Radioligand and computational insight in structure – Activity relationship of saccharin derivatives being ipsapirone and revospirone analogues

DOI: 10.1016/j.bmcl.2021.128028

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Bioorganic and Medicinal Chemistry Letters (2021)

Update date:2022-08-12

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Authors:

Ku?aga, Damian

Ja?kowska, Jolanta

Sata?a, Grzegorz

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Article abstract of DOI:10.1016/j.bmcl.2021.128028

Schizophrenia and depression are diseases that significantly impede human functioning in society. Current antidepressant drugs are not fully effective. According to literature data, the effect on D₂R or 5-HT_1AR can effectively reduce

Full text of DOI:10.1016/j.bmcl.2021.128028

Bioorg. Med. Chem. Lett. xxx (xxxx) xxx

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

Radioligand and computational insight in structure – Activity relationship

of saccharin derivatives being ipsapirone and revospirone analogues

Damian Kułaga^a, Jolanta Jaskowska , Grzegorz Satała

^aFaculty of Chemical Engineering and Technology, Institute of Organic Chemistry and Technology, Cracow University of Technology, 24 Warszawska Street, 31-155

Krakow, Poland

Department of Medicinal Chemistry, Maj Institute of Pharmacology, Polish Academy of Sciences, 12 Smętna Street, 31-343 Krakow, Poland

A R T I C L E I N F O

A B S T R A C T

Keywords:

Schizophrenia and depression are diseases that signiﬁcantly impede human functioning in society. Current an-

tidepressant drugs are not fully effective. According to literature data, the effect on D₂R or 5-HT_1AR can effec-

tively reduce the symptoms of depression or schizophrenia. Recent research hypothetized that the synergism of

both of these receptors can improve the effectiveness of therapy. Ipsapirone, a representative of long-chain

arylpiperazines, is a known 5-HT_1AR ligand that has antidepressant effect. This compound has no afﬁnity for

the D₂R. Bearing in mind, we decided to design ligands with improved afﬁnity to D₂R and conﬁrmed that in some

cases elongation of the carbon linker or arylpiperazine exchange may have beneﬁcial inﬂuence on the binding to

D2R and 5-HT1AR. Four groups of ligands being ipsapirone analogues with butyl, pentyl, hexyl and stiffened

xylene chains were designed. All compounds were obtained in solvent-free reactions supported by a microwave

irradiation with an efﬁciency mainly above 60%. All ligands containing 1-(2-pyrimidinyl)piperazine exhibited

high afﬁnity to 5-HT_1AR. In this case, chemical modiﬁcations within the chain did not affect the afﬁnity to D₂R.

In the case of ligands containing 1-phenylpiperazine, 1-(3-triﬂuoromethylphenyl)piperazine, 1-(1-naphthyl)

piperazine, and 1-(4-chlorophenyl)piperazine, elongation of carbon linker increases of afﬁnity to D₂R. For li-

gands containing 1- (2-pyridyl) piperazine, and 1-(2,3-dichlorophenyl)piperazine, we observed an opposite ef-

fect. For ligands containing 1-phenylpiperazine, 1-(2-methoxyphenyl)piperazine and 1-(2-pyridyl)piperazine,

chain elongation had no effect on 5-HT_1AR binding. In turn of ligands containing 1-(3-triﬂuoromethylphenyl)

piperazine and 1- (2,3-dichlorophenyl)piperazine, we observed that elongation of carbon linker has a positive

inﬂuence to 5-HT_1AR. Molecular modelling was used to support the SAR study.

Ipsapirone

Microwave chemistry

Docking

5-HT ligands

₂ligands

SAR study

Central nervous system (CNS) disorders such as depression or

the 5-HT_1AR was shown to increase dopamine secretion in prefrontal

cortex resulting in a reduction of schizophrenia-speciﬁc symptoms.

Sumiyoshi et al.¹⁰showed that buspirone or tandospirone in combina-

tion with haloperidol signiﬁcantly improved the cognitive abilities of

patients suffering from schizophrenia. Thus dual D2R/5-HT1AR ligands

seem to be much more promising, with a broader spectrum of activity

than single ones, however this hypothesis must be conﬁrmed in further

research.⁴

schizophrenia in the near future may become one of the main dysfunc-

tions that impede human functioning in society. Despite the advances

made in pharmacy in the context of CNS disorders, the currently used

drugs still have numerous side effects.¹Treatment of depression or

schizophrenia is related with affecting on D₂R, 5-HT_1AR as well as 5-

HT₆R or 5-HT₇R.^2–65-HT_1AR and D₂R are involved in depression or

schizophrenia disorders.^7–9In major depressive disorders (MDD) pa-

tients, 5-HT_1AR agonists (such as buspirone or gepirone) are thought to

act synergistically to increase 5-HT neurotransmission in postsynaptic

structures (in the cortex and the limbic areas) and thus may exhibit

antidepressant properties.^4,10Nevertheless, improvement in therapy can

be obtained by acting additionally on D₂R. Dual D₂R/5-HT_1AR action

seems to be more effective than single one. In a rat study, stimulation of

Ipsapirone belongs to a well-characterized group of azapirones. As it

was mentioned above an anti-depressant effect is related to the release of

serotonin in synaptic cleft in corticolimbic structures due to the sub-

stance’s high afﬁnity to postsynaptic 5-HT_1AR. Affecting these receptors

may have additional modulatory effect on other neurotransmitter sys-

tems such as the glutamatergic system in various parts of brain.¹¹

* Corresponding author.

E-mail address: damian.kulaga@doktorant.pk.edu.pl (D. Kułaga).

https://doi.org/10.1016/j.bmcl.2021.128028

Received 22 December 2020; Received in revised form 25 March 2021; Accepted 4 April 2021

Available online 9 April 2021

Please cite this article as: Damian Kułaga, Bioorg. Med. Chem. Lett., https://doi.org/10.1016/j.bmcl.2021.128028

D. Kułaga et al.

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx

Ipsapirone exhibits anxiolytic, anti-depressant, and anti-aggressive

properties in animal models.¹²as it acts as a postsynaptic partial

agonist to 5-HT_1AR with K_i= 7.9 nM and no activity to D₂R.^13–15In the

literature^16–18there is described only one analogue of ipsapirone with

ethyl linker (A) that does not bind to 5-HT_1AR, two analogues of ipsa-

pirone with propyl linker that bind to 5-HT_1AR with K_i= 400 nM for B

and K_i= 2 nM for C (revospirone) and four four-carbon analogues of

ipsapirone with known afﬁnity to D₂R (four analogues) as well as 5-

HT_1AR/5-HT_2AR (one analogue) (Table 1). In our previous research¹⁹

with saccharin being a terminal moiety of LCAP, we demonstrated that

carbon linker extended up to six carbons may have a beneﬁcial inﬂuence

on afﬁnity mostly to D₂R (Table 1). Taking into account that acting on

both D₂R and 5-HT_1AR may have a beneﬁcial inﬂuence in treatment of

depression or schizophrenia, the aim of our work is to perform the full

structure activity relationship analysis for ipsapirone analogues to

conﬁrm our hypothesis that elongation of carbon linker has a beneﬁcial

inﬂuence on afﬁnity to D₂R/5-HT_1AR.

In light of the above consideration, to fully perform structure activity

relationship study of ipsapirone, we designed four groups of compounds.

Compounds with ﬂexible butyl, pentyl, hexyl chain and more rigid

xylene linker have been chosen to ﬁnd out the optimal length of carbon

linker. Additionally, these four groups include various N-arylpiperazines

in order to ﬁnd their inﬂuence on binding to selected receptors (Fig. 1).

In this study, we focused mostly on dual D₂R/5-HT_1AR effect, however,

to examine their selectivity, binding to 5-HT_2AR, 5-HT₆R and 5-HT₇R

were examined as well. We used molecular modelling to support the SAR

study.

Designed compounds were obtained in solvent-free reactions with

microwave irradiation. The procedure has already been described in our

previous publications.^19,31Ligands from groups I, II and IV were ob-

tained in three independent synthetic routes using two-step reactions

(Scheme 1 and Scheme 2). For SAR study, ligands from group of III were

taken from our previous publication.¹⁹

In the ﬁrst step, a saccharin 1 is N-alkylated with appropriate alkyl

halide 2i-2k. The ﬁrst step was carried out in the presence of 3 equiv-

alents of potassium carbonate, 0.1 equivalents of tetrabutylammonium

bromide (TBAB), and 2–3 drops of N,N-dimethylformamide (DMF) or

acetonitrile (ACN). Intermediates 3i and 3k were puriﬁed via crystalli-

zation from methanol, while 3j was puriﬁed using column chromatog-

raphy. Reaction conditions for the second step were similar to those for

the ﬁrst one. Final products were puriﬁed using column chromatog-

raphy. Pure ligands were transformed into HCl salts and subjected to

radioligand assays.

In the medicinal chemistry, hit-to-lead is a well-known approach,

where a desirable pharmacological proﬁle for protein/receptor is ex-

pected via various chemical modiﬁcations.²⁰In the literature there are

numerous reports about SAR (Structure Activity Relationship) study

within LCAPs (Long Chain Arylpiperazines) including carbon linker and

various arylpiperazine exchange (Table 2).^7,21–24In general, N-arylpi-

perazine itself exhibits moderate afﬁnity and selectivity to the following

receptors: serotonin²¹, dopamine²⁵, noradrenaline²⁶as well as mono-

amine transporters.^9–10The functionalization of N-arylpiperazine by

introducing a long chain core attached to the basic nitrogen of pipera-

zine core may have beneﬁcial inﬂuence on afﬁnity to receptors.²¹For

example, 1-(2-methoxyphenyl)piperazine binds to 5-HT_1AR with K_i=

68 nM. Introducing phthalimide with ethyl linker (I)to basic nitrogen

atom resulted in loss of activity to 5-HT_1AR (K_i> 1000 nM) (Table 2).

Further elongation of carbon linker resulted in gain of afﬁnity to 5-

HT_1AR in the following pattern: propyl linker (J) K_i= 13 nM²¹, butyl

The aim of this research was to elucidate SAR of LACPs being

saccharin derivatives with increased afﬁnity to D₂R/5-HT_1AR. All li-

gands were tested in radioligand binding assays according to a known

procedure.³²The assays were performed via the displacement of the

respective radioligands from cloned human receptors, all stably

expressed in HEK293 cells (except for 5-HT_2AR which was expressed in

CHO cells): [³H]-8-OH-DPAT for 5-HT_1AR, [³H]-ketanserin for 5-HT_2AR,

[³H]-LSD for 5-HT₆R, [³H]-5-CT for 5-HT₇R and [³H]-raclopride for

D₂R.

linker (K – NAN-190) K_i= 0.6 nM^21,pentyl linker (L) K_i= 7.2 nM²⁷

hexyl linker (M) K_i= 22 nM.²⁷According to the NAN-190 SAR study,

optimal carbon linker length is around 4 atoms. Shorter linker decreases

afﬁnity, and similar effects are observed for longer linkers as well. For

salicylamide and 1,3-benzoxazine-2,4-dione in terminal position, the

same trend has been observed (Table 2). Considering the SAR study for

trazodone, elongation of carbon linker up to 6 atoms resulted in a near 5-

fold increase of afﬁnity to 5-HT_1AR.²³Further research revealed that

using other N-arylpiperazines such as 1-(2-methoxyphenyl)piperazine

or 1-(3-methoxyphenyl)piperazine instead of 1-(3-chlorophenyl)piper-

azine dramatically increased afﬁnity to D₂R/5-HT_1AR (Table 3).

According to Structure – Linker Relationship, most of the obtained

ligands exhibited afﬁnity to the mixed D₂R/5-HT_1AR proﬁle. In the

group of ligands with no substituents of the N-arylpiperazine moiety

(5a, 5b, 5c), slightly decreased activity was observed upon linker chain

elongation. Ligands still exhibited high afﬁnity to 5-HT_1AR with K_i≤ 20

nM. The opposite pattern was observed with respect to D₂R. We

observed moderate or no afﬁnity to the remainder of tested receptors.

Because saccharin core is structurally similar to phthalimide, we ex-

pected that in case of 1-(2-methoxyphenyl)piperazine (ligands 5b, 6b

Table 1

Saccharin carbon linker comparison on afﬁnity (K_i[nM]) to D₂R/5-HT_1AR/5-HT_2AR.^16–19

Ligand No.

h-D1

h-D2

h-D3

h-D4

h-D5

h-5-HT1A

h-5-HT2A

h-5-HT2C

h-α1

3-CF3-Ph

2-Pyrimidinyl

136

400

700

1629

230

1756

1132

4-Cl-Ph

177

113

360

1600

287

107

187

F = 5e

3-CF3-Ph

2-OMe-Ph

2-Pyrimidinyl

100

2250

1500

H**

=5f

7.9

14,980

229

321

4-Cl-Ph

3-CF3-Ph

2-OMe-Ph

2-Pyrimidinyl

167

183

NA – not active (Ki > 5

μM) nd - not determined, * - revospirone, ** - ipsapirone, h-human receptor type

D. Kułaga et al.

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx

Table 2

Phthalimide, benzoxazine and salicylamide effects of carbon linker on afﬁnity to h-5-HT_1AR^27–30.

Ligand No

K_i[nM]

Ligand No

K_i[nM]

Ligand No

K_i[nM]

>1000

I-1

I-2

155

J-1

3.2

J-2

0.6*

7.2

K-1

L-1

M¡1

N-1

O-1

P-1

R-2

S-1

K-2

L-2

M¡2

N-2

O-2

P-2

R-2

S-2

>1000

200

143

402

2.8

8.5

*NAN-190, na – not available, h – human receptor type.

Table 3

K_i[nM] ± SEM of radioligand binding for all synthetized compounds for tested receptors.

Ligand No.

linker

h-D₂R

h-5-HT_1A

h-5-HT_2A

h-5-HT₆R

h-5-HT₇R

butyl group I

249

474

4664

161

631

2-OMe

2,3-diCl

4-Cl

382

2580

318

2662

2729

372

536

571

113**

1224

165

100**

3-CF₃

2250**

14,980

313

2-Pyrimidyl

16,800

1836

1991

197

1406

148

125

149

191

pentyl group II

hexyl* group III

xylene group IV

2-OMe

2,3-diCl

4-Cl

584

101

126

254

139

703

3-CF₃

162

1354

7097

2626

101

2-Pyrimidyl

2-Pyridyl

1-Naphtyl

954

6306

1438

105

2673

179

6 g

6 h

20*

58*

84*

25*

13*

48*

321*

764

971*

779

192*

105

119*

487

58*

2515

552*

23*

2-OMe

2,3-diCl

4-Cl

132*

28*

209*

299

920*

3-CF₃

167*

3530

1290*

44*

52*

2-Pyrimidyl

2-Pyridyl

1-Naphtyl

2-OMe

3274

239*

12*

5708

2352*

77*

7 g

7 h

4754

10,090

na - not available, * - Ref.¹⁹; ** - Ref.³³; high afﬁnity K_i< 50 nM, moderate afﬁnity 50 nM < K_i< 500 nM, low afﬁnity 500 nM < K_i< 1000 nM, not active K_i> 1000 nM.

Each compound was tested in triplicate at 7 concentrations (0.1 nM – 100 µM). The inhibition constants (K_i) were calculated from the Chenge-Prusoff equation³⁷

Results were expressed as means of at least two separate experiments (SEM ≤ 25%; h – human receptor type.

Fig. 1. Structure – Activity Relationship analysis plan leading to dual D₂R/5-HT_1AR ligands.

D. Kułaga et al.

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx

4a-4f

5a-5f

4a-4h

6a-6h

Scheme 1. Synthetic pathway leading to new compounds with ﬂexible linker.

Scheme 2. Synthetic pathway leading to new compounds with rigid linker.

and 7b) the binding pattern to 5-HT_1AR would be similar to I-M. Ligands

with four (5b) or ﬁve (6b) carbon atoms in the linker showed the highest

afﬁnity with K_i= 8 nM and K_i= 3 nM, respectively. Adding an extra

carbon to the linker (6 carbons in total) resulted in slight loss of afﬁnity

(7b, K_i= 10 nM). Increased rigidity of the carbon linker by introducing

xylene moiety resulted in further loss of activity to 5-HT_1AR (8b, K_i= 77

nM). In this case increasing rigidity did not have any effect on afﬁnity to

D₂R. Ligands 5b, 6b, 7b and 8b did not exhibit afﬁnity to 5-HT_2AR and

5-HT₆R, however, exhibited moderate afﬁnity to 5-HT₇R. A different

binding pattern was observed when 1-(2,3-dichlorophenyl)piperazine

was present in the ligands. Elongation of the carbon linker resulted in a

decrease of afﬁnity to 5-HT_1AR in the following pattern: 5c K_i= 1 nM, 6c

K_i= 29 nM and 7c K_i= 58 nM. According to D₂R, ligand with propyl

linker was the most active in this group (6c, K_i= 17 nM). Shortening

(5c) as well as elongation (7c) of the carbon linker resulted in a decrease

of activity when compared to 6c with K_i= 26 nM and K_i= 132 nM,

respectively. Ligands with para-Cl substituent of arylpiperazine moiety

were less active than those with 2,3-dichloro substituents. Ligand with

four carbons (5d) as well as six carbons (7d) exhibits similar afﬁnity to

5-HT_1AR, while ligand with ﬁve carbons (6d) exhibits slight worse af-

ﬁnity with K_i= 101 nM. Elongation of the carbon linker resulted in

increased of afﬁnity only in term to D₂R. The same ligands exhibited

moderate afﬁnity to 5-HT_2AR, 5-HT₆R and 5-HT₇R or were not active

towards these receptors. With respect to 1-(3-triﬂuoromethylphenyl)

piperazine ligands, elongation of carbon linker resulted in increased

afﬁnity to

the mentioned afﬁnity, making them highly selective towards tested

receptors. For 1-(2-pyridinyl)piperazine ligands (6g and 7g, K_i= 13

nM), we did not observe differences in the binding to

5-HT_1AR. During conducted research, we found that ligands 6h and

7h showed multifunctional binding, where the structure-carbon length

relationship may be difﬁcult to analyse. According to Structure – Aryl-

piperazine Relationship, in the ﬁrst group of ligands (5a-5c, 5f) where

four-carbon linker was present, high afﬁnity to 5-HT_1AR was observed in

the range of K_ibetween 1 and 15 nM. Introduction of para-Cl substituent

to N-arylpiperazine core (5d, K_i= 78 nM) as well as meta-CF₃substit-

uent (5e, K_i= 100 nM) resulted in considerable decrease of afﬁnity.

Generally, we observed less afﬁnity of the ligands 5a-5f to D₂R than to 5-

HT_1AR. Ligands with ortho-OCH₃(5b) and 2,3-dichloro (5c) substituent

only exhibited high afﬁnity with K_i= 7 nM and K_i= 26 nM, respectively.

In most cases, these ligands were not active toward 5-HT_2AR, 5-HT₆R

and 5-HT₇R. In the second group of ligands where ﬁve-carbon linker was

present, the afﬁnity to 5-HT_1AR was also very high – K_i≤ 29 nM. One

exception was 6d, where introducing of para-Cl substituent resulted in

the decrease of activity with K_i= 101 nM. For D₂R, an interesting

relationship was observed with respect to ligands with only ortho and/or

meta positions occupied (6b, 6c, 6e and 6i). In that case, ligands

exhibited high afﬁnity with K_i≤ 34 nM. We indicated that most of li-

gands with pentyl linker (6a-6i) did not exhibit high afﬁnity to 5-HT_2AR,

5-HT₆R and 5-HT₇R except of ligand 6i which behaved as a multifunc-

tional ligand. Ligands with six-carbon linker seem to be less selective

than those with ﬁve or four carbons. Ligand 7e exhibits high afﬁnity to

four types of receptors: D₂, 5-HT_1A, 5-HT₆and 5-HT₇. Ligands 6h and 7h

exhibit afﬁnity to three type of receptors:D₂, 5-HT_1Aand 5-HT₇.

We conﬁrmed a fully conducted SAR study based on in vitro assays via

molecular modelling approach using docking for selected ligands (Fig.2,

Fig.3) Due to lack of crystalline structure of 5-HT_1AR, a homology model

based on 5-HT_1BR (PDB 4IAR) template with ergotamine was created

using Swiss-Model server.³⁴The 5-HT_1AR homology model was vali-

dated using also Swiss-Model server as well as QMEAN by parameter

determination. The amino acids sequence identity was>41% and the

coverage was 0.89 – such values correspond with literature.^35–36The

percent of residues in favoured regions of the Ramanchadron plot was

also determined (Supplementary Material). For D₂R, we used the crystal

5-HT_1AR. Ligand with short ethyl chain (A) linker was inactive.^16–18

Elongation of ethyl chain to 3 atoms (i.e., propyl) (B) resulted in

increased afﬁnity with K_i= 400 nM.^16–18Further elongation caused

increase of afﬁnity in the following pattern: 5e K_i= 100 nM, 6e K_i= 25

nM and 7e K_i= 21 nM. The same pattern was observed in term of D₂R.

Ligands exhibited high afﬁnity to 5-HT₇R as well. With respect to 5-

HT_1AR, the binding pattern of ligands with 1-(2-Pyrimidinyl)piperazine

was similar to those with 1-(2-methoxyphenyl)piperazine. Ligands with

short linkers: propyl (C), butyl (5f), and pentyl (6f), exhibited high af-

ﬁnity to 5-HT_1AR with the K_i= 2 nM, K_i= 15 nM and K_i= 6 nM,

respectively. So far, we reported that investigated ligands exhibit af-

ﬁnity to D₂R. Ligands with 1-(2-pyrimidinyl)piperazine do not exhibit

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Article Doi

Radioligand and computational insight in structure – Activity relationship of saccharin derivatives being ipsapirone and revospirone analogues

DOI: 10.1016/j.bmcl.2021.128028

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Article abstract of DOI:10.1016/j.bmcl.2021.128028

Full text of DOI:10.1016/j.bmcl.2021.128028

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