Design and synthesis of new potent PTP1B inhibitors with the skeleton of 2-substituted imino-3-substituted-5-heteroarylidene-1,3-thiazolidine-4-one: Part I

Accepted Manuscript

Design and synthesis of new potent PTP1B inhibitors with the skeleton of 2-

substituted imino-3-substituted-5-heteroarylidene-1,3-thiazolidine-4-one: Part I

Ge Meng, Meilin Zheng, Mei Wang, Jing Tong, Weijuan Ge, Jiehe Zhang, Aqun

Zheng, Jingya Li, Lixin Gao, Jia Li

PII:

S0223-5234(16)30458-5

10.1016/j.ejmech.2016.05.060

EJMECH 8652

DOI:

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 29 January 2016

Revised Date: 26 May 2016

Accepted Date: 26 May 2016

Please cite this article as: G. Meng, M. Zheng, M. Wang, J. Tong, W. Ge, J. Zhang, A. Zheng, J. Li,

L. Gao, J. Li, Design and synthesis of new potent PTP1B inhibitors with the skeleton of 2-substituted

imino-3-substituted-5-heteroarylidene-1,3-thiazolidine-4-one: Part I, European Journal of Medicinal

Chemistry (2016), doi: 10.1016/j.ejmech.2016.05.060.

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ACCEPTED MANUSCRIPT

Graphic abstract

Design and synthesis of new potent PTP1B inhibitors with the skeleton of

2-substituted imino-3-substituted-5-heterocyclidene-1,3-thiazolidine-4-one: Part

I

Ge Meng^a, Meilin Zheng^a, Mei Wang^a, Jing Tong^a, Weijuan Ge^a, Jiehe Zhang^a, Aqun Zheng^b, Jingya Li^c, Lixin Gao^cand Jia Li^c*

^aSchool of Pharmacy, Health Science Center, Xi’an Jiaotong University, No. 76, Yanta West Road, Xi’an, Shaanxi, 710061, P. R. China

^bSchool of Science, Xi’an Jiaotong University, No. 28, Xianning West Road, Xi’an, Shaanxi, 710049, P. R. China

^cState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P. R. China

ACCEPTED MANUSCRIPT

Design and synthesis of new potent PTP1B inhibitors with the skeleton of 2-

substituted imino-3-substituted-5-heteroarylidene-1,3-thiazolidine-4-one: Part I

Ge Meng^a, Meilin Zheng^a, Mei Wang^a, Jing Tong^a, Weijuan Ge^a, Jiehe Zhang^a, Aqun Zheng^b, Jingya Li^c,

Lixin Gao^cand Jia Li^c*

^aSchool of Pharmacy, Health Science Center, Xi’an Jiaotong University, No. 76, Yanta West Road, Xi’an, Shaanxi, 710061, P. R. China

^bSchool of Science, Xi’an Jiaotong University, No. 28, Xianning West Road, Xi’an, Shaanxi, 710049, P. R. China

^cState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, P. R. China

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

A new series of 2-substituted imino-3-substituted-5- heteroarylidene-1,3-thiazolidine-4-ones as

the potent bidentate PTP1B inhibitors were designed and synthesized in this paper. All of the

new compounds were characterized and identified by spectra analysis. The biological screening

test against PTP1B showed that some of these compounds have the positive inhibitory activity

against PTP1B. The activity of the compounds with 5-substituted pyrrole on 5-postion of 1,3-

thiazolidine-4-one are more potent than that of those compounds with 5-substituted pyridine

group. Compound 14b, 14h and 14i showed IC₅₀values of 8.66 ꢀM, 6.83 ꢀM and 6.09 ꢀM

against PTP1B, respectively. Docking analysis of these active compounds with PTP1B showed

the possible interaction modes of these biheterocyclic compounds with the active sites of

PTP1B. The inhibition tests against oncogenetic CDC25B were also conducted on this set of

compounds to evaluate the selectivity and possible anti-neoplastic activity. Compound 14b also

showed the lowest IC₅₀of 1.66 ꢀM against CDC25B among all the possible inhibitors, including

14g, 14h, 14i and 15c. Some pharmacological parameters including VolSurf, steric and electric

descriptors of all the compounds were calculated to give some hints about the relative

relationship with the biological activity. The result of this study might give some light on

designing the possible anti-cancer drugs targeting at phosphatases. The most active compound

14i might be used as the lead compound for further structure modification of the new low

molecular weight PTP1B inhibitors with the N-containing heterocyclic skeleton.

Available online

Keywords:

1,3-Thiazolidine-4-one

PTP1B

Pyrrole

Pyridine

Dock

SAR

1. Introduction

diseases[2, 7, 8], Therefore, many researchers’ efforts have been

focused and progressed on PTP1B inhibitors as the multiple

Being dynamically controlled by the coordinated action of both

protein tyrosine kinases (PTKs)[1] and protein tyrosine

phosphatases (PTPs) [2], the reversible protein tyrosine

phosphorylation is a common post-translational modification,

which plays a pivotal role in the structure and proper function of

a variety of protein by creating novel recognition motifs for

protein-protein interaction and cellular localization, controlling

protein stability and regulating enzyme activity[3]. As a

consequence, an appropriate level of protein tyrosine

phosphorylation is essential for many aspects of cell physiology

especially in the signal transduction process for regulating a

variety of cellular functions, including proliferation,

differentiation, migration, metabolism, gene transcription, cell-

cell communication, ion channel activity, immune response and

apoptosis/survival decisions[4]. Working in concert with

PTKs[5], PTPs comprises more than 100 enzymes to constituting

a large family[6], among which intracellular PTP1B is highly

coveted by both the pharmaceutical industry and the academic

institutions and therefore constitutes the first example of PTP

with many physiological functions that makes it an ideal drug

target for the therapeutic intervention in many common human

potential therapeutic agents for the purpose of treating the

important disease including diabetes, obesity[9] and cancer[10-

12]. The chemical structures of both PTP1B and the various

kinds of complexes with the different ligands have also been

clearly analyzed by the crystallography researches or NMR

explorations[13-16].

Over the last decade, numerous different PTP1B inhibitors

with various structure skeletons have been reported (Fig. 1) [17-

25], but to date, unfortunately, none of which have passed

clinical trials except for one candidate with a thiophen-fused

tricyclic ring in the structure skeleton (Ertiprotafib), the only one

that has been terminated at clinical trials[26] for its insufficient

efficacy and strong unwanted side effects. Generally speaking,

the reasons for the failure in trials is due to either the side effects

or the insufficient efficacy[27], which could be the consequence

of either the scarce selectivity or the limited cell permeability of

the molecules[7]. The reasons for both of these two dilemmas

might be analyzed in details from enzymatic levels in the

following paragraph.

———

Corresponding author. Tel.: +86-029-82656327; fax: +86-29-82655424; e-mail: mengge@mail.xjtu.edu.cn (G. Meng); jli@mail.shcnc.ac.cn(J.

Li)

ACCEPTED MANUSCRIPT

Figure 1. The chemical structures of some of the reported PTP1B inhibitors with the relatively low IC₅₀values.

Firstly, developing selective and potent inhibitors of PTP1B is

appropriate PTP1B inhibitors is currently still a great challenge

a major challenge because of its high homology to other cellular

PTPs and the polar nature of the active binding pockets. Also the

inhibitors binding to the catalytic site bear high charge densities

hence exhibit the limited selectivity and poor cell permeability.

On the other hand, efforts to obtain the selective PTP1B

inhibitors have often resulted in the synthesis of peptidic or large

highly charged molecules with high in vitro potency but

potentially low bioavailability. However, in the case of PTP1B,

the identification of a second noncatalytic arylphosphate binding

site, which is not present in all PTPs has provided a further

structural basis for targeted drug design[15, 28, 29]. Therefore

developing the bidentate PTP1B inhibitors that could occupy

simultaneously at both the active site (Site-A) and the adjacent

pTyr binding site (Site-B) partially addressed the selectivity

issue.[30] The hydrophobic character critical for the compound’s

cell permeability could be also be enhanced by creating the small

molecules binding at the site-C with the reduced negative charges

(Fig. 2) [31-34].

in this research area.

Among the aromatic PTP1B inhibitors with the heterocyclic

skeleton, 5-arylidene-1,3-thiazolidine-2,4-diones have been

designed and synthesized as the potent PTP1B inhibitors[36].

Albeit, the unsubstituted oxygen atom on 2-position of this set of

1,3-thiazolidine-2,4-diones was almost impossible to modify

further to achieve the sufficiently proper pKa or LogP values (eg.

logP≤5) for the good pharmacological profiles. Therefore 2-

substituted-imino-1,3-thiazolidine-4-ones might offer a possible

alternative skeleton providing the further modification chance on

the structure with many suitable substituted groups(R¹and R²)

(Fig. 3).

O

R¹

N

S

transformed into

S

HN

R²

N

O

2-imino-1,3-thiazolidine-4-ones

1,3-thiazolidine-2,4-diones

could not be

modified at 2-oxygen group

Site C

are flexible in modulating

the substituted groups (R¹and R²)

Figure 3. 1,3-thiazolidine-2,4-diones versus 1,3-thiazolidine-4-diones

Based on many crystals of the structure of PTP1B and possible

analysis of interaction mechanism between the ligand and the

active sites of enzyme, using the basic principle of the fragments-

based drug design, we have designed and synthesize a new series

of compounds (14a-14i and 15a-15h). The skeleton of thiazolidi-

4-one was also designed to suit for the active site A of PTP1B,

which is similar to the catalytic intermediates during the catalytic

process of PTP1B attaching to the phosphate substrate.

Linker

Site A

Site B

pTyr Site

Figure 2. A strategy for creating the selective and high-affinity PTP1B

inhibitors. Based on the principle of additivity of free energy of binding,

high-affinity ligands can be obtained by linking several functional groups that

bind to the active site (pTyr binding site) and the peripheral site Xs (Site B

and Site C). The added specificity arises from the fact that site X is not

conserved and from the fact that the tethered ligand has to bind all these sites

simultaneously.

Therefore, the current thrust of research in this field was shift

to the identification of the low molecular weight, nonphosphorus

and nonpeptide heterocyclic inhibitors that are potentially

endowed with the favorable pharmacokinetics. Guided by the

above strategies, many cell permeable inhibitors of this

phosphatase have been designed and synthesized[20, 23, 35].

Any way, there was no one candidate yet passed successfully the

clinical trial by now, therefore the discovery of pharmaceutically

All of these new type of PTP1B inhibitors shared the

bisheterocyclic rings including both 1,3-thiazolidine-4-ones and

another N-containing heterocyclic ring attached together with a

suitable linker to accommodate with the double or triple sites of

PTP1B (Fig.4). The form and length of the linker groups in each

set of the molecules could also been adjusted via using the

different atoms or groups, which could be provided by various

raw materials via the different synthetic methods. We therefore in

this paper reported the characterization and the biological

activities of these new heterocyclic compounds against PTP1B as

well as the related analysis about the molecular modeling and the

calculation of the parameters of these molecules.

Indeed, several carboxylic acids with the in vitro PTP1B

ACCEPTED MANUSCRIPT

inhibitory properties and the good cellular activity or the oral

bioavailability have been reported[23, 24, 35, 37, 38]. The

carboxylate group on the 3-substituted pyrrole ring of 14 has

been designed as the tentative bioisosteric monoanionic group of

the phosphate group, which have been considered as a valid

method to obtain the potential inhibitors with the low

polarity[37].

2. Results and discussions

2.1. Chemistry

Figure 4. Design strategies of the bidentate ligands targeting at the active

sites of PTP1B.

N

F

H

N

Cl

O

N

OH

N

OH

O

2

OHC

4

N

O

OHC

a

5

3

b

d

1

O

e

c

O

HOOC

NH

O

HN

7

6

8

R¹

O

N

S

R²

O

f

O

OHC

N

O

HN

O

g

9

10

HN

O

14

S

R²

N

O

h

R²

R¹

N

Cl

R²

+

N

H

N

5

N

OH

R¹

H

N

S

O

g

12

11

N

13

N

R¹

15

12,13,15

R¹

Et

R²

Et

14

R¹

OH

Phenyl

R²

i-Pr

a

n-Bu (for 12 and 13);

b

n-Bu

i-Pr

b

Phenyl

R¹N = O (only for 15)

i-Pr (only for 12);

OH (for 13,15)

Phenyl

c

4-Methylphenyl

4-Methoxyphenyl

4-Methylphenyl

d

e

Phenyl

4-Methylphenyl

d

e

4-Methoxyphenyl

3-Chloro-2-methylphenyl

4-Methylphenyl

3-Chloro-2-methylphenyl

f

4-Methoxyphenyl

f

3-Trifluoromethylphenyl

g

h

3-Chloro-2-methylphenyl

3-Trifluoromethylphenyl

3-Chloro-2-methylphenyl

3-Trifluoromethylphenyl

g

h

2-Fluorophenyl

3-Fluorophenyl

2-Fluorophenyl

3-Fluorophenyl

2-Fluorophenyl

(only for 12,13)

3-Fluorophenyl

(only for 12,13)

4-Fluorophenyl

(only for 12,13)

2-Fluorophenyl

(only for 12,13)

3-Fluorophenyl

(only for 12,13)

4-Fluorophenyl

(only for 12,13)

i

j

i

4-Fluorophenyl

k

Scheme 1. Synthesis route to the target molecules 14 and 15. Reagents and conditions: (a) Refluxing; (b) NaH, or KOH, TEBA; (c) CH₃COOH, NaNO₂, 0~10ꢀ;

then zinc powder, 70~100ꢀ, and then refluxing, 5 h; (d) EtOH, KOH (aq); then HCl; (e) Heat under N₂atmosphere; (f) POCl₃, DMF, DCM; (g) EtOH,

piperidine, refluxing; (h) H₂O or EtOH refluxing (The newly formed groups or bonds were colored in blue.).

The synthesis route of the title compounds 14a-14i and 15a-

15h was shown in Scheme 1. The structural skeleton of the target

molecules includes the following two basic types: 2-substituted

imino-3-substituted-5-(3,5-dimethyl-4-ethoxylcarbonyl-1H-py

rrole-2-yl)-methylene-1,3-thiazolidine-4-ones (14a-14i) and 2-

substitutedimino-3-substituted-5-[4-[2-[N-methyl-N-(2-

pyridinyl)] amino]ethoxyl]benzylethylene-1,3-thiazolidine-4-one

(15a-15h). The synthesis method of both of the two structural

types shared the same key intermediates, i.e. 2-imino-1,3-

thiazolidine-4-ones (13a-13k), which could be synthesized

conveniently from 2-chloroacetic acid (11) and N,N’-

disubstituted thioureas (12a-12k) via an eco-friendly method

according to our improved procedures[39-41].

Used as the important intermediate to synthesize target

compound

15,

4-[2-[N-Methyl-N-(2-pyridyl)]amido]ethoxy

benzaldehyde (5) was obtained by the sequential two-step

reactions (amidation and etherification) with the excellent yields

from the commercially available materials[42], including 2-

chloropyridine (1), 2-(N-methylamido)ethanol (2) and 4-

fluorobenzaldehyde (4) (Scheme 1). These materials and

intermediates have been widely used to synthesize the FDA-

approved anti-diabetic drugs, such as Rosiglitazone, Pioglitazone,

etc.

oncogenetic reagent. Among all tested drug candidates,

compound 14b also showed the highest IC₅₀of 1.66 ꢀM against

CDC25B. Other compounds, such as 14g, 14h, 14i and 15c also

showed some moderated anti-CDC25B activity.

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2.3. Molecular docking analysis

As for the synthesis of the pyrrole substituted target

compounds,

ethyl

5-formyl-2,4-dimethyl-1H-pyrrole-3-

Compound 14b, 14h and 14i were docked into the active

binding sites of PTP1B, respectively, the result of which might

offer a comparison of the different interaction model between the

ligand and the binding site of PTP1B. Since there were already

more than 100 items of PTP1B crystal structure data in PDB,

selecting a suitable crystal might be a great task to get some

valuable information from the docking analysis. Among all the

available 124 crystal structures, 2VEY was chosen for the final

analysis discussion about the docking result with the target

compounds for the following three reasons.

carboxylate (10) was also used as a similar aldehyde to

intermediate 5. Compound 10 was obtained from acetate ethyl

ester by Knorr reaction via several steps of reactions, including

cyclization, hydrolysis, decarboxylation and Vilsmeier-Haack

formulation. Several important intermediates, including 7, 8 and

9, were sequentially obtained during this synthetic course

(Scheme 1). While controlling the mole ratio of compound 7 to

KOH, the concentration of the base in water, the stirring method,

the temperature and the reaction time, the regioselective

transformation from 7 to 8 could be achieved accordingly[43].

These intermediates were also used to synthesize an FDA-

approved anti-neoplastic drug Sunitinib, which is a first orally

active inhibitor against the multitargets including tyrosine kinase

and angio-genesis, etc[44].

1. The chemical structure of the ligand in the complex (2VEY) is

shown in Fig. 5 with the chemical name of N-((S)-2-(4-((S)-1,1-

dioxido-3-oxoiso-1,2-thiazolidine-5-yl)phenyl)-1-(4-(3-

phenylpropyl)-1H-imidazol-2-yl)ethyl)-3-fluorobenzene

sulfonamide [16]. This structure belongs to

a kind of

In order to connect both of the designed heterocyclic

fragments in one molecule with a newly formed double bond,

Knoevenagel condensation (KC) was adopted in the final step

between the formyl group of the aldehyde intermediate (5 or 10)

and the active methylene group on 5-position of various

substituted 1,3-thiazolidine-4-one intermediates 13 to afford the

desired target molecules (14, 15)[45, 46].

isothiazolidinone heterocycle, which is similar to the 1,3-

thiazolidine-4-one skeleton of the target molecules synthesized in

the article (Scheme 1, 14, 15).

2.2. Biological activity

The anti-PTP1B activity of the target molecules was

measured using the traditional screening method for tyrosine

phosphatase and comparing with already well-known PTP1B

inhibitor oleanolic acid. The biological testing results of these

target molecules together with the controlling compound are

listed in Table 1. Some of the target compounds showed

moderate to potent activities against PTP1B with IC₅₀at ꢀM

level. The structure containing phenylimino group, phenyl group

and carboxylate ethyl ester substituted 1H-pyrrole group on the

2-, 3- and 5-positions of 1,3-thiazolidine-4-one skeleton (14b)

showed the relatively higher activity against PTP1B with IC₅₀of

8.88ꢀM than other compounds. Compounds 14i showed the

highest activity against PTP1B with IC₅₀of 6.09 ꢀM among all

the tested samples. Compounds 14h showed the moderated

activity with IC₅₀of 6.83 ꢀM.

Figure 5. The chemical structure of the ligand in PTP1B (PDB ID: 2VEY)

2. The average molecule weight of all the target molecules and

the molecule weight of the ligand of 2VEY are all around 500

with value of 497.0 and 596.5- respectively.

3. The sequence of the total scores obtained from Surflex Dock

via SYBYLX-2.0 workstation is in agreement with the sequence

of IC₅₀values (ie, 14i<14h<14b) obtained the traditional pNPP

enzyme assay evaluating PTP1B inhibitory activity.

Therefore, the docking score of the most active compound

14i with 2VEY ranked as the highest value, and the docking

score of the least active 14b with 2VEY ranked as the lowest

value. The docking results of these three compounds with 2VEY

are discussed individually based on these pictures shown in the

following three figures (Fig. 6~8).

CDC25 test results of the target compounds showed that

some of the compounds might also served as the possible anti-

Figure 6. Docking analysis of compound 14b with PTP1B (2VEY). The total score is 4.4639. The crash is -0.8702. The polar is 1.8660. The similarity is 0.248.

(Left: Whole enzyme, Right: Dock active site, Red: Site A, Blue: WPD loop).

Arg 254 in Fig. 6 and Fig. 7), successfully verifying the design

^{Both 14b and 14h could reached from}A^theC^aC^ctE^ivePT^sitE^eD^AMANUSCRIPT

(which is represented by the red portion in Fig. 6 and Fig7) to the

second binding site B (which is represented by the amino residue

strategy of the bidentic inhibitor based on the structure features

of PTP1B[47].

Figure 7. Docking analysis of compound 14h with PTP1B (2VEY). The total score is 4.6400. The crash is -1.0312. The polar is 1.8677. The similarity is 0.301.

(Left: Whole enzyme, Right: Dock active site, Red: Site A, Blue: WPD loop).

As shown in Fig. 6 and Fig. 7, the carbonyl oxygen on the

4-carboxylate group on the pyrrole ring of both compound 14b

and 14h pointed directly to the second phosphate binding site of

PTP1B. A relatively stable six-member ring was formed between

the ligand and the target enzyme via two hydrogen bonds

between the guanidine NH groups on the amino residue Arg254.

This interaction mode predicted by Surflex dock could also be

inferred from the estimated total score of 4.64 obtained for the

best docked conformation with PTP1B on SYBYL workstation.

From crystallographic analysis of PTP1B, Arg254 is one of

the important amino residues on the second aryl phosphate-

binding site adjacent to the catalytic site, including Arg24,

Arg254, Met258, Phe52 and Gln262, etc[48]. Although this

secondary site is catalytically inactive, it could still provide

weaker interactions compared with the primary active site,

owing to its more open structure to the solvent.

Figure 8. Docking analysis of compound 14i with PTP1B (2VEY). The total score is 5.1028. The crash is -1.8277. The polar is 1.7484. The similarity is 0.306.

(Left: Whole enzyme, Right: Dock active site, Red: Site A, Blue: WPD loop)

The docking result showed that the interactions between the

compound 14i and PTP1B catalytic domains might adopt a new

binding mode different from 14b and 14h. The docked

conformation of the compound 14i could stretch from the site A

(which is represented by the red portion in Fig. 8) to another

active YRD motif (which is represented by the amino residue

Arg 48 in Fig. 8). A hydrogen bond was formed between NH on

pyrrole ring of the most active compound 14i and oxygen on

carboxylic acid group of amino acid residue Asp48 of PTP1B

(Fig. 8). Asp48 is situated on the YRD motif (including Tyr46,

Arg47, and Asp48), which is a proximal site near the primary

binding site providing the specificity of PTP1B for

phosphotyrosine over phosphoserine or phosphothreonine[15,

49]. Among all the 350 amino residues of PTP1B, Arg47, Asp48,

Val49, Lys120, Ala217, Ile219, Gly220, Met258, and Thr263

were found to be important in modulating the activities, which

was verified by both calculation and experiments[50].

This new binding mode might explain the higher biological

activity of 14i over 14b and 14h, the interaction between 14i and

RGD motif might be induced by the assistance of the two

fluoride atoms on 4-position the two phenyl rings of the

compound 14i. The different binding modes of compounds 14i,

14b and 14h might offer the possible combining strategies in

further design of new potent PTP 1B inhibitors.

Compared with the docking result of 2VEY with another set

of PTP1B inhibitor with the structure feature of sulfamide[51],

the target molecules in this article showed less hydrogen bond

with the active site. This gave some light on the possible

structural modification of this series of compounds with the

substituent contained the similar structures of the reported

literature[52]. Therefore introduction of some sulfonamide group,

difluoromethylenephosphonic acids, hydroxyl group into the

skeleton of these compounds will be benefit for forming more

hydrogen bonds with the target enzyme so to enhance the affinity

with the substrate target as well as the biological activity of the

target molecules.

the level of 80% required for the druggable molecules, except for

the compound 15c. This means that most of the series 15

compounds might show a good binding affinity with human

serum albumin (HSA), conforming to the PB standard required

by the druggable compounds. VD values of compounds 15e, 15f

and 15g are lower than -0.8, meaning the volume distribution in

plasma of these three compounds are more than 6.3 L/Kg,

implying some possible excretory problem. VD values of

compounds 15a, 15b, 15d and 15h are between -0.8 and -0.5,

meaning the volume distribution in plasma of these four

compounds are between 3.2 and 6.3L/Kg, showing a good

distribution in vivo. VD value of compound 15c is between -0.5

and -0.2, meaning the volume distribution in plasma of these four

compounds are between 3.2 and 6.3 L/Kg, showing a good in

vivo distribution with good excretory ability than other

compounds. BBB indicators of the compounds 15a to 15g are

between -0.3~0.3, denoting the moderate ability to across the

blood brain barrier of the seven compounds. While BBB value (-

0.756) of the compound 15h is less than -0.3 showing no

permeability crossing BBB with two trifluoromethyl groups on

the two benzene rings of structure skeleton. MS values of

compounds 15d, 15e, 15f, 15g and 15h are lower than -1,

suggesting the possible low metabolic stability of these five

compounds in vivo. This could be associated with decomposition

in the metabolic enzyme system, leading to a short action time in

vivo. On the contrary, compounds 15a, 15b and 15c are relatively

more stable than the above five compound based on MS value

prediction. The hERG values of compounds 15d, 15e, 15f, 15g

and 15h are less than -0.5, implying the block performance

ability of the hERG potassium ion channel of these five

compounds, leading to the high risk of heart toxic effect of the

compounds. The hERG values of compounds 15a and 15b are

between -0.5 and 0, implying the possible blocking ability of the

hERG of these two compounds, leading to the possible heart

toxic effect of the compounds. Only the hERG value (0.214) of

compound 15c is more than 0, denoting the relatively low

blocking ability to the potassium ion channel. Therefore, series

compounds 15 might show more potential toxic effects to the

heart than series compound 14. Generally speaking, the

pharmacokinetic properties are less affected by the substituted

groups in the chemical structures of the compounds 15a-15h than

in the chemical structures of the compounds 14a-14i.

ACCEPTED MANUSCRIPT

2.5. Molecular property descriptors prediction

The pharmacokinetic properties of compounds 14a~14i and

compounds 15a~15h are analyzed in the following two

paragraphs, respectively.

LogS values of compounds 14a to 14i are between -6 to -3,

showing the relatively higher solubility than compounds 15a-15h.

LogS values of all of the target compounds are in the scope of -8

to 2 required by the druggable molecules. Papp values of

compounds 14b, 14c, 14d and 14e are higher than the threshold

value of 0.4, showing good trans-membrane ability in Caco-2 cell.

Papp values of compound 14a, 14f, 14g, 14h and 14i are between

-0.1 and 0.3, showing relatively low trans-membrane ability.

PB(%) percentages of compounds 14b, 14c, 14d, 14e and 14f are

all above the level of 80% required for the druggable molecules.

This means that these five compounds might show a good

binding affinity with human serum albumin (HSA), conforming

to the PB standard required by the druggable compounds. PB(%)

percentages of compounds 14g, 14h and 14i are between 70%

and 80%. Only PB(%) of 14a is low than 55%, showing the

necessity for structure modifications. VD values of compounds

14b~14i are between -0.8 and -0.2, which denotes the volume

distribution in plasma of these eight compounds are between

6.3L/Kg and 1.6L/Kg, showing a good distribution in vivo. BBB

indicators of compounds 14b~14e are between-0.3-0.3, denoting

the moderate ability to across the blood brain barrier of these four

compounds. While BBB values (-0.756) of compounds 14a and

14f~14i are less than -0.3, showing no permeability crossing BBB

of these five compounds. MS values of compounds 14a~14i are

between -1 and 1, suggesting the moderate metabolic stability of

these nine compounds in vivo, among which compound 14a

might be the most stable compounds. The widely spreading

hERG values of compounds 14a~14i are more than -0.5, outside

the scope for blocking the hERG potassium ion channel. The

hERG values of compound 14a, 14f and 14h are 0.515, 0.511,

0.913, respectively, which is far away from the scope for

blocking the hERG potassium ion channel. These compounds

might be much more high safer for the heart than the compounds

15. From the structure analysis, the electric potential effects of

the substituents of compounds 14a~14i might have more impacts

on the related pharmacokinetic properties than that of compounds

15a~14g. The substituted groups on the benzene ring of

compounds 14f~14i are CF₃, 2-F, 3-F and 4-F, respectively.

These substituted groups are all electron withdrawing groups,

compared with the electron donating groups such as Me, OMe in

compounds 14b~14e. The BBB values, MS value and hERG

values have been improved in compounds with electron

withdrawing groups than in compounds with electron donating

groups. The Caco-2 cell permeability and the protein binding

ability are still not very favorable for the compounds 14f~14i.

As a summary for the VolSurf descriptor prediction, the

predicted BBB permeability, metabolic stability and hERG

blocking ability could be improved a lot for the unexpected

obtained compounds including 15b, 15c and 14a. Albeit, the

Caco-2 cell permeability and the protein binding ability were less

favorable for the molecules with the carbonyl group on 2-

position of 1,3-thiazolidine-2,4-dione than for the original

expected compounds. The pharmacokinetic properties of

compounds 14a~14i are much better than that of compounds

15a~14g. Compounds 14a~14i have some drug like properties,

such as less heart toxic effects, although other properties of these

compounds still need to be optimized.

LogS values of compounds 15a to 15h are between -8 to -6,

showing the relatively low solubility in the scope of -8 to 2 of the

druggability compounds. Papp values of compounds 15a, 15b,

15d, 15e, 15f and 15g are around 1.0, which is high above the

threshold value of 0.4 for crossing Caco-2 cell membrane, except

for the compound 15c and 15h. This means that the cell

membrane permeability of compounds 15a, 15b, 15d, 15e, 15f

and 15g are around 8*10^-6cm/s, which is much higher than that

of compounds 15c and 15h. Only if the compound had the

reasonable cell membrane permeability, an orally administrated

drug could be developed successfully. PB(%) percentages of

compounds 15a, 15b, 15d, 15e, 15f , 15g and 15h are all above

Some valuable implications might also be summarized from

the descriptors calculated via Chemdraw software. Firstly, all of

the active compounds 14b, 14h, and 14i, have one hydrogen

bond donors (HBD) and 4~6 hydrogen bond acceptors (HBA).

The relationship between the activity and these descriptors

implied that the possibility to forming hydrogen bonds might be

benefit for enhancing the biological activity. Secondly, the three

active compounds against PTP1B have the LogP values of 5.037

or 5.353, which fallen into the druggable scope of the Lipinski

rule[53]. On the other hand, this was also supported by another

set of descriptor values, which were shown by the partition

internal standard tetramethylsilane (TMS). Mass spectra were

^{coefficient (PC) of the three active compoun}A^dsC⁽⁶C^.4E⁰P^orT⁶E^.6D^8).MANUSCRIPT

Thirdly, the polar surface areas (PSA) of all the three active

compounds are all 71.00 Å², which is a moderate value among all

the compounds (from 57.53 to 91.23). This might suggest the

polar interactions between the ligand and PTP1B active site

might be needed to some certain extents for achieving the desired

activity.

obtained on a Waters Quattro Micro-mass instrument using electron

spray ionization (ESI) techniques. Elemental analyses were

performed on a Carlo Erba1106 instrumental analyzer.

4.1.1. Synthesis of intermediates 3 and 5

The important intermediates including 2-[N-methyl-N-(2-

pyridyl)amidoethanol (3) and 4-[2-[N-methyl-N-(2-pyridyl)]

amido]ethoxybenzaldehyde (5) were synthesized from 2-

chloropyridine (1), 2-(N-methylamido)ethanol (2) and 4-

fluorobenzaldehyde (4) according to the reported procedures[42].

3. Conclusion

As a summary, a series of new compounds with the chemical

skeleton

of

2-imino-3-substituted-5-heterocyclidene-1,3-

4.1.2. Synthesis of intermediates 7~10

thiazolidine-4-ones as the possible PTP1B inhibitors were

designed and successfully synthesized via several steps of

reaction. The biological evaluation tests of the target compounds

indicated that the biological activities of the compounds with the

substituted pyrrole groups on the 5-positon of the 1,3-

thiazolidine-4-one are much more potent than that of the

compounds with the substituted pyridine groups on the 5-positon

of the 1,3-thiazolidine-4-one. Some of the target molecules with

the 1H-pyrrole heterocycle on the 1,3-thiazolidine-4-one ring,

including compounds 14b, 14h and 14i displayed good activity

against PTP1B, among which the most active compound 14i

showed the IC₅₀of 6.37 ꢀM. Beside, compound 14b also display

the interesting activity against CDC25B with an IC₅₀of 1.67 ꢀM,

which showed the possible anti-cancer abilities of this series of

compounds. Therefore, this study clearly highlighted the

necessary to keep a proper heterocyclic ring with polar group

such as hydroxyl group and a satisfactory lipophilic ability of the

whole molecule to achieve a higher level of inhibition rate on

PTP1B. The tentative SAR analysis of this series of heterocyclic

compounds as possible PTP1B inhibitors might provide further

modification of the possible. The phenyl or 2,3-fluorophenyl

groups are much more potent in enhancing the anti-PTP1B

inhitory activity than the polar groups. The most active

compound 14i might be used as the lead compound for further

structure modification of the new low molecular weight PTP1B

inhibitors with the N-containing heterocyclic skeleton.

The important intermediates including diethyl-3,5-dimethyl-

1H-pyrrole-2,4-dicarboxylate

(7),

4-ethoxycarbonyl-3,5-

dimethyl-1H-pyrrole-2-carboxylic acid (8), ethyl 2,4-dimethyl-

1H-pyrrole-3-carboxylate (9) and ethyl 5-formyl-2,4-dimethyl-

1H-pyrrole-3-carboxylate (10) were synthesized via the improved

procedures from the commercially available materials, such as

ethyl acetyl acetate ester[43].

4.1.3. Synthetic procedures of the intermediates

13a~13k

The various kinds of intermediates with the structure skeleton

of 2-substituted-3-substituted-1,3-thiazolidine-4-ones (13a~13k)

were also synthesized from various kinds of substituted aromatic

amines according to the improved methods[39, 40].

4.1.4. Synthetic procedures of the target molecules

14

4.1.4.1. Synthesis of 5-(3,5-dimethyl-4-ethoxycar-

bonyl-1H-pyrrolyl-2-)methylene-3-iso-propyl-2-

(hydroxyimino)-1,3-thiazolidine-4-one(14a)

3-iso-Propyl-2-hydroxyimino-1,3-thiazolidine-4-one

(13c,

0.25 g, 1.50 mmol) and 2,4-dimethyl-5-formyl-1H-pyrrole-3-

carboxylic ethyl ester (10, 0.21 g, 1.10 mmol) were mixed

together with absolute ethanol (10.0 mL), to which anhydrous

pyridine (0.30 mL) was added with stirring. The reaction mixture

was heating to 85 under refluxing for 3.0 h and stopped heating.

The residue was purified with column chromatography (silica gel,

eluent: petroleum ether/ethyl acetate, v/v = 10:1 ~ 5:1, stepwise

elution) to give the crude product (0.27 g, 69.8%), which was

recrystallized from dichloromethane/petroleum ether to afford

some yellow solids as the pure product 14a (0.13 g, 48.2% for

The different binding modes of the different molecules have

important implications in the design of new type of potent

inhibitors. The most important amino residue for these active

molecules to bind with is Arg254, while other amino residue

(Asp48) might be also implicated in the interaction. These

multiple binding modes might provide a possibility of using the

strategy of independently finding molecules that bind to each site,

and then linking them together to get a much potent inhibitor in

further molecular design. The structure of most potent target

compounds 14i, the preliminary SAR analysis and the new

binding mode could provide valuable information for the rational

design of effective inhibitors with the potent therapeutic profiles

against PTP1B. The further design and synthesis of new possible

new PTP1B inhibitors based on the available biological data and

SAR analysis is being undertaken in our research group.

1

recrystallization), m.p. 175-178 . H NMR (400 MHz, CDCl₃):

δ 12.70 (s, 1H, thizolidine-5-CH=), 6.84 (s, 1H, pyrrole-NH),

5.50 (m, 1H, R¹R²NCH(CH₃)₂), 4.33 (m, 2H, CH₃CH₂O), 2.65 (s,

3H, pyrrole-5-CH₃), 2.44 (s, 3H, pyrrole-3-CH₃), 1.59 (m, 7H,

R¹R²NCH(CH₃)₂), 1.40 (t, 3H, J = 8.0 Hz, CH₃CH₂O-); ¹³C NMR

(400 MHz, CDCl₃): δ 11.85, 14.17, 17.98, 50.36, 59.85, 122.32,

125.69, 134.13, 141.34, 167.63; EI MS m/z (%): 351.9 ([M+H]⁺¹,

50), 223.0 (90), 193.9 (70), 149.0 (65). Anal. Calcd for

C₁₆H₂₁N₃O₄S: C, 54.68; H, 6.02; N, 11.96; S, 9.12. Found: C,

54.71; H, 5.99; N, 11.98; S, 9.10.

4. Experimental section

4.1.4.2. 5-(3,5-Dimethyl -4-ethoxylcarbonyl-1H-py-

rroly-2-)methylene-3-phenyl-2-(phenylimino)-1,3-

thiazolidine-4-one (14b)

4.1. Chemistry

Chemical reagents and solvents, purchased from commercial

sources, were of analytical grade and were used without further

purification. Melting points were measured on a SGW X-1

microscopic melting point apparatus. TLC analyses were run on pre-

coated silica gel G plates at 254 nm under a UV lamp using a variety

3-Phenyl-2-phenylimino-1,3-thiazolidine-4-one (13d, 0.27 g,

1.10 mmol) and 2,4-dimethyl-5-formyl-1H-pyrrole-3-carboxylate

ethyl ester (10, 0.20 g, 1.10 mmol) were mixed together with

absolute ethanol (5.0 mL), to which anhydrous pyridine (0.20

mL) was added with stirring. The reaction mixture was heating to

85 under refluxing for 3.0 h and stopped heating. The residue

was purified with column chromatography (silica gel, eluent:

petroleum ether/ethyl acetate, v/v = 10:1 ~ 5:1, stepwise elution)

to give the product 14b, which was recrystallized from

of solvent systems. Column chromatography separations were

1

performed with silica gel (300

~

400 mesh). H NMR and ¹³C NMR

spectra were recorded on a Bruker AV400 MHz spectrometer in

CDCl₃. Chemical shifts are reported in δ (ppm) units relative to the

dichloromethane/n-hexane to give the silk-shinny yellow solids

Anal. Calcd for C₂₇H₂₇N₃O₅S: C, 64.14; H, 5.38; N, 8.31; S, 6.34.

Found: C, 64.16; H, 5.39; N, 8.35; S, 6.35.

ACCEPTED MANUSCRIPT

as the pure state of product (0.08 g, 18.0%), m.p. 164-165 ,

another portion of 14b in the filtrate was recovered from the

mother liquor (0.10 g, 22.5%). The overall yield of the product

4.1.4.5. Synthesis of 5-(3,5-dimethyl-4-ethoxycar-

bonyl-1H-pyrrolyl-2-)methylene-3-(3-chloro-2-

methylphenyl)-2-(3-chloro-2-methylphenylimino)-

1,3-thiazolidine-4-one(14e)

1

14b is 40.4%. H NMR (400 MHz, CDCl₃): δ 12.41 (s, 1H,

thiazolidine-5-CH=), 7.59 (m, 2H, R¹R²NArH), 7.49 (m, 3H,

ArH), 7.33 (m, 2H, ArH), 7.13(t, 1H, ArH), 6.98 (d, 2H, ArH),

6.79 (s, 1H, pyrrole-1-H), 4.26 (q, 2H, CH₃CH₂O-), 2.51 (s, 3H,

pyrrole-5-CH₃), 2.36 (s, 3H, pyrrole-3-CH₃), 1.34 (t, 2H, -

CH₃CH₂O); ¹³C NMR (400 MHz, CDCl₃): δ 11.37, 14.42, 14.73,

59.50, 110.22, 113.63, 121.11, 122.44, 124.66, 125.05, 128.26,

129.51, 135.01, 140.48, 148.46, 152.06, 165.31, 166.82; EI MS

m/z (%): 446.1 ([M+H]⁺¹, 60). Anal. Calcd for C₂₅H₂₃N₃O₃S: C,

67.40; H, 5.20; N, 9.43; S, 7.20. Found: C, 67.42; H, 5.19; N,

9.46; S, 7.19.

3-(3-Chloro-2-methylphenyl)-2-(3-chloro-2-methylphenyl-

imino)-1,3-thiazolidine-4-one (13g, 0.37 g, 1.10 mmol), 2,4-

dimethyl-5-formyl-1H-pyrrole-3-carboxylic ethyl ester (10, 0.20

g, 1.10 mmol), absolute ethanol (6.0 mL) and anhydrous pyridine

(0.30 mL) were used. The synthetic method was similar to that of

14a and 14b. Yellow solid was obtained as 14e (0.12 g, 20.1%),

which was recrystallized from dichloromethane/petroleum ether

to give 14e as yellow crystals (0.01 g, 8.3% for recrystallization).

Another portion of product 14d was recovered from the mother

4.1.4.3. Synthesis of 5-(3,5-Dimethyl-4-ethoxycar-

bonyl-1H-pyrrolyl-2-)methylene-3-(4-methylphenyl)

-2-(4-methylphenylimino)-1,3-thiazolidine-4-one

(14c)

1

liquor as yellow solid (0.10 g). m.p. 175-176 . H NMR (400

MHz, CDCl₃): δ 12.37 (s, 1H, thiazolidine-5-CH=), 7.56 (d, 1H,

J = 8.0 Hz, ArH), 7.36 (t, 1H, J = 8.0 Hz, ArH), 7.30 (m, 1H,

ArH), 7.17 (d, 1H, J = 8.0 Hz, ArH), 7.10 (t, 1H, J = 8.0 Hz,

ArH), 6.81 (m, 2H, pyrrole-1-H & ArH), 4.30 (q, 2H, J = 8.0 Hz,

CH₃CH₂O-), 2.54 (s, 3H, pyrrole-5-CH₃), 2.39 (d, 6H, 2×ArCH₃),

2.17 (s, 3H, pyrrole-3-CH₃), 1.38 (t, 3H, J=8.0 Hz, CH₃CH₂O-);

¹³C NMR (400 MHz, CDCl₃): δ 10.69, 14.00, 14.11, 15.08 (2C),

60.89, 114.78, 118.98, 121.97, 124.28, 125.89, 126.07, 126.38,

127.16, 127.28, 128.39, 130.66, 131.67, 134.49, 135.38, 135.57,

136.88, 138.49, 148.26, 158.28, 165.88, 166.87. EI MS m/z (%):

541.0 ([M]⁺¹, 30), 223.1 (100), 194.0 (45). Anal. Calcd for

C₂₇H₂₅Cl₂N₃O₃S: C, 59.78; H, 4.65; Cl, 13.07; N, 7.75; S, 5.91.

Found: C, 59.82; H, 4.66; Cl, 13.10; N, 7.74; S, 5.89.

3-(4-Methylphenyl)-2-(4-methylphenylimino)-1,3-

thiazolidine-4-one (13e, 0.24 g, 0.80 mmol), 2,4-dimethyl-5-

formyl-1H-pyrrole-3-carboxylate ethyl ester (10, 0.20 g, 1.10

mmol), absolute ethanol (10.0 mL) and anhydrous pyridine (0.30

mL) were used in the reaction with the similar synthetic

procedures of 14a and 14b. Yellow solid was obtained (0.11 g,

29.0%), which was recrystallized from dichloromethane/

petroleum ether to afford some yellow crystals as the desired

compound 14c (0.11 g, 100% for recrystallization), m.p. 132-

134 . ¹H NMR (400 MHz, CDCl₃): δ 12.46 (s, 1H, thiazolidine-

5-CH=), 7.38 (q, 4H, J = 8.0 Hz, =NArH), 7.15 (d, 2H, J = 8.0

Hz, ArH), 6.88 (d, 2H, J = 8.0 Hz, ArH), 6.79 (s, 1H, pyrrole-1-

H), 4.31 (q, 2H, CH₃CH₂O), 2.53 (s, 3H, CH₃ArNR¹R²), 2.45 (s,

3H, CH₃ArN=), 2.39 (s, 3H, pyrrole-5-CH₃), 2.35 (s, 3H, pyrole-

3-CH₃), 1.38 (t, 2H, CH₃CH₂O-); ¹³C NMR (400 MHz, CDCl₃): δ

11.36, 14.41, 14.69, 20.97, 21.36, 59.05, 110.68, 113.64, 120.96,

122.27, 125.05, 127.81, 129.72, 130.20, 131.02, 132.54, 134.49,

139.03, 145.86, 151.65, 164.54, 166.84; EI MS m/z (%): 473.1

([M]⁺¹, 50), 223.0 (100), 194.0 (50). Anal. Calcd for

C₂₇H₂₇N₃O₃S: C, 68.48; H, 5.75; N, 8.87; S, 6.77. Found: C,

68.51; H, 5.76; N, 8.89; S, 6.79.

4.1.4.6. Synthesis of 5-(3,5-dimethyl-4-ethoxycar-

bonyl-1H-pyrrolyl-2-)methylene-3-(3-trifluorome-

thylphenyl)-2-(3-trifluoromethylphenyl)-1,3-

thiazolidine-4-one(14f)

3-(3-Trifluoromethylphenyl)-2-(3-trifluoromethylphenyl)-1,3-

thiazolidine-4-one (13h, 0.20 g, 0.50 mmol) and 2, 4-dimethyl-5-

formyl-1H-pyrrole-3-carboxylate ethyl ester (10, 0.10 g, 0.60

mmol), absolute ethanol (6.0 mL), and anhydrous pyridine (0.30

mL) were used. The synthetic method was similar to that of 14a

and 14b. Yellow solid was obtained as 14f (0.20 g, 68.8 %), m.p.

123-125 . ¹H NMR (400 MHz, CDCl₃): δ 12.35 (s, 1H,

thiazolidine-5-CH=), 8.10 (m, 1H, ArH), 7.79 (m, 2H, ArH), 7.71

(m, 2H, ArH), 7.53 (t, 1H, J = 8.0 Hz, ArH), 7.47 (d, 1H, J = 8.0

Hz, ArH), 7.27 (s, 1H, pyrrole-1-H), 7.19 (d, 1H, J = 8.0 Hz,

ArH), 4.32 (q, 2H, J = 4.0 Hz, CH₃CH₂O), 2.59 (d, 3H, pyrrole-

5-CH₃), 2.44 (d, 3H, pyrrole-3-CH₃), 1.38 (t, 3H, J = 4.0 Hz,

CH₃CH₂O-); ¹³C NMR (400 MHz, CDCl₃): δ 11.58, 14.42, 14.83,

29.71, 59.84, 110.12, 114.53, 118.24, 120.02, 121.70, 122.99,

124.44, 125.39, 125.96, 129.69, 130.03, 131.45, 132.54, 135.04,

142.09, 148.39, 150.55, 164.85, 165.84, 177.27; EI MS m/z (%):

581.0 ([M]⁺¹, 50), 223.1 (100), 194.0 (45). Anal. Calcd for

C₂₇H₂₁F₆N₃O₃S: C, 55.76; H, 3.64; F, 19.60; N, 7.23; S, 5.51.

Found: C, 55.78; H, 3.66; F, 19.61; N, 7.27; S, 5.55.

4.1.4.4. Synthesis of 5-(3,5-dimethyl-4-ethoxycar-

bonyl-1H-pyrrolyl-2-)methylene-3-(4-methoxy

phenyl)-2-(4-methoxyphenylimino)-1,3-thiazolidine-

4-one(14d)

3-(4-Methoxyphenyl)-2-(4-methoxyphenylimino)-1,3-thiazoli-

dine-4-one (13f, 0.33 g, 1.00 mmol), 2,4-dimethyl-5-formyl-1H

pyrrole-3-carboxylic ethyl ester (10, 0.20 g, 1.10 mmol), absolute

ethanol (6 mL) and anhydrouspyridine (0.30 mL) were used in

via the similar synthetic procedures of 14a and 14b. Yellow solid

was obtained as 14d (0.20 g, 39.6%), which was recrystallized

from dichloromethane/petroleum ether (0.13 g, 65.0% for

recrystallization). Second portion of product 14d was recovered

from the recrystalization filtrate as the yellow solid (0.07 g). m.p.

113-115 . ¹H NMR (400 MHz, CDCl₃): δ 12.47 (s, 1H,

thiazolidine-5-CH=), 7.39 (d, J = 12.0 Hz, 2H, ArH), 7.09 (d, 2H,

J = 8.0 Hz, ArH), 6.90 (q, 4H, J = 8.0 Hz, =NArH), 6.80 (s, 1H,

pyrrole-1-H), 4.30 (q, 2H, J = 8.0 Hz, CH₃CH₂O-), 3.88 (s, 3H,

CH₃OArNR¹R²), 3.82 (s, 3H, CH₃OArN=), 2.53 (s, 3H, pyrrole-

5-CH₃), 2.39 (s, 3H, pyrrole-3-CH₃), 1.37 (t, 2H, J=8.0 Hz,

CH₃CH₂O-); ¹³C NMR (400 MHz, CDCl₃): δ 11.05, 14.51, 55.20,

59.03, 110.57, 113.60, 114.33, 114.88, 122.16, 122.36, 125.05,

127.56, 129.25, 131.01, 140.53, 141.69, 152.01, 156.98, 164.95,

166.85; EI MS m/z (%): 505.2 ([M]⁺¹, 2), 155.2 (8), 127.2 (20).

4.1.4.7. Synthesis of 5-(3,5-dimethyl-4-

ethoxycarbonyl-1H-pyrrolyl-2-)methylene-3-(2-

fluorophenyl)-2-(2-fluorophenylimino)-1,3-

thiazolidine-4-one(14g)

3-(2-Fluorophenyl)-2-(2-fluorophenylimino)-1,3-thiazolidine-

4-one (13i, 0.30 g, 1.00 mmol), 2, 4-dimethyl-5-formyl-1H-

pyrrole-3-carboxylate ethyl ester (10, 0.20 g, 1.10 mmol),

absolute ethanol (10.0 mL) and anhydrous pyridine (0.40 mL)

were used. The synthetic method was similar to that of 14a and

14b. Yellow solid was obtained as 14g (0.36 g, 74.8%), m.p.

166-167 . ¹H NMR (400 MHz, CDCl₃): δ 8.14 (s, 1H,

thiazolidine-5-CH=), 7.76 (s, 1H, pyrrole-1-H), 7.50 (m, 2H,

2-Ethylimino-3-ethyl-1,3-thiazolidine-4-one (13a, 0.17 g, 1.00

ACCEPTED MANUSCRIPT

ArH), 7.33 (m, 2H, ArH), 7.16 (m, 3H, ArH), 7.03 (m, 1H, ArH),

4.32 (q, 2H, CH₃CH₂O-), 2.57 (s, 3H, pyrrole-5-CH₃), 2.42 (s,

3H, pyrrole-3-CH₃), 1.38 (t, 3H, CH₃CH₂O-); ¹³C NMR (400

MHz, CDCl₃): δ 11.55, 14.42, 14.76, 59.04,110.86, 114.14,

119.78, 123.27, 124.71, 125.98, 130.31, 132.03, 141.72, 164.96,

165.50; EI MS m/z (%): 481.1 ([M]⁺¹, 50), 223.1 (100), 194.1

(60). Anal. Calcd for C₂₅H₂₁F₂N₃O₃S: C, 62.36; H, 4.40; F, 7.89;

N, 8.73; S, 6.66. Found: C, 62.35; H, 4.39; F, 7.86; N, 8.72; S,

6.67.

mmol)

and

4-[2-[N-methyl-N-(2-pyridyl)]amido]ethoxy

benzaldehyde (5, 0.26 g, 1.10 mmol) were mixed in absolute

ethanol (8.0 mL). Anhydrous pyridine (0.20 mL) was added

under stirring to the reaction mixture and was refluxed for 9.0 h,

when the TLC monitor show that the material has been consumed

completely. The stirring was then stopped and some yellow

crystal-like solid precipitated from the reaction mixture while

being cooled down to room temperature. The main pure product

was obtained by column chromatography (silica gel, eluent:

petroleum ether/ethyl acetate, v/v = 10 : 1 ~ 1 : 1, system

stepwise elution) as the pale yellow solid (0.12 g, 29.2%), which

was recrystallized from ethanol to afford the pale yellow crystals

as the pure product 15a (0.04 g, 33.3% for recrystallization). m.p.

89-90℃. ¹H NMR (400 MHz, CDCl₃): δ 8.18 (d, J = 8.0 Hz, 1H,

pyridine-6-H), 7.67 (s, 1H, =CH-Ar), 7.48 (m, 3H, pyridine-4-H

& 2×(ArH)), 6.98 (m, 2H, ArH), 6.59 (t, 1H, J = 4.0 Hz,

pyridine-3-H), 6.55 (d, 1H, J = 8.0 Hz, pyridine-5-H), 4.26 (t,

2H, J = 12.0 Hz, -OCH₂CH₂N), 4.03 (t, 2H, J = 12.0 Hz, -

OCH₂CH₂N), 3.93 (q, 2H, J = 8.0 Hz, CH₃CH₂N-), 3.47 (q, 2H, J

= 8.0 Hz, CH₃CH₂-N=), 3.17 (s, 3H, CH₃N), 1.31 (t, 3H, J = 8.0

Hz, CH₃CH₂N-), 1.27 (t, 3H, J = 8.0 Hz, CH₃CH₂-N=); ¹³C NMR

(400 MHz, CDCl₃): δ 11.85, 16.03, 37.00, 46.86, 49.51, 66.32,

105.51, 111.94, 119.17, 126.79, 131.57, 137.33, 147.48, 147.92,

148.02, 157.16, 157.55, 159.80, 166.76; EI MS m/z (%): 410.1

([M]⁺¹, 3), 135.1 (100), 121.1 (35). Anal. Calcd for C₂₂H₂₆N₄O₂S:

C, 64.36; H, 6.38; N, 13.65; S, 7.81. Found: C, 64.39; H, 6.40; N,

13.68; S, 7.85.

4.1.4.8. Synthesis of 5-(3,5-dimethyl-4-

ethoxycarbonyl-1H-pyrrolyl-2-)methylene-3-(3-

fluorophenyl)-2-(3-fluorophenylimino)-1,3-

thiazolidine-4-one(14h)

3-(3-Fluorophenyl)-2-(3-fluorophenylimino)-1,3-thiazolidine-

4-one (13j, 0.70 g, 2.30 mmol), 2,4-dimethyl-5-formyl-1H-

pyrrole-3-carboxylate ethyl ester (10, 0.45 g, 2.30 mmol),

absolute ethanol (15.0 mL) and anhydrous pyridine (0.40 mL)

were used. The synthetic method was similar to that of 14a and

14b. Yellow solid was obtained as the crude product 14h (0.24 g,

21.7%), which was recrystallized from dichloromethane/

petroleum ether (v/v=1:1) to give 14h in the crystal state (0.12 g,

1

50% for recrystallization). m.p. 136-137 . H NMR (400 MHz,

CDCl₃): δ 12.37 (s, 1H, thiazolidine-5-CH=), 7.58 (q, 1H, J = 8.0

Hz, ArH), 7.32 (t, 2H, J = 8.0 Hz, ArH), 7.23 (t, 2H, J = 8.0 Hz,

ArH), 6.88 (m, 1H, ArH), 6.86 (s, 1H, pyrrole-1-H), 6.78 (d, 1H,

J = 8.0 Hz, ArH), 6.73 (m, 1H, ArH), 4.31 (q, 2H, CH₃CH₂O),

2.55 (s, 3H, pyrrole-5-CH₃), 2.40 (s, 3H, pyrrole-3-CH₃), 1.38 (t,

3H, CH₃CH₂O-); ¹³C NMR (400 MHz, CDCl₃): δ 11.41, 14.44,

14.82, 59.84, 108.60, 109.13, 111.52, 113.90, 116.47, 123.37,

124.09, 124.96, 130.62, 132.12, 140.97, 149.78, 152.52, 165.17,

166.21; EI MS m/z (%): 481.1 ([M]⁺¹, 60), 223.1 (100), 194.1

(60). Anal. Calcd for C₂₅H₂₁F₂N₃O₃S: C, 62.36; H, 4.40; F, 7.89;

N, 8.73; S, 6.66. Found: C, 62.38; H, 4.41; F, 7.90; N, 8.76; S,

6.69.

4.1.5.2. Synthesis

of

5-[[4-[2-[N-methyl-N-(2-

pyridyl)]amido] ethoxy]phenylmethylene] -3-n-butyl-

-1,3-thiazolidine-2,4-dione(15b)

3-n-Butyl-2-(n-butylimino)-1,3-thiazolidine-4-one (13b, 0.23

g,

1.00

mmol),

4-[2-[N-methyl-N-(2-pyridyl)]amido]

ethoxybenzaldehyde (5, 0.26 g, 1.10 mmol), anhydrous pyridine

(0.20 mL) and absolute ethanol (10.0 mL) were added into a

flask. The synthetic method was similar to that of 15a. Yellow

solid was obtained as 15b (0.31 g, 72.7%), which was

recrystallized from ethanol to afford pale yellowish powder (0.10

4.1.4.9. Synthesis

of

5-(3,5-dimethyl-4-

ethoxycarbonyl-1H-pyrrolyl-2-)methylene-3-(4-

fluorophenyl)-2-(4-fluorophenylimino)-1,3-

thiazolidine-4-one (14i)

1

g, 32.3% for recrystallization), m.p. 117-118℃. H NMR (400

MHz, CDCl₃): δ 8.16 (d, 1H, J = 8 Hz, pyridine-6-H), 7.84 (m,

1H, pyridine-4-H), 7.48 (m, 3H, =CHAr, & ArH), 6.98 (d, 2H, J

= 8.0 Hz, ArH), 6.58 (t, 1H, J = 4.0 Hz, pyridine-3-H), 6.52 (d,

1H, J = 8.0 Hz, pyridine-5-H), 4.26 (m, 2H, -OCH₂CH₂N-), 4.02

(m, 2H, J = 12.0 Hz, -OCH₂CH₂N-), 3.75 (t, 2H, J = 8.0 Hz,

CH₃CH₂CH₂CH₂N-), 3.15 (s, 3H, CH₃N-), 1.60 (m, 2H,

CH₃CH₂CH₂CH₂N-), 1.36 (m, 2H, CH₃CH₂CH₂CH₂N-), 0.95 (t,

3H, J = 8.0 Hz, CH₃CH₂CH₂CH₂N-); ¹³C NMR (400 MHz,

CDCl₃): δ 13.89, 19.87, 29.56, 35.76, 44.47, 61.38, 66.16,

106.25, 114.36(2C), 116.05, 117.96, 129.86(2C), 132.36, 143.36,

148.17, 154.16, 158.66, 164.15, 173.06. 173.06; EI MS m/z (%):

411.0 ([M]⁺¹, 1), 135.1 (100), 121.1 (60). Anal. Calcd for

C₂₂H₂₅N₃O₃S: C, 64.21; H, 6.12; N, 10.21; S, 7.79, Found: C,

64.23; H, 6.13; N, 10.22; S, 7.76.

3-(4-Fluorophenyl)-2-(4-fluorophenylimino)-1,3-thiazolidine-

4-one (13k, 0.51 g, 1.60 mmol), 2,4-dimethyl-5-formyl-1H-

pyrrole-3-carboxylate ethyl ester (10, 0.33 g, 1.60 mmol),

absolute ethanol (10.0 mL) and anhydrous pyridine (0.30 mL)

were used with the similar synthetic method to that of 14a and

14b. Yellow solid was obtained as 14i (0.42 g, 51.3%), which

was recrystallized from dichloromethane/petroleum ether to give

14i as crystals (0.17 g, 40.5% for recrystallization). Another

portion of the product 14i was recovered from the mother liquor

as the yellow solid (0.23 g). m.p. 170-171 . ¹H NMR (400 MHz,

CDCl₃): δ 12.39 (s, 1H, thiazolidine-5-CH=), 7.46 (m, 2H, ArH),

7.28 (m, 2H, ArH), 7.05 (t, 2H, J = 8.0 Hz, ArH), 6.93 (m, 2H,

ArH), 6.83 (s, 1H, pyrrole-1-H), 4.31 (q, 2H, CH₃CH₂O-), 2.55

(s, 3H, pyrrole-5-CH₃), 2.40 (s, 3H, pyrrole-3-CH₃), 1.38 (t, 3H,

CH₃CH₂O-); ¹³C NMR (400 MHz, CDCl₃): δ 11.40, 14.45, 14.80,

59.85, 109.58, 113.92, 115.87, 116.51, 122.47, 123.17, 125.04,

130.06, 131.69, 140.71, 165.14, 166.66ꢁEI MS m/z (%): 481.1

([M]⁺¹, 60), 223.0 (100), 194.0 (60). Anal. Calcd for

C₂₅H₂₁F₂N₃O₃S: C, 62.36; H, 4.40; F, 7.89; N, 8.73; S, 6.66.

Found: C, 62.38; H, 4.42; F, 7.93; N, 8.75; S, 6.69.

4.1.5.3. Synthesis

of

5-[[4-[2-[N-methyl-N-(2-

pyridyl)]amido] ethoxy]phenylmethylene]-3-iso-

propyl-2-(hydroxylimino)-1,3-thiazolidine-4-

one(15c)

3-iso-Propyl-2-hydroxyimino-1,3-thiazolidine-4-one

(13c,

0.30 g, 1.80 mmol), 4-[2-[N-methyl-N-(2-pyridyl)]amido]ethoxy

benzaldehyde (5, 0.40 g, 1.50 mmol) were mixed together in a

flask, to which was added with absolute ethanol (10.0 mL) and

anhydrous pyridine (0.20 mL) under stirring. The reaction

mixture was heated under refluxing for 3.5 h before cooling

down to r. t. Large amount of yellow solid appeared at the bottom

4.1.5. Synthetic procedures of target molecules 15

4.1.5.1. Synthesis of 3-ethyl-2-(ethylimino)-5-[4-

[2-[N-methyl-N-(pyridine-2-yl)amino] ethoxy]

benzylidene] -1,3-thiazolidine-4-one (15a)

1

of the flask, therefore the upper clear liquid could be removed by

decanting as much as possible. The residue was added with a

small amount of ethanol (3.0 mL) to recrystallize the crude

product. Large amount of brown powder solid was obtained by

filitration and washed with small amount of ethanol, dried under

vacuo to give solid as the first portion of 15c (0.30 g). The

second portion of 15c (0.10 g) could be recovered from the

filtrate (Total amount of crude 15c is 0.40 g, 64.7%). The

obtained solid were combined and recrystallized from ethanol to

give yellow powder 15c (0.35 g, 87.5% for recrystallization).

(0.31 g, 52.7%), m.p. 89-90 . H NMR (400 MHz, CDCl₃): δ

ACCEPTED MANUSCRIPT

8.15 (d, J=4.0 Hz, 1H, pyridine-6-H), 7.76 (s, 1H, =CHAr), 7.47

(d, 1H, pyridine-4-H), 7.42 (d, 2H, ArH), 7.34 (m, 4H, ArH),

7.16 (d, 2H, ArH), 6.92 (d, 2H, J = 8.0 Hz, ArH), 6.87 (d, 2H, J =

8.0 Hz, ArH), 6.58 (m, 1H, J = 4.0 Hz, pyridine-3-H), 6.52 (d,

1H, pyridine-5-H), 4.23 (m, 2H, -OCH₂CH₂N-), 4.01 (t, 2H, -

OCH₂CH₂N-), 3.15 (s, 3H, CH₃N-), 2.41 (s, 3H, CH₃ArN-), 2.36

(s, 3H, CH₃ArN=); ¹³C NMR (400 MHz, CDCl₃): δ 20.65, 37.01,

49.53, 66.33, 105.54, 112.01, 114.67, 118.15, 126.12, 129.51,

130.66, 131.84, 145.87, 151.64, 158.12, 166.49; EI MS m/z (%):

535 ([M+H]⁺¹, 95). Anal. Calcd for C₃₂H₃₀N₄O₂S: C, 71.88; H,

5.66; N, 10.48; S, 6.00. Found: C, 71.87; H, 5.63; N, 10.49; S,

6.02.

1

m.p. 120-121 . H NMR (400 MHz, CDCl₃): δ 8.18 (d, J = 4.0

Hz, 1H, pyridine-6-H), 7.59 (s, 1H, =CHAr), 7.48 (t, 1H,

pyridine-4-H), 7.41 (d, 2H, ArH), 7.00 (d, 2H, J = 12.0 Hz, ArH),

6.60 (m, 1H, pyridine-3-H), 6.54 (d, 1H, pyridine-5-H), 5.44 (m,

1H, R¹R²NCH(CH₃)₂), 4.28 (t, 2H, J = 4.0 Hz, -OCH₂CH₂N-),

4.03 (t, 2H, J = 4.0 Hz, -OCH₂CH₂N-), 3.16 (s, 3H, -CH₃N-),

1.57 (d, 6H, J = 8.0 Hz, R¹R²NCH(CH₃)₂); ¹³C NMR (400 MHz,

CDCl₃): δ 18.68, 38.15, 49.36, 50.00, 66.31, 105.63, 111.96,

115.35, 119.41, 126.11, 132.13, 132.68, 137.37, 147.98, 158.09,

160.83, 168.11, 193.90; EI MS m/z (%): 413.1 ([M+H]⁺¹, 2),

135.2 (100), 121.1 (50). Anal. Calcd for C₂₁H₂₄N₄O₃S: C, 61.14;

H, 5.86; N, 13.58; S, 7.77. Found: C, 61.11; H, 5.83; N, 13.64; S,

7.79.

4.1.5.6. Synthesis

of

5-[[4-[2-[N-methyl-N-(2-

pyridyl) amido]ethoxy]phenylmethylene] -3-(4-

methoxyphenyl)-2-(4-methoxyphenylimino)-1,3-

thiazolidine-4-one(15f)

3-(4-Methoxyphenyl)-2-(4-methoxyphenylimino)-1,3-

thiazolidine-4-one (0.40 g, 1.30 mmol), 4-[2-[N-methyl-N-(2-

pyridyl)]amido]ethoxybenzaldehyde (0.26 g, 1.10 mmol),

anhydrous pyridine (0.20 mL) and absolute ethanol (10.0 mL)

were put into a clean flask. The following procedures are similar

to that of the compound 15a. The crude product was obtained as

the crude product 15f (0.33 g, 52.9%), recrystallized with ethanol

to obtain pale yellow solid as 15f (0.10 g, 30.3% for

recrystallization), m.p. 138-139 .¹H NMR (400 MHz, CDCl₃): δ

8.16 (d, J = 4.0 Hz, 1H, 6-pyridine-H), 7.76 (s, 1H, 4-pyridine-

H), 7.48 (s, 1H, -CH=), 7.42 (d, 2H, ArH), 7.37 (d, 2H, 2,6-

ArHC=), 7.04 (d, 2H, 2,6-ArHN=), 6.93 (d, 2H, 3,5-ArHN-),

6.91 (d, 2H, 3,5-ArHC=), 6.88 (d, 2H, 3,5-ArHN=), 6.62 (s, 1H,

3-pyridine-H), 6.58 (s, 1H, 5-pyridine-H), 4.22 (m, 2H, -CH₂O-),

4.03 (m, 2H, -CH₂N-), 3.85 (s, 3H, CH₃OArN-), 3.83 (s, 3H,

CH₃OArN=), 3.15 (s, 3H, CH₃N-); ¹³C NMR (400 MHz, CDCl₃):

δ 37.01, 49.18, 55.21, 66.33, 105.53, 111.65, 114.67, 118.13,

121.85, 126.14, 127.23, 128.79, 130.66, 137.53, 141.33, 147.46,

151.66, 156.78, 158.27, 159.68, 160.32, 166.50; EI MS m/z (%):

566.2 ([M]⁺¹, 2), 254.2 (100), 240.1 (70), 150.1 (65), 135.2 (30).

Anal. Calcd for C₃₂H₃₀N₄O₄S: C, 67.82; H, 5.34; N, 9.89; S, 5.66.

Found: C, 67.80; H, 5.37; N, 9.92; S, 5.68.

4.1.5.4. Synthesis

of

5-[[4-[2-[N-methyl-N-(2-

pyridyl)]amido] ethoxy]phenylmethylene] -3-phenyl-

2-(phenylimino)-1,3-thiazolidine-4-one(15d)

3-Phenyl-2-phenylimino-1,3-thiazolidine-4-one (13f, 0.40 g,

1.50

mmol),

4-[2-[N-methyl-N-(2-pyridyl)]amido]ethoxy

benzaldehyde (5, 0.38 g, 1.50 mmol), anhydrous pyridine (0.20

mL) and absolute ethanol (10.0 mL) were mixed together in a

clean flask. The reaction system was heated under refluxing for

1h and then cooled down to r. t. until a large amount of yellow

substances appeared from bottom of solution. The upper clear

solution was then decanted, to which was added with ethanol (3.0

mL) to recrystallize the crude product. Brown powder like solid

was filtered and washed with small amount of ethanol, dried

under vacuo to give the product 15d (0.35 g). The second portion

of compound 15d (0.30 g) could be recovered from the filtrate

(Total amount of crude 15c is 0.60 g, 85.5%). These two portions

of solid were combined and recrystallized from ethanol to afford

yellow powder as 15d (0.58 g, 89.2% for recrystallization), m.p.

155-157 . ¹H NMR (400 MHz, CDCl₃): δ 8.15 (d, J=4.0 Hz, 1H,

pyridine-6-H), 7.78 (s, 1H, pyridine-4-H), 7.55 (t, 2H, ArH), 7.42

(m, 3H, ArH & =CHAr), 7.37 (m, 4H, ArH), 7.18 (t, 1H, ArH),

6.98 (d, 2H, J = 8.0 Hz, ArH), 6.93 (d, 2H, J = 8.0 Hz, ArH),

6.57 (m, 1H, J = 4.0 Hz, pyridine-3-H), 6.53 (d, 1H, pyridine-5-

H), 4.23 (m, 2H, -OCH₂CH₂N-), 4.01 (t, 2H, -OCH₂CH₂N-), 3.14

(s, 3H, CH₃N-); ¹³C NMR (400 MHz, CDCl₃): δ 32.38, 37.36,

49.52, 66.69, 105.79, 111.84, 115.05, 118.55, 120.76, 120.99,

124.75, 126.40, 128.20, 131.41, 131.82, 134.87, 137.38, 147.85,

148.47, 151.18, 158.14, 160.33, 166.80, 171.45; EI MS m/z (%):

507 ([M+H]⁺¹, 98). Anal. Calcd for C₃₀H₂₆N₄O₂S: C, 71.12; H,

5.17; N, 11.06; S, 6.33. Found: C, 71.15; H, 5.19; N, 11.09; S,

6.36.

4.1.5.7. Synthesis

pyridyl)]amido] ethoxy]phenylmethylene] -3-(3-

chloro-2-methyl phenyl-2-(3-chloro-2-methyl

of

5-[[4-[2-[N-methyl-N-(2-

phenylimino)-1,3-thiazolidine-4-one(15g)

3-(3-Chloro-2-methylphenyl)-2-(3-chloro-2-methylphenyl

imino)-1,3-thiazolidine-4-one (13, 0.50 g, 1.40 mmol), 4-[2-[N-

methyl-N-(2-pyridyl)]amido]ethoxybenzaldehyde (10, 0.40 g,

1.50 mmol) and anhydrous pyridine (0.20 mL) were mixed in

absolute ethanol (10.0 mL) in a flask. The mixture was

conducted with the similar method to that of the compound 15d.

The yellow powder like solid was obtained as 15g (0.30 g,

1

35.5%.), m.p. 63-66 , H NMR (400 MHz, CDCl₃): δ 8.16 (m,

1H, 6-pyridine-H), 7.79 (m, 1H, 4-pyridine-H), 7.50 (m, 1H,

ArH), 7.45 (m, 1H, ArH), 7.40 (d, 2H, ArH), 7.32 (t, 1H, ArH),

7.25 (m, 1H, ArCH=), 7.18 (m, 1H, ArH), 7.11 (t, 1H, ArH), 6.95

(d, 2H, ArH), 6.77 (d, 1H, ArH), 6.56 (m, 1H, pyridine-3-H),

6.50 (d, 1H, pyridine-5-H), 4.24 (t, 2H, -NCH₂CH₂O-), 3.99 (t,

2H, -OCH₂CH₂N-), 3.13 (s, 3H, CH₃N-), 2.33 (s, 3H, CH₃ArN-),

2.15 (s, 3H, CH₃ArN=); ¹³C NMR (400 MHz, CDCl₃): δ 14.25,

15.86, 36.06, 61.55, 66.35, 106.43, 114.56 (2C), 116.27, 118.16,

119.28, 124.52, 126.36, 126.66, 126.99, 127.46, 127.55, 128.64,

130.05 (2C), 134.77, 135.66, 135.85, 137.15, 138.53, 143.51,

148.38, 148.57, 154.32, 158.51, 158.88, 167.01. EI MS m/z (%):

605 ([M]⁺¹, 90). Anal. Calcd for C₃₂H₂₈Cl₂N₄O₂S: C, 63.68; H,

4.68; N, 9.28; S, 5.31. Found: C, 63.70; H, 4.72; N, 9.30; S, 5.30.

4.1.5.5. Synthesis

of

5-[[4-[2-[N-methyl-N-(2-

pyridyl)]amido] ethoxy]phenylmethylene]-3-(4-

methylphenyl)-2-(4-methylphenylimino)-1,3-

thiazolidine-4-one(15e)

3-(4-Methylphenyl)-2-(4-methylphenylimino)-1,3-thiazolidine

-4-one (13e, 0.30 g, 1.10 mmol), 4-[2-[N-methyl-N-(2-

pyridyl)]amido]ethoxybenzaldehyde (5, 0.26 g, 1.10 mmol),

anhydrous pyridine (0.20 mL) and absolute ethanol (10.0 mL)

were reaction with the similar method to that of the compound

15d. The yellow powder was obtained as the desired product 15e

4.1.5.8. Synthesis of 5-[[4-[2-[N-methyl-N-(2-pyri-

As an evaluation of the selectivity of these PTP1B inhibitor,

CDC25B phosphatase test were also conducted on some of these

_{dyl)]amido] ethoxy]phenylmethylene] -}A_3-C₍₃C_-tE_riP_flT_uoE_rD_oMANUSCRIPT

methylphenyl-2-(3-trifluoromethyl phenyl)-1,3-

thiazolidine-4-one (15h)

new molecules to evaluate the selectivity as well their activities

as the possible anti-neoplastic reagents. The substances were

evaluated as inhibitors of the recombinant human CDC25B.

Phosphatase activity of a purified GST-CDC25B fusion protein

was determined by measuring the rate of dephosphorylation of

the fluorigenic, generic phosphatase substrate fluoresce in

diphosphate in 384-well microtiter plates[65]. The compounds

were preincubated with the enzyme for 1 h and subsequently the

reaction was initiated by addition of the substrate (5mM).

Fluorescence intensity was determined in the linear range of the

reaction after 1 h of incubation at room temperature. Na₃VO₄

were used as the positive controls and DMSO as negative control

to evaluate the biological activity of the target compounds for

CDC25B tests.

3-(3-Trifluoromethylphenyl)-2-(3-trifluoromethylphenyl)-1,3-

thiazolidine-4-one (13h, 0.67 g, 1.60 mmol), 4-[2-[N-methyl-N-

(2-pyridyl)]amido]ethoxybenzaldehyde (10, 0.43 g, 1.60 mmol),

bsolute ethanol (10.0 mL), anhydrous pyridine (0.20 mL) were

mixed together into a clean flask. The synthetic procedures are

similar to that of the compound 15a. The oily substance was

obtained as the crude product 15h (0.65 g, 59.5%), which was

then recrystallized with ethanol to afford the pale yellow solid as

the pure product 15h (0.45 g, 69.2% for recrystallization), m.p.

41-43 . ¹H NMR (400 MHz, CDCl₃): δ 8.15 (m, 1H, pyridine-6-

H), 7.84 (s, 1H, R¹R²NArH), 7.79 (s, 1H, ArCH=), 7.50 (m, 1H,

ArH), 7.42 (d, 2H, ArH), 7.33 (t, 3H, ArH), 7.29 (m, 1H,

pyridine-4-H), 7.03 (m, 3H, ArH), 6.98 (m, 2H, ArH), 6.59 (m,

1H, pyridine-3-H), 6.53 (d, 1H, pyridine-5-H), 4.26 (t, 2H, -

NCH₂CH₂O-), 4.02 (t, 2H, -OCH₂CH₂N-), 3.13 (s, 3H, CH₃N-);

¹³C NMR (400 MHz, CDCl₃): δ 35.82, 61.32, 66.12, 106.22,

114.33 (2C), 116.02, 117.93, 118.62, 120.71, 123.61, 124.13

(2C), 125.62, 125.92, 126.83, 129.21, 129.83(2C), 130.32,

131.21, 131.43, 132.31, 133.01, 138.32, 143.33, 148.12, 149.31,

154.13, 158.32, 166.92, 158.61. EI MS m/z (%): 566.2 ([M]⁺¹, 2),

254.2 (100), 240.1 (70), 150.1 (65), 135.2 (30); EI MS m/z (%):

643.60 ([M+H]⁺¹, 60). Anal. Calcd for C₃₂H₂₄F₆N₄O₂S: C, 59.81;

H, 3.76; N, 8.72; S, 4.99. Found: C, 59.82; H, 3.75; N, 8.75; S,

5.02.

4.3. Molecular modeling

Molecular modeling was carried out with the Tripos molecular

modeling packages SYBYLX-2.0. All the molecules for docking

were built using standard bond lengths and angles from SYBYL-

X2.0/base Builder and were then optimized using the Tripos

force field for 2000 generations two times or more, until the

minimized conformers of the ligand were the same. The flexible

docking method, called Surflex-Dock, docks the ligand

automatically into the active binding site of the receptor enzyme

by using a protocol-based approach and an empirically derived

scoring function.[66] The protocol is

a

computational

representation of a putative ligand that binds to the intended

binding site and is a unique and essential element of the docking

algorithm. [67] The scoring function in Surflex-Dock, which

contains hydrophobic, polar, repulsive, entropic and solvation

terms, was trained to estimate the dissociation constant (Kd)

expressed in -log (Kd)².[68]

4.2. Biological activity assay against phosphatases

4.2.1. PTP1B inhibition test

PTP1B inhibitory activity of these PTP1B inhibitors in vitro

may be determined via screening assay described as the

following. GST-fusion human PTP1B was cloned into pGEX-KG

plasmid. PTPs were over expressed as GST-fusion proteins in

Escherichia coli BL21-Conden plus (DE3) and purified through

affinity chromatography. The enzymatic activities of PTP1B

catalytic domain were determined at 30ꢀ by monitoring the

hydrolysis of pNPP. Dephosphorylation of pNPP generates the

product pNP, which can be monitored at an absorbance for 405

nm. For the 50% inhibition concentration (IC₅₀) calculation,

inhibition assays were performed with 30 nmol/L recombinant

enzyme, 2.0 mmol/L pNPP in 50 mmol/L MOPs (3-[N-

morpholino]propane-sulfonic acid) at pH = 6.5, and the inhibitors

diluted around the estimated IC₅₀values. The IC₅₀was calculated

with Prism 4 software according to the formula: % Inhibition =

100/(1+[IC₅₀/[I]]k), where k is the Hill coefficient. Oleanolic acid

was used as the positive controls and DMSO as the negative

control to evaluate the biological activities of the target

compounds against PTP1B.

4.3.1. Molecular Docking

Prior to docking, the protein was prepared by removing water

molecules, the ligands, and other unnecessary small molecules

from the crystal structure of PTP1B (PDB code: 2VEY[16]); and

then the hydrogen atoms were added to the protein also. Surflex-

Dock default settings were used for other parameters, such as the

number of starting conformations per molecule (set to 0), the size

to expand search grid (set to 8Å), the maximum number of

rotatable bonds per molecule (set to 100), and the maximum

number of poses per ligand (set to 20).

During the docking procedure, all of the single bonds in

residue side chains inside the defined PTP1B binding pocket

were regarded as rotatable or flexible, and the ligand was allowed

to be rotated on all single bonds and move flexibly within the

tentative binding pocket. The atomic charges were recalculated

using the Kollman all-atom approach for the protein and the

Gasteiger Hückel approach for the ligand. The binding

interaction energy was calculated to include van der Waals,

electrostatic, and torsional energy terms defined in the Tripos

force field. The structure optimization was performed for 20,000

generations using a genetic algorithm, and the 20-best-scoring

ligand-protein complexes were kept for further analyses. The -log

(Kd)²values of the 20-best-scoring complexes, which represented

the binding affinities of ligand with the target enzyme, ranged

over a wide scope of functional classes (10^-2~10^-9). Therefore,

only the highest-scoring 3D structural model of the ligand-bound

PTP1B was chosen to define the binding interaction.

4.2.2. CDC25B inhibition test

CDC25B has attracted considerable interest, as its

overexpression has been correlated to the malignancy of

tumors[54]. CDC25B are potential oncogenes for the over

expression in a large number of human carcinomas, such as

colorectal[55], oesophagus[56], gastric[57], prostate[58],

ovary[59], lymphomas and melanomas[60], cutaneous malignant

melanoma[60] and other tumor cell lines including breast, lung,

head and neck tumors[61]. Transgenic mice over expressing

CDC25B in the mammary and saliva glands of developed

hyperplasia or showed increasing susceptibility to chemically-

induced mammary tumors[62, 63]. Therefore, selective inhibitors

of CDC25B could offer potential interesting reagents for treating

the proliferation of tumor cells[64].

4.3.2. Prediction of descriptors of target molecules

The pharmacokinetic descriptors for predicting ADMET

features of the target compounds were predicted via VolSurf tool

packaged in SYBYLX-2.0 workstation (Table 2).[69] The

development of cardiac arrhythmias. As a consequence, a number

of drugs associated with QT prolongation have been removed

from the market over the past decade. All cases of drug-induced

QT prolongation are associated with a particular ion channel

known as hERG.

ACCEPTED MANUSCRIPT

compounds were sketched in Chemdraw, saved as the *.cdx files,

and then input into Chem3D, minimization via AM method

before being saved as *.mol files. The mol file of each molecule

was then input into the sibyl workstation, minimized after being

added with Tripos force field and Gasteiger-Hückel charge. All

the named molecules were saved in a database before being

calculated using the VolSurf package.

Some other descriptors of the target molecules were also

predicated using calculation method of via the ChemBioDraw

software imbedded Excel software (Table 3). The various

parameters including both the steric properties and the electric

descriptors were calculated by the options available modules in

Cambridge software package, which was imbedded in an

worksheet of Excel. To obtain these parameters, molecular

dynamics calculation and energy minimization were sequentially

run on each molecule with the default values (Step interval = 2.0

fs, Frame interval = 10 fs, Terminate after 1000 steps) at first, the

parameters of compounds 14a-14i, 15a-15h were then computed

via Chem3D Ultra (Cambridge software), respectively.

The meanings of these descriptors were explained in the

following context, respectively. MW means the Molecular

weight of the compounds. LogS value refers to the aqueous

solubility, which has long been recognized as a key molecular

property in pharmaceutical science. The P_appvalue means the

apparent permeability coefficients of the compounds crossing the

Caco-2 cell membrane. Caco-2 is the human intestinal epithelial

cell line derived from a colorectal carcinoma, which has gained

in popularity in vivo human absorption surrogate evaluation.

PB(%) mean the protein binding rate via the “in silico”

quantitative models to predict the binding affinity with human

serum albumin (HSA), which are very useful in pharmaceutical

industries to provide the pharmacokinetic properties in the early

drug discovery stage. VD value denotes the abbreviation for

volume distribution, which is the volume that accounts for the

total dose administration of a drug based on the observed plasma

concentration. BBB indicator denotes the ability to cross the

blood-brain barrier (BBB). The prediction of blood-brain barrier

(BBB) permeation is a big challenge in drug design[70]. MS

stand for the abbreviation for the metabolic stability of a drug in

human CYP3A4 cDNA-expressed microsomal preparation,

which offers a suitable approach to predict the metabolic stability

of an external compound. The hERG inhibitory effect represents

an important safety consideration in drug discovery. Although

hERG inhibition seems to be dependent on 3D-pharmacophoric

features disposition of a drug, VolSurf descriptors are widely

used to simulate hERG inhibition, with amazingly good results.

Since QT prolongation is an important biomarker for the

Table 1. Biological activity against PTP1B and CDC25B of the target compounds

The explanations and category of the calculated parameters

are listed as the following. General descriptors included the

molecular weight (MW). Hydrophobic parameters included Log

value of partition coefficient (LogP) and partition coefficient

(PC). Steric parameters included the following descriptors: molar

refraction (MR), molecular shape index (Ovality), the number of

hydrogen bond donor (HBD), the number of hydrogen bond

acceptor (HBA), connolly accessible area (CAA), connolly

molecular area (CMA) and connolly solvent excluded volume

(CSEV). Molecular topology index included the following:

balaban index (BI), number of rotatable bonds (NRB), polar

surface area (PSA), radius (R), shape coefficient (SC) and total

valence connectivity (TVC). Energy related index including the

following items: energy of the higher occupied molecular orbit

(HOMO), energy of the lowest unoccupied molecular orbit

(LUMO) and total energy after energy minimization by MM

semi-empirical method (TE). All these calculated descriptors

mentioned above were summarized in Table 3.

PTP1B

CDC25B

Inhibition rate at 20

ꢀg/mL (%) (±

deviations)

Inhibition rate at

5ꢀg/mL (%) (±

deviations)

IC₅₀(ꢀg/mL)

(±deviations)

IC₅₀(ꢀmol/mL)

Inhibition rate at

5ꢀg/mL (%) (±

deviations)

IC₅₀(ꢀg/mL)

IC₅₀(ꢀmol/mL)

Compd

(±deviations)

14a

14b

14c

14d

14e

14f

95.76 (±0.34)

99.93 (±0.04)

28.43 (±15.26)

98.69 (±0.84)

NA

48.99 (±6.13)

81.14 (±1.87)

NA

3.86 (±0.70)

NA

8.66 (±1.57)

NA

80.17 (±1.86)

83.66 (±0.75)

NA

2.05 (±0.48)

0.74 (±0.09)

NA

5.83

1.66 (±0.20)

NA

47.66 (±8.41)

NA

76.51 (±5.40)

NA

1.30 (±0.20)

NA

2.74

NA

14g

14h

14i

98.28 (±1.55)

96.30 (±0.51)

94.45 (±0.34)

42.12 (±0.72)

NA

44.96 (±11.03)

74.18 (±7.95)

82.66 (±9.22)

NA

64.20 (±1.41)

82.79 (±2.11)

84.68 (±1.50)

NA

1.76 (±0.16)

1.63 (±0.29)

1.38 (±0.14)

NA

3.66 (±0.33)

3.39 (±0.60)

2.87 (±0.29)

NA

3.29 (±0.63)

2.93 (±0.29)

NA

6.83 (±1.31)

6.09 (±0.60)

NA

15a

15b

15c

15d

15e

15f

NA

67.55 (±1.67)

NA

19.26 (±4.81)

NA

98.55 (±1.67)

NA

1.02 (±0.09)

NA

2.47 (±0.22)

NA

15g

15h

NA

OA

SV

NA

1.09 (±0.07)

2.39 (±0.15)

NA

ACCEPTED MANUSCRIPT

NA

0.17 (±0.03)

0.91 (±0.15)

a

NA: Not available; ^bOA: Oleanolic acid was used as the reference compound for PTP1B inhibitor evaluation test; ^cSV: Sodium vanadate was used as the

reference compound for CDC25B inhibitor evaluation test.

Table 2. The predicted pharmacological parameters of the target compounds via the tool of VolSurf models in SYBYLX-2.0 software package.

Compds

14a

14b

14c

14d

14e

14f

MW

LogS

-3.992

-5.828

-6.155

-6.078

-6.433

-5.701

-5.407

-5.342

-5.510

-6.087

-5.409

-4.833

-7.305

-7.733

-7.436

-7.953

-7.329

P_app.

0.153

0.62

PB(%)

54.968

85.855

89.407

85.605

94.930

90.664

71.822

77.999

79.488

95.537

93.103

70.413

103.600

111.089

113.916

114.618

123.165

VD

BBB

MS

hERG

-0.148

-0.541

-0.604

-0.574

-0.663

-0.441

-0.428

-0.374

-0.389

-0.652

-0.567

-0.214

-0.774

-0.830

-0.822

-0.874

-0.750

-0.544

-0.041

-0.021

-0.271

-0.080

-0.825

-0.338

-0.533

-0.455

0.148

0.523

0.913

-0.155

-0.300

-0.022

-0.454

0.515

0.425

0.511

0.379

-0.463

-0.273

0.214

-1.207

-1.419

-1.028

-1.418

-0.715

?

351.42

445.53

473.59

505.59

542.48

581.53

481.51

410.53

411.52

412.51

506.62

534.67

566.67

603.56

642.61

-0.650

-0.785

-0.670

-0.862

-0.265

-0.341

-0.299

-0.351

-0.893

-0.384

0.091

0.638

0.428

0.611

-0.052

0.264

0.206

0.235

1.002

0.818

0.271

0.937

0.957

0.766

0.944

0.318

14g

14h

14i

15a

15b

15c

15d

15e

15f

0.018

-0.575

-0.031

-0.053

-0.226

-0.104

-0.756

-1.265

-1.421

-1.242

-1.508

-1.021

15g

15h

Medicinal

range:

Medicinal

range:

Low to Medium

values

Medicinal range: ꢂ8*10-

Medicinal range: ꢂ

About

500

Criteria

Medium

ꢃꢄꢅꢆ

ꢇ

6 cm/s

97.412

ꢂ6.3 L/Kg

Table 3. The predicted parameters of the target compounds and the reference compound via Chem3D Ultra software in the Cambridge software package.

Compds

14a

14b

14c

14d

14e

14f

LogP

2.297

MR

PC

Ovality

1.58

1.32

1.34

1.32

1.34

1.67

1.68

1.72

1.36

1.70

1.79

1.83

1.86

1.79

HBD HBA CAA

CMA

CSEV

BI

NRB

PSA

Radium SC

TVC

HOF

94.87

3.21

2

1

0

1

0

5

4

6

4

10

6

5

6

5

7

5

622.10 333.52 288.08

539246

5.00 91.23

6.00 71.00

8.00 89.46

6.00 71.00

8.00 71.00

6.00 71.00

8.00 57.50

9.00 62.21

7.00 77.73

8.00 57.50

6.00 1.00 0.000

8.00 0.00 0.000

8.00 1.00 0.000

9.00 0.00 0.000

8.00 0.00 0.000

8.00 1.00 0.000

8.00 0.00 0.000

8.00 1.00 0.000

9.00 1.00 0.000

0

5.037 128.68 6.40

6.011 138.76 7.39

4.784 141.61 6.23

7.127 148.37 8.82

6.879 140.63 8.16

5.353 129.11 6.68

4.188 120.65 4.60

4.296 118.08 5.48

4.099 116.91 3.73

6.838 150.71 6.92

7.813 160.80 7.92

6.586 163.64 6.76

8.929 170.41 9.34

391.34 188.76 160.16 1316979

418.62 205.81 178.85 1757944

428.88 212.60 186.96 2324268

416.71 206.21 182.65 2199597

407.19 198.77 170.63 3646429

726.22 401.16 349.34 1705816

734.29 404.27 350.64 1732185

731.52 404.50 351.20 1757944

787.16 423.59 362.78 1249859

428.24 206.54 176.65 1267914

760.46 407.38 347.57 1238333

875.81 493.08 429.66 2599862

939.41 531.38 464.30 3304729

0

14g

14h

14i

0

15a

15b

15c

15d

15e

15f

111

10.00 0.00 0.000 56.02

9.00 0.00 0.000

10.00 1.00 0.00

-25.5

419

11.00 0.00 0.000

11.00 1.00 0.000

10.00 1.00 0.000

355

974.26 551.28 481.46 4171781 10.00 75.96

928.70 535.22 484.72 4010193 8.00 57.50

90.7

301

15g

15h

OA

ACCEP₉T₅₃E_.5D₁MANUSCRIPT

8.681 162.66 8.69

1.84

1.20

0

2

0

11

547.87 481.29 6141450 10.00 57.50

11.00 0.00 0.000

6.00 1.00 0.000

1.00 1.00 1.383

-839

-808

0

7.475 133.62 5.28

-

2

358.25 174.88 166.46 1072241

0.00 0.00 0.00 5442

1.00 57.53

0.00 68.28

SV

0.000

0.00

########

4

0.92

Lee, Bicyclic and tricyclic thiophenes as protein tyrosine phosphatase 1B inhibitors, Bioorg.

Med. Chem., 14 (2006) 2162-2177.

Acknowledgments

[19] S.R. Klopfenstein, A.G. Evdokimov, A.O. Colson, N.T. Fairweather, J.J. Neuman, M.B.

Maier, J.L. Gray, G.S. Gerwe, G.E. Stake, B.W. Howard, J.A. Farmer, M.E. Pokross, T.R.

Downs, B. Kasibhatla, K.G. Peters, 1,2,3,4-Tetrahydroisoquinolinyl sulfamic acids as

phosphatase PTP1B inhibitors, Bioorg. Med. Chem. Lett., 16 (2006) 1574-1578.

[20] A.P. Combs, W. Zhu, M.L. Crawley, B. Glass, P. Polam, R.B. Sparks, D. Modi, A.

Takvorian, E. McLaughlin, E.W. Yue, Z. Wasserman, M. Bower, M. Wei, M. Rupar, P.J. Ala,

B.M. Reid, D. Ellis, L. Gonneville, T. Emm, N. Taylor, S. Yeleswaram, Y. Li, R. Wynn, T.C.

Burn, G. Hollis, P.C.C. Liu, B. Metcalf, Potent benzimidazole sulfonamide protein tyrosine

phosphatase 1B inhibitors containing the heterocyclic (S)-isothiazolidinone phosphotyrosine

mimetic, J. Med. Chem., 49 (2006) 3774-3789.

The authors are grateful to the Scientific Research Foundation

for the Returned Overseas Chinese Scholars, State Education

Ministry, P. R. China (2011), the Shaanxi Province Science and

Technology Research and Development Program of China,

International Cooperation (2013KW31-04).

Supplementary Material

Supplementary data including color figures and docking

information associated with this article can be found in the online

version at web site.

[21] S.Y. Cho, J.Y. Baek, S.S. Han, S.K. Kang, J.D. Ha, J.H. Ahn, J.D. Lee, K.R. Kim, H.G.

Cheon, S.D. Rhee, S.D. Yang, G.H. Yon, C.S. Pak, J.K. Choi, PTP-1B inhibitors:

cyclopenta[d][1,2]-oxazine derivatives, Bioorg. Med. Chem. Lett., 16 (2006) 499-502.

[22] X. Li, A. Bhandari, C.P. Holmes, A.K. Szardenings, α,α-Difluoro-β-ketophosphonates as

potent inhibitors of protein tyrosine phosphatase 1B, Bioorg. Med. Chem. Lett., 14 (2004)

4301-4306.

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ACCEPTED MANUSCRIPT

Highlight

1. 17 new compounds with two heterocycles were designed, synthesized as PTP1B inhibitor.

2. All the molecules were tested for the inhibitory activities against PTP1B and CDC25B.

3. Compound 14i containing pyrrole and thiazolidine-4-one showed IC₅₀=6.09µM for PTP1B.

4. Compound 14b displayed IC₅₀of 8.66µM and 1.66µM for PTP1B and CDC25B respectively.

5. Dock modes between 14b/14h/14i and PTP1B, ADMET properties and SAR were analyzed.

Article Doi

Design and synthesis of new potent PTP1B inhibitors with the skeleton of 2-substituted imino-3-substituted-5-heteroarylidene-1,3-thiazolidine-4-one: Part I

DOI: 10.1016/j.ejmech.2016.05.060

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Article abstract of DOI:10.1016/j.ejmech.2016.05.060

Full text of DOI:10.1016/j.ejmech.2016.05.060

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