Aryloxyalkanoic acids as non-covalent modifiers of the allosteric properties of hemoglobin

molecules

Article

Aryloxyalkanoic Acids as Non-Covalent Modiﬁers of

the Allosteric Properties of Hemoglobin

Abdelsattar M. Omar ^1,2,*, Mona A. Mahran ³, Mohini S. Ghatge ⁴, Faida H. A. Bamane ⁵,

Mostafa H. Ahmed ⁴, Moustafa E. El-Araby ^1,6, Osheiza Abdulmalik ⁷and Martin K. Safo ^4,

*

1

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Alsulaymanyah,

Jeddah 21589, Saudi Arabia; alamka@gmail.com

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria 21521,

Egypt; mahranmona@yahoo.com

Department of Medicinal Chemistry, and The Institute for Structural Biology, Drug Discovery and

Development, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298, USA;

msghatge@vcu.edu (M.S.G.); ahmedmh@vcu.edu (M.H.A.)

2

3

4

5

6

7

Department of Biochemistry, Faculty of Medicine, King Abdulaziz University, Alsulaymanyah,

Jeddah 21589, Saudi Arabia; fbamane@yahoo.com

Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Helwan University,

Cairo 11790, Egypt

Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA;

abdulmalik@email.chop.edu

*

Correspondence: asmansour@kau.edu.sa (A.M.O.); msafo@vcu.edu (M.K.S.);

Tel.: +966-567-68-14-66 (A.M.O.); +1-804-828-7291 (M.K.S.)

Academic Editor: Derek J. McPhee

Received: 27 June 2016; Accepted: 9 August 2016; Published: 13 August 2016

Abstract: Hemoglobin (Hb) modiﬁers that stereospeciﬁcally inhibit sickle hemoglobin polymer

formation and/or allosterically increase Hb afﬁnity for oxygen have been shown to prevent

the primary pathophysiology of sickle cell disease (SCD), speciﬁcally, Hb polymerization and

red blood cell sickling. Several such compounds are currently being clinically studied for the

treatment of SCD. Based on the previously reported non-covalent Hb binding characteristics of

substituted aryloxyalkanoic acids that exhibited antisickling properties, we designed, synthesized

and evaluated 18 new compounds (KAUS II series) for enhanced antisickling activities. Surprisingly,

select test compounds showed no antisickling effects or promoted erythrocyte sickling. Additionally,

the compounds showed no signiﬁcant effect on Hb oxygen afﬁnity (or in some cases, even decreased

the afﬁnity for oxygen). The X-ray structure of deoxygenated Hb in complex with a prototype

compound, KAUS-23, revealed that the effector bound in the central water cavity of the protein,

providing atomic level explanations for the observed functional and biological activities. Although

the structural modiﬁcation did not lead to the anticipated biological effects, the ﬁndings provide

important direction for designing candidate antisickling agents, as well as a framework for novel Hb

allosteric effectors that conversely, decrease the protein afﬁnity for oxygen for potential therapeutic

use for hypoxic- and/or ischemic-related diseases.

Keywords: hemoglobin; sickle cell disease; aryloxyalkanoic acids; halogenated benzene; imidazole;

antisickling; oxygen equilibrium curve; high afﬁnity; low afﬁnity; crystal structure

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1. Introduction

In its function of transporting oxygen from the lungs to tissues, hemoglobin (Hb) alternates

between tense or T-state (unliganded or deoxygenated Hb) and the relaxed or R-state (liganded or

oxygenated Hb). The equilibrium between the two states can be regulated by synthetic allosteric

effectors that bind to the central water cavity or the surface of the protein [1,2]. Stabilization

of the R-state and/or destabilization of the T-state shifts Hb oxygen equilibrium curve (OEC)

to the left, producing a high-afﬁnity Hb that is known to be beneﬁcial for sickle cell disease

(SCD) therapy [

compounds that are also potentially useful for the treatment of hypoxic- and/or ischemic-related

diseases is also true [ ]. Under hypoxia sickle Hb (Hb S) polymerizes into long, rigid, and

1,2]. The converse, where a low-afﬁnity Hb is produced with OEC right-shifting

1,2

insoluble ﬁbers causing the primary pathophysiology associated with SCD, which leads to RBC

sickling, vaso-occlusion, painful crises, organ damage, oxidative stress, hemolysis of RBCs, decreased

vascular NO bioavailability, inﬂammation, impaired microvascular blood ﬂow, morbidity and even

mortality [

presumably as a result of unusually high concentration of 2,3-diphosphoglycerate and/or sphingosine

phosphate in sickle red blood cells (RBC) [ 10]. The polymer is initiated by a primary interaction

between the pathological 2Val6 from one Hb S molecule and a hydrophobic acceptor pocket in the

region of 1Ala70, 1Phe85 and 1Leu88 of another Hb molecule, and further stabilized by several

secondary contacts between the Hb molecules [11 15]. Disruption or weakening of the secondary

3–6]. The polymerization process is exacerbated by the low oxygen afﬁnity of Hb S,

7–

β

–

contacts is known to reduce Hb S polymerization and RBC sickling as demonstrated by a large number

of naturally occurring mutations that are known to mitigate the severity of SCD [16,17].

There have been several efforts to develop pharmacological interventions that increase Hb oxygen

afﬁnity to prevent the hypoxia-induced Hb S polymerization and/or directly destabilize polymer

contacts [

1

,2

,10]. The most promising are covalent modiﬁers of Hb, with examples such as aromatic

aldehydes [18

–

21], ethacrynic acid derivatives [22], isothiocynates [23], and thiols [24]. Nonetheless

nonspeciﬁc binding to other proteins and the resulting toxic effects has precluded several of these

covalent modiﬁers from being considered as therapeutic agents [10]. Abraham and co-workers in

the eighties showed that halogenated benzene derivatives or substituted aryloxyalkanoic acids bind

non-covalently to Hb at multiple Hb sites, including the central water cavity or the CD-corner or the

α

Trp14 binding pocket at the surface of the protein [25–29]. Those compounds that bind exclusively

to the CD-corner or Trp14 site, e.g., 3,4-(dichlorobenzyl)oxyacetic acid or p-(bromobenzyl)oxyacetic

α

acid showed no signiﬁcant effect on Hb oxygen binding properties, however they exhibited weak

antisickling effect, which was speculated to be due to weakening or destabilization of the polymer

contacts [25

that bind to the central water cavity decreased Hb afﬁnity for oxygen due to stabilization of T-state

Hb [25 32]. Although, compounds that decrease Hb afﬁnity for oxygen are expected to promote

hypoxia-induced sickling, cloﬁbrate was reported to exhibit a weak antisickling effect, which was

–28]. On the other hand, compounds, e.g., cloﬁbrate and several of its analogs or derivatives

–

attributed to the compound also binding to the

α

Trp14 site beside the central water cavity to destabilize

the polymer [27].

Even though the reported

αTrp14 binders showed marginal antisickling effects, the αTrp14

site remains an attractive target for drug design, which we decided to investigate. Consequently,

we have structurally modiﬁed the previously studied aryloxyalkanoic acid pharmacophore into two

series of compounds, 4-(imidazole-2-carbonyl)phenoxyalkanoic acids (7a

and 4-(imidazol-2-yl)phenoxyalkanoic acids (13 17a 21a ; KAUS-20 to KAUS-27) that are expected

to bind to the Trp14 site. The compounds have been tested for their effect on both sickle RBC

–I; KAUS-1 to KAUS-8)

,

–

e,

–c

α

morphology and Hb oxygen binding properties. One of the compounds, KAUS-23 was co-crystallized

with deoxygenated Hb and its binding mode studied to gain insight into the compound’s functional

and biological activities.

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2. Results and Discussion

2.1. Compounds Designed to Bind at the αTrp14 Site of Hemoglobin

Substituted aromatic compounds, e.g., aryloxyalkanoic acids, have been shown to bind

non-covalently to Hb at the central water cavity or the surface of the protein, e.g., Trp14 or CD-corner

binding sites and display moderate or no antisickling effect, and in some instances even prosickling

effects [22 25 29]. In most cases, the compounds that bind exclusively to the central water cavity

showed no antisickling activity, due to stabilization of T-state Hb and the concomitant decrease in

Hb afﬁnity for oxygen [25 27 28]. In contrast, compounds that bind to the surface of the protein, e.g.,

the Trp14 binding pocket exhibited moderate antisickling activities, which was attributed to direct

polymer destabilization [25 28]. Other aryloxyalkanoic acids, e.g., cloﬁbrate (Figure 1) which is known

α

–

,

α

–

to bind to both the central water cavity and the

afﬁnity and antisickling properties [27].

αTrp14 site was reported to show both low-oxygen

Figure 1. Example of previously studied aryloxyalkanoic acid derivatives.

The

α

Trp14 binding pocket is lined with several hydrophobic residues, including αTrp14, αVal17,

α

Ala21,

α

Tyr24,

α

Phe128,

α

Leu105,

Thr67, Lys11 and

in complex with 3,4-(dichlorobenzyl)oxyacetic acid or cloﬁbrate or other aryloxyalkanoic acids that

bind to the Trp14 have been described previously [25 29]. Due to the low resolution nature of the

α

Leu109 and

αLeu125, and hydrophilic residues at the mouth

of the cavity including,

α

Lys60. The crystal structures of deoxygenated Hb

α

–

structures, detailed interactions between the protein and compounds were not described, however

the interactions with the surrounding hydrophobic residues were suggested to be predominantly

hydrophobic and dipolar van der Waals in nature [25

to the Trp14 pocket like the toluene molecule whose coordinates are available from a more recently

determined high-resolution structure (PDB code 1R1X)[33]. In this structure, the Trp14 pocket shows

two bound toluene molecules making hydrophobic interactions with Trp14, Val17, Tyr24, Thr67,

αLue105, αLeu109, αLeu125, αPhe128, αVal10, αVal70 and αLeu66 (Figure 2a).

Based on the Trp14 binding interactions with 3,4-[(dichlorobenzyl)oxy]acetic acid or cloﬁbrate

–28]. These compounds presumably bind similarly

α

and the fact that two toluene molecules are able to ﬁt the binding cavity (Figure 2a), we hypothesized

that increasing the size of the aryloxyalkanoic acids for additional hydrophobic interactions with the

α

Trp14 pocket residues might lead to stronger compound binding, resulting in increased Hb afﬁnity

for oxygen and antisickling potency. We therefore embarked on a series of structural modiﬁcations

of the aryloxyalkanoic acid pharmacophore by introducing an imidazole to the aryloxyalkanoic ring

that we anticipate will increase hydrophobic interaction with the αTrp14 non-polar pocket residues

as putatively illustrated in Figure 2b (please see Supplementary Material for a description of the

docking process).

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(a)

(b)

Figure 2. The

α

Trp14 binding pocket. (

a) Binding of two molecules of toluene at the αTrp14 binding

pocket of carbonmonoxy Hb (PDB ID: 1R1X). As noted in the text, CFA binds in a similar fashion but

the coordinate is not available [27]; (b) Putative binding of KAUS-23 at the Trp14 binding pocket of

deoxygenated Hb (PDB ID: 2DN2).

Two classes of compounds, (imidazole-2-carbonyl)phenoxyalkanoic acids (7a

–I; KAUS-1 to

KAUS-8) and (imidazol-2-yl)phenoxyalkanoic acids (13 17a 21a ; KAUS-20 to KAUS-27) were

,

–

e,

–c

synthesized. The proposed compounds, like 3,4-(dichlorobenzyl)oxyacetic acid and cloﬁbrate also

contain carboxylate moieties that could potentially form salt-bridge/hydrogen-bond interactions with

α

Lys60 and/or αThr67 located at the mouth of the pocket (Figure 2b). We should point out that there

is no indication to suggest that the carboxylate of 3,4-(dichlorobenzyl)oxyacetic acid or cloﬁbrate form

such polar interactions. It was also anticipated that the compounds will exhibit minimal toxicity effect

due to their non-covalent interactions with Hb compared to compounds that bind covalently to Hb.

Each class of the proposed compound can further be subdivided into three subclasses that differ in

their carboxylate arm length. Compounds in each subclass also differ in the number or position of the

chloro substituents on the phenoxyalkanoic ring. The synthetic pathways adopted for the preparation

of the intermediate and target compounds are outlined in Schemes 1–5 and detailed syntheses are

described in the Experimental Section. The compounds were then tested for their effect on sickle RBC

morphology and Hb oxygen afﬁnity.

Scheme 1. Synthesis of {[2,3-Di/mono substituted-4-(1H-imidazol-2yl)carbonyl]-phenoxy}acetic/butyric acids.

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R₁

R₂

OH

CrO₃/AcOH

R₁

R₂

R₁

R₂

CO₂H

OH

HO

Br

HO

O

8

OH

O

N

K₂CO₃

N

tr

N

tr

NH

9a-c

7g-i

3a-c

7g R¹= R²= Cl

7h: R¹= H, R²= Cl

7i: R¹= Cl, R²= H

9a: R¹= R²= Cl

9b: R¹= H, R²= Cl

9c: R¹= Cl, R²= H

Scheme 2. Synthesis of {[2,3-Di/mono substituted-4-(1H-imidazol-2yl)carbonyl]-phenoxy}propionic acids.

Scheme 3. Synthesis of 2-[3-chloro-4-(1H-imidazol-2-yl)phenoxy]acetic acid.

Scheme 4. Synthesis of 2-[Di/monochloro-4-(1H-imidazol-2-yl)phenoxy]acetic/butyric acids.

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Scheme 5. Synthesis of 2-[Di/monochloro-4-(1H-imidazol-2-yl)phenoxy]propionic acids.

2.2. Test Compounds Showed no Antisickling Effect

The KAUS compounds were expected to bind to the

α

Trp14 site and prevent sickling of red blood

cells through polymer destabilization. We therefore tested selected compounds, including KAUS-4,

KAUS-23 and KAUS-24, and the control cloﬁbrate (2 mM) on their effect on sickle RBC morphology as

previously reported [18,19,22]. Unexpectedly, the antisickling study showed KAUS-4 to have almost no

effect on the RBC morphology, while both KAUS-23 and KAUS-24 actually induced sickling (Figure 3).

Control (buffer)

KAUS-4

KAUS-24

Figure 3. RBC morphological antisickling results of compounds KAUS-4 and KAUS-24. Note that

cloﬁbrate and KAUS-23 showed similar prosickling effect as KAUS-24.

Cloﬁbrate acted similarly to KAUS-23 and KAUS-24. It is apparent that our structural

modiﬁcations failed to have the desired biological effect. Based on this result we speculated that

the KAUS compounds instead of binding to the αTrp14 site, are probably binding to the central water

cavity of the protein to decrease the protein afﬁnity for oxygen, resulting in the observed biological

activities. We therefore undertook both oxygen equilibrium curve and crystallographic studies to

understand the biological behavior of the compounds.

2.3. Compounds Either Showed no Allosteric Activity or Produced Low-Afﬁnity Hemoglobin

All the KAUS compounds, as well as cloﬁbrate, were studied at 2 mM concentration to

determine their effect on Hb oxygen binding property using normal whole blood following published

procedures [18,19,22]. The results are shown in Tables 1 and 2.

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Table 1. The Effect of Compounds 7a–i on Hb Afﬁnity for Oxygen Using Normal Whole Blood ^a.

c

b

Comp.

Name

R₁

R₂

N

∆P₅₀

Mean P₅₀

DMSO ^d

Cloﬁbrate

KAUS-1

KAUS-4

KAUS-7

KAUS-3

KAUS-6

KAUS-9

KAUS-2

KAUS-5

KAUS-8

Ctr

7a

7b

7c

7d

7e

7f

38.3 ± 0.0

40.3 ± 0.1

38.3 ± 0.4

37.5 ± 0.3

39.3 ± 0.6

38.6 ± 0.1

38.6 ± 0.6

38.2 ± 0.5

38.6 ± 0.1

39.2 ± 0.4

38.6 ± 0.4

0.0

2.0 ± 0.1

0.0 ± 0.4

−0.8 ± 0.3

1.0 ± 0.6

0.3 ± 0.1

0.3 ± 0.6

−0.1 ± 0.5

0.3 ± 0.1

0.9 ± 0.4

0.3 ± 0.4

Cl

H

Cl

H

Cl

H

Cl

H

Cl

H

Cl

H

1

3

2

7g

7h

7i

Cl

a

b

Results are the means of two or three measurements. P₅₀is the oxygen pressure in mmHg at which normal

RBC (22% hematocrit) is 50% saturated with oxygen.

control. The ﬁnal concentration of DMSO was <2% in all samples.

c

∆

P₅₀(mm Hg) is P₅₀of compound treated cells

−

P₅₀of

d

Table 2. The Effect of Compounds (13, 17a–e, 21a–c) on Hb Afﬁnity for Oxygen Using Normal

Whole Blood ^a.

b

c

Comp.

Name

R₁

R₂

N

Mean P₅₀

∆P₅₀

Ctr

13

DMSO ^d

KAUS-25

KAUS-19

KAUS-22

KAUS-26

KAUS-20

KAUS-23

KAUS-27

KAUS-21

KAUS-24

38.3 ± 0.0

39.7 ± 0.3

39.2 ± 0.2

41.3 ± 0.05

39.7 ± 0.1

38.9 ± 0.3

40.8 ± 0.02

39.1 ± 0.3

39.6 ± 0.6

41.7 ± 0.3

0.0

Cl

H

Cl

H

Cl

H

Cl

H

Cl

H

1

2

3

1.4 ± 0.3

0.9 ± 0.2

2.9 ± 0.05

1.4 ± 0.1

0.7 ± 0.3

2.5 ± 0.02

0.8 ± 0.3

1.3 ± 0.6

3.4 ± 0.3

17a

17b

21c

21a

21b

17e

17c

17d

Cl

a

b

The results are the means of two or three measurements. P₅₀is the oxygen pressure at which normal

RBC (22% hematocrit) is 50% saturated with oxygen.

The ﬁnal concentration of DMSO was <2% in all samples.

c

∆P₅₀is P₅₀of compound treated cells—P₅₀of control.

d

Compounds that increase or decrease the oxygen afﬁnity of Hb are expected to shift the OEC to

the left or right, respectively, and the degree of shift is reported as a decrease or increase in P₅₀(the

oxygen tension at 50% Hb O₂saturation). Cloﬁbrate slightly decreased the protein afﬁnity for oxygen

(∆P₅₀of 2 mmHg) consistent with its prosickling activity. The KAUS compounds either showed no

signiﬁcant effect on Hb allosteric activity, as observed with the (imidazole-2-carbonyl)phenoxy-alkanoic

acids KAUS 1–8 (Table 1) or marginally decreased the protein afﬁnity for oxygen as observed with

the (imidazol-2-yl)phenoxyalkanoic acids KAUS 20–27 (Table 2). Among the three subclasses of the

(imidazol-2-yl)phenoxyalkanoic acids (different carboxylate chain lengths), the compounds with chloro

substitution at the 2-position (KAUS-22, KAUS-23 and KAUS-24) produced the most low-afﬁnity Hb

with

∆P₅₀shift of 2.9, 2.5 and 3.4 mmHg, respectively, with no apparent correlation between the

varying carboxylic arm length and the P₅₀shift. It is evident that the KAUS compounds generally

did not increase Hb oxygen afﬁnity and thus not expected to prevent hypoxia-induced RBC sickling.

It is also apparent that the compounds that produced low-afﬁnity Hb, e.g., KAUS-23 and KAUS-24

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are the ones that promoted sickling, while KAUS-4 which showed almost no effect on Hb oxygen

binding properties virtually have no effect on RBC morphology. These observations in addition to

the antisickling results conﬁrm our suspicion that the compounds do not bind to the αTrp14 site as

predicted by our design, but most likely bind to the central water cavity. X-ray crystallographic study

was then conducted to determine the actual binding site of these compounds.

2.4. Structural Studies Showed KAUS-23 Bound Exclusively to the Central Water Cavity of Hemoglobin

Attempts were made to co-crystallize KAUS-4, KAUS-23, and KAUS-24 with deoxygenated Hb

using high-salt conditions as previously published [22,34].

Table 3. Data collection and reﬁnement statistics of deoxygenated Hb in complex with KAUS-23.

Numbers in parentheses are for the highest resolution shell.

Data

KAUS-23

Space group

Unit-cell a, b, c (Å)

Resolution (Å)

Unique reﬂections

Redundancy

P2₁

62.2, 80.8, 53.4, 90.0, 99.2, 90.0

29.64–2.15 (2.23–2.15)

27383

6.75

96.2 (94.1)

21.0 (8.4)

6.4 (14.6)

Completeness (%)

Average I/σ(I)

R_merge(%) ^a

Reﬁnement ^b

Resolution (Å)

No. of reﬂections

R_work(%)

29.64–2.15 (2.23–2.15)

27358 (2521)

19.24 (24.73)

25.71 (35.69)

0.007

R_free(%)

R.m.s.d. bonds (Å)

◦

R.m.s.d. angles ( )

1.28

Dihedral angles

Most favored (%)

Allowed (%)

Average B (Å²) / atoms

All atoms

95.6

3.7

29.4

29.0

Protein

Hemes

KAUS-23

27.2

40.7

Phosphate

Water

54.6

34.7

PDB ID code

5KDQ

a

b

R_merge

= Σ_hklΣ_i|I_i(hkl) – <I(hkl)>|/Σ_hklΣ_iI_i(hkl). R_freewas calculated from 5% randomly selected reﬂection

for cross-validation. All other measured reﬂections were used during reﬁnement.

X-ray quality T-state crystals were obtained only for KAUS-23, which crystallized with a tetramer

in the asymmetric unit and in space group P2₁. The crystals are isomorphous with the native

deoxygenated Hb crystal (PDB code 2DN2) [35], necessitating the use of 2DN2 as the starting model

for the reﬁnement. Structural statistics are summarized in Table 3. The structure has been deposited in

the PDB with the ID code 5KDQ.

As suspected from the OEC and antisickling results, the complex structure showed exclusive

binding of KAUS-23 in the central water cavity, close to the

α-cleft with no apparent binding at

the expected Trp14 binding site (Figure 4a). We expected two molecules of KAUS-23 to bind,

α

however, only one of the compounds was included in the ﬁnal structural model as the density at

the symmetry-related site was too weak and broken to allow ﬁtting of the second compound. Water

molecules were instead modelled into the broken densities (Figure S1).

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(b)

(a)

(c)

Figure 4. Tetrameric structure of deoxygenated Hb (ribbon) in complex with KAUS-23 (K23; sticks).

(a) KAUS-23 bound in the central water cavity. Note that only one of the symmetry-related molecules

was modelled since the electron density of the second site was too weak and disordered. The

are colored in yellow and -subunits in cyan; ( ) Close-up view of KAUS-23 binding with the ﬁnal

2Fo-Fc reﬁned electron density map contoured at 0.8 . For clarity, not all binding site residues shown

but described in the text; ( ) Schematic representation of the interactions between KAUS-23 and the

α-subunits

β

b

σ

c

protein at the central water cavity.

Although the imidazole and chlorophenoxy moieties show relatively well-deﬁned density,

the carboxylate group was characterized by weak and highly disordered electron densities (Figure 4b).

The imidazole moiety is located in a hydrophobic pocket formed by

1Ser131 and 1Ala130 (Figure 4c). One of the nitrogen atoms of the imidazole forms a water-mediated

hydrogen-bond interaction with the side chain of 1Thr134, as well as a direct hydrogen-bond

interaction with the main-chain nitrogen atom. Note that all these reactions involve residues from

the same -subunit. The chlorophenoxycarboxylic acid moiety directs further down the central water

cavity, where the chlorophenyl group makes inter-subunit hydrophobic interactions with 2Arg141,

2Thr137 and 2Ser138. As noted above, the carboxylate is characterized by very weak density,

consistent with the apparent lack of interaction with the protein. It is notable that the KAUS-23 binding

α1Val1, α1Leu2, α1Lys127,

α

site overlaps the chloride ion binding sites at the

binding and inﬂuence the Bohr effect [36].

α-cleft and could potentially inhibit chloride ion

Interestingly, when KAUS-23 is compared to cloﬁbrate, the two compounds bind at different

sites in the central water cavity. Note that the coordinate for the deoxygenated Hb-cloﬁbrate structure

is not available, but cloﬁbrate binding at the central water cavity has been described in published

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articles at a site surrounded by the

α

Tyr140,

α

Pro95,

α

Val96,

βTrp37,

βAsn108 and

α

Lys99 [27].

Bezaﬁbrate, urea derivatives of bezaﬁbrate and RSR13 (Efaproxiral) are potent allosteric effectors that

were designed based on cloﬁbrate binding to the central water cavity [ 30 31 37 38]. Expectedly,

2,6, , , ,

the high-resolutions structures of these compounds in complex with Hb show them overlapping

with cloﬁbrate, allowing for better atomic level understanding of how cloﬁbrate binds and stabilizes

the T-state Hb. Speciﬁcally, these effectors interact with one

His103, Lys99, Asn108, Tyr35, Trp37 and Val96; residues that are known to surround cloﬁbrate

binding. Notably, these residues are known to shift signiﬁcantly during the T R transition and their

β-chain and two α-chains that involve

α

β

α

→

interactions with the effectors freeze them from moving to the R-state position [1,2].

Like cloﬁbrate, bezaﬁbrate, RSR13 and several of their analogs and derivatives, the inter-molecular

mediated interaction of KAUS-23 with the protein is expected to stabilize the T-state [1,2], consistent

with its observed allosteric activity of decreasing the protein afﬁnity for oxygen. We expect other

KAUS compounds to bind in a similar fashion in the central water cavity, although subtle differences in

their binding modes may have different stabilization effect on the T-state, explaining the differences in

their allosteric properties. This is consistent with several oxygen binding and crystallographic studies

that show effectors binding at the same site of Hb yet shift the oxygen equilibrium curve by different

degrees [1,2,39]. We are currently working to co-crystallize Hb and the KAUS compounds that exhibit

a wide range of activity that are likely to provide additional explanation for their properties.

3. Experimental Section

3.1. Materials

Normal whole blood was collected from adult donors at the Virginia Commonwealth University

(Richmond, VA, USA) after informed consent. Hb was puriﬁed from discarded normal blood samples

following a published procedure [34]. The use of these human samples is in accordance with regulations

of the IRB for Protection of Human Subjects. For the sickling assays, leftover blood samples from

patients with homozygous SS were utilized, based on an approved IRB protocol at the Children’s

Hospital of Philadelphia (Philadelphia, PA, USA), with informed consent.

3.2. Chemistry

Except as otherwise indicated, all reactions were carried out under a nitrogen atmosphere in

ﬂame- or oven-dried glassware. Reagents and solvents for chemical synthesis were purchased

from Sigma-Aldrich (St. Louis, MO, USA; through Bayouni Imp. Inc., Dist., Jeddah, Saudi

Arabia), Alfa Aesr (Haverhill, MA, USA; through Saggaf Co., Dist, Jeddah, Saudi Arabia) or Acros

Organics (Geel, Belgium; through Abdullatif H. Abuljadayel Est., Dist, Jeddah, Saudi Arabia) as

ACS-reagent grade; and used without further puriﬁcation. Tetrahydrofuran (THF) was distilled

from sodium/benzophenone-ketyl. Reactions were monitored by thin layer chromatography (TLC)

1

with 0.25-mm pre-coated silica gel plates (E. Merck, Billerica, MA, USA). H-NMR spectra were

recorded on an AV-300 NMR spectrometer (Bruker Billerica, MA, USA) equipped with the Top Spin

software. Infrared spectra were recorded on a Bruker ATIR spectrometer. Melting points were recorded

on a Buchi (Flawil, Switzerland; Bayouni Imp. Inc.) melting point apparatus and were uncorrected.

LCMS were run on an Agilent 6130 Series (Santa Clara, CA, USA), single quad instrument. HPLC

separation was run on 1200 SERIES HPLC using Chemstation Software (B.02, Agilent). Purities were

assessed by HPLC and were conﬁrmed to be >95% for all ﬁnal compounds. Elemental analyses were

performed using Vario, an Elementary apparatus (Shimadzu, Kyoto, Japan), located at the Organic

Microanalysis Unit, Cairo University (Giza, Egypt). Column chromatography was performed on silica

gel 60 (particle size 0.06–0.20 mm). Jones’ reagent was prepared by adding glacial acetic acid (6 mL)

drop wise to an ice cold solution of CrO₃(6.7 g) in water (50 mL). The orange solution was stirred

for 30 min at the same temperature. Freshly prepared solution was used for oxidation reactions.

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3.3. General Procedure for the Synthesis of 2,3-Di/monosubstituted-4-[(1-trityl-1H-imidazol-2-yl)-

hydroxymethyl]phenols 3a–c [40]

To a solution of 1-tritylimidazole (8.12 g, 26.160 mmol) in anhydrous THF (165 mL) was added

◦

n-BuLi (1.28 M in THF, 20.0 mL, 1.67 g, 13.08 mmol) at

−

20 C over a period of 20 min under nitrogen

atmosphere. The red solution was allowed to attain room temperature and stirred for 1 h, then cooled

◦

to

−

78 C. In a separate ﬂask the appropriate aldehyde 1a

–

c

(10.47 mmol) was dissolved in anhydrous

THF (4 mL) and added to the red solution dropwise at −78 ^◦C. The reaction mixture was stirred

at −78 ^◦C for 1 h and slowly brought to room temperature during which red color tuned to yellow

and then to colorless. After complete reaction, saturated NH₄Cl (250 mL) was added to the reaction

mixture at

−

78 ^◦C. The resulting mixture was extracted with EtOAc (3

×

100 mL); the organic layer

was separated, washed with water, saturated NaCl, and dried over anhydrous Na₂SO₄. The organic

layer was evaporated in vacuo and the residue washed with cold CH₂Cl₂.

2,3-Dichloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenol (3a): white solid (3.99 g, 76%),

m.p. 190.0–191.8 ^◦C. ¹H-NMR (DMSO-d₆)

δ 10.42 (s, 1H, OH), 7.25–7.21 (m, 9H, trityl-H), 7.08

(d, 2H, J = 8.7 Hz, Ar-H), 7.00–6.98 (m, 6H, trityl-H), 6.67 (d, 2H, J = 4.8 Hz, imidazole-H), 5.25 (d, 1H,

J = 7.8 Hz, OH), 5.18 (d, 1H, J = 7.8 Hz, CH).

2-Chloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenol (3b): white solid (2.76 g 93%), m.p. 165.2–166.5 ^◦C.

¹H-NMR (DMSO-d₆)

δ 9.83 (s, 1H, OH), 7.35–7.33 (m, 9H, trityl-H), 7.09–7.07 (m, 6H, trityl-H), 6.96

(s, 1H, Ar-H3), 6.57–6.39 (m, 3H, Ar-H and imidazole-H), 6.37 (d, 1H, J = 8.7 Hz, imidazole-H), 5.38

(d, 1H, J = 6.6 Hz, OH), 4.82 (d, 1H, J = 6.3 Hz, CH).

3-Chloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenol (3c): white solid (2.1 g, 70.4%), m.p. 196.0–197.7 ^◦C.

¹H-NMR (DMSO-d₆)

δ 9.59 (s, 1H, OH), 7.24–7.00 (m, 10H, Ar-H and trityl-H), 6.99–6.65 (m, 6H,

trityl-H), 6.64 (s, 1H, Ar-H2), 6.49–6.43 (m, 3H, Ar-H and imidazole-H), 5.23 (d, 1H, J = 6.6 Hz,

OH), 4.90 (d, 1H, J = 6.6 Hz, CH).

3.4. General Procedure for the Synthesis of Ethyl {2,3-Di/monosubstituted-4-[(1-trityl-1H-imidazol-2-yl)-

hydroxymethyl]phenoxy}esters 5a–f [41]

To a solution of the appropriate alcohol

K₂CO₃(3.082 g, 22.300 mmol) at room temperature and stirred for 1 h. To the reaction mixture bromo

ethyl esters (10.70 mmol) was added and stirred for 5–6 h at room temperature. Water (450 mL) was

3 (7.75 mmol) in anhydrous DMF (45 mL) was added

4

added, and the precipitated solid was ﬁltered and dried. The crude products were used as such in the

next step without further puriﬁcation.

Ethyl {2,3-dichloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}acetate (5a): white solid (4.82 g, 94%),

m.p. 169.1–171.5 ^◦C. The ¹H-NMR (DMSO-d₆)

δ 7.39–7.10 (9H, m, trityl-H), 6.98–6.81 (m, 7H, trityl-H

and Ar-H), 6.80 (d, 1H, J = 9 Hz, Ar-H), 5.56 (d, 1H, J = 6.9 Hz, OH), 4.80 (d,1H, J = 6.9 Hz, CH), 4.75 (s,

2H, OCH₂), 4.18 (q, 2H, J = 6.9 Hz, CH₂), 1.24 (t, 3H, J = 6.9 Hz, CH₃).

Ethyl {2-chloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}acetate (5b): white solid (2.0 g, 88.8%),

◦

m.p. 161.5–163.9 C. ¹H-NMR (DMSO-d₆)

δ 7.39–7.33 (m, 9H, trityl-H), 7.10–7.07 (m, 6H, trityl-H), 6.98

(s, 1H, Ar-H3), 6.66 (d, 1H, J = 8.7 Hz, Ar-H6), 6.57–6.49 (m, 3H, Ar-H5 and imidazole-H), 5.55 (s, 1H,

OH), 4.87 (s, 1H, CH), 4.75 (s, 1H, OCH₂), 4.16 (q, 2H, J = 7.1 Hz, CH₂), 1.22 (t, 3H, J = 6.9 Hz, CH₃).

Ethyl {3-chloro-4-[_◦1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}acetate (5c): white solid (1.5 g, 84.4%),

m.p. 189.3–192.3 C. ¹H-NMR (DMSO-d₆)

δ 7.30–7.22 (m, 10H, Ar-H and trityl-H), 7.08 (s, 1H, Ar-H2),

7.01–6.98 (m, 6H, trityl-H), 6.68–6.63 (m, 3H, Ar-H and imidazole-H), 5.25 (d, 1H, J = 6.9 Hz, OH), 5.12

(d, 1H, J = 6.9 Hz, CH), 4.75 (s, 2H, OCH₂), 4.16 (q, 2H, J = 7.2 Hz, CH₂), 1.23 (t, 3H, J = 6 Hz, CH₃).

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Ethyl 4-{[2,3-Dichloro-4-(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}butyrate (5d): white solid

◦

1

(3.0 g, 97.7%), m.p. 192.1–193.6 C. H-NMR (DMSO-d₆)

δ 7.21–7.17 (m, 19H, trityl-H, Ar-H and

imidazole-H), 5.8 (s, 1H, OH), 5.33 (t, 2H, J = 9 Hz, OCH₂), 4.07 (m, 3H, CH and OCH₂), 2.5 (2H, under

DMSO, CH₂), 1.98 (t, 2H, J = 6.6 Hz, CH₂), 1.17 (t, 3H, J = 6.9 Hz, CH₃).

Ethyl 4-{2-chloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}butyrate (5e): white solid (4.1 g, 66%),

m.p. 161.5–164.9 ^◦C.¹H-NMR (DMSO-d₆) δ_H7.33–7.20 (m, 9H, trityl-H), 7.09–7.08(m, 7H, trityl-H and

Ar-H), 6.97 (s, 1H, Ar-H3), 6.73 (d, 1H, J = 8.7 Hz, Ar-H6), 6.56–6.54 (m, 2H, imidazole-H), 5.52 (s, 1H,

OH), 4.86 (s, 1H, CH), 4.05 (q, 2H, J = 6.9 Hz, OCH₂), 3.97 (t, 2H, J = 7.2, 6.9 Hz, OCH₂), 2.45 (2H under

DMSO, CH₂), 1.93 (t, 2H, J = 6.6 Hz, CH₂), 1.17 (t, 3H, J = 6. 9 Hz, CH₃).

Ethyl 4-{3-Chloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}butyrate (5f): white solid (3.7 g, 84.95%),

◦

m.p. 147.5–148.8 C. ¹H-NMR (DMSO-d₆) δ_H7.31–7.21 (m, 10H, trityl-H and ArH), 7.08 (s, 1H, Ar-H2),

7.01–6.99 (m, 6H, trityl-H), 6.66–6.61 (m, 3H, Ar-H and imidazole-H), 5.26 (s, 1H, OH), 5.06 (s, 1H, CH),

4.07 (q, 2H, J = 7.2, 6.9 Hz, CH₂), 3.94(t, 2H, J = 6, 5.7 Hz, OCH₂), 2.46 (under DMSO, CH₂), 1.94(t, 2H,

J = 6.6, 6.3 Hz, CH₂), 1.19 (t, 3H, J = 6.9 Hz, CH₃).

3.5. General Procedure for the Synthesis of Ethyl {[2,3-Di/monosubstituted-4-(1-trityl-1H-imidazol-2-yl)

carbonyl]phenoxy}esters 6a–f [42]

◦

To a solution of alcohol

5

(7.659 mmol) in acetone (45 mL), Jones reagent (30.6 mL) at 0 C,

was added. The reaction mixture was allowed to attain room temperature and stirred for 7–8 h. Then,

water (50 mL) was added to the reaction mixture and extracted with CH₂Cl₂(3 100 mL). The organic

×

layer was separated, washed with water, followed by saturated NaCl. After drying over anhydrous

Na₂SO₄, the organic layer was evaporated in vacuo; the obtained solid was collected and dried.

The crude product was used as such in the next step without further puriﬁcation.

Ethyl {[2,3-dichloro-4-(1-trityl-1H-imidazol-2-yl)carbonyl]phenoxy}acetate (6a): pale yellow solid (2.8 g,

quantitative), m.p. 116.0–120.5 ^◦C. ¹H-NMR (DMSO-d₆) δ_H7.62 (d, 2H, J = 8.7 Hz, Ar-H), 7.30–7.19

(m, 15H, trityl-H), 6.45 (d, 2H, J = 1. 2 Hz, imidazole-H), 5.07 (s, 2H, OCH₂), 4.19 (q, 2H, J = 7.2, 6.9 Hz,

CH₂), 1.23 (t, 3H, J = 7.2, 6.9 Hz, CH₃).

Ethyl {[2-Chloro-4-(1-trityl-1H-imidazol-2-yl)carbonyl]phenoxy}acetate (6b): white solid (1.1 g, quantitative),

◦

m.p. 160.3–161.6 C. ¹H-NMR (DMSO-d₆) δ_H7.08–7.53 (m, 19H, trityl-H, Ar-H and imidazole-H), 6.46

(s, 1H, Ar-H3), 5.07 (s, 2H, OCH₂), 4.20 (q, 2H, J = 7.2, 6.9 Hz, CH₂), 1.23 (t, 3H, J = 7.2, 6.9 Hz, CH₃).

Ethyl {[3-Chloro-4-(1H-imidazol-2-yl)carbonyl]phenoxy}acetate (6c): white solid (0.9 g, quantitative),

m.p. 171.5–172.0 ^◦C. ¹H-NMR (DMSO-d₆) δ_H13.56 (s, 1H, NH), 7.74 (d, 1H, J = 6.9 Hz, Ar-H5),

7.54 (d, 1H, J = 1.2 Hz, Ar-H2), 7.22–7.01 (m, 3H, imidazole-H and Ar-H6), 4.95 (s, 2H, OCH₂), 4.20

(q, 2H, J = 7.2, 6.9 Hz, CH₂), 1.24 (t, 3H, J = 7.2, 6.9 Hz, CH₃).

Ethyl 4-{[2,3-Dichloro-4-(1-trityl-1H-imidazol-2-yl)carbonyl]phenoxy}butyrate (6d): pale yellow solid (1.8 g,

quantitative). The crude product was used as such in the next step without further puriﬁcation.

¹H-NMR (DMSO-d₆) δ_H7.33–7.19 (m, 19H, trityl-H, Ar-H and imidazole-H), 4.22 (t, 2H, J = 6.3, 6 Hz,

OCH₂), 4.07 (q, 2H, J = 7.2, 3.9 Hz, CH₂), 3.55 (under DMSO, CH₂), 3.05 (m, 2H,CH₂), 1.18 (t, 3H

,

J = 7.2 Hz, CH₃).

Ethyl 4-{[2-Chloro-4-(1-trityl-1H-imidazol-2-yl)carbonyl]phenoxy}butyrate (6e): white solid (3.7 g, quantitative),

◦

m.p. 169.1–171.5 C. ¹H-NMR (DMSO-d₆) δ_H7.30–7.19 (m, 19H, trityl-H, Ar-H and imidazole-H), 6.46

(s, 1H, Ar-H3), 4.22 (t, 2H, J = 6.3, 6 Hz, OCH₂), 4.07 (q, 2H, J = 7.2, 6.9 Hz, OCH₂), 3.55 (under DMSO,

CH₂), 2.07-2.03 (m, 2H,CH₂), 1.18 (t, 3H, J = 7.2, 6.9 Hz, CH₃).

Ethyl 4-{[3-Chloro-4-(1H-imidazol-2-yl)carbonyl]phenoxy}butyrate (6f): white solid (1.8 g, 89.74%),

1

m.p. 112.1–114.0 ^◦C. H-NMR (DMSO-d₆) δ_H13.52 (s, 1H, NH), 7.75 (d, 1H, J = 8.4 Hz, Ar-H5),

7.53 (s, 1H, Ar-H2), 7.22–6.99 (m, 3H, ArH6 and imidazole-H), 4.11–4.10 (m, 4H, two OCH₂), 2.51

(under DMSO, CH₂), 1.99 (t, 2H, J = 6.6 Hz, CH₂), 1.19 (t, 3H, J = 6.9 Hz, CH₃).

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3.6. General Procedure for the Synthesis of {[2,3-Di/monosubstituted-4-(1H-imidazol-2yl)carbonyl]-phenoxy}

acetic/butyric acids (7a–f) [43]

To solution of

6

(8.159 mmol) in aqueous THF (12 mL/ water 6 mL) was added LiOH·H₂O (0.687 g,

16.37 mmol) at room temperature. The reaction mixture was stirred for 1 h during which the

solution turned to green. The reaction was monitored with TLC. After complete reaction, the solvent

was evaporated in vacuo. The crude material was then dissolved in minimum amount of water.

The precipitated solid was ﬁltered and the pH of the ﬁltrate was adjusted to 6–7 using aqueous 1.5 N

HCl, and the obtained solid product was collected by ﬁltration and dried. The crude material was

puriﬁed by column chromatography over silica gel (230–400 mesh) using 40 to 50% ethyl acetate in

hexane as eluent to afford the desired ﬁnal products 7a–i.

{[2,3-Dichloro-4-(1H-imidazol-2-yl)carbonyl]phenoxy}acetic acid, (7a): white solid (1.22 g, 51%),

m.p. 240.2–243 ^◦C. IR (KBr, _maxcm⁻¹): 3527 (OH), 3200 (NH), 1662 (CO), 1269, 1077 (C-O-C); ¹H-NMR

ν

(DMSO-d₆) δ_H13.64 (s, 1H, NH), 7.64–7.57 (m, 2H, Ar-H), 7.22–7.13 (m, 2H, imidazole-H), 4.95 (s, 2H,

OCH₂); LC/MS (ESI) m/z 315 (M + 1), 316 (M + 2), 317 (M + 3), 318 (M + 4).

{[2-Chloro-4-(1H-imidazol-2-yl)carbonyl]phenoxy}acetic acid (7b): pale yellow solid (1.04 g, 81.7%),

m.p. 303–306 ^◦C. ¹H-NMR (DMSO-d₆) δ_H13.42 (brs, 1H, NH), 8.76 (d, 1H, J = 1.8 Hz, Ar-H3), 8.39

(t, 1H, J = 9, 1.8 Hz, Ar-H5), 7.38 (s, 2H, imidazole-H), 6.97 (d, 1H, J = 9 Hz, Ar-H6), 4.38 (s, 2H, OCH₂);

LC/MS (ESI) m/z 280.1 (M + ), 282.1 (M + 2).

{3-Chloro-4-[(1H-imidazol-2-yl)carbonyl]phenoxy}acetic acid (7c): white solid (0.64 g, 64.0%),

m.p. 230.8–233.9 ^◦C. IR (KBr,

ν

cm⁻¹): 3497 (NH), 3306 (OH), 1647 (CO), 1269, 1085 (C-O-C);

max

¹H-NMR (DMSO-d₆) δ_H13.56 (s, 1H, NH), 7.72 (d, 1H, J = 8.7 Hz, Ar-H5), 7.51 (s, 1H, imidazole-H),

7.20 (s, 1H, imidazole-H), 6.96 (d, 1H, J = 2.1 Hz, Ar-H2), 6.88 (t, 1H, J = 6.3, 2.4 Hz Ar-H6), 4.40 (s, 2H,

OCH₂); LC/MS (ESI) m/z 280.1 (M + )⁻, 282.1 (M + 2)⁻.

4-{2,3-Dichloro-4-[(1H-imidazol-2-yl)carbonyl]phenoxy}butyric acid (7d): white solid (1.15 g, 69%),

m.p. 167.5–169.4 ^◦C. IR (KBr,

ν

cm⁻¹): 3527 (NH), 3247 (OH), 1643 (CO), 1274, 1041 (C-O-C).

max

¹H-NMR (DMSO-d₆) δ_H13.61 (s, 1H, NH), 7.64 (d, 1H, J = 8.4 Hz, Ar-H5), 7.36 (s, 2H, imidazole-H),

7.29 (d, 1H, J = 8.7 Hz, Ar-H6), 4.19 (t, 2H, J = 6.6 Hz, OCH₂), 2.13 (t, 2H, J = 6.6 Hz, OCH₂), 1.94 (q, 2H,

J = 6.6, 6.3 Hz, CH₂). LC/MS (ESI) m/z 341 (M − 1)⁻, 343 (M + 1)⁻, 344 (M + 2)⁻, 346 (M + 4)⁻.

4-{2-Chloro-4-[(1H-imidazol-2-yl)carbonyl]phenoxy}butyric acid (7e): white solid (2.4 g, 71%),

1

m.p. 169.1–171.5 ^◦C. H-NMR (DMSO-d₆) δ_H13.52 (brs, 1H, NH), 8.73 (s, 1H, Ar-H3), 8.52 (d, 1H,

J = 7.8 Hz, Ar-H5), 7.41–7.32 (m, 3H, Ar-H6 and imidazole-H), 4.20 (t, 2H, J = 6 Hz, ₋OCH₂), 2.3 (t, 2H,

− −

1) , 308 (M + ) , 309.1 (M + 1) ,

J = 6 Hz, CH₂), 1.99–1.97(m, 2H, CH₂). LC/MS (ESI) m/z 307.1 (M

−

310 (M + 2)⁻, 312 (M + 4)⁻.

4-{3-Chloro-4-[(1H-imidazole-2-yl)carbonyl]phenoxy}butyric acid (7f): white solid (0.8 g, 48.5%),

m.p. 188.4–191.1 ^◦C. ¹H-NMR (DMSO-d₆) δ_H13.53 (s, 1H, NH), 12.19 (s,1H,OH), 7.76 (d, 1H, J = 8.7 Hz,

Ar-H5), 7.52 (s, 1H, Ar-H2), 7.23 (s, 1H, imidazole-H), 7.13(s, 1H, imidazole-H), 7.02 (d, 1H, J = 8.4 Hz,

Ar-H6), 4.10 (t, 2H, J = 6.3, 6 Hz, OCH₂), 2.40 (2H, under DMSO, CH₂), 1.97 (m, 2H, CH₂); LC/MS

(ESI) m/z 307 (M − 1)⁻, 309.1 (M + 1)⁻, 310.1 (M + 2)⁻, 312 (M + 4)⁻.

3.7. General Procedure for the Synthesis of 3-{2,3-Di/monochloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxy-

methyl]phenoxy}propanols 9a–c [41]

To a solution of the appropriate alcohol 3a–c (12.76 mmol) in anhydrous DMF (65 mL) was

added K₂CO₃(4.4 g, 31.84 mmol) at room temperature and stirred for 1 h. To the reaction mixture,

3-bromopropanol (2.12 g, 1.4 mL, 15.31 mmol) was added and stirred for 16 h at room temperature.

The solid formed after addition of water (650 mL) was ﬁltered, treated with diethyl ether and dried

in vacuo.

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3-{2,3-Dichloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}propanol (9a): white solid (6.34 g, 88.78%),

m.p. 174.7–177.7 ^◦C. ¹H-NMR (DMSO-d₆) δ_H7.25–7.10 (m, 11H, trityl-H and Ar-H), 7.0–6.9

(m, 7H, trityl-H and imidazole-H), 6.87 (d, 1H, J = 8.4 Hz, imidazole-H), 5.29 (s, 2H, OH and CH),

4.6 (brs, 1H, OH), 4.09 (t, 2H, J = 6, 6.3 Hz, OCH₂), 3.57 (t, 2H, J = 6, 6.3 Hz, OCH₂), 1.83 (quint, 2H,

J = 6.3 Hz, CH₂).

3-{2-Chloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}propanol (9b): white solid (3.9 g, 88.94%),

m.p. 155.9–157.5 ^◦C. ¹H-NMR (DMSO-d₆) δ_H7.34–7.33 (m, 9H, trityl-H), 7.10–7.08 (m, 6H, trityl-H),

6.97 (s, 1H, Ar-H3), 6.74 (d, 1H, J = 8.4 Hz, Ar-H6), 6.57–6.51 (m, 3H, ArH and imidazole-H), 5.57 (brs,

1H, OH), 4.86 (s, 1H, CH), 4.59 (brs, 1H, OH), 4.01 (t, 2H, J = 6, 6.3 Hz, OCH₂), 3.54 (t, 2H, J = 6, 6.3 Hz,

OCH₂), 1.83 (quint, 2H, J = 6.3 Hz, CH₂).

3-{3-Chloro-4-[(1-trityl-1H-imidazol-2-yl)hydroxymethyl]phenoxy}propanol (9c): white solid (4.5 g, 87.0%),

◦

m.p. 182.3–183.9 C. ¹H-NMR (DMSO-d₆) δ_H7.31–7.28 (m, 10H, Ar-H and trityl-H), 7.22 (s, 1H, Ar-H2),

7.08–7.00 (m, 6H, trityl-H), 6.66–6.60 (m, 3H, ArH and imidazole-H), 5.26 (s, 1H, CH), 5.04 (brs, 1H,

OH), 4.56 (brs, 1H, OH), 3.97 (t, 2H, J = 6, 5.4 Hz, OCH₂), 3. 49 (t, 2H, J = 6, 6.3 Hz, OCH₂), 1.81 (quint,

2H, J = 6, 5.7 Hz, CH₂).

3.8. General Procedure for the Synthesis of 3-{2,3-Di/monochloro-4-[(1H-imidazol-2-yl)carbonyl]phenoxy}-

propionic Acids 7g–i [42]

To a solution of alcohol 9a

at 0 ^◦C. The reaction mixture was allowed to attain room temperature and stirred for 48 h and ﬁltered

to remove insoluble impurities. The ﬁltrate was extracted with diethyl ether (6 50 mL). The organic

–c (10.72 mmol) in acetone (60 mL), Jones reagent (25 mL) was added

×

layer was separated, dried over anhydrous Na₂SO₄and evaporated in vacuo to get a pale yellow solid.

The product was dissolved in 5% NaHCO₃solution (minimum amount) and washed with diethyl

ether (2

×

50 mL) followed by ethyl acetate (2

×

50 mL). The pH of the aqueous layer was carefully

adjusted to 6 using 1.5 N aqueous HCl. The solid product formed was ﬁltered, washed with diethyl

ether and dried.

3-{2,3-dichloro-4-[(1H-imidazol-2-yl)carbonyl]phenoxy}propionic acid, 7g: white solid (0.45 g, 12.75%),

m.p. 207 ^◦C (decomposed). IR (KBr,

1270, 1035 (C-O-C). H-NMR (DMSO-d₆)

ν

_maxcm⁻¹): 3497(NH), 3306 (OH), 1647(CO), 1600, 1409 (CO₂⁻),

1

δ 13.64 (s, 1H, NH), 7.67 (d, 1H, J = 8.7 Hz, Ar-H5),

7.40 (s, 2H, imidazole-H), 7.28 (d, 1H, J = 8.7 Hz, Ar-H6), 4.36 (t, 2H, J = 6 Hz, OCH₂), 2.68 (2H,

J = 6.3, 5.7 Hz, CH₂).

3-{2-Chloro-4-[(1H-imidazol-2-yl)carbonyl]phenoxy}propionic acid (7h): white solid (1.0 g, 24.08%),

m.p. 195.0–195.4 ^◦C. IR (KBr,

ν

cm⁻¹): 3294 (NH), 3000–2800 (OH), 1703 (CO), 1613(CO), 1583

max

(C=N), 1254, 1037 (C-O-C). ¹H-NMR (DMSO-d₆) δ_H13.47 (s, 1H, NH), 12.49(s, 1H, OH), 8.72 (d, 1H,

J = 1.8 Hz, Ar-H3), 8.53 (t, 1H, J = 6.6, 2.1 Hz, Ar-H5), 7.52 (s, 1H, imidazole-H), 7.35(d, 1H, J = 8.7 Hz,

Ar-H6), 7.31 (s, 1H, imidazole-H), 4.38 (t, 2H, J = 6, 5.7 Hz, OCH₂), 2.78 (t, 2H, J = 6, 5.7 Hz, CH₂).

LC/MS (ESI) m/z 293 (M − 1), 295 (M + 1), 296 (M + 2).

3-{3-Chloro-4-(1H-imidazol-2-yl)carbonyl]phenoxy}propionic acid (7i): white solid (0.536 g, 21.22%),

◦

m.p. 193.0–194.2 C. IR (KBr,

ν

cm⁻¹): 3301 (NH), 3100–2600 (OH), 1717 (CO), 1645 (CO), 1598

max

(C=N), 1294, 1033 (C-O-C). ¹H-NMR (DMSO-d₆) δ_H13.53 (s, 1H, NH), 12.43 (s, 1H, OH), 7.75 (d, 1H,

J = 8.7 Hz, Ar-H5), 7.53 (s, 1H, imidazole-H), 7.22 (s, 1H, imidazole-H) 7.14 (d, 1H, J = 2.1 Hz, Ar-H2),

7.02 (dd, 1H, J = 6.3, 2.4 Hz, Ar-H6), 4.28 (t, 2H, J = 6, 5.7 Hz, OCH₂), 2.73 (t, 2H, J = 6, 5.7 Hz, CH₂).

LC/MS (ESI) m/z 293 (M − 1), 294 (M + ), 295 (M + 1), 296 (M + 2).

3.9. 3-Chloro-4-(1H-imidazol-2-yl)phenol (11) [30]

Glyoxal 10 (40% in H₂O, 14 mL, 0.095 mol) was placed in water (20 mL), then added to a cooled

solution (about 5 ^◦C) of 2-chloro-4-hydroxybenzaldehyde 1c (5 g, 0.031 mmol) in methanol (10 mL)

to afford a white turbid solution. Ammonium hydroxide (25% in H₂O, 67 mL, 0.478 mmol) was

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added drop wise over a period of 1 h at 0–5 ^◦C and the reaction mixture was stirred for 3 h at 0–5 ^◦C.

The mixture was allowed to warm to room temperature and stirred further for 24 h. The solution

was concentrated in vacuo and the crude was puriﬁed by column chromatography using mixture of

DCM/MeOH (95:5) as an eluent to obtain 11 as a beige solid (6.4 g, quantitative), m.p. 155–158 ^◦C.

¹H-NMR (300 MHz, DMSO-d₆) δ_H12.07 (brs, 1H, NH), 10.20 (brs, 1H, OH), 7.52–7.63 (m, 1H, Ar-H6),

7.10 (s, 1H, Ar-H2), 6.91 (s, 2H, imidazole-H), 6.83 (d, J = 8.50 Hz, 1H, Ar-H5).

3.10. Ethyl 2-[3-Chloro-4-(1H-imidazol-2-yl)phenoxy]acetate (12) [42]

◦

To a cold (5–10 C) solution of 11 (9.0 g, 0.049 mol) and K₂CO₃(1 g, 0.070 mol), KI (0.37 g, 0.005 mol)

in DMF (90 mL), was added ethyl bromoacetate (7 g, 0.049 mol) while stirring. The stirring was

continued for 1 h at room temperature. Then the reaction was treated with water (150 mL) and

extracted in DCM (2

×

100 mL). The combined organic layer was washed with water and brine, dried

with anhydrous Na₂SO₄, and evaporated under vacuum to give the crude product. Puriﬁcation was

carried out using ﬂash chromatography (hexane–EtOAC 3:7) to furnish a white solid of 12 (1.4 g, 11%),

m.p. 131–133 ^◦C. ¹H-NMR (300 MHz, DMSO-d₆) δ_Hppm 12.29 (brs, 1H, NH) 7.67 (d, J = 8.78 Hz, 1H,

Ar-H5), 7.07–7.21 (m, 3H, Ar-H2 and imidazole-H), 7.03 (dd, J = 8.69, 2.55 Hz, 1H, Ar-H6), 4.90 (s, 2H,

OCH₂), 4.19 (q, J = 7.08 Hz, 2H, CH₂), 1.22 (t, J = 7.08 Hz, 3H, CH₃).

3.11. 2-[3-Chloro-4-(1H-imidazol-2-yl)phenoxy]acetic acid (13) [43]

To a solution of 12 (1.4 g, 0.005 mol) in THF (5.6 mL) and water (2.8 mL) was added LiOH H₂O

·

(0.36 g, 0.015 mol) at room temperature and stirred for 30 min until consumption of the starting

material. The reaction was treated with 1.5N HCl until the pH of the reaction mixture became 6.5.

The precipitated product was ﬁltered and dried under vacuum as an off-white solid, (1.15 g, 92%),

m.p. 313–316 ^◦C.¹H-NMR (300 MHz, DMSO-d₆) δ_Hppm 14.75 (brs,1H, NH), 7.81 (s, 2H, imidazole-H),

7.71 (d, J =8.69 Hz, 1H, Ar-H5), 7.34 (d, J = 2.46 Hz, 1H, Ar-H2), 7.18 (dd, J = 8.69, 2.46 Hz, 1H, Ar-H6),

4.88 (s, 2H, OCH₂); LC-MS (ESI) m/z 251.9 (M + )⁻, 253.9 (M + 2)⁻; Anal. Calcd for (C₁₁H₉ClN₂O₃):

C, 52.29; H, 3.59; Cl, 14.03; N, 11.09; Found: C, 52.15; H, 3.24; Cl, 14.40; N, 11.12.

3.12. General Procedure for the Synthesis of Ethyl [(2,3-Di/monochloro-4-formyl)phenoxy]acetates 14a

To a cold mixture of 4-hydroxybenzaldehydes 1a (0.01 mol) and K₂CO₃(0.015 mol) in DMF (20 mL),

was added ethyl bromoacetate (0.012 mol) at 5–10 ^◦C. The reaction mixture was stirred for 1hr then

treated with water (20 mL) and extracted in DCM (2 25 mL). The combined organic extract was

,b [41]

,b

×

washed with water and brine then dried over anhydrous MgSO₄. After evaporation in vacuo, the

residue was treated with hexane to give the esters 14a,b.

◦

Ethyl [(2,3-dichloro-4-formyl)phenoxy]acetate (14a): pale brown solid (2.4 g, 82%), m.p. 134–138 C.

¹H-NMR (300 MHz, CDCl₃) δ_Hppm 10.39 (s, 1H, CHO); 7.87 (d, J = 8.78 Hz, 1H, Ar-H), 6.84 (d,

J = 8.78 Hz, 1H, Ar-H), 4.83 (s, 2H, OCH₂), 4.31 (q, J = 7.14 Hz, 2H, CH₂), 1.32 (t, J = 7.13 Hz, 3H, CH₃).

1

Ethyl [(2-chloro-4-formyl)phenoxy]acetate (14b): clear liquid (4.3 g, 94% yield); H-NMR (300 MHz,

DMSO-d₆) δ_Hppm 9.88 (s, 1H, CHO), 7.93–8.05 (m, 1H, Ar-H3), 7.86 (d, J = 8.50 Hz, 1H, Ar-H5), 7.30

(d, J = 8.50 Hz, 1H, Ar-H6), 5.09 (s, 2H, CH₂), 4.25 (q, J = 7.04 Hz, 2H), 1.23 (t, J = 7.06 Hz, 3H, CH₃).

3.13. General Procedure for the Synthesis of Ethyl 4-[(Di/monochloro-4-formyl)phenoxy]butanoates 14c

–e [41]

To a solution of 1a (0.018 mol) in N-methylpyrrolidone (NMP, 30 mL) was added K₂CO₃

–

c

(0.0275 mol) and KI (1.8 mmol) at room temperature. After stirring for 30 min, ethyl 4-bromobutyrate

(3.5 g, 0.0183 mol) was added, and the reaction was continued for 7 h at 60 ^◦C. The reaction mixture

was diluted with water (50 mL) and extracted with DCM (3

×

75 mL). The combined organic extract

was washed with water then brine and dried over anhydrous Na₂SO₄. The solvent was removed

under reduced pressure, and the crude product was puriﬁed by column chromatography (6% EtOAc

in hexane) to give the products 14c–e.

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◦

Ethyl 4-[(2,3-dichloro-4-formyl)phenoxy]butanoate (14c): white solid (4 g, 72%), m.p. 163–165 C. ¹H-NMR

(300 MHz, DMSO-d₆) δ_Hppm 10.19 (s, 1H, CHO), 7.84 (d, J = 8.88 Hz, 1H, Ar-H5), 7.32 (d, J = 8.78 Hz,

1H, Ar-H6), 4.25 (t, J = 6.23 Hz, 2H, CH₂), 4.07 (q, J = 7.08 Hz, 2H, CH₂), 2.51 (t, 2H, Under DMSO),

2.04 (quin, J = 6.77 Hz, 2H, CH₂), 1.14 (q, J = 6.31, 3H, CH₃).

Ethyl 4-[(2-chloro-4-formyl)phenoxy]butanoate (14d): clear liquid (7.1 g, 82%); ¹H-NMR (300 MHz,

DMSO-d₆) δ_Hppm 9.86 (s, 1H, CHO), 7.94 (s, 1H, Ar-H3), 7.88 (d, J = 8.50 Hz, 1H, Ar-H6), 7.35

(d, J = 8.50 Hz, 1H, Ar-H5), 4.22 (t, J = 6.23 Hz, 2H, CH₂), 4.07 (q, J = 7.08 Hz, 2H, CH₂), 2.39 (2H, under

DMSO), 1.96–2.12 (m, 2H, CH₂), 1.18 (t, J = 7.13 Hz, 3H, CH₃).

Ethyl 4-[(3-chloro-4-formyl)phenoxy]butanoate (14e): white solid, (5 g, 82%), m.p. 118–121 ^◦C. ¹H-NMR

(300 MHz, DMSO-d₆) δ_H10.19 (s, 1H, CHO), 7.83 (d, J = 8.78 Hz, 1H, Ar-H5), 7.18 (d, J = 1.70 Hz,

1H, Ar-H3), 7.08 (dd, J = 8.78, 2.27 Hz, 1H, Ar-H6), 4.10–4.20 (m, 2H, CH₂), 4.02–4.10 (m, 2H, CH₂),

2.41–2.49 (2H, under DMSO, CH₂), 1.99 (t, J = 6.80 Hz, 2H, CH₂), 1.18 (t, J = 7.13 Hz, 3H, CH₃)

3.14. General Procedure for the Synthesis of Ethyl [Di/monochloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]-

acetate/butanoates 15a–e [44]

To a stirred solution_◦of ethyl esters 14a

–e (8.6 mmol) in chloroform (25 mL), was added

ethylenediamine at 0 to 5 C. After stirring for 30 min at room temperature, N-bromosuccinimide

was added at 0 to 5 ^◦C over 20 min. The reaction mixture was slowly warmed to room temperature

and stirred for 16 h. NaHCO₃10% solution (25 mL) was added to quench the reaction, followed by

extraction with chloroform (3

crude product 15a–e.

×

50 mL). The combined organic extract was concentrated to afford

Ethyl [2,3-dichloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]acetate (15a): pale yellow solid (2.5 g, 91%),

m.p. 132–134 ^◦C. ¹H-NMR (300 MHz, DMSO-d₆) δ_H7.45 (d, J = 8.69 Hz,1H, Ar-H5), 7.12 (d, J = 8.78 Hz,

1H, Ar-H6), 6.79 (brs, 1H, NH), 5.03 (s, 2H, OCH₂), 4.18 (q, J = 7.08 Hz, 2H, CH₂), 3.59 (brs, 4H,

imidazoline-H), 1.22 (t, J = 7.03 Hz, 3H, CH₃).

Ethyl [2-chloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]acetate (15b): greyish white solid (3.8 g, 66%),

m.p. 124–126 ^◦C. ¹H-NMR (300 MHz, DMSO-d₆) δ_H7.89 (s, 1H, NH), 7.73 (d, J = 8.69 Hz, 1H, Ar-H6),

7.11 (brs, 1H, Ar-H3), 7.12 (d, J = 8.69 Hz, 1H, Ar-H5), 4.98 (s, 2H, OCH₂) 4.18 (q, J = 7.02 Hz, 2H, CH₂),

3.60 (s, 4H, imidazoline-H) 1.22 (t, J = 7.08 Hz, 3H, CH₃).

Ethyl 4-[2,3-dichloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]butanoate (15c): pale greyish white

solid (4 g, 89%); ¹H-NMR (300 MHz, DMSO-d₆) δ_H7.48 (d, J = 8.59 Hz, 1H, Ar-H6), 7.19 (d, J = 8.88 Hz,

1H, Ar-H5) 4.13–4.25 (m, 2H, CH₂) 4.07 (q, J = 6.89 Hz, 2H, CH₂), 3.60 (m, 4H, imidazoline-H), 2.51

(t, 2H, under DMSO), 1.94–2.10 (m, 2H, CH₂), 1.18 (t, J = 6.76 Hz, 3H, CH₃).

Ethyl 4-[2-chloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]butanoate (15d): T pale greyish white solid

(6.2 g, 76%), m.p. 138–140 ^◦C.¹H-NMR (300 MHz, DMSO-d₆) δ_H7.88 (d, J = 1.51 Hz, 1H, Ar-H3), 7.77

(dd, J = 8.64, 1.65 Hz, 1H, Ar-H5), 7.22(d, J = 8.59 Hz, 1H, Ar-H6), 4.14 (t, J = 6.98 Hz, 2H, CH₂), 4.05

(q, J = 7.12 Hz, 2H,CH₂), 3.62 (m, 4H, imidazoline-H), 2.47 (under DMSO, 2H, CH₂), 2.01 (m, 2H, CH₂),

1.18 (t, J = 7.08 Hz,3H, CH₃).

Ethyl 4-[3-chloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]butanoate (15e): pale yellow solid (5 g, 87%),

◦

m.p. 137–139 C. ¹H-NMR (300 MHz, DMSO-d₆) δ_H7.52 (d, J = 8.69 Hz, 1H, Ar-H5), 7.07 (d, J = 2.27 Hz,

1H, Ar-H2), 6.95 (dd, J = 8.64, 2.31 Hz, 1H, Ar-H6), 3.99–4.10 (m, 4H, imidazoline-H), 3.60 (m, 4H,

2CH₂), 2.45 (t, J = 7.32 Hz, 2H, CH₂), 1.97 (t, J = 6.80 Hz, 2H, CH₂),1.18 (t, J = 7.08 Hz, 3H, CH₃).

3.15. General Procedure for the Synthesis of Ethyl [2,3-Dichloro-4-(1H-imidazol-2-yl)phenoxy]acetate/

butanoates 16a–e [44]

To a solution of 15a

–e (7.8 mmol) in DMSO (25 mL) was added K₂CO₃(8.6 mmol) and

diacetoxyiodobenzene (8.6 mmol) at room temperature. The reaction mixture was stirred for 60 min in

Molecules 2016, 21, 1057

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a dark place, and then water was added followed by extraction with DCM (3

layer was dried over Na₂SO₄and concentrated under reduced pressure. The residue was subjected to

×

50 mL). The organic

ﬂash column chromatography (EtOAc 2:hexane 1) to afford the products 16a–e.

Ethyl [2,3-dichloro-4-(1H-imidazol-2-yl)phenoxy]acetate (16a): off-white solid (1.1g, 44%), m.p. 193–194 ^◦C.

¹H-NMR (300 MHz, DMSO-d₆) δ_H12.31 (brs, 1H, NH), 7.62 (d, J = 8.78 Hz, 1H, Ar-H6), 7.06–7.26

(m, 3H, Ar-H and imidazole-H), 5.04 (s, 2H, OCH₂), 4.19 (q, J = 7.05 Hz, 2H, CH₂), 1.22 (t, J = 7.08 Hz,

3H, CH₃).

Ethyl [2-chloro-4-(1H-imidazol-2-yl)phenoxy]acetate (16b): greyish white solid (1.5 g, 43%), m.p. 164–166 ^◦C.

¹H-NMR (300 MHz, DMSO-d₆) δ_H12.47 (brs, 1H, NH), 8.00 (d, J = 2.08 Hz, 1H, Ar-H3), 7.82 (dd, J = 8.59,

2.17 Hz, 1H, Ar-H5), 4.96 (s, 2H, OCH₂), 7.15 (m, 3H, Ar-H and imidazole-H), 4.19 (q, J = 7.08 Hz, 2H,

CH₂) 1.22 (t, J = 7.13 Hz, 3H, CH₃).

Ethyl 4-[2,3-dichloro-4-(1H-imidazol-2-yl)phenoxy]butanoate (16c): off-white solid (2.3 g, 55.8%),

m.p. 226–227 ^◦C. ¹H-NMR (300 MHz, CDCl₃) δ_H10.19 (brs, 1H, NH), 8.14 (d, J = 8.88 Hz, 1H, Ar-H6),

7.21 (s, 2H, imidazole-H), 6.98 (d, J = 8.97 Hz, 1H, Ar-H5), 4.00–4.34 (m, 4H, 2CH₂), 2.61 (t, J = 7.18 Hz,

2H, CH₂), 2.21 (quin, J = 6.61 Hz, 2H, CH₂), 1.16–1.44 (m, 3H, CH₃).

Ethyl 4-[2-chloro-4-(1H-imidazol-2-yl)phenoxy]butanoate (16d): greyish white solid (3.4 g, 57%),

m.p. 179–182 ^◦C. ¹H-NMR (300 MHz, DMSO-d₆) δ_Hppm 12.44 (brs, 1H, NH), 7.98 (d, J = 1.98 Hz,

1H, Ar-H3), 7.85 (dd, J = 8.59, 1.98 Hz, 1H, Ar-H5), 7.23(d, J = 8.69 Hz, 1H, Ar-H6), 7.10 (m, 2H,

imidazole-H), 4.03–4.17 (m, 4H, 2CH₂), 2.48 (under DMSO, 2H, CH₂), 1.94–2.08 (m, 2H, CH₂), 1.18 (t,

J = 7.08 Hz, 3H, CH₃).

Ethyl 4-[3-chloro-4-(1H-imidazol-2-yl)phenoxy]butanoate (16e): greyish white solid (2.58 g, 50%),

m.p. 168–172 ^◦C. ¹H-NMR (300 MHz, DMSO-d₆) δ_H7.68 (d, J = 8.69 Hz, 1H, Ar-H5), 7.05–7.19 (m, 3H,

Ar-H2 and imidazole-H), 7.00 (dd, J = 8.69, 2.55 Hz, 1H, Ar-H6), 3.99–4.15 (m, 4H, 2CH₂), 2.43–2.50

(under DMSO, 2H, CH₂), 1.99 (quin, J = 6.75 Hz, 2H, CH₂) 1.19 (t, J = 7.13 Hz, 3H, CH₃).

3.16. General Procedure for the Synthesis of 2-[Di/monochloro-4-(1H-imidazol-2-yl)phenoxy]acetic/butyric

Acids 17a–e [43]

These compounds were prepared according to procedure described above for preparation of 7a

starting with esters 16a–e (3.2 mmol).

–f,

2-[2,3-Dichloro-4-(1H-imidazol-2-yl)phenoxy]acetic acid (17a): off-white solid (0.87 g, 82%), m.p. >300 ^◦C

1

(dec.). H-NMR (300 MHz, DMSO-d₆) δ_H15.08 (brs, 1H, OH), 7.81 (s, 1H, NH), 7.70 (d, J = 8.88 Hz, 1H,

Ar-H6) 7.34 (d, J = 9.06 Hz, 1H, Ar-H5), 7.0–7.01 (m, 2H, imidazole-H), 5.00 (s, 2H,OCH₂), LC/MS,

m/z 286.9 (M + 1)⁺, 288.9 (M + 3)⁺, 291.1 (M + 5)⁺. Anal. Calcd for (C₁₁H₈Cl₂N₂O₃): C, 46.02; H, 2.81;

Cl, 24.70; N, 9.76; Found: C, 46.21; H, 3.02; Cl, 24.34; N, 9.49.

◦

[2-Chloro-4-(1H-imidazol-2-yl)phenoxy]acetic acid (17b): off-white solid (1.2 g, 88.8%), m.p. >300 C

(dec.); ¹H-NMR (300 MHz, DMSO-d₆) δ_H7.99 (d, J = 2.08 Hz, 1H, Ar-H3), 7.82 (dd, J = 8.69, 1.89 Hz,

1H, Ar-H5), 7.02–7.20 (m, 3H, Ar-H6 and imidazole-H), 4.85 (s, 2H, OCH₂); LC/MS, m/z 251.0 (M⁻),

253.1 (M + 2)⁻.

4-[2,3-Dichloro-4-(1H-imidazol-2-yl)phenoxy]butanoic acid (17c): off-white solid (0.87 g, 82%), m.p. >300 ^◦C

1

(dec.); H-NMR (300 MHz, DMSO-d₆) δ_H12.23 (brs, 2H, NH and OH), 7.65 (d, J = 8.69 Hz, 1H,

Ar-H6), 7.25 (d, J = 8.69 Hz, 1H, Ar-H5), 7.15 (s, 2H, imidazoline-H), 4.18 (t, J = 5.95 Hz, 2H, OCH₂),

2.44 (t, J = 7.18 Hz, 2H, CH₂), 1.93–2.08 (m, 2H, CH₂); LC/MS, m/z 316.0 (M + 1)⁺, 318.0 (M + 3)⁺,

320.0 (M + 5)⁺.

4-[2-Chloro-4-(1H-imidazol-2-yl)phenoxy]butanoic acid (17d): pale brown solid (1.5 g, 83%), m.p. 219–220 ^◦C;

¹H-NMR (300 MHz, DMSO-d₆) δ_H12.47 (brs, 1H, OH), 12.20 (brs, 1H, NH), 7.98 (d, J = 1.89 Hz, 1H,

Ar-H3), 7.85 (d, J = 8.59 Hz, 1H, Ar-H6), 7.23 (d, J = 8.69 Hz, 1H, Ar-H5), 7.11 (s, 2H, imidazole-H), 4.12

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(t, J = 6.23 Hz, 2H, OCH₂), 2.42 (t, J = 7.08 Hz, 2H, CH₂), 1.95 (quin, J = 6.93 Hz, 2H, CH₂); LC/MS

(ESI), m/z 281.1 (M + 1)⁺, 283.1 (M + 3)⁺.

4-[3-Chloro-4-(1H-imidazol-2-yl)phenoxy]butanoic acid (17e): yellow solid (1.7 g, quantitative), m.p. 103–106 ^◦C;

¹H-NMR (300 MHz, DMSO-d₆) δ_H12.14 (brs, 1H, NH), 7.81 (d, J = 2.50 Hz, 1H, Ar-H2), 7.67

(d, J = 8.69 Hz, 1H, Ar-H5), 7.12 (d, J = 2.36 Hz, 2H, imidazole-H), 7.01 (dd, J = 8.73, 2.50 Hz, 1H,

Ar-H6), 4.06 (t, J = 6.42 Hz, 2H, OCH₂), 2.39 (t, J = 7.27 Hz, 2H, CH₂), 1.95 (quin, J = 6.80 Hz, 2H,

CH₂); LC/MS, m/z 281.0 (M + 1)⁺, 283.1 (M + 3)⁺; Anal. Calcd for (C₁₃H₁₃ClN₂O₃): C, 55.62; H, 4.67;

Cl, 12.63; N, 9.98; Found: C, 55.87; H, 5.01; Cl, 12.99; N, 9.88.

3.17. General Procedure for the Synthesis of 2,3-Di/monochloro-4-(3-hydroxypropoxy)benzaldehydes 18a

–c [42]

To a cooled stirred solution of the appropriate 4-hydroxybenzaldehyde (1a ) (0.031 mol) and

–

c

K₂CO₃(0.157 mol), KI (0.003 mol) in DMF (60 mL), was added 3-chloro-1-propanol (6 g, 0.062 mol)

at 5–10 ^◦C. The mixture was stirred for 16 h at 70 ^◦C, then left to attain room temperature. Ice and

water were added and the crude product was extracted with DCM (3

extract was washed with water and brine, dried over anhydrous MgSO₄. The crude compound was

×

100 mL). The combined organic

puriﬁed by ﬂash chromatography using (EtOAc 1:hexane 4) to afford ethers 18a–c.

2,3-Dichloro-4-(3-hydroxypropoxy)benzaldehyde (18a): pale yellow liquid (6.2 g, 79%); ¹H-NMR (300 MHz,

DMSO-d₆) δ_H10.19 (brs, 1H, CHO), 7.95 (brs, 1H, OH), 7.32 (d, J = 8.14 Hz, 1H, Ar-H6), 4.59–4.70 (m,

1H, Ar-H5), 4.28 (m, 2H, OCH₂), 3.60 (q, J = 5.19 Hz, 2H, CH₂), 1.84–2.02 (m, 2H, CH₂).

2-Chloro-4-(3-hydroxypropoxy)benzaldehyde (18b): pale yellow liquid (9.5 g, 86.6%); ¹H-NMR (300 MHz,

DMSO-d₆) δ_H10.20 (s, 1H, CHO), 7.83 (d, J = 8.69 Hz, 1H, Ar-H6); 7.18 (d, J = 2.27 Hz, 1H, Ar-H3),

7.09 (dd, J = 8.73, 2.31 Hz, 1H, CH₅), 4.61 (t, J = 5.15 Hz, 1H, OH), 4.19 (t, J = 6.37 Hz, 2H, OCH₂), 3.55

(q, J = 5.89 Hz, 2H, CH₂), 1.88 (quin, J = 6.26 Hz, 2H, CH₂).

1

3-Chloro-4-(3-hydroxypropoxy)benzaldehyde (18c): pale yellow liquid (9.5 g, 86.6%). H-NMR (300 MHz,

DMSO-d₆) δ_H9.85 (s, 1H, CHO), 7.70–8.08 (m, 2H, Ar-H2, Ar-H6), 7.34 (d, J = 8.50 Hz, 1H, Ar-H5),

4.55–4.69 (brs, 1H, OH), 4.24 (t, J = 6.18 Hz, 2H, OCH₂), 3.53–3.68 (m, 2H, CH₂), 1.92 (quin, J = 6.09 Hz,

2H, CH₂).

3.18. General Procedure for the Synthesis of 3-[Di/monochloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]

propan-1-ols 19a–c [44]

These intermediates were prepared according to procedure described above for the preparation of

15a–e starting with the appropriate 4-(3-hydroxypropoxy)benzaldehyde derivatives 18a–c (0.016 mol).

3-[2,3-Dichloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]propan-1-ol (19a): pale yellow liquid (4 g, 86%);

¹H-NMR (300 MHz, DMSO-d₆) δ_H7.48 (d, J = 8.69 Hz, 1H, Ar-H6), 7.20 (d, J = 8.78 Hz, 1H, Ar-H5),

6.73 (brs, 1H, NH), 4.62 (brs, 1H, OH), 4.20 (t, J = 6.00 Hz, 2H, imidazoline-H), 3.78 (t, J = 8.31 Hz, 2H,

imidazoline-H), 3.59 (brs, 2H, OCH₂), 3.44 (t, J = 7.77 Hz, 2H, CH₂),1.93 (quin, J = 6.72 Hz, 2H, CH₂).

3-[2-Chloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]propan-1-ol (19b): pale yellow solid (7.74 g, 69.2%),

m.p. 88–91 ^◦C. ¹H-NMR (300 MHz, DMSO-d₆) δ_H7.85 (d, J = 1.98 Hz, 1H, Ar-H3), 7.75 (dd, J = 8.59,

1.98 Hz, 1H, Ar-H5) 7.20 (d, J = 8.69 Hz, 1H, Ar-H6), 6.93 (brs, 1H, NH), 4.61 (brs, 1H, OH), 4.17

(t, J = 6.18 Hz, 2H, imidazoline-H), 3.75 (brs, 2H, imidazoline-H), 3.59 (t, J = 6.00 Hz, 2H, OCH₂), 3.41

(brs, 2H, CH₂), 1.90 (quin, J = 6.18 Hz, 2H, CH₂).

3-[3-Chloro-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]propan-1-ol (19c): pale yellow liquid (10.3 g, 96.5%);

¹H-NMR (300 MHz, DMSO-d₆) δ_H7.52 (d, J = 8.69 Hz, 1H, Ar-H5), 7.05 (d, J = 2.27 Hz, 1H, Ar-H2), 6.94

(dd, J = 8.64, 2.41 Hz, 1H, Ar-H6), 6.63 (brs, 1H, NH), 4.59 (d, J = 4.44 Hz, 1H, OH) 4.09 (t, J = 6.33 Hz,

2H, imidazoline-H), 3.54 (t, J = 6.18 Hz, 2H, imidazoline-H), 3.48 (t, J = 6.00 Hz, 2H, OCH₂), 3.41 (brs,

2H, CH₂), 1.85 (quin, J = 6.28 Hz, 2H, CH₂).

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3.19. General Procedure for the Synthesis of 3-[Di/monochloro-4-(1H-imidazol-2-yl)phenoxy]propan-1-ols

20a–c [44]

These intermediates were prepared following the same procedure described above for the

preparation of 16a–c starting with the appropriate 19a–c (0.027 mol).

3-[2,3-Dichloro-4-(1H-imidazol-2-yl)phenoxy]propan-1-ol (20a): pale yellow liquid (3.8 g, 50 %); ¹H-NMR

(300 MHz, DMSO-d₆) δ_H12.29 (brs, 1H, NH) 7.66 (d, J = 8.78 Hz, 1H, Ar-H6), 7.26 (d, J = 8.88 Hz, 1H,

Ar-H5), 7.15 (m, 2H, imidazole-H), 4.62 (brs, 1H, OH), 4.22 (t, J = 6.04 Hz, 2H, OCH₂), 3.53–3.61 (m, 2H,

CH₂), 1.92 (t, J = 6.09 Hz, 2H, CH₂).

1

3-[2-Chloro-4-(1H-imidazol-2-yl)phenoxy]propan-1-ol (20b): pale yellow liquid (3.2 g, 42%); H-NMR

(300 MHz, DMSO-d₆) δ_H12.43 (brs, 1H, NH), 7.98 (d, J = 1.98 Hz, 1H, Ar-H3), 7.85 (dd, J = 8.59, 1.98 Hz,

1H, Ar-H5), 7.24 (d, J = 8.69 Hz, 1H, Ar-H6), 7.10 (brs, 2H, imidazole-H), 4.59 (t, J = 5.10 Hz, 1H, OH),

4.17 (t, J = 6.23 Hz, 2H, OCH₂) 3.60 (q, J = 5.92 Hz, 2H, CH₂), 1.90 (quin, J = 6.1 Hz, 2H, CH₂).

3-[3-Chloro-4-(1H-imidazol-2-yl)phenoxy]propan-1-ol (20c): wine red liquid (7.1 g, 70%); ¹H-NMR

(300 MHz, DMSO-d₆) δ_H12.16 (brs, 1H, NH), 7.93 (d, J = 1.98 Hz, 1H, Ar-H2), 7.67 (d, J = 8.69 Hz, 1H,

Ar-H5), 7.07–7.17 (m, 2H, imidazole-H), 7.01 (dd, J = 8.69, 2.36 Hz, 1H, Ar-H6), 4.60 (brs, 1H, OH), 4.11

(t, J = 6.28 Hz, 2H, OCH₂), 3.56 (d, J = 4.25 Hz, 2H, CH₂), 1.87 (quin, J = 6.2 Hz, 2H, CH₂).

3.20. General Procedure for the Synthesis of 3-[2,3-Di/monochloro-4-(1H-imidazol-2-yl)phenoxy]propanoic

Acids 21a–c [42]

To a stirred solution of the appropriate alcohol 20a–c (12.8 mmol) in acetone (40 mL), a solution

of chromium trioxide (68 mmol) in H₂O (50 mL) and AcOH (6 mL) was added while keeping the

◦

temperature between 5–10 C. The mixture was stirred for 30 min at the same temperature then

warmed up to room temperature for 16 h. The reaction was monitored by LC-MS till complete

disappearance of the starting material. Then the reaction mixture was treated with NaHCO₃and

the pH was adjusted to 8. The mixture was washed with DCM (3

×

50 mL) and the aqueous layer

was concentrated under reduced pressure to 50 mL. Again, the pH was adjusted to pH 5 (1.5 N HCl).

The precipitated solid product was collected by ﬁltration, washed with water and dried. Puriﬁcation

by crystallization from MeOH gave the required products (21a–c).

3-[2,3-Dichloro-4-(1H-imidazol-2-yl)phenoxy]propanoic acids (21a): off-white solid (0.73 g, 18%),

m.p. 193–194 ^◦C. IR (KBr,

ν

cm⁻¹): 3585(NH), 3100 (OH), 1620 (CO), 1294, 1023 (C-O-C); ¹H-NMR

max

(300 MHz, DMSO-d₆) δ_H13.1(brs, 1H, OH), 12.4 (brs, 1H, NH), 7.64 (d, J = 8.50 Hz, 1H, Ar-H6), 7.27

(d, J = 8.69 Hz, 1H, Ar-H5), 7.15 (s, 2H, imidazole-H), 4.32 (t, J = 8.2 Hz, 2H, OCH₂), 2.67 (t, J = 8.2 Hz,

2H, CH₂); LC/MS: m/z 300.0 (M)⁻, 302.0 (M + 2)⁻, 304.0 (M + 4)⁻.

3-[2-Chloro-4-(1H-imidazol-2-yl)phenoxy]propanoic acid (21b): off-white solid (0.23 g, 44%), m.p. 210–213 ^◦C.

1

IR (KBr,

ν

cm⁻¹): 3443 (NH), 3142 (OH), 1687 (CO), 1269, 1065 (C-O-C); H-NMR (300 MHz,

max

DMSO-d₆) δ_H12.35 (brs, 1H, OH), 7.97 (brs, 1H, NH), 7.86 (d, J = 8.59 Hz, 1H, Ar-H5), 7.27

(d, J = 8.40 Hz, 1H, Ar-H6), 7.11 (m, 3H, Ar-H3 and imidazole-H), 4.30 (t, J = 7.1 Hz, 2H, CH₂), 2.74

(t, J = 6.2 Hz, 2H, CH₂); LC/MS: m/z 267.0 (M + 1), 269.1 (M + 2); Anal. Calcd for (C₁₃H₁₁ClN₂O₃):

C, 54.05; H, 4.16; Cl, 13.29; N, 10.50; Found: C, 53.79; H, 3.82; Cl, 13.61; N, 10.55.

3-[3-Chloro-4-(1H-imidazol-2-yl)phenoxy]propanoic acid (21c): off-white solid (0.6 g, 9.5%), m.p. 208–210 ^◦C.

1

IR (KBr,

ν

cm⁻¹): 3400 (NH), 3139 (OH), 1694 (CO), 1296, 1092 (C-O-C); H-NMR (300 MHz,

max

DMSO-d₆) δ_H13.0, 12.21 (two brs, each 1H, OH and NH), 7.67 (d, J = 8.69 Hz, 1H, Ar-H5), 7.12

(m, 3H, Ar-H2 and imidazole-H), 7.01 (d, J = 7.08 Hz, 1H, Ar-H6), 4.24 (t, J = 5.17 Hz, 2H, OCH₂), 2.70

(t, J = 5.43 Hz, 2H, CH₂); LC/MS: m/z 267.0 (M + 1)⁻, 269.0 (M + 3)⁻.

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3.21. RBC Morphological Antisickling Studies

KAUS-4, KAUS-23 and KAUS-24, and cloﬁbrate were tested for their effect on sickle RBC

morphology as previously reported [18 19 22]. Brieﬂy sickle RBCs were incubated under air in

,

the absence or presence of 2 mM concentration of test compound (solubilized in DMSO) at room

temperature for 1 h. Following, the suspension was incubated under hypoxic condition (4%

oxygen/96% nitrogen) at 37 ^◦C for 3 h. The suspension was ﬁxed with 2% glutaraldehyde solution

without exposure to air and then subjected to microscopic morphological analysis.

3.22. Oxygen Equilibrium Curve Studies

Normal blood samples (hematocrit 22%) in the absence (control) or presence of 2 mM concentration

of the test KAUS compounds (solubilized in DMSO) were incubated at 37 ^◦C for 1.5 h and

then subjected to OEC analysis using tonometry as previously described [18,19,22]. Brieﬂy, the

compound-treated blood samples is incubated in IL 237 tonometers (Instrumentation Laboratories,

Inc. Lexington, MA, USA) for approximately 10 min at 37 ^◦C, and allowed to equilibrate at oxygen

tensions 7, 20, 40 and 60 mmHg. The samples were then aspirated into an ABL 700 Automated Blood

Gas Analyzer (Radiometer) to determine the pH, partial pressure of CO₂(pCO₂), partial pressure of

oxygen (pO₂), and Hb oxygen saturation values (SO₂). The measured values of pO₂(mmHg) and SO₂

at each pO₂value were then subjected to a non-linear regression analysis using the program Scientist

(Micromath, Salt Lake City, UT, USA) to estimate P₅₀and Hill coefﬁcient values (n₅₀). Cloﬁbrate was

tested as a positive control, while DMSO was tested as negative control.

3.23. Crystallization, Data Collection and Structure Determination of Deoxygenated Hb in Complex

with KAUS-23

Freshly prepared solution of KAUS-23 in DMSO was incubated with deoxygenated

Hb (40 mg/mL) for 30 min at Hb tetramer:KAUS-23 molar ratio of 1:10 at room temperature and then

crystallized with 3.2 M sulfate/phosphate precipitant, pH 6.8 using the batch method as previously

described [18,19,22]. Diffraction data was collected at 100 K on an R-axis IV++ image plate detector

using CuKα X-ray (λ = 1.5417) from a Rigaku Micro-Max™ -007 X-ray source equipped with Varimax

confocal optics operating at 40 kV and 20 mA (Rigaku, The Woodlands, TX, USA). Crystals were

cryoprotected with 15% glycerol. The dataset was processed with the d*trek software (v9.9.9.7, Rigaku

Corporation, Tokyo, Japan) and the CCP4 suite of programs [45].

The isomorphous native human deoxygenated Hb tetramer structure (PDB code 2DN2) was used

as the starting model to reﬁne the structure, using both Phenix and CNS reﬁnement programs [46

,47].

Model building and correction were carried out using COOT [48 49]. A round of reﬁnement showed a

,

bound KAUS-23 molecule at the central water cavity. The symmetry-related site was poorly deﬁned.

Therefore, only one KAUS-23 molecule was built into the model and reﬁned. Also, included in the

ﬁnal model are two molecules of sulfate ions, and 400 water molecules. The structure reﬁned to a

ﬁnal Rfactor/Rfree of 19.68/24.92% at a resolution of 2.15 Å. The atomic coordinate and structure

factor ﬁles have been deposited in the RCSB Protein Data Bank with accession codes 5KDQ. Detailed

crystallographic and structural analysis parameters are reported in Table 3.

4. Conclusions

Aryloxyalkanoic acids that bind non-covalently to the

been shown to exhibit antisickling activities, while those that bind to the central water cavity of the

protein in most instances showed the opposite effect [25 29]. The novel KAUS molecules were designed

to have an imidazole pharmacophore, which was expected to increase hydrophobic interactions at the

Trp14 binding site and result in enhanced antisickling activity. However, our structural modiﬁcations

αTrp14 hydrophobic pocket of Hb have

–

α

did not work as anticipated, as these compounds appear to bind to the central water cavity instead,

stabilizing the T-state Hb and resulting mostly in a decrease in Hb afﬁnity for oxygen. Considering that

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compounds that decrease Hb afﬁnity for oxygen and enhance O2 delivery to tissues have been studied

to treat hypoxic- or ischemic-related diseases [50 57], this series of compounds may potentially be

useful. For example, RSR-13 and several of its analogs that bind to the central water cavity of Hb and

signiﬁcantly decrease Hb afﬁnity for oxygen ( P₅₀of 20–30 mmHg at 2 mM) have been the subject of

–

∆

investigation for stroke, wound healing, and as a radiation enhancer in the radiotherapy of hypoxic

tumors, adjuncts for Hb based oxygen carriers, nitric oxide, carbon monoxide and other non-oxygen

therapeutic gases [50 57]. These compounds were designed based on cloﬁbrate atomic interaction

–

with Hb [ ,30–32,37,57,58]. Like cloﬁbrate, the KAUS compounds bind to the central water cavity

2

of Hb and weakly decrease Hb afﬁnity for oxygen by stabilizing the T-state. The structure provides

direction and approach for designing a new class of potent Hb allosteric compounds that decrease Hb

afﬁnity for oxygen. We note that the alkanoic acid moiety of KAUS-23 is close to the side-chains of

α

Tyr140,

the T R transition. By incorporating another phenyl group between the methoxyphenyl and the

alkanoic acid not only would allow increased hydrophobic interactions with these residues, but

also extend the carboxylate to potentially form salt-bridge/hydrogen-bond interactions with Lys99.

αPro95, αVal96, βTrp37 and αLys99; residues that are known to move signiﬁcantly during

↔

α

We therefore anticipate these interactions to lock the residues in the T-state conformation and lead to

further stabilization of the T-state with a concomitant decrease in Hb afﬁnity for oxygen.

Besides aryloxyalkanoic acids, non-acidic halogenated benzene derivatives or toluene are also

known to bind to the

acids, the halogenated benzenes or toluene bind exclusively to the

is directing the KAUS compounds into the central water cavity because modelling KAUS-23 at the

Trp14 binding pocket (Figure 2 b) suggested a more favorable binding when compared to its actual

binding in the central water cavity (see Supplementary Material). We speculate that like the large

number of Hb effectors with carboxylate moiety [29 32], the central water cavity which is lined with

α

Trp14 hydrophobic pocket [26

,33

,59]. However, unlike the aryloxyalkanoic

α

Trp14 site. It is not obvious what

α

–

several positive residues is acting like a sink to attract these carboxylate compounds. As part of our

future investigations, we plan to modify or eliminate the acidic moieties from the KAUS compounds,

which would likely lead to the desired interactions at the

antisickling activity.

αTrp14 site, and result in corresponding

Supplementary Materials: Supplementary materials can be accessed at: http://ww w .mdpi.com/1420-3049/21/

8/1057/s1.

Acknowledgments: This project was funded by the National Plan for Science, Technology and Innovation

(MAARIFAH)-King Abdulaziz City for Science and Technology-the Kingdom of Saudi Arabia award number

(BIO1253-03-10), NIH/NIMHD grant MD009124 (MKS, OA); NIH/NIDDK grant R01DK084188 (OA). Structural

biology resources were provided in part by NIH grant CA16059 to the VCU Massey Cancer Center. The authors

also, acknowledge with thanks Science and Technology Unit, King Abdulaziz University for technical support.

Author Contributions: M.K.S., A.M.O., M.E.E.-A., M.A.M. and M.H.A. conceived and designed the experiments;

A.M.O., F.H.A.B. and M.E.E.-A. synthesized the compounds; M.S.G. and O.A. performed the Oxygen Equilibrium

Curve Studies and antisickling experiments; M.H.A. and M.K.S. determined the crystal structure; M.K.S., A.M.O.

M.E.E.-A. and O.A. analyzed the data; M.K.S., A.M.O., M.E.E.-A., O.A., F.H.A.B. and M.H.A. contributed

reagents/materials/analysis tools; M.K.S., A.M.O., M.E.E.-A., M.A.M., O.A. and M.H.A. wrote the paper.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of the compounds are available from the authors.

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Aryloxyalkanoic acids as non-covalent modifiers of the allosteric properties of hemoglobin

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