Group Ltd

the R2-state Hb) becomes predominant, while for KAUS-10, (Basel, Switzerland). Mouse normal fibroblast cells (C-166)

which slightly right-shifts the OEC, the T-state stabilization were a generous gift from Dr Ahmed M. Al-Abd, National

may be more dominant. These studies support the general Research Center, Cairo, Egypt. Cells were maintained in

principle that allosteric eﬀectors can bind to the same site but RPMI-1640 containing 100 U mL⁻¹penicillin, 100 μg mL⁻¹

produce opposite allosteric eﬀects.

streptomycin, and 0.025 μg mL⁻¹amphotericin B, sup-

GSH plays a major cellular role in removing toxic electro- plemented with 10% heat-inactivated fetal bovine serum (FBS).

philic xenobiotics, and thus compounds with a reactive β-unsa- Cell lines were incubated under standard conditions in a

turated carbon like KAUS could pose a toxicity risk to the humidified 5% (v/v) CO₂atmosphere at 37 °C.

cell.⁵¹However, unlike ECA, the KAUS compounds showed

Compounds 1a–1i (Fig. 1) were synthesized as described

weak reactivity towards GSH. Minimal cellular toxicity was also below. Except as otherwise indicated, all synthetic reactions

observed for the tested compounds, KAUS-12 and KAUS-15 were carried out under nitrogen atmosphere in flame- or oven-

(IC₅₀> 1.6 mM) when compared to ECA (IC₅₀of 22 μM) with dried glassware, and solvents were freshly distilled. Tetrahydro-

the C-166 mouse fibroblast cell line. These studies suggest that furan (THF) was distilled from sodium/benzophenone. Reac-

the KAUS compounds may have a more favorable therapeutic tions were monitored by thin layer chromatography (TLC) with

index than ECA.

0.25 mm E. Merck pre-coated silica gel plates. ¹H-NMR spectra

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were recorded on a Bruker AV-300 NMR spectrometer with

Synthesis of 2-(4-acetyl-2,3-dichlorophenoxy)acetic acid

TopSpin software. Infrared spectra were recorded on a Bruker (4a). A solution of 2,3-dichloro-4-hydroxyacetophenone 2a

FT-IR spectrometer. Melting points were recorded on a Buchi (3.5 g, 17.073 mmol) in N,N-dimethylformamide (DMF)

melting point apparatus and are uncorrected. LC/MS were run (35 mL) was cooled to 0 °C, followed by addition of potassium

on an Agilent 6130 Series, single quad.

carbonate (7.09 g, 51.219 mmol). The mixture was stirred at 25

De-identified, leftover EDTA venous blood samples from to 27 °C for 15 min and ethyl bromoacetate (3.4 g,

patients with SCD, who visited The Children’s Hospital of Phi- 20.48 mmol) was added drop-wise over a period of 15 min.

ladelphia for routine clinic visits, were obtained after informed The reaction mass was heated to 80 °C and stirred at the same

consent. Normal whole blood was collected from adult donors temperature for 2 h, then cooled to room temperature and

at the Virginia Commonwealth University after informed quenched with water (100 mL). The product was extracted

consent. Hb was purified from discarded normal blood from the aqueous phase using methyl tert-butyl ether (MeOtB)

samples following a published procedure.⁴⁶The use of these (2 × 100 mL). The combined organic layer was washed with

human samples is in accordance with the regulations of the water (100 mL) and brine (100 mL), dried over anhydrous

IRB for Protection of Human Subjects.

sodium sulfate, and concentrated under vacuum to aﬀord 3a

as a pale yellow liquid (4.97 g, 100%). The crude product was

taken into the next step without further purification. H NMR

Preparation of imidazolylacryloyl derivatives 1a–1i

1

Synthesis of 1-(2,3-dichloro-4-hydroxyphenyl)ethan-1-one (CDCl₃) δ_Hppm 7.50 (d, 1H, J = 8.7 Hz, Ar-H5), 6.79 (d. 1H, J =

(2a). A solution of 2,3-dichloroanisole (75 g, 423.68 mmol) in 8.7 Hz, Ar-H6), 4.78 (s, 2H, OCH₂), 4.29 (q, 2H, J = 6.9 Hz,

dichloromethane (1.5 L) was cooled to 0 °C, followed by CH₂), 2.66 (d, 3H, COCH₃). 1.32 (t, 3H, J = 6.9 Hz, CH₃). MS

portion-wise addition of aluminum chloride (112.98 g, (ESI) m/z = 202.9 (M − CH₂COOCH₂CH₃), 205, 207.

847.36 mmol), and was stirred for 10 min. Acetyl chloride

A solution of 3a (1.8 g, 6.18 mmol) in tetrahydrofuran

(39.90 g, 508.416 mmol) was added drop-wise over a period of (THF) (7.2 mL) was cooled to 0 °C and lithium hydroxide

25 min, and stirred at 25 to 27 °C for 30 min. The reaction (1.29 g, 30.91 mmol) in water (1.8 mL) was added. The reaction

mass was cooled to 0 °C and then quenched by addition of mixture was stirred at 25 to 27 °C for 5 h, and then acidified

water (200 mL), and the product extracted from the aqueous with aqueous 1.5 N hydrochloric acid (25 mL). The product

phase using dichloromethane (2 × 500 mL). The combined was extracted from the aqueous phase using dichloromethane

organic layer was washed with water (200 mL) and brine (2 × 25 mL). The organic layer was dried using anhydrous

(250 mL), dried over anhydrous sodium sulfate and concen- sodium sulfate, and concentrated under vacuum to obtain a

trated under vacuum to obtain the crude product as an oﬀ- brown solid. The crude solid was basified with 10% aqueous

white solid. The crude solid was triturated with n-hexane sodium bicarbonate (20 mL), the aqueous layer washed with

(500 mL) and the solid filtered to aﬀord the intermediate ethyl acetate (EtOAC) (25 mL), then cooled to 4 °C and acidi-

4-acetyl-2,3-dichloroanisole as an oﬀ-white solid (61 g, 65.7%). fied using aqueous 1.5 N hydrochloric acid (HCl) (50 mL). The

Mp 81.8–82.6 °C; ¹H-NMR (300 MHz, CDCl₃-d) δ_Hppm 7.55 (d, product was extracted from the aqueous phase using dichloro-

1H, J = 8.7 Hz Ar-H5), 6.9 (d, 1H, J = 8.7 Hz, Ar-H6) 3.98 (s, 3H, methane (DCM) (2 × 25 mL). The organic layer was washed

OCH₃), 2.65 (s, 3H, COH₃).

with water (50 mL) and brine (50 mL), dried over anhydrous

A

solution of 4-acetyl-2,3-dichloroanisole (50 g, sodium sulfate and concentrated under vacuum to give 4a as a

1

228.31 mmol) in acetic acid (150 mL) was warmed to 60° C pale brown solid (1.5 g, 94.33%), mp 150.6–153.5 °C. H NMR

and added to hydrobromic acid (47% in water, 3.3 L) drop-wise (300 MHz, DMSO-d₆) δ_Hppm 13.3 (br s, 1H, OH), 7.72 (d, 2H,

over a period of 1 h. The reaction mass was heated to 100 °C J = 8.4 Hz, Ar-H5), 7.16 (d, 2H, J = 9 Hz, Ar-H6), 4.96 (s. 2H,

for 16 h, and then cooled to about 0° C, followed by quenching CH₂), 2.57 (s, 3H, CH₃). LC/MS (ESI) m/z = 261 (M − 1), 263 (M

with water (1 L). The product was extracted from the aqueous + ), 245 (M + 2), purity = 99.55%.

phase using dichloromethane (2 × 1 L). The organic layer was

Synthesis

of

4-(4-acetyl-2,3-dichlorophenoxy)butanoate

dried over anhydrous sodium sulfate and concentrated under (4c). A solution of 2,3-dichloro-4-methoxyacetophenone (4.5 g,

vacuum to obtain a yellow solid. The crude product was dis- 21.95 mmol) in DMF (45 mL) was cooled to 0 °C, then potass-

solved in 10% aqueous sodium hydroxide solution (500 mL), ium carbonate (9.08 g, 65.85 mmol) added, and the mixture

and the aqueous layer washed with ethyl acetate (2 × 250 mL). was stirred at 25 to 27 °C for 15 min. Ethyl 4-bromobutyrate

The aqueous layer, which was maintained between 0 and 5 °C, (5.141 g, 26.34 mmol) was added drop-wise over a period of

was acidified with HCl (1.5 N, 750 mL), and the product 15 min, and then the mixture heated to 80 °C for 2 h. The reac-

extracted using dichloromethane (2 × 500 mL). The combined tion mass was cooled to ambient temperature and quenched

organic layer was washed with water (500 mL) followed by with water (100 mL). The product was extracted from the

brine (500 mL), dried over anhydrous sodium sulfate, and con- aqueous phase using MeOtB (2 × 100 mL). The combined

centrated under vacuum to aﬀord 2a as a white solid (10 g, organic layer was washed with water (100 mL), followed by

21.3%), mp 151.2–153.5 °C. ¹H NMR (300 MHz, CDCl₃) brine (100 mL). The organic layer was dried over anhydrous

δ_Hppm 7.55 (d, 1H, J = 8.7 Hz Ar-H5), 7.03 (d, 1H, J = 8.7 Hz, sodium sulfate and concentrated under vacuum to get a pale

Ar-H6), 6.03 (br, 1H, OH), 2.66 (s, 3H, COCH₃). LC/MS (ESI) yellow liquid. The crude material was purified by column

m/z = 205 (M + 1), purity = 95.8%.

chromatography over silica gel (230–400 mesh) using 40 to

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50% ethyl acetate in hexane as eluent to aﬀord 3c as a pale DMSO, COCH₃); LC/MS (ESI) m/z = 227 (M − 1), 229 (M + 1);

brown liquid (3.8 g, 58.4%). ¹H NMR (300 MHz, DMSO-d₆) purity = 99.45%.

δ_H7.52 (d, 1H, J = 9.3 Hz, Ar-H5), 6.89 (d. 1H, J = 10.8 Hz, Ar-

Synthesis of 4-(4-acetyl-3-chlorophenoxy)butanoic acid (4i).

H6), 4.20–4.13 (m, 4H, two OCH₂), 2.65 (s, 3H, COCH₂), 2.57 This compound was prepared following the procedure

(d, 2H, COCH₂), 2.19 (q, 2H, J = 6.3 Hz, CH₂), 1.28 (t, 3H, J = described above for the synthesis of the similar analogue 4c

6.9, 7.2 Hz, CH₃); LC/MS (ESI) m/z = 319 (M + 1), 321 (M + 3), and was obtained as an oﬀ-white solid (5.5 g, 73.3%), mp

323 (M + 5), purity = 99.13%.

105.9–109.0 °C. ¹H NMR (300 MHz, DMSO-d₆) δ_H12.2 (br s,

A solution of 4-(4-acetyl-2,3-dichlorophenoxy)butyric acid 1H, OH), 7.75 (d, 1H, J = 8.7 Hz, Ar-H5), 7.10 (d, 1H, J = 2.4 Hz,

ethyl ester 3c (3.4 g, 10.65 mmol) in THF (13.6 mL) was cooled Ar-H2), 7.01 (q, 1H, J = 2.4, 8.7 Hz, Ar-H6), 4.09 (t, 2H, J = 6.6,

to 0 °C and 3.4 mL lithium hydroxide (2.23 g, 53.26 mmol) was 6.3 Hz, OCH₂), 2.50 (under DMSO, COCH₃), 2.38 (t, 2H, J = 7.5,

added. The above suspension was stirred at 25 to 27 °C for 4 h, 7.2 Hz, CH₂), 1.49 (t, 2H, J = 6.6, 6.9 Hz, CH₂); IR (FT-IR,

and the reaction mass cooled to 0 °C and quenched with 1.5 N cm⁻¹): 2914.9, 1668.2, 1614.4, 1591.6, 1564.7, 1473.6, 1409.5,

hydrochloric acid (25 mL). The product was extracted from the 1373.4, 1257.9; LC/MS (ESI) m/z = 196 (M − (CH₂)₃COOH), 171;

aqueous phase using DCM (2 × 50 mL). The combined organic purity = 98.94%.

layer was washed with water (50 mL) and brine (25 mL). The

Synthesis of 1-(triphenylmethyl)-1H-imidazole-2-carbalde-

organic layer was dried over anhydrous sodium sulfate, and hyde (5). Sodium hydride (7.4 g, 312.29 mmol) in DMF

concentrated under vacuum to obtain an oﬀ-white solid. The (125 mL, 5 vol.) was cooled to 0 °C, 2-imidazolecarboxaldehyde

crude product was dissolved in 10% sodium bicarbonate (25 g, 260.19 mmol) was added portion-wise, and the reaction

(25 mL) and the aqueous layer washed with EtOAc (50 mL), mass was stirred at 28 °C for 30 min. The reaction mass was

cooled and acidified with 1.5 N HCl (50 mL). The product was cooled to 0 °C followed by drop-wise addition of a solution of

extracted from the aqueous phase using DCM (2 × 50 mL), and trityl chloride (87 g, 312.29 mmol) in DMF (175 mL) and was

the combined organic layer washed with water (50 mL) and stirred for 1 h under the same cooling conditions until com-

brine (25 mL). The organic layer was dried over anhydrous pletion (TLC monitoring). The reaction mixture was quenched

sodium sulfate and concentrated under vacuum to aﬀord 4c as with ammonium chloride solution (500 mL), and extracted

a white solid (2.6 g, 76.47%), mp 151.2–154.6 °C. ¹H NMR with MeOtB (250 mL). The organic layer was dried over sodium

(300 MHz, DMSO-d₆) δ_Hppm 1.94–2.05 (m, 2H) 2.39–2.48 sulfate, and the solvent removed under reduced pressure to

(m, 3H) 2.57 (d, J = 1.89 Hz, 4 H) 4.20 (t, J = 6.33 Hz, 2H) 7.25 obtain the crude product, which was purified by column

(dd, J = 8.78, 1.61 Hz, 1H) 7.74 (dd, J = 8.69, 1.70 Hz, 1H) 12.20 chromatography over neutral alumina by using 0 to 7% ethyl

(br s, 1H); LC/MS (ESI) m/z = 203 (M − (CH₂)₃COOH), 205, 207, acetate in hexane as eluent. The product 1-(triphenylmethyl)-

purity = 96.94%.

1H-imidazole-2-carbaldehyde was a white solid (33 g, 37%),

Synthesis of 2-(4-acetyl-2-chlorophenoxy) acetic acid (4d). mp 165.9–160.7 °C. ¹H NMR (300 MHz, DMSO-d₆) δ_Hppm

This compound was prepared following the procedure detailed 6.97–7.14 (m, 9H) 7.32–7.49 (m, 13H) 9.14 (s, 1H). LC/MS (ESI)

above for the synthesis of the similar analogue 4a. The crude m/z = 337 (M − 1), purity = 86.79%.

product was purified by acid–base workup to aﬀord 4d as an

Synthesis of {2,3-dichloro-4-[(E)-3-1H-imidazol-2-ylacryloyl]-

oﬀ-white solid (5.5 g, 82.0%), mp 147.8–149.2 °C. ¹H NMR phenoxy}acetic acid (1a). 1-Trityl-1H-imidazole-2-carbaldehyde

(300 MHz, DMSO-d₆) δ_H13.24 (s, 1H, OH), 7.99 (d, 1H, J = 5 (2.89 g, 8.55 mmol) and acetophenone derivative 4a (1.5 g,

2.1 Hz Ar-H3), 7.89 (r, 1H, J = 6.6, 2.1 Hz, Ar-H5), 7.15 (d, 1H, 5.7 mmol) were dissolved in ethanol (51 mL). The reaction

J = 8.7 Hz, Ar-H6), 4.94 (s. 2H, OCH₂), 2.51 (under DMSO, mixture was cooled to 0 °C and 1 N aqueous NaOH solution

COCH₃); LC/MS (ESI) m/z = 227 (M − 1), 229 (M + 1); purity = (51.75 mL) was added drop-wise over a period of 30 min while

99.43%.

keeping the temperature between 0 to 5 °C. The mixture was

Synthesis of 4-(4-acetyl-2-chlorophenoxy)butyric acid (4f). then stirred at 25 to 27 °C for 48 h, followed by dilution with

This compound was prepared following the procedure water (50 mL). The aqueous layer was washed with MeOtB (2 ×

described above for the synthesis of the similar analogue 4c 200 mL), cooled to 0 to 5 °C, and 1.5 N HCl (50 mL) added

and was obtained as an oﬀ-white solid (5.5 g, 82.08%), mp drop-wise to adjust the pH to 4. The solid formed was filtered

129.2–135.8 °C. ¹H NMR (300 MHz, DMSO-d₆) δ_Hppm 12.38 and dried under vacuum to aﬀord a pale yellow solid of 6a.

(br s, 1H, OH), 7.98–7.25 (m, 2H, Ar-H3, 5), 7.26 (d, 1H, J = 8.7 The crude product was taken into the next step without further

Hz, Ar-H6), 4.19 (t, 2H, J = 6.3 Hz, OCH₂), 2.54–2.41 (m, 5H, purification (2.5 g, 75.75%).

CH₃& CH₂), 1.99 (t, 2H, J = 6.3, 6.9 Hz, CH₂); LC/MS (ESI) m/z

= 169 (M − (CH₂)₃COOH), purity = 97.71%.

A solution of 6a (2.5 g, 4.284 mmol) in DCM (12.5 mL) was

cooled 0° C and trifluoroacetic acid (5 mL) in DCM (12.5 mL)

Synthesis of ethyl 2-(4-acetyl-3-chlorophenoxy) acetic acid added drop-wise over a period of 15 min. The reaction mass

(4g). This compound was prepared following the procedure was stirred at 25 to 27 °C for 1 h, then concentrated under

described above for the synthesis of the similar analogue 4a vacuum to aﬀord a yellow solid. To the crude solid, MeOtB

and was obtained as an oﬀ-white solid (5.5 g, 82.08%), mp (100 mL) was added, stirred at 25 to 27 °C for 15 min, filtered

1

103.7–106.5 °C. H NMR (300 MHz, DMSO-d₆) δ_H13.18 (s, 1H, and dried under vacuum to aﬀord a yellow solid. Water

OH), 7.77 (d, 1H, J = 8.7 Hz, Ar-H5), 7.10 (d, 1H, J = 2.7 Hz, (20 mL) was added to the crude product and cooled to 0 °C

Ar-H2), 7.02 (s, 1H, Ar-H6), 4.83 (s, 2H, OCH₂), 2.51 (under and basified with 5% sodium hydroxide solution (10 mL) to

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pH 9. The suspension was stirred at 25 to 27 °C for 15 min, DMSO-d₆) δ_H12.84 (br, 1H, OH), 12.23 (s, 1H, NH), 8.10 (s, 1H,

and then cooled and acidified using 1.5 N HCl (20 mL) until Ar-H3), 8.04(d, 1H, J = 9 Hz, Ar-H5), 7.89 (d, 1H, J = 15.6 Hz,

the pH was adjusted to 4. The solid was filtered and dried CH), 7.48 (d, 1H, J = 15.6 Hz, CH), 7.37–7.35 (m, 3H, Ar-H6 &

under vacuum to aﬀord 1a as a pale yellow solid product (1 g, imidazole-H), 4.22 (t. 2H, OCH₂), 2.45 (under DMSO, CH₂),

71.42%), mp 273.0–274.1 °C. ¹H NMR (300 MHz, DMSO-d₆) 2.01 (t, 2H, CH₂). LC/MS (ESI) m/z = 333 (M − 1), 335 (M + 1),

δ_H12.70 (br, 2H, NH & OH), 7.53 (d, 1H, J = 9.3 Hz, Ar-H5), purity = 97.62%. Anal. Calcd for (C₁₆H₁₅ClN₂O₄): C, 57.41; H,

7.33–7.12 (m, 5H, Ar-H6 & imidazole-H & 2CH), 4.98 (s, 2H, 4.52; Cl, 10.59; N, 8.37. Found C, 57.46; H, 4.50; Cl, 10.55; N,

CH₂); LC/MS (ESI) m/z = 339 (M − 1), 341 (M + 1), 343 (M + 3), 8.34.

purity = 98.08%. Anal. Calcd for (C₁₄H₁₀Cl₂N₂O₄): C, 49.29; H,

2.95; Cl, 20.78; N, 8.21. Found C, 49.17; H, 2.96; Cl, 20.74; N, 2-enoyl]phenoxy}acetic acid (1g). This compound was pre-

8.18. pared according to the procedure described for the preparation

Synthesis of 2-{3-chloro-4-[(2E)-3-(1H-imidazol-2-yl)prop-

Synthesis of 4-{2,3-dichloro-4-[(2E)-3-(1H-imidazol-2-yl)prop- of the similar analogue 1a and was obtained as a yellow solid

2-enoyl]phenoxy}butanoic acid (1c). To a cooled solution of (1.2 g, 75.08%), mp 258.3 °C; ¹H NMR (300 MHz, DMSO-d₆)

1-(triphenylmethyl)-1H-imidazole-2-carbaldehyde

5

(2.6 g, δ_H13.02 (br, 1H, NH), 7.57 (d, 1H, J = 8.4 Hz, Ar-H5), 7.31–7.15

7.72 mmol) and 4-(4-acetyl-2,3-dichloro-phenoxy)-butyric acid (m, 5H, Ar-H2 & imidazole-H & 2CH), 7.05 (d, 1H, J = 8.4 Hz,

4c (1.5 g, 1.51 mmol) in ethanol (51.7 mL) at 0 to 5° C, 1 N Ar-H6), 4.85 (s, 2H, OCH₂); IR (FT-IR, cm⁻¹): 3142.3, 1672.7,

NaOH solution (52 mL) was added drop-wise over a period of 1590.8, 1553.46, 1413.3, 1329.8, 1251.4, 1222.7; LC/MS (ESI)

15 min. The reaction mass was stirred at 25 to 27° C for 24 h m/z = 307 (M + 1), 309 (M + 3); purity = 98.19%. Anal. Calcd for

while monitoring by TLC. The reaction mass was diluted with (C₁₄H₁₁ClN₂O₄): C, 54.83; H, 3.62; Cl, 11.56; N, 9.13. Found C,

water (10 mL), washed with MeOtB (25 mL), and cooled to 0 to 54.72; H, 3.63; Cl, 11.58; N, 9.14.

5 °C. HCl (1.5 N, 5 mL) added drop-wise to adjust the pH

to 4. The solid formed was filtered and dried under vacuum to 2-enoyl]phenoxy}butanoic acid (1i). This compound was pre-

aﬀord 6c a pale yellow solid. pared according to the procedure described for the preparation

Synthesis of 4-{3-chloro-4-[(2E)-3-(1H-imidazol-2-yl)prop-

The crude 6c (2.8 g, 4.59 mmol) was dissolved in DCM of the similar analogue 1c and was obtained as a yellow solid

(14 mL), cooled to 0 °C, and trifluroacetic acid (5.6 mL) in (2.2 g, 73.08%), mp 219.4–222.8 °C. ¹H NMR (300 MHz,

DCM (14 mL) added drop-wise while keeping the temperature DMSO-d₆) δ_H12.23 (br, 1H, OH), 7.59 (d, 1H, J = 8.7 Hz,

between 0 to 5 °C. The reaction mass was allowed to stir at Ar-H5), 7.36–7.17(m, 5H, 2CH, 2imidazole-H & Ar-H), 7.06 (d,

25 to 27 °C for 1 h. The solvent was removed under reduced 1H, J = 8.7 Hz, Ar-H6), 4.11 (t, 2H, J = 6, 6.3 Hz, OCH₂), 2.40 (t,

pressure and the crude solid was triturated with MeOtB 2H, J = 7.2 Hz, CH₂), 1.79 (q, 2H, CH₂); IR (FT-IR, cm⁻¹):

(20 mL), filtered and dried to aﬀord the product as a yellow 3236.0, 1695.8, 1660.8, 1585.6, 1550.6, 1444.8, 1345.6, 1321.3,

solid (1.2 g, 75.0%), mp 214.9–217.2 °C; ¹H NMR (300 MHz, 1296.6; LC/MS (ESI) m/z = 335 (M + 1) and 337 (M + 3), purity =

DMSO-d₆) δ_H12.98 (br, 1H, OH), 12.22 (s, 1H, NH), 7.33–7.13 98.74%.

(m, 5H, Ar-H & imidazole-H & 2CH), 4.21 (t, 2H, J = 5.7 Hz,

Synthesis of 1-[2,3-dichloro-4-(2-[1,3]dioxolan-2-yl-ethoxy)-

OCH₂), 2.45 (under DMSO, CH₂), 2.01 (t, 2H, J = 6.3 Hz, CH₂); phenyl]ethanone (7a). A solution of 2,3-dichloro-4-methoxy-

IR (FT-IR, cm⁻¹): 2938.8, 1681.1, 1609.7, 1584.6, 1446.0, acetophenone (5.5 g, 26.82 mmol) in DMF (55 mL) was cooled

1387.6, 1303.0; LC/MS (ESI) m/z = 367 (M − (CH₂)₃COOH), 369, to 0 to 5 °C, potassium carbonate (11.12 g, 80.51 mmol)

371; purity = 99.76%. Anal. Calcd for (C₁₆H₁₂Cl₄N₂O₄): C, added, and the mixture stirred at 25 to 27 °C for 15 min. 2-(2-

43.87; H, 2.76; Cl, 32.37; N, 6.39. Found: C, 43.76; H, 2.75; Cl, Bromoethyl)-1,3-dioxalane (5.82 g, 32.19 mmol) was added to

32.44; N, 6.38.

the above suspension drop-wise over a period of 15 min. The

Synthesis of 2-{2-chloro-4-[(2E)-3-(1H-imidazol-2-yl)prop- reaction mass was heated to 80 °C and maintained at the same

2-enoyl]phenoxy}acetic acid (1d). This compound was pre- temperature for 2 h, followed by cooling to ambient tempera-

pared according to the procedure described for the preparation ture and quenching with water (100 mL). The product was

of the similar analogue 1a and was obtained as a yellow solid extracted from the aqueous phase using MeOtB (2 × 100 mL),

1

(1 g , 76%), mp 246.9–248.7 °C; H NMR (300 MHz, DMSO-d₆) and the combined organic layer washed with water (100 mL)

δ 8.01 (s, 1H, Ar-H3), 7.93(t, 2H, J = 6, 10.5 Hz, CH & Ar-H5), and brine (100 mL), dried over anhydrous sodium sulfate and

7.45 (d, 1H, J = 15.6 Hz, CH), 7.28 (s, 2H, imidazole-H), 7.07 (d, concentrated under vacuum to obtain a pale brown gum.

1H, J = 8.7 Hz, Ar-H6), 4.59 (s, 2H, OCH₂); IR (FT-IR, cm⁻¹): Hexane (70 mL) was added to the residue and the suspension

3143.8, 2918.5, 1670.3, 1588.0, 1483.0, 1409.9, 1259.2, 1238.7; stirred at 25 to 27° C for 30 min, filtered and dried under

LC/MS (ESI) m/z = 307 (M − 1), 307 (M + 1), purity = 97.92%. vacuum to obtain 7a as a brown solid (6.8 g, 84.69%). ¹H NMR

Anal. Calcd for (C₁₆H₁₂Cl₄N₂O₄): C, 43.87; H, 2.76; Cl, 32.37; N, (300 MHz, CDCl₃) δ_Hppm 7.53 (m, J = 8.88 Hz, 2H), 7.28 (s,

6.39. Found: C, 43.76; H, 2.75; Cl, 32.44; N, 6.38.

1H), 6.91 (m, J = 8.69 Hz, 2H), 5.16 (t, J = 4.72 Hz, 2H), 4.26 (t,

Synthesis of 4-{2-chloro-4-[(2E)-3-(1H-imidazol-2-yl)prop-2- J = 6.52 Hz, 5H), 3.85–4.08 (m, 10H),), 2.65 (s, 7H), 2.25 (td, J =

enoyl]phenoxy}butanoic acid (1f). This compound was pre- 6.47, 4.82 Hz, 5H).

pared according to the procedure described for the preparation

Synthesis

of

1-{3-chloro-4-[2-(1,3-dioxolan-2-yl)ethoxy]-

of the similar analogue 1c and was obtained as a pale yellow phenyl}ethanone (7b). This compound was prepared accord-

1

solid (1.2 g, 68.95%), mp 210.2–214.2 °C; H NMR (300 MHz, ing to the procedure described above for the preparation of 7a.

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The crude product, obtained as an oﬀ-white solid (7.9 g, obtained as an oﬀ-white solid (3.2 g, 45.13%), mp

100%), was used for the next step without further purification. 95.9–98.2 °C. ¹H NMR (300 MHz, methanol-d₄) δ ppm 12.44

¹H NMR (300 MHz, CDCl₃) δ_Hppm 8.00 (1H, d, J = 1.89 Hz), (1H, br s), 7.77 (1H, d, J = 8.50 Hz), 6.90–7.16 (1H, m), 4.26

7.86 (1H, dd, J = 8.59, 1.98 Hz), 6.98 (1H, d, J = 8.69 Hz), 5.17 (1H, t, J = 5.85 Hz), 3.33 (1H, s), 2.71 (1H, t, J = 5.67 Hz), 2.52

(1H, t, J = 4.82 Hz), 4.28 (2H, t, J = 6.52 Hz), 3.78–4.08 (4H, m), (4H, d,

2.57 (3H, s), 2.36–2.16 (2H, m), 1.65 (1H, br s). LC/MS (ESI) m/z (CH₂)₂COOH), 171, purity = 93.54%.

= 271 (M + 1), purity = 95.46%. Synthesis of 3-{2,3-dichloro-4-[(2E)-3-(1H-imidazol-2-yl)prop-

Synthesis of 1-{2-chloro-4-[2-(1,3-dioxolan-2-yl)ethoxy]- 2-enoyl]phenoxy}propionic acid (1b). 1 NaOH solution

phenyl}ethanone (7c). This compound was prepared accord- (93.15 mL) was added drop-wise to a cooled solution of

ing to the procedure described above for the preparation of 7a. 1-(triphenylmethyl)-1H-imidazole-2-carbaldehyde (5.2 g,

J = 15.30 Hz). LC/MS (ESI) m/z = 169 (M −

N

The crude product was obtained as a pale brown liquid (7.9 g, 15.5 mmol) 5 and 8a (2.7 g, 9.72 mmol) in ethanol (93 mL) at

100%) and used for the next step without further purification. 0 to 5 °C. The reaction mass was stirred at 25 to 27 °C for 24 h,

¹H NMR (300 MHz, CDCl₃) δ_Hppm 7.68 (1H, d, J = 8.50 Hz), and monitored by TLC. After completion of the reaction, the

6.96 (1H, br s), 6.85 (1H, d, J = 8.50 Hz), 5.13–4.95 (1H, m), mixture was diluted with water (10 mL), and the aqueous layer

4.2–4.10 (1H, m), 3.76–4.10 (5H, m), 3.39–3.54 (1H, m), 2.65 was washed with MeOtB (30 mL). The aqueous layer was

(2H, s), 2.29–2.07 (2H, m). LC/MS (ESI) m/z = 271 (M + 1), cooled to 0 to 5 °C and 1.5 N HCl was added drop-wise to

purity = 93.88%.

Synthesis of 3-(4-acetyl-2,3-dichlorophenoxy)propionic acid under vacuum to aﬀord 9a as a pale yellow solid.

(8a). To a solution of 7a (6.8 g, 22.84 mmol) in acetone The crude intermediate 9a was dissolved in DCM (25 mL),

adjust the pH to 4. The solid formed was filtered and dried

(68 mL) was added 1.5 N HCl (68 mL) drop-wise at 0 to 5 °C cooled to 0 °C, trifluroacetic acid (10 mL, 2 vol.) in dichloro-

over a period of 20 min. The reaction mixture was stirred at 25 methane (25 mL) added at 0 to 5 °C and the reaction allowed

to 27 °C for 16 h, and then concentrated under vacuum. The to stir at 25 to 27 °C for 1 h. The solvent was removed under

crude product was diluted with water (50 mL) and extracted reduced pressure, and the crude solid triturated with MeOtB

with DCM (2 × 50 mL). The combined organic layer was (50 mL), filtered and dried under vacuum to aﬀord the crude

washed with water (50 mL) and brine (50 mL), dried over anhy- product as a yellow solid. The solid was suspended in water

drous sodium sulfate, and concentrated under vacuum to give (40 mL), and the pH adjusted to 9 using 5% aqueous NaOH

a pale yellow gum. The above material was dissolved in DMF solution at 0 to 5 °C. The reaction mass was allowed to stir at

(58 mL), oxone (7.012 g, 22.841 mmol) was added, and the 25 to 27 °C for 15 min, and then acidified to pH 4 using 1.5 N

mixture stirred at 25 to 27° C for 6 h. The reaction mass was HCl. The crude product was further purified by crystallization

diluted with water (50 mL) and the product extracted from the from DMF–acetonitrile mixture to aﬀord 1b as a yellow solid

aqueous layer using dichloromethane (2 × 100 mL). The com- (0.8 g, 23.52%), mp 223.2–226.6 °C. ¹H NMR (300 MHz,

bined organic layer was dried over anhydrous sodium sulfate, DMSO-d₆) δ_Hppm 7.56 (1H, d, J = 8.69 Hz), 7.28–7.45 (3H, m),

and concentrated under vacuum to yield a pale brown gum. 7.06–7.28 (2H, m), 4.38 (2H, t, J = 5.76 Hz), 2.70–2.85 (2H, m);

The crude solid was dissolved in ethyl acetate (100 mL) and IR (FT-IR, cm⁻¹): 3261.2, 1692.1, 1669.3, 1605.8, 1581.9,

washed with 10% sodium bicarbonate (75 mL). The aqueous 1547.7, 1388.7, 1302.7, 1249.4; LC/MS (ESI) m/z = 283 (M −

layer was cooled to 5 °C and acidified using 1.5 N HCl (50 mL) (CH₂)₂COOH), 285, 287; purity = 95.97%. Anal. Calcd for

to a pH of 3. The product was extracted from the aqueous (C₁₅H₁₂Cl₂N₂O₄): C, 50.72; H, 3.41; Cl, 19.96; N, 7.89. Found C,

phase using MeOtB (2 × 250 mL). The combined organic layer 50.63; H, 3.40; Cl, 19.92; N, 7.87.

was washed with water (2 × 250 mL) and brine (100 mL), dried

Synthesis of 3-{2-chloro-4-[(E)-3-(1H-imidazol-2-yl)acryloyl]-

over anhydrous sodium sulfate, and concentrated under phenoxy}propionic acid (1e). This compound was prepared

vacuum to give 8a as an oﬀ-white solid product (2.9 g, 47%), according to the procedure described above for the preparation

mp 142.2–144.9 °C. ¹H NMR (300 MHz, DMSO-d₆) δ_Hppm 7.75 of 1b and was obtained as a yellow solid (0.53 g, 16.6%), mp

(1H, d, J = 8.88 Hz), 7.29 (1H, d, J = 8.69 Hz), 4.36 (2H, t, J = 220–223.5 °C. ¹H NMR (300 MHz, DMSO-d₆) δ_Hppm 12.49

5.85 Hz), 2.77 (2H, t, J = 5.76 Hz), 2.58 (3H, s). LC/MS (ESI) m/z (1H, br s), 7.99–8.14 (2H, m), 7.89 (1H, d, J = 15.67 Hz),

= 203 (M − (CH₂)₂COOH), 205, 207, purity = 100%.

7.26–7.55 (4H, m), 4.39 (2H, t, J = 5.76 Hz), 2.80 (2H, t, J = 5.76

Synthesis of 3-(4-acetyl-2-chlorophenoxy)propionic acid Hz); IR (FT-IR, cm⁻¹): 3258.2, 1708.7, 1671.6, 1660.5, 1607.9,

(8b). This compound was prepared according to the procedure 1590.2, 1503.1, 1412.5, 1260.9, 1207.0; LC/MS (ESI) m/z = 247

described above for the preparation of 8a. The product was (M − (CH₂)₂COOH), 249, purity = 96.13%. Anal. Calcd for

obtained as an oﬀ-white solid (3 g, 49.18%), mp (C₁₅H₁₃ClN₂O₄): C, 56.17; H, 4.09; Cl, 11.05; N, 8.73. Found C,

124.7–127.9 °C. ¹H NMR (300 MHz, DMSO-d₆) δ_Hppm 56.03; H, 4.10; Cl, 11.07; N, 8.75.

7.85–8.02 (2H, m), 7.30 (1H, d, J = 8.50 Hz),4.35 (2H, t, J = 5.85

Hz), 3.34 (1H, br s), 2.64–2.84 (2H, m), 2.54 (2H, s); LC/MS phenoxy}propionic acid (1h). This compound was prepared

(ESI) m/z = 169 (M − (CH₂)₂COOH), 171, purity = 97.85%. according to the procedure described above for the preparation

Synthesis of 3-{3-chloro-4-[(E)-3-(1H-imidazol-2-yl)acryloyl]-

Synthesis of 3-(4-acetyl-3-chlorophenoxy)propionic acid of 1b and was obtained as a yellow solid (1.2 g, 70%), mp

(8c). This compound was prepared according to the procedure 237.3–238.2 °C; ¹H NMR (300 MHz, DMSO-d₆) δ_Hppm, 7.57

described above for the preparation of 8a. The product was (1H, d, J = 8.50 Hz), 7.12–7.44 (5H, m), 7.07 (1H, d, J = 8.50

6366 | Org. Biomol. Chem., 2015, 13, 6353–6370

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Paper

Hz), 4.28 (2H, t, J = 5.57 Hz), 2.74 (2H, t, J = 5.57 Hz); tallized with 3.0–3.4 M sodium monobasic phosphate and pot-

IR (FT-IR, cm⁻¹): 3255.0, 1696.1, 1667.7, 1599.3, 1449.0 1329.6, assium phosphate dibasic, at pH values ranging from 6.4 to 7.2.

1305.2, 1246.73, 1246.7, 1223.7; LC/MS (ESI) m/z = 247 (M − One or two drops of toluene was added to the solution in each

(CH₂)₂COOH), 249, purity = 98.2%.

tube to facilitate crystallization. X-ray-quality crystals grew in 2–3

days as trigonal bipyrimidal crystals in the space group P4₁2₁2

with typical cell constants of 53, 53, 193 Å. The crystals diﬀr-

Biological evaluation

Oxygen equilibrium curve studies. The ability of the nine acted to 2.8 and 2.0 Å for KAUS-12 and KAUS-15, respectively.

KAUS compounds (Fig. 1; Table 1) to left-shift the OEC (stabil- Diﬀraction data for all crystals were obtained at ∼100 K on

ize the R-state or increase the oxygen aﬃnity of Hb) in normal an R-axis IV++ image plate detector using CuKα X-rays (λ =

whole blood was determined using multipoint tonometry as 1.5417) from a Rigaku Micro-Max^™-007 X-ray source equipped

previously reported.¹⁸Stock solutions of all compounds were with Varimax confocal optics operating at 40 kV and 20 mA

made at 250 mM or 125 mM concentration in DMSO. Blood (Rigaku, The Woodlands, TX). Crystals were cryoprotected in

(hematocrit of 22%) was incubated with the compound at their mother liquor supplemented with 15–25% glycerol. The

37 °C for 1.5 h. OEC studies were performed in duplicate at data set was processed and scaled with Rigaku D*TREK soft-

2 mM final test compound concentrations. Ethacrynic acid ware and the CCP4 suite of programs.⁵²

(ECA) was used as the positive control.

Structure determination of deoxyHb in complex with

RBC morphological antisickling studies. Six compounds, KAUS-12 or KAUS-15. The two deoxyHb structures in complex

including KAUS-12, KAUS-13, KAUS-15, KAUS-16, KAUS-17, with KAUS-12 and KAUS-15 were refined independently using

and the positive control ECA, were tested for their antisickling the isomorphous T-state native deoxyHb structure (PDB code

potencies using SS cells that had been pre-incubated with 4% 2DN2). Cycles of refinement with COOT and CNS.^53,54identi-

O₂, as previously reported.^11,18Briefly, SS cells were suspended fied two-symmetry-related bound compounds at the α-cleft

in Hemox buﬀer (TCS Scientific Corp, Southampton, PA), pH that appeared to form a covalent interaction with the N-term-

7.4, that contained 10 mM glucose and 0.2% bovine serum inal αVal1 nitrogens of the Hb. For both complexes, symmetry-

albumin. The solution was incubated under air in the absence related molecules were built into the model at the two α-sub-

(control) or presence of a 2 mM concentration of test com- units. Several water molecules and sulfate molecules were

pound at 37 °C for 1 h. Following this, the suspension was added and the structures refined to final R_factor/R_freeof 18.2/

incubated under hypoxic conditions (4% oxygen/96% nitrogen) 20.6% and 21.3/24.8% for KAUS-12 and KAUS-15, respectively.

at 37 °C for 5 h. The suspension was fixed with 2% glutaralde- The two KAUS-12 and KAUS-15 coordinates have been de-

hyde solution without exposure to air and then subjected to posited at the PDB with accession codes of 4ROL and 4ROM,

microscopic morphological analysis as previously reported.^11,18

respectively.

Structure determination of COHb in complex with KAUS-12

or KAUS-15. The isomorphous α1β1 dimer classical R-state

X-ray crystallography

Co-crystallization of deoxygenated Hb or carbonmonoxy Hb structure (1LJW) was used as the starting model to refine the

with KAUS compounds. A freshly prepared solution of KAUS-12 and KAUS-15 complexes. Unlike the T-state crystals,

KAUS-12 or KAUS-15 in DMSO was either directly incubated repeated refinements with model building and addition of

with deoxyHb (50 mg mL⁻¹) for 1 h or incubated with oxyHb water showed no observable eﬀector binding, and as a result

for 1 h (followed by deoxygenation to obtain the deoxyHb-drug the two structures were not refined to conclusion. The 2.0 Å

complex) at a Hb tetramer–compound molar ratio of 1 : 5 at structure of KAUS-15 was refined to R_factor/R_freeof 23.5/26.3,

37 °C, and then crystallized with 3.2 M sulfate/phosphate pre- while the lower resolution 2.8 Å structure of KAUS-12 was

cipitant, pH 6.8, using the batch method as previously refined to 26.4/33.5, and the refinement terminated.

described, to obtain T-state crystals in about 4 days.^11,18,22,46

Reactivity of KAUS toward glutathione (GSH) and the free

The crystals obtained by first incubating the compounds with amino acids ^L-Cys, L-Val, L-His and L-Lys. A freshly prepared

oxyHb followed by deoxygenation were poorly formed, result- 10 mM solution of glutathione (50 µL) was mixed thoroughly

ing in unusable diﬀraction data for structure determination, with 1.9 mL 0.1 M TRIS-citrate buﬀer (pH 7.5), which was then

while crystals obtained from directly incubating the com- added to 50 µL of freshly prepared KAUS-12, KAUS-15 or ECA

pounds with deoxyHb diﬀracted to 1.7 and 1.9 Å for KAUS-12 (10 mM in DMSO) to initiate the reaction. After a specified

and KAUS-15, respectively. The two crystals are isomorphous time (0 min, 60 min and 180 min), 0.4 mL of the reaction

with each other, crystallizing in the space group P2₁with mixture was transferred to a vessel containing 40 µL phospho-

typical cell constants of 60, 80, 53 Å, and 98°. Diﬀraction data ric acid (10% in water) and mixed thoroughly. The GSH conju-

statistics are shown in Table 4.

gate with KAUS-12, KAUS-15 or ECA was then detected using

The compound–Hb complexes were also crystallized in the LC-MS. A similar reaction procedure, but only studied at one

classical R-state form using COHb, following a previously time point of 3 hour, was performed for the free amino acids

described procedure.^18,22,46Briefly, a solution of KAUS-12 or (^L-Cys, L-Val, L-His and L-Lys), and the conjugate formation fol-

KAUS-15 in DMSO was incubated with oxyHb for 1 h at 37 °C lowed using UPLC-MS.

at a Hb tetramer : compound molar ratio of 1 : 5. The mixture

The LC-MS system was composed of an Agilent 1200 HPLC

was then saturated with CO to generate COHb, and then crys- system, a solvent delivery module, a quaternary pump, an auto-

Org. Biomol. Chem., 2015, 13, 6353–6370 | 6367

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Organic & Biomolecular Chemistry

sampler, and a column compartment (Agilent Technology, benzoate), which was quantified by measuring absorbance at

Germany). The column eﬄuent was connected to an Agilent 412 nm.

6320 Ion Trap-ESI-MS. The column heater was set to 25 2 °C.

Cytotoxicity assessment against C-166 mouse fibroblast cell

The control of the HPLC system and data processing were per- line. The cytotoxicities of the test compounds, including

formed using ChemStation (Rev. B.01.03 SR2-204) and 6300 KAUS-12, KAUS-15 and ECA, were tested against C-166 cells by

Series Trap Control version 6.2 Build no. 62.24 (Bruker Dalto- sulforhodamine B (SRB) colorimetric assay as previously

nik GmbH). The analytes were separated using an Agilent described.⁵⁵Exponentially growing cells were collected using

Zorbax Extend-C18 column (80 Å, 150 mm length × 4.6 mm, 0.25% Trypsin-EDTA and plated in 96-well plates at 1000–2000

i.d., 5 μm) and an Agilent-Zorbax Extend-C18 pre-column cells per well. Cells were exposed to test compounds for 72 h

(Agilent Technologies, Palo Alto, CA, USA). General MS adjust- and subsequently fixed with TCA (10%) for 1 h at 4 °C. After

ments were set as follows: capillary voltage, 4000 V; nebulizer, several washings, the cells were exposed to 0.4% SRB solution

35 psi; drying gas, 12 L min⁻¹; desolvation temperature, for 10 min in a dark place and subsequently washed with 1%

350 °C; ion charge control (ICC) smart target, 150 000; and glacial acetic acid. After drying overnight, Tris-HCl was used to

max accumulation time, 150 ms. Auto-MS positive mode was dissolve the SRB-stained cells and the color intensity was

applied. Mobile system: isocratic elution using 55% aceto- measured at 540 nm. All cytotoxicity measurements were

nitrile and 45% water containing 0.1% formic acid (w/v).

repeated thrice (n = 3). The dose response curve of the com-

The UPLC-MS analysis was performed using an Acquity pounds was analyzed using the E_maxmodel.

ꢀ

ꢁ

H-Class UPLC connected to a PDA detector and an Acquity

TQD detector. The column used was an Acquity UPLC BEH

C18 1.7 µm, 2.1 × 50 mm, with a Vanguard pre-column

attached. Solvent A consisted of 90 : 10 water : acetonitrile with

m

½Dꢂ

% Cell viability ¼ ð100 ꢀ RÞ ꢁ 1ꢀ

þ R

m

K_dþ ½Dꢂ

where R is the residual unaﬀected fraction (the resistance frac-

tion), [D] is the drug concentration used, K_dis the drug con-

centration that produces a 50% reduction of the maximum

inhibition rate and m is a Hill-type coeﬃcient. The IC₅₀was

defined as the drug concentration required to reduce the fluo-

rescence to 50% of that of the control (i.e., K_d= IC₅₀when R =

0 and E_max= 100 − R).⁵²

0.1% formic acid, while solvent

B consisted of 90 : 10

acetonitrile : water with 0.1% formic acid. A gradient run was

performed such that solvent B was increased from 0% B to

100% B from time 0–7 min, followed by washing with 100% B

for 5 min and then a return and re-equilibration at 100% A in

the final 3 min (total run time = 15 min). 10 µL of sample was

injected per run. The eluent of the column was connected to a

PDA UV detector which scanned from 250–350 nm and a 2D

channel of 280 nm was used for quantification of compound

and adduct. The eluent was then introduced into the TQD

detector to confirm the masses of adducts formed. The TQD

detector was set to positive ionization mode with a capillary

voltage of 3.20 kV, cone voltage of 20 V, extractor voltage of 3 V,

and RF lens voltage of 0.1 V. The source temperature was set at

150 °C, while the desolvation temperature was set at 350 °C

and the desolvation and cone gas flows were set at 650 and 50

L h⁻¹, respectively. Scans were made from 100–700 m/z with a

scan duration of 0.5 seconds to obtain mass spectra at

diﬀerent time points.

Reactivity of KAUS-15 toward βCys93 of Hb. The accessible

sulfhydryl groups in Hb, and their reactivity with the test com-

pounds, were quantified by observing the results of the di-

sulfide exchange reaction of the thiols of βCys93 and DTNB at

412 nm (ε = 14 150 M⁻¹cm⁻¹). An aqueous solution of Hb (50

µM tetramer) was mixed with KAUS-15 or ECA (2 mM) in PBS

or no compound, and the mixture incubated for four hours at

25 °C. The mixture was centrifuged with washing (PBS) to sep-

arate Hb from excess reagents using centrifugal filters (cut oﬀ:

10 kDa, Millipore) at 5000 rpm for 30 min at 4 °C. The washed

hemoglobin was stored at 4 °C. 10 μl of each Hb solution was

diluted to 500 μl in 0.1 M potassium phosphate buﬀer at pH

8.0 and incubated at 25 °C for 1 h (non-DTNB control). To

another set, 10 μl of 10 mM DTNB was added and incubated

under similar conditions. Both sets of tubes were centrifuged

using centrifugal filters to collect the yellow filtrate (2-nitro-

Acknowledgements

The authors thank Dr Ahmed Al-Abd, Prof. Dr Alaa Khedr and

Dr Ali Alhalawany, Faculty of Pharmacy, King Abdulaziz Univer-

sity, for generous support in performing the cell viability

(A. Al-Abd) and GSH reactivity (A. Khedr for LC-MS and

A. Alhalawany for HPLC) tests, and Dr Akul Mehta at the Virgi-

nia Commonwealth University for UPLC-MS analysis of free

amino acid conjugates of the test compounds. The authors

also thank the Science and Technology Unit, King Abdulaziz

University for technical support. This work was supported by

the NSTIP strategic technologies program in the Kingdom of

Saudi Arabia, Project No. 10-BIO1253-03, NIH/NIMHD grant

MD009124 (MKS, OA); NIH/NHLBI grant K01HL103186 (OA).

Structural biology resources were provided in part by NIH

grant CA16059 to the VCU Massey Cancer Center.

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Article Doi

Identification of a novel class of covalent modifiers of hemoglobin as potential antisickling agents

DOI: 10.1039/c5ob00367a

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Article abstract of DOI:10.1039/c5ob00367a

Full text of DOI:10.1039/c5ob00367a

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