Design and synthesis of novel 3,5-bis-N-(aryl/heteroaryl) carbamoyl-4-aryl-1,4-dihydropyridines as small molecule BACE-1 inhibitors

Bioorganic & Medicinal Chemistry 21 (2013) 6893–6909

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Design and synthesis of novel 3,5-bis-N-(aryl/heteroaryl)

carbamoyl-4-aryl-1,4-dihydropyridines as small molecule

BACE-1 inhibitors

Nima Razzaghi-Asl ^a, Omidreza Firuzi ^a, Bahram Hemmateenejad ^a,c, Katayoun Javidnia ^a,b

,

Najmeh Edraki ^a, Ramin Miri ^a,b,

⇑

^aMedicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, PO Box 3288-71345, Shiraz, Iran

^bDepartment of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, PO Box 3288-71345, Shiraz, Iran

^cChemistry Department, Shiraz University, Shiraz, Iran

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 8 July 2013

Revised 4 September 2013

Accepted 12 September 2013

Available online 20 September 2013

Alzheimer disease (AD) is a neuronal dementia for which no treatment has been consolidated yet. Major

pathologic hallmark of AD is the aggregated extracellular amyloid-b plaques in the brains of disease suf-

ferers. Ab-peptide is a major component of amyloid plaques and is produced from amyloid precursor pro-

tein (APP) via the proteolysis action. An aspartyl protease known as b-site amyloid precursor protein

cleaving enzyme (BACE-1) is responsible for this proteolytic action. Distinctive role of BACE-1 in AD path-

ogenesis has made it a validated target to develop anti-Alzheimer agents. Our structure-based virtual

screening method led to the synthesis of novel 3,5-bis-N-(aryl/heteroaryl) carbamoyl-4-aryl-1,4-dihydro-

pyridine BACE-1 inhibitors (6a–6p; in vitro hits). Molecular docking and DFT-based ab initio studies using

B3LYP functional in association with triple-f basis set (TZV) proposed binding mode and binding energies

of ligands in the active site of the receptor. In vitro BACE-1 inhibitory activities were determined by enzy-

matic ﬂuorescence resonance energy transfer (FRET) assay. Most of the synthesized dihydropyridine scaf-

folds were active against BACE-1 while 6d, 6k, 6n and 6a were found to be the most potent molecules

Keywords:

Alzheimer

BACE-1

Dihydropyridine

Virtual screening

with IC₅₀values of 4.21, 4.27, 4.66 and 6.78 lM, respectively. Superior BACE-1 inhibitory activities were

observed for dihydropyridine derivatives containing fused/nonfused thiazole containing groups, possibly

attributing to the additional interactions with S2–S3 subpocket residues. Relatively reliable correlation

between calculated binding energies and experimental BACE-1 inhibitory activities was achieved

(R²= 0.51). Moreover, compounds 6d, 6k, 6n and 6a exhibited relatively no calcium channel blocking

activity with regard to nifedipine suggesting them as appropriate candidates for further modiﬁcation(s)

to BACE-1 inhibitory scaffolds.

1. Introduction

By 1999, various independent researchers discovered BACE-1 as

the aspartyl protease b-site APP-cleaving enzyme. BACE-1 initiates

Alzheimer disease (AD) is a neurodegenerative disorder clini-

cally recognized by progressive decline in memory and cognition.¹

AD accounts for most cases of dementia in the elderly popula-

tion.^2,3Histological examination of the brains of affected individu-

als has revealed the existence of signiﬁcant synaptic loss.⁴The

main neuropathological characteristics of AD are the presence of

extracellular amyloid plaques and intracellular Neuroﬁbrillary tan-

gles (NFT) in the brain. The amyloid plaques result from the depo-

sition of aggregated amyloid beta (Ab) peptides. Ab peptides

(mainly as Ab₄₀and Ab₄₂) are the product of two consecutive pro-

teolytic cleavages on a large trans-membrane protein; amyloid

precursor protein (APP).

the amyloid cascade by cleavage of APP.^5,6Subsequent cleavage of

the C-terminus of APP by

Ab₄₀and Ab₄₂. In the nonamyloidogenic pathway,

cleaves APP leading to the generation of neuroprotective peptide

sAPPa ⁷

Inhibition of b and

c-secretase results in the formation of

a

-secretase

.

c

-secretase enzymes by small molecule

blockers is a promising approach to ﬁnd therapeutic agents for

AD. In this regard, inhibition of b-secretase is very importance

since several studies have shown that inhibition of

c-secretase

may have some mechanism-based adverse effects.^8,9The strategy

of BACE-1 blockade for AD-therapy has been further supported

by the observation of normal phenotype in BACE-1 knockout

mice.¹⁰

Many research groups have focused on the development of

⇑

Corresponding author. Tel./fax: +98 711 2303872.

E-mail address: mirir@sums.ac.ir (R. Miri).

BACE-1 inhibitors.^11,12BACE-1 inhibitors were primarily designed

http://dx.doi.org/10.1016/j.bmc.2013.09.033

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N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

Table 1

mimicking the structures of substrate and transition-state analogs

with sub-nanomolar activities,¹¹but poor pharmacokinetic proﬁle

of peptidic inhibitors prevented them from being further devel-

oped to oral bioavailable CNS drugs. Recently signiﬁcant numbers

of BACE-1 inhibitors have been developed as small molecule candi-

dates possessing drug-like features.^3,13In this regard, a diverse set

of chemical scaffolds have been proposed as BACE-1 inhibitors.^14,15

For more information, readers are referred to the CheMBL

database.¹⁶

Docking validation results for different holo PDB structures of BACE-1 using

AutoDock4.2

RMSD from

reference

structure (Å) cluster (%)

Population in

the optimum

Maximum

no. of energy runs (Å)

evaluations

GA

Resolution

PDB

code

0.515

0.533

0.478

0.414

1.157

0.479

0.845

0.756

0.486

1.616

47

80

88

27

18

34

16

35

31

25

5.0 ꢀ 10⁶

100

1.70

1.80

2.20

1.75

1.60

1.80

1.79

1.80

2.05

2B8L

2B8V

2IRZ

2IS0

2QMF

2VJ9

2QZL

2VNM

2P8H

2VKM

Among nitrogen containing heterocy1es, 1,4-dihydropyridine

(DHP) is a privileged scaffold with a broad range of pharmacolog-

ical activities. Several DHPs have found their way to the drug mar-

ket as calcium channel blockers.^17,18However, little attention has

been directed towards the BACE-1 blocking activity of these com-

pounds.^19,20Choi and co-workers have reported novel N-methyl-

sulfonamide-1,4-dihydropyridine derivatives with IC₅₀values of

8–30 l

M in a cell-based assay.¹⁹

population in the top-ranked cluster of AutoDock output ﬁle, we

selected 2IRZ as the most appropriate crystallographic structure

for further modeling studies. Results of docking validation are

summarized in Table 1.

DHPs have many features that make them privileged chemical

scaffolds. They can be converted to the quaternary amines leading

to their retention in the brain.^21,22Another characteristic feature of

DHP scaffold is the possibility of structural modiﬁcations via intro-

ducing various chemical substituents in different positions of the

DHP ring and hence providing remarkable changes in pharmaco-

logical proﬁle.^19,23

2.1.2. Virtual binding afﬁnities of DHP molecules

The focus of this study was to obtain novel DHP-based BACE-1

inhibitors with the aim of introducing important structural

prerequisites for the optimal BACE-1 inhibitory activity. Our initial

design strategy was on the basis of developing nonesteriﬁed car-

boxamide-based 1,4-DHP derivatives with possibly less calcium

channel blocking activity (less side effect)³³while at the same time

mimicking the previously analyzed BACE-1 binding patterns.^34,35

To this end, a pool of 3,5-bis-N-(aryl/alkyl) carbamoyl-4-aryl/

alkyl-1,4-DHP structures was constructed and subjected to docking

simulation (2IRZ; http://www.rcsb.org/) Scheme 1.

Docking free energy of binding and the presence of key ligand–

residue binding interactions in the BACE-1 active site were consid-

ered as screening ﬁlters. In the next step, candidate structures

were subjected to synthesis followed by enzymatic assessment

for their BACE-1 inhibitory activity.

Docking simulations provided some insight into the ligand–

receptor interactions. Predicted docking afﬁnities for in vitro hits

are summarized in Table 2. To get more information, 2D schematic

representation of ligand–receptor interactions for compound 6d is

displayed in Figure 1.

Moreover; ligand–amino acid systems were subjected to opti-

mization process with functional B3LYP in association with tri-

ple-f basis set (TZV) to obtain the geometry of H-bonds (Table 3).

In continuation to our interest in novel BACE-1 inhibitors,²⁴and

also to further extend the scope of 1,4-DHPs as privileged small

molecule medicinal scaffolds,^25,26we hereby report molecular

modeling, synthesis, and in vitro FRET-based BACE-1 inhibitory

activities of some new 2,6-dimethyl-3,5-bis-N-(aryl) carbamoyl-

4-aryl-(aryl/heteroaryl)-1,4-dihydropyridine derivatives.

2. Result and discussions

2.1. Molecular docking

Modern drug discovery strategies greatly rely on crystallo-

graphic 3D structural information of the biomolecular targets.

Available X-ray crystallographic data on Protein Data Bank facili-

tated the performance of virtual structure based drug discovery

projects aiming at BACE-1 as a molecular target for Alzheimer dis-

ease. In this regard, docking simulation is a key tool in structural

molecular biology and computer-assisted drug design (CADD). To

explain more, a process to mimic the stereoelectronic ﬁt of ligand

and receptor via a lowest energy pathway may be best represented

by docking simulations.²⁷Numerous successful applications of

molecular docking in drug discovery efforts have been

reported.^28,29One of the most popular docking packages is Auto-

Dock. Literature review shows that AutoDock has offered several

fruitful advantages in the ﬁeld of drug design (The AutoDock web-

site. http://autodock.scripps.edu).³⁰

2.2. Amino acid decomposition analysis

Determination of individual ligand–amino acid binding ener-

gies in the active site of the receptor (amino acid decomposition

analysis) is a valuable technique in understanding the key interac-

tions between enzyme and inhibitor. Moreover; the process of

pharmacophore design might be much more efﬁcient regarding de-

tailed binding energetics of the system. Molecular docking is a

popular method for ﬁnding probable binding mode and binding

afﬁnity of ligand in the active site of the protein with known 3D

structure. However, with all advantageous features, docking simu-

lation provides no information on individual ligand–amino acid

binding energies in the active site of the biomolecular target. In

this regard, ab initio-based amino acid decomposition analysis is

a useful approach to the energetics of intermolecular interactions.

According to this rationale and also to compare the effect of li-

gand structural changes on the contribution of individual residues

of BACE-1 active site to the binding energy, we decided to estimate

the ligand–residue interaction energies for designed 1,4-DHPs via

2.1.1. Docking validation

It is a commonly accepted practice to test a performance of a

typical docking protocol via checking its ability in predicting pre-

dominant binding mode of

a cognate (co-crystallographic)

ligand.³¹This procedure is performed via extracting the

structure of a cognate ligand and re-docking it into its receptor (self-

docking). Success is often interpreted via comparison of root mean

square deviation (RMSD) of the Cartesian coordinates of the atoms of

the ligand in the docked and crystallographic conformations.

Ten PDB derived BACE-1 structures were subjected to docking

validation procedure. PDB structures were chosen on the basis of

crystallographic resolutions and also considering relative similar-

ity of co-crystallized ligands to the DHP structures (http://

www.rcsb.org/). Regarding RMSD values³²and also conformation

N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

6895

Scheme 1. Structure based virtual screening of designed DHP library for BACE-1 inhibitory activity

Table 2

the comparative evaluation possible. It is well understood from

the diagram of binding energies (Fig. 2) that nearly similar trends

are captured by various designed molecules. This distribution pat-

tern might indicate relatively similar binding poses of 3,5-dicarba-

moylated DHPs in the BACE-1 active site.

Docking simulation results for BACE-1 inhibitors (PDB deposited code: 2IRZ)

a

Compd. code

D

G_b(kcal/mol)

6a

6b

6c

6d

6e

6f

6g

6h

6i

6j

6k

6l

6m

6n

6o

6p

ꢁ11.31

ꢁ11.27

ꢁ11.07

ꢁ11.14

ꢁ11.25

ꢁ11.01

ꢁ11.23

ꢁ11.21

ꢁ11.28

ꢁ11.01

ꢁ11.25

ꢁ11.06

ꢁ11.03

ꢁ11.30

ꢁ11.19

ꢁ11.33

2.3. Conformational analysis

The difference of ligand electronic energies in the optimized

and docked conformers may be indicative of conformational insta-

bility gain upon binding to the receptor. Accordingly, we decided to

model the conformational distortions occurred in the DHP struc-

tures as a result of binding to the active site of BACE-1. To this

end, compound 6d (IC₅₀= 4.21 lM) was chosen as a model system

for determining the imposed conformational deviation in the

BACE-1 active site. In all calculations, dielectric effects of water

(i.e. 80) were considered using COSMO model³⁶

.

Different energies of the evaluated ligand conformations may

be a direct outcome of varied internal energies of ligand in its

docked and optimized conditions within

E_instability). In this regard, E_instabilityis indicative of the torsional

instability and should be considered in estimation of binding ener-

gies. For calculation of E_instability, optimized conformation of 6d

was obtained in water and relevant energy was assigned to this un-

bound conformation.

a biological media

quantum mechanical methods. To this end, BACE-1 residues sitting

within a deﬁnite cut off radius from mass center of the ligands

(regarding previous crystallographic data; http://www.rcsb.org/)

were selected as the model system. Our proposed model included

DHP molecules and amino acids constructing the active site of

BACE-1 (thirty-four residues). Relevant residues were as follows:

Gly11, Gln12, Gly13, Tyr14, Leu30, Asp32, Gly34, Ser35, Val69,

Pro70, Tyr71, Thr72, Gln73, Phe108, Phe109, Ile110, Trp115,

Ile118, Tyr198, Lys224, Ile226, Asp228, Ser229, Gly230, Thr231,

Thr232, Asn233, Arg235, Arg307, Ser325, Thr329, Val332, Ala335,

and Glu339. All selected amino acids have been previously de-

tected as important residues in binding patterns of various crystal-

lographic BACE-1 inhibitors (http://www.rcsb.org)³.

(D

D

E_instabilitymay be well related with the free energy of binding

via following equations:

D

G_b¼

D

H_bꢁ T

E_tꢁ P

E_bþ

E_instabilityvalues provides lower binding energies (

D

S

ð2Þ

ð3Þ

H_b

E_tb

¼

D

V ꢂ

DE_tb

¼

D

E_inst:

ð4Þ

E_tb)

Higher

D

Ligand–residue binding energies (

Eq. 1:

D

E_b) were calculated by the

and subsequently weaker ligand–receptor interactions

(DG_b)

would be expected. Conformational instability (kcal/mol) of 6d

upon binding to the BACE-1 active site was found to be

28.59 kcal/mol. The estimated instability value might be indicative

of noticeable conformational shifts toward less stable geometric

pose. Different geometric poses of 6d in the docked and minimum

energy conformations are shown in Figure 3.

It seemed that functional groups being involved in key interac-

tions with receptor, exhibited sensible torsional distortions and

1,4-DHPs need not necessarily interact with the BACE-1 active

site in their optimized conformation. To acquire a quantitative

D

E_b¼ E_LRꢁ E_Rꢁ E_L:

In the above equation, E_LRstands for ligand–residue binding en-

ð1Þ

ergy, while E_Rand E_Lindicate the electronic energies of unbound

residues and ligands, respectively. Various binding energies be-

tween 1,4-DHPs and key residues surrounding the active site of

BACE-1 were obtained independently at the B3LYP functional in

association with triple-f basis set (TZV) level of calculation

(summarized in Fig. 2). All ab initio calculations were performed

within a ﬁxed number of involved amino acids in order to make

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N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

Figure 1. 2D scheme of binding interactions between 6d and BACE-1 active site generated by LIGPLOT, PDB deposition code: 2IRZ.

view on induced conformational changes, estimated geometric

parameters (dihedral angles for compound 6d) are summarized

in Table 4.

angles (Table 4). These conformational distortions afforded a prop-

er orientation of amide functionalities to make key H-bonds with

Asp228, Gly230 and Thr72 residues of the BACE-1 active site.

Appropriate spatial orientation of thiazole ring in the S2 sub-

pocket may be a direct outcome of twisted N12–C14 bond (ꢂ80°

distortion). In our interaction maps, the acquired binding pose sup-

ported key H-bond with NH of Thr232 backbone (Fig. 3). Similar

dihedral shifts could be observed around a N30–C33 bond (ꢂ96°

distortion) leading to the lipophilic contacts of another thiazole

ring with Lys224 (ꢁ0.76 kcal/mol) and Tyr198 (ꢁ1.22 kcal/mol)

residues of the BACE-1 active site.

Our calculations demonstrated that binding of 6d to the BACE-1

active site was associated with perceptible torsional distortions

conﬁrming the obtained instability gain (28.59 kcal/mol). For more

elucidation, these changes are highlighted in Table 4. Obtained

data demonstrated that C1–C10 and C4–C29 bonds were the

molecular sites bearing signiﬁcant rotations with regard to DHP

ring (P100° distortion) exhibited via altered C2–C1–C10–O11,

C2–C1–C10–O12, C2–C4–C29–N30, and C2–C4–C29–N32 dihedral

N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

6897

Table 3

Estimated hydrogen bond distances (Å) for docked DHP BACE-1 inhibitors

Compd. code

Participant amino acids^a

Optimized hydrogen bond distances (Å)

Involved atom of the ligand

Involved atom of the amino acid

6a

Thr72

Gln73

2.36

2.49

2.38

2.02

2.57

2.27

2.45

2.63

2.51

2.09

2.29

2.23

2.01

2.58

2.06

2.13

2.17

2.50

2.34

2.30

2.02

2.43

2.18

2.44

2.59

2.46

2.05

2.34

2.08

2.16

2.49

2.05

2.14

2.46

2.15

2.50

2.38

2.17

2.54

2.31

2.36

2.54

2.36

2.00

2.33

2.32

2.31

2.42

2.43

1.89

2.70

2.79

2.19

2.21

2.61

2.13

2.04

2.52

2.29

2.65

2.43

2.11

2.21

2.30

2.56

2.37

2.25

2.19

2.79

2.19

2.50

2.44

2.16

2.25

(Amide) O

(DHP) NH

(Amide) NH

(Furyl) O

(Thiazole) N

(Amide) O

(DHP) NH

(Amide) NH

(Thiazole) N

(Nitro) O

(Amide) O

(DHP) NH

(Amide) NH

(Nitro) O

(Amide) O

(DHP) NH

(Amide) NH

(Thiazole) N

(Nitro) O

(Amide) O

(DHP) NH

(Amide) NH

(Benzothiazole) N

(Nitro) O

(Amide) O

(DHP) NH

(Amide) NH

(Nitro) O

(Amide) O

(DHP) NH

(Amide) NH

(Thiazole) N

(Nitro) O

(Amide) O

(DHP) NH

(Amide) NH

(Benzothiazole) O

(Nitro) O

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

(Side chain) OH

(Backbone) NH

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

(Backbone) OH

(Side chain) NH₂

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

Asp228

Gly230

Thr231

Thr232

Thr72

6b

Gln73

Asp228

Gly230

Thr232

Arg235

Thr72

6c

Gln73

Asp228

Gly230

Arg235

Thr72

+

(Side chain) NH₂

6d

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

(Backbone) NH

Gln73

Asp228

Gly230

Thr232

Arg235

Thr72

+

(Side chain) NH₂

6e

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

(Backbone) NH

Gln73

Asp228

Gly230

Thr232

Arg235

Thr72

+

(Side chain) NH₂

6f

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

(Backbone) NH

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

(Backbone) NH

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

(Backbone) NH

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

(Backbone) NH

Gln73

Asp228

Gly230

Asn233

Thr72

6g

Gln73

Asp228

Gly230

Thr232

Asn233

Thr72

6h

6i

Gln73

Asp228

Gly230

Thr232

Asn233

Thr72

(Amide) O

(DHP) NH

(Amide) NH

(benzothiazole) N

(Nitro) O

(Ethoxy) O

(Amide) O

(DHP) NH

(Amide) NH

(Nitro) O

(Amide) O

(DHP) NH

(Amide) NH

(Benzothiazole) N

(Nitro) O

Gln73

Asp228

Gly230

Thr232

Asn233

Arg307

Thr72

+

(Side chain) NH₂

6j

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

(Backbone) NH

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

(Backbone) NH

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

(Backbone) NH

Gln73

Asp228

Gly230

Asn233

Thr72

6k

Gln73

Asp228

Gly230

Thr232

Asn233

Thr72

6l

(Amide) O

(DHP) NH

Gln73

Asp228

Gly230

Thr231

Thr72

(Amide) NH

(Pyrrole) NH

(Amide) O

(DHP) NH

(Amide) NH

(Benzothiazole) N

6m

Gln73

Asp228

Gly230

Thr232

(continued on next page)

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N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

Table 3 (continued)

Compd. code

Participant amino acids^a

Optimized hydrogen bond distances (Å)

Involved atom of the ligand

Involved atom of the amino acid

6n

Thr72

Gln73

Asp228

Gly230

Thr232

Thr72

1.99

2.68

2.71

2.17

2.84

2.29

2.46

2.32

2.06

2.45

2.53

(Amide) O

(DHP) NH

(Amide) NH

(Benzothiazole) N

(Amide) O

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

(Backbone) NH

(Side chain) OH

(Backbone) C@O

(Side chain) COO^ꢁ

(Backbone) C@O

(Backbone) NH

6o

Gln73

(DHP) NH

Asp228

Gly230

Thr232

Arg235

(Amide) NH

(Thiazole) N

(Methoxy) O

+

(Side chain) NH₂

a

H-bond distances have been reported as (H. . ...acceptor) lengths.

Figure 2. DFT-based binding energies of 3,5-dicarbamoylated DHPs into the BACE-1 active site residues, binding energies attributed to H-bonds are bold-highlighted in the

upper table. In the lower chart, most favored binding energies are addressed toward their site of interactions.

Other noticeable rotations occurred around a C2–C20 (ꢂ30°)

and C23–N26 (ꢂ50°) bonds leading to the disturbed co-planarity

of nitro substituent with the phenyl ring and appropriate H-bond

interaction with guanidinium side chain of Arg235. The results of

this study may be best explained by this statement that ligands tol-

erated some bond torsions to achieve key binding features in the

receptor active site leading to the stabilization of ligand–receptor

complex.

3,5-bis-N-(aryl/heteroaryl)

carbamoyl-1,4-dihydropyridines

(6a–p) was achieved via molecular condensation of corresponding

aldehydes (4), appropriate N-aryl-acetoacetamide derivatives

(3a–e) and ammonium acetate (5) in absolute ethanol (Scheme 2).

Our results showed that corresponding DHPs were obtained in

good yields and shorter reaction times using nano-Fe₃O₄catalyst

in EtOH (Table 7). Several other results for nano-catalyzed synthe-

sis of DHPs also indicated a higher synthetic yields and shorter

reaction times in ethanol.^37,38An efﬁcient synthesis of some ester-

iﬁed 1,4-DHP scaffolds by nano-Fe₃O₄particles was previously re-

ported in the literature.³⁹

2.4. Chemistry

In an attempt to provide the structural features for BACE-1 inhib-

itory activity, a series of DHP derivatives were synthesized (6a–p,

Table 7). Synthesis of desired 2,6-dimethyl-4-aryl/heteroaryl-

On the basis of general knowledge, following mechanistic

hypothesis may be proposed for nano-catalyzed cyclocondensation

reaction (Scheme 3).³⁸It is believed that nano-Fe₃O₄particles

N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

6899

Figure 3. Conformation of 6d in (a) DFT-optimized and (b) docked pose (BACE-1 active site).

possess efﬁcient Lewis acid sites incorporating higher electrophi-

licity into N-arylacetoacetamides and enabling facile approaching

of ammonia and enamines.

and sulfur atom of thiazole/benzothiazole rings in DHPs.

Moreover; methyl substituent of the thiazole ring might be

responsible for further hydrophobic interactions with

Lys224 in the S⁰2 subpocket. It should be noted that theses

hydrophobic contacts might be weaker for 6b-Lys224 and

6b-Val332 interactions (Fig. 2). Similar reports can be found

in the literature.⁴¹

2.5. Biological assessments

2.5.1. BACE-1 inhibitory activity and structure activity

relationship (SAR)

(3) As it could be clearly understood from the data in Table 5

and 4-nitrophenyl and 2-nitrophenyl aromatic groups on

the C4 position of DHP ring might be better tolerated

without signiﬁcant loss of activity when compared to

BACE-1 inhibitory activities of synthesized 1,4-DHPs were

determined using a ﬂuorescence resonance energy transfer (FRET)

based BACE-1 kit that included BACE-1 enzyme and speciﬁc APP-

based peptide substrate (Rh-EVNLDAEFK-quencher). IC₅₀values

were determined for compounds exhibiting minimum 50%

3-nitrophenyl substituent (6d; IC₅₀= 4.21

lM; 6k; IC₅₀=

4.27 M). Our theoretical studies showed that this priority

l

BACE-1 inhibitory activity at 10

for three to four times for each compound under study. Results

are summarized in Table 5.

A relatively reliable correlation could be obtained between

docking afﬁnities and in vitro BACE-1 inhibitory activities in terms

of logI₁₀(I₁₀: BACE-1 inhibition% at 10 lM) (Fig. 4).

Generally, the active site of BACE-1 is composed of 8 sub-

pockets that are designated as S1, S2, S3, S4, S⁰1, S⁰2, S⁰3 and S⁰4.

These subsites are the correspondent binding pockets of BACE-1

active site for the peptidic inhibitors (P1, P2, P3, P4, P⁰1, P⁰2, P⁰3

and P⁰4).⁴⁰SAR analysis of DHP molecules in accordance with

molecular modeling results indicated that designed DHP molecules

made H-bonds and hydrophobic contacts with some of these

sub-pockets. For more information on sub-pockets, readers may

be referred to the previous publication.³⁴Regarding this, following

points may be concluded:

l

M. Experiments were repeated

might be attributed to efﬁcient H-bonds with Arg235 and

Asn233 in 4-nitrophenyl and 2-nitrophenyl-substituted

series, respectively. However the introduction of polar het-

eroaryl rings to the C4 position of DHP ring maintained

the binding afﬁnity (6a and 6l). Compound 6l (containing

2-pyrrole ring) was the most potent compound in the

3,5-bis-N-(5-methylisoxazol-3-yl) carbamoyl substituted

series. Similar trend could be observed for compound 6a

which possessed IC₅₀value of 6.78 lM (Table 5).

(4) Contribution of H-bonds to the binding afﬁnity of more polar

DHP molecules (6c, 6f, 6i, 6j, and 6l) might be greatly

depended on solvation/desolvation effects. This could be

apparently detected considering lower binding afﬁnity of

6i when compared to 6h and 6k (despite close docking afﬁn-

ities, Table 2). Indeed, larger polarities may be paid for by

higher desolvation penalties.

(5) 3,5-Bis-N-(5-methyl-3-isoxazolyl) carbamoylated DHPs (6c,

6f, 6j and 6l) had lower BACE-1 inhibitory activities (Table 5).

For these derivatives, modeling studies showed no sign of

key H-bond(s) to Thr232. This might be attributed to the

geometric orientation of isoxazole ring in the active site of

BACE-1. Compounds 6a, 6b, 6d, 6e, 6g, 6h, 6i, 6k, 6m, 6n

and 6o interacted with S2 subpocket through H-bonds

between Thr232 backbone NH and thiazole/benzothiazole

nitrogen atom.

(6) A charge-assisted H-bond interaction with Arg235 guanidi-

nium group might be envisaged through H-acceptor moie-

ties (ꢁNO₂) of the para position of C4-substituted aryl ring.

It should be noted that 6o interacted with Arg235 via its

para-methoxy substituent. While compared to 6b, 6c, 6d

and 6e, this interaction was supported by lower binding

energy (ꢁ8.85 kcal/mol). Moreover, binding poses indicated

that aromatic ring of DHPs stacked near to the guanidinium

(1) Among studied molecules, achieving a desirable BACE-1

inhibitory activity might be possible through electrostatic

interactions including H-bonds. All the 3,5-dicarbamoylated

DHP hits participated in key H-bond interactions with

Asp228 (supported by favorable binding energies; Fig. 2)

and Gly230 sitting in the region between S1 and S2 subsites.

In this regard, diagram of DFT-based binding energies (Fig. 2)

might be characterized by two distinguishing minima that

were related to the charge-assisted H-bonds of DHP mole-

cules with Asp228 and Arg235 residues of the BACE-1 active

site (Fig. 5).

(2) The most potent compound (6d) had an IC₅₀value of

4.21 lM. Docking/ab initio studies revealed that compound

6d might participate in key H-bonds with Arg235, Thr232,

Thr72, Asp228, and Gly230 in the S1 and S2 subsites.

Detailed analysis of hydrophobic binding patterns showed

that these contacts might be envisaged between Val332

group so that its

p electrons might tend to stabilize a partial

6900

N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

Table 4

Dihedral angles of 6d in the DFT-optimized and docked conformers (BACE-1 active site)

Dihedral angle

Angle (degree)

docked pose

Dihedral angle

Angle (degree)

docked pose

Optimized pose

C(3)–C(1)–C(2)–C(4)

C(3)–C(1)–C(2)–C(20)

C(10)–C(1)–C(2)–C(4)

C(10)–C(1)–C(2)–C(20)

C(2)–C(1)–C(3)–C(7)

C(2)–C(1)–C(3)–N(9)

C(10)–C(1)–C(3)–C(7)

C(10)–C(1)–C(3)–N(9)

C(2)–C(1)–C(10)–O(11)

C(2)–C(1)–C(10)–N(12)

C(3)–C(1)–C(10)–O(11)

C(3)–C(1)–C(10)–N(12)

C(1)–C(2)–C(4)–C(5)

C(1)–C(2)–C(4)–C(29)

C(20)–C(2)–C(4)–C(5)

C(20)–C(2)–C(4)–C(29)

C(1)–C(2)–C(20)–C(21)

C(1)–C(2)–C(20)–C(25)

C(4)–C(2)–C(20)–C(21)

C(4)–C(2)–C(20)–C(25)

C(1)–C(3)–N(9)–C(5)

C(1)–C(3)–N(9)–H(8)

C(7)–C(3)–N(9)–C(5)

C(7)–C(3)–N(9)–H(8)

C(2)–C(4)–C(5)–C(6)

C(2)–C(4)–C(5)–N(9)

C(29)–C(4)–C(5)–C(6)

C(29)–C(4)–C(5)–N(9)

C(2)–C(4)–C(29)–N(30)

C(2)–C(4)–C(29)–O(32)

C(5)–C(4)–C(29)–N(30)

C(5)–C(4)–C(29)–O(32)

C(4)–C(5)–N(9)–C(3)

C(4)–C(5)–N(9)–H(8)

C(6)–C(5)–N(9)–C(3)

C(6)–C(5)–N(9)–H(8)

C(1)–C(10)–N(12)–C(14)

C(1)–C(10)–N(12)–H(13)

O(11)–C(10)–N(12)–C(14)

O(11)–C(10)–N(12)–H(13)

C(10)–N(12)–C(14)–S(15)

C(10)–N(12)–C(14)–N(19)

8.480

ꢁ116.802

ꢁ167.550

67.240

8.435

ꢁ116.795

ꢁ167.562

67.208

H(13)–N(12)–C(14)–S(15)

H(13)–N(12)–C(14)–N(19)

N(12)–C(14)–S(15)–C(16)

N(19)–C(14)–S(15)–C(16)

N(12)–C(14)–N(19)–C(17)

S(15)–C(14)–N(19)–C(17)

C(14)–S(15)–C(16)–C(17)

S(15)–C(16)–C(17)–C(18)

S(15)–C(16)–C(17)–N(19)

C(16)–C(17)–N(19)–C(14)

C(18)–C(17)–N(19)–C(14)

C(2)–C(20)–C(21)–C(22)

C(25)–C(20)–C(21)–C(22)

C(2)–C(20)–C(25)–C(24)

C(21)–C(20)–C(25)–C(24)

C(20)–C(21)–C(22)–C(23)

C(21)–C(22)–C(23)–C(24)

C(21)–C(22)–C(23)–N(26)

C(22)–C(23)–C(24)–C(25)

N(26)–C(23)–C(24)–C(25)

C(22)–C(23)–N(26)–O(27)

C(22)–C(23)–N(26)–O(28)

C(24)–C(23)–N(26)–O(27)

C(24)–C(23)–N(26)–O(28)

C(23)–C(24)–C(25)–C(20)

C(4)–C(29)–N(30)–C(33)

C(4)–C(29)–N(30)–H(31)

O(32)–C(29)–N(30)–C(33)

O(32)–C(29)–N(30)–H(31)

C(29)–N(30)–C(33)–N(34)

C(29)–N(30)–C(33)–S(38)

H(31)–N(30)–C(33)–N(34)

H(31)–N(30)–C(33)–S(38)

N(30)–C(33)–N(34)–C(35)

S(38)–C(33)–N(34)–C(35)

C(30)–C(33)–C(38)–H(37)

N(34)–C(33)–C(38)–C(37)

C(33)–N(34)–C(35)–C(36)

C(33)–N(34)–C(35)–C(37)

N(34)–C(35)–C(37)–S(38)

C(36)–C(35)–C(37)–S(38)

C(35)–C(37)–S(38)–C(33)

94.890

ꢁ85.402

179.717

ꢁ0.013

ꢁ179.819

ꢁ0.086

0.109

179.774

ꢁ0.186

0.172

ꢁ179.879

ꢁ179.810

0.417

179.811

ꢁ0.410

0.017

175.968

ꢁ4.372

179.687

0.001

ꢁ179.785

ꢁ0.096

0.095

179.739

ꢁ0.174

0.171

ꢁ179.746

ꢁ179.840

0.448

179.818

ꢁ0.461

ꢁ0.024

ꢁ0.394

179.819

0.381

ꢁ179.833

ꢁ1.577

178.501

178.633

ꢁ1.288

0.051

177.491

0.791

ꢁ3.034

ꢁ179.734

ꢁ178.765

1.528

ꢁ179.212

ꢁ179.183

0.730

0.677

ꢁ3.599

176.342

ꢁ50.182

131.126

133.860

ꢁ44.832

ꢁ9.530

ꢁ3.529

176.133

56.622

ꢁ126.098

ꢁ123.375

57.905

ꢁ9.503

172.017

114.770

ꢁ63.682

99.915

172.07

114.804

ꢁ63.675

97.451

ꢁ82.835

ꢁ27.444

152.270

ꢁ9.940

ꢁ175.040

169.927

4.827

ꢁ0.468

179.818

0.475

ꢁ113.311

ꢁ57.957

121.818

ꢁ9.970

ꢁ179.810

ꢁ52.516

127.433

127.766

ꢁ52.285

ꢁ0.033

177.486

0.857

ꢁ3.050

ꢁ179.679

84.511

ꢁ95.175

ꢁ98.803

81.510

ꢁ179.743

ꢁ0.030

179.692

ꢁ0.018

0.078

ꢁ179.856

ꢁ0.093

179.839

0.061

ꢁ175.136

169.975

4.809

ꢁ179.524

ꢁ17.539

1.535

1.482

ꢁ1.133

179.926

ꢁ125.518

55.029

ꢁ1.119

179.902

ꢁ62.974

117.562

118.513

ꢁ60.951

8.876

174.009

ꢁ170.65

ꢁ5.032

179.021

3.051

55.955

ꢁ123.458

ꢁ2.014

178.279

ꢁ179.782

ꢁ0.050

179.732

0.002

8.853

174.048

ꢁ170.152

ꢁ4.958

179.054

3.119

ꢁ179.898

0.350

0.289

0.087

ꢁ175.586

ꢁ81.093

98.614

ꢁ175.682

ꢁ0.047

179.613

ꢁ0.088

179.897

0.047

positive charge on the NH of guanidinium group providing a

possible -cation interaction. Binding pose studies and

interaction energies (Fig. 2) indicated that in the case of

Asn233, these interactions were nonbonded contacts (van

der Waals) (except for 6j and 6k that contributed to H-bonds

with Asn233).

(8) In the C4 position of the DHP ring, incorporation of ﬁve-

member heterocycles (furan and pyrrol) instead of phenyl

rings might be well tolerated without signiﬁcant loss of

BACE-1 inhibitory activity (6a; 66.38% inhibition and 6d;

p

77.22% inhibition at 10 lM).

(9) Tyr71 (ﬂap residue) contributed to hydrophobic contacts

with C2 and/or C6 methyl groups of all DHP rings via its side

chain benzyl site. These interactions were associated with

binding energies in the range of ꢁ1.52 to ꢁ1.98 kcal/mol.

(10) Ligand–residue binding energies and docked poses indicated

that compounds 6e, 6h, 6i, 6k, and 6m might participate in

(7) All of the docked DHP structures represented a boat like con-

formation in the active site of the BACE-1. This boat like con-

formation of DHP ring in the active site of the BACE-1 might

take part in key H-bond with carbonyl oxygen of Gln73

backbone (Fig. 6).

N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

6901

Scheme 2. Synthesis of compounds (6a–p). Reagents and conditions: (i) xylene, reﬂux, 2–4 h, (ii) EtOH, reﬂux, 24–48 h, (iii) EtOH, 20 mol% nano-Fe₃O₄, 1–10 h.

Scheme 3. Possible mechanistic view of nano-Fe₃O₄catalyzed cyclocondensation of ammonia, N-arylacetoacetamides and aldehydes leading to DHP molecules.

favorable

p

–p

stacking contacts with Tyr198 via their fused

2.5.2. Calcium channel blocking activity

aromatic rings (supported by ꢁ2.41 to ꢁ2.99 kcal/mol).

Nearly parallel orientation of aromatic rings (Tyr198 phenyl

ring and ligand benzothiazole ring) might be responsible for

In the present work, developed compounds had essential

requirement for calcium channel blocking activity as belonging

to the DHP class. Due to this rationale, superior BACE-1 inhibitors

(6a, 6d, 6k and 6n) were evaluated as calcium channel antagonists

using high potassium ion concentration in guinea-pig ileal longitu-

dinal smooth muscle. The calcium channel blocking activities were

determined as the concentration needed to produce 50% inhibition

of the guinea-pig ileal longitudinal smooth muscle contractility

(Summarized in Table 6).

detected p–p stacking interaction (Fig. 7).

(11) Arg307 made weak to moderate lipophilic contacts with

benzothiazole containing structures. Extended well oriented

structure of 6i (C6⁰-ethoxy group) provided additional

H-bond interaction with Arg307 (NH-2 of guanidine side

chain, Fig. 7) in the S4 subpocket.

6902

N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

Table 5

In vitro BACE-1 inhibitory activities of compounds 6a–p

M^a(%)

Inhibition at 10

lM (%)

IC₅₀(lmol/L)

b

Compd.

Ar

Ar⁰

Inhibition at 50

l

6a

6b

6c

6d

6e

6f

6g

6h

6i

6j

6k

6l

6m

6n

6o

4-Methyl-2-thiazolyl

2-Thiazolyl

5-Methyl-3-isoxazolyl

4-Methyl-2-thiazolyl

2-Benzothiazolyl

5-Methyl-3-isoxazolyl

4-Methyl-2-thiazolyl

2-Benzothiazolyl

6-Ethoxy-2-benzothiazolyl

5-Methyl-3-isoxazolyl

2-Benzothiazolyl

5-Methyl-3-isoxazolyl

2-Benzothiazolyl

4-Methyl-2-thiazolyl

—

2-Furyl

95.12 4.29

46.76 6.69

42.98 11.56

88.01 3.97

90.83 6.59

ND^c

70.37 6.26

56.16 3.05

93.97 4.63

53.96 11.13

87.15 2.22

57.39 1.33

ND

66.38 5.15

28.14 6.03

23.50 5.18

77.22 3.64

49.84 2.99

11.63 3.34

34.76 6.32

49.35 2.23

49.06 5.02

12.72 1.54

65.56 4.29

24.13 8.31

44.93 2.20

69.38 7.59

44.42 10.71

ND

6.78 0.54

—

4.21 0.11

—

4.27 0.65

—

4.66 2.24

—

4-Nitrophenyl

3-Nitrophenyl

2-Nitrophenyl

2-Pyrrolyl

Phenyl

2-Nitrophenyl

4-Methoxyphenyl

6-Methyl-2-pyridyl

—

94.95 3.70

86.35 7.92

ND

6p

OM99-2^d

—

0.003( 0.00)

a

b

c

Values represent means standard error (S.E.) of three to ﬁve independent experiments.

IC₅₀values determined for compounds achieving over % 50 BACE-1inhibition at 10 M.

There was an interference of compound with ﬂuorescence resonance energy transfer (FRET) assay.

OM99 was tested at 10, 1 and 0.1 nM.

l

d

Figure 4. Correlation of docking binding energies with BACE-1 inhibitory

activities).

Figure 5. Superimposed docked poses of DHP molecules in the active site of BACE-1.

According to the obtained data, all of the assessed compounds

exhibited low calcium channel blocking activity (IC₃₀= 3.48 ꢀ 10^ꢁ6

to 2.66 ꢀ 10^ꢁ5) when compared to nifedipine (IC₃₀= 9.27 ꢀ 10^ꢁ9).

role in BACE-1 inhibitory activity of 3,5-bis-N-(aryl) carbamoylated

DHPs. Results demonstrated that the solvation/desolvation effects

should be considered in interpretation of binding afﬁnity results

of more polar DHP derivatives. SAR exploration of developed

BACE-1 inhibitors demonstrated the vital role of two carbamoyl

NHs of 3,5-disubstituted aryl/heteroaryl-carbamoyl-1,4-DHPs in

making key H-bonds with Asp228 and Gly230. Residing in the

same plane with Asp228, the amide groups of DHP molecules con-

tributed to monodentate H-bonds with aspartate oxygens of

Asp228 (H-bond distance range = 2.05ꢁ2.71 Å) and backbone car-

bonyl oxygen of Gly230 (H-bond distance range = 1.89–2.19 Å).

In conﬁrmation of previous results, our biological data suggested

that although much favoured, the direct H-bonding interactions

3. Conclusion

Small molecule BACE-1 inhibitors could be successfully devel-

oped as in vitro hits via structure-based virtual screening of a li-

brary of 3,5-bis-N-(aryl) carbamoylated DHPs. Ab initio

calculations of interaction energies were complementary to the

molecular docking simulations in modeling of DHP-based BACE-1

inhibitors. The most potent compound (6d) had an IC₅₀of

4.21

lM in the FRET assay. Molecular modeling studies showed

that well-oriented H-bond donors/acceptors might play a central

N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

6903

(http://www.rcsb.org/). The protein structure was subjected to

optimization step in order to minimize the crystallographic in-

duced bond clashes using steepest descent method by Gromacs

package.⁴³For preparation of a target protein as a template (PDB

code: 2IRZ), cognate ligand (IO2) and all crystallographic water

molecules were removed from the original receptor structure. All

the pre-processing steps for receptor crystallographic structure

were performed via WHAT IF server (http://swift.cmbi.ru.nl/

servers/html/prepdock.html/ European Molecular Laboratory

Heidelberg, Germany) and AutoDock Tools 1.5.4 program

(ADT).⁴²ADT program was used to merge nonpolar hydrogens into

related carbon atoms of the receptor and Kollman charges were

also assigned. For docked ligands, nonpolar hydrogens were added;

Gasteiger charges assigned and torsions degrees of freedom were

also allocated by ADT program. Lamarckian genetic algorithm

(LGA) was applied to model the interaction/binding between DHPs

and BACE-1 active site. Desirable independent genetic algorithm

(GA) runs were considered for each ligand under study. For

Lamarckian GA; 27,000 maximum generations; a gene mutation

rate of 0.02; and a crossover rate of 0.8 were used.

Figure 6. Possible H-binding pattern of docked DHP (6d) in the BACE-1 active site.

The grid maps characterizing the protein in the actual docking

procedure were calculated with AutoGrid (part of the AutoDock

package). The size of grid (one for each atom type in the ligand,

plus one for electrostatic interactions) was set in a way to include

not only the active site but also considerable portions of the sur-

rounding surface. For this purpose, a grid of 60 ꢀ 60 ꢀ 60 points

in x, y, and z directions was built centered on the center of mass

of the catalytic site of b-secretase with a spacing of 0.375 Å. Cluster

analysis was performed on the docked results using an RMSD tol-

erance of 2 Å.

between the ligand and two components of Asp dyad might not be

necessarily important for BACE-1 inhibition and an adaptable level

of inhibition might be expected through H-bond with Asp228.¹⁵

3,5-Bis-N-4-methyl-2-thiazolyl and 3,5-bis-N-2-benzothiazolyl

substituted DHP structures (bearing well oriented H-acceptors on

their C4-substituted aromatic rings) are ideal cases for further

extension to S4, S3, S1 and S2 subpockets with the aim of achieving

more potent small molecule BACE-1 inhibitors. Moreover, further

biological assessment of the DHP compounds (6a, 6d, 6k and 6n)

revealed signiﬁcantly lower calcium channel blocking activity with

regard to nifedipine. Therefore the offered DHP structures might be

judged as potential BACE-1 inhibitors without possible side ef-

fect(s). Further work will focus on more extended decorations

within N³- and N⁵-substituents of DHP molecules in the symmetric/

nonsymmetric framework.

Schematic 2D representations of the ligand-receptor interac-

tions were all generated using LIGPLOT.⁴⁴

4.1.2. Ab initio study

Detailed analysis of ligand–receptor interactions were per-

formed using amino acid decomposition analysis (ADA). The con-

tribution of individual ligand–amino acid interaction energies in

total binding energy was estimated using this method. Previously,

ADA has been applied by our group in analyzing BACE-1 interac-

tions with an isophthalamide-based scaffold.³⁴

Amino acid residues involved in binding of 1,4-DHP ligands to

the BACE-1 active site were chosen as a model on the basis of ob-

tained ligand–receptor binding proﬁles and various crystallo-

graphic data (http://www.rcsb.org/). For this purpose, all

involved amino acids (residues) were considered in their real elec-

trostatic state. Each residue was truncated in its C and N terminals.

4. Materials and methods

4.1. Computational section

4.1.1. Molecular docking study

Flexible-ligand docking studies were carried out using Auto-

Dock version 4.2.⁴²All the X-ray crystallographic holo structures

of BACE-1 were retrieved from the Brookhaven protein data bank

Figure 7. (a) Nearly parallel orientated aromatic rings of 6m and Tyr198 and possible

p–p stacking interaction, (b) Extended p-acceptor substituted aromatic ring of 6i

supported H-bond interaction with Arg307 in the S4 subpocket.

6904

N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

Table 6

Calcium channel antagonist activities (IC50) of compounds 6a, 6d, 6k and 6n

a

IC₁₅(M)

IC₃₀(M)

Ar’

Ar

Compd. code

(2.26 1.19) ꢀ 10^ꢁ6

10^ꢁ5

<

2-furyl

4-methyl-2-thiazolyl

2-benzothiazolyl

4-methyl-2-thiazolyl

—

6a

6d

6k

6n

(4.63 1.43) ꢀ 10^ꢁ6

(6.47 1.46) ꢀ 10^ꢁ7

(1.82 0.45) ꢀ 10^ꢁ6

(2.02 0.76 ꢀ 10^ꢁ9

(2.66 1.35) ꢀ 10^ꢁ5

4-nitrophenyl

2-nitrophenyl

—

(3.48 0.97) ꢀ 10^ꢁ6

10^ꢁ5

<

(9.27 1.24) ꢀ 10^ꢁ9

Nifedipine

a

Muscle relaxation effects have been reported as the average of at least 3 trials ( S.E.).

Table 7

Physical and analytical data of intermediates (3a–e)

Compd.

Substituent Ar

Mol. formula (Mol. Wt.)

C₇H₈N₂O₂S (184.03)

mp^a(°C)

Yield^a(%)

89

3a

158–160

3b

C₈H₁₀N₂O₃(182.07)

C₈H₁₀N₂O₂S (198.05)

120–121

173–175

91

86

3c

3d

C₁₁H₁₀N₂O₂S (234.05)

C₁₃H₁₄N₂O₃S (278.07)

214–216

206–208

65

41

3e

a

Isolated yield.

To mimic the original electron density, N-terminal was acetylated

and C-terminal was methyl amidated. All conformational and con-

ﬁgurational features were held as original X-ray structure.

Hydrogen bonds are not clearly recognized in a typical X-ray crys-

tallographic ﬁle, and due to this limitation, we further optimized

the heavy atom-hydrogen bonds by B3LYP/TZV (2d,2p) method

using heavy atom ﬁxing (HAF) approximation (constrained optimi-

zations).⁴⁵Constrained optimization was used to prevent irrational

movement of the side chains.⁴⁶All the interaction energies were

estimated by the same method and basis set. In energy calcula-

tions, long-range dielectric effects of protein were considered using

COSMO model³⁶with a dielectric constant of 4.8.⁴⁷The whole cal-

culations were done with the ORCA quantum chemistry package.⁴⁸

Melting points were determined with a Reichert-Jung hot-stage

microscope and were uncorrected. FT-IR (KBr) spectra were re-

corded on a Nicolet FT-IR Magna 550 spectrophotometer. ¹H

NMR spectra were determined by a Bruker FT-500 MHz spectrom-

eter in chloroform-d₁or DMSO-d₆. All the chemical shifts were re-

ported as (d) values (ppm) against tetramethylsilane as an internal

standard. The MS spectra were recorded using Agilent 7890A

spectrometer at 70 eV. CHN/CHNS analysis was performed using

CHNS-932 Leco analyzer and the results were within 0.4% of

the theoretical values.

The progress of the reactions and purity of the products were

checked by analytical thin-layer chromatography (TLC) on pre-

coated silica gel 60 F254 aluminum plates (Merck, Germany). Pre-

parative column chromatographies were carried out on silica gel

(230–400 mesh, G60 Merck). Preparative thin-layer chromatogra-

phy was done with prepared glass-backed plates (20 ꢀ 20 cm²,

4.2. Chemistry

All the reagents were purchased from Merck chemical company

and used without further puriﬁcation. Nano particles of Fe₃O₄were

prepared according to the co-precipitation procedure and were

freshly used.⁴⁹Prepared super paramagnetic nanoparticles

(Fe₃O₄) were characterized by transmission electron microscopy

(TEM) (Philips), the zeta-potential particle size distributing

method (Horiba LB-550) and FT-IR spectrum (Nicolet FT-IR Magna

550).

500 l) using silica gel (Merk Kieselgel 60 HF254, Art. 7739).

4.2.1. General procedure for the synthesis of N-(aryl)-3-

oxobutanamides (3a–e)

N-(Aryl)-3-oxobutanamides (3a–e) were synthesized according

to the modiﬁed method of Clemens via condensation of 2,2,6-

trimethyl-1,3-dioxine-4-one with the appropriate arylamine.⁵⁰

The results are represented in Table 7.

N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

6905

4.2.1.1. N-(2-Thiazolyl) 3-oxo-butanamide (3a).

Pale yellow

MS m/z (%): 198(29) [M⁺], 141(8), 114(100), 72(23), 43(65); Anal.

Calcd (%) for C₈H₁₀N₂O₂S: C, 48.47; H, 5.08; N, 14.13; S, 16.17.

Found: C, 48.39; H, 5.14; N, 14.26; S, 16.30.

crystals; Yield 89%; ¹H NMR (DMSO-d₆) d (ppm) 11.32 (s, 2H, NH-

amide), 8.93 (s, 1H, NH-DHP), 7.46 (d, J = 4 Hz, 1H, CH-furyl), 6.71

(t, J = 3.6 Hz, 1H, CH-furyl), 6.40 (s, 2H, CH₃-thiazole), 5.90 (d,

J = 3.2 Hz, 1H, CH-furyl), 5.34 (s, 1H, C4H-DHP), 2.29 (s, 6H, CH₃-

4.2.1.4. N-(2-Benzothiazolyl)-3-oxobutanamide (3d).

Pale

thiazole), 2.25 (s, 6H, CH₃-DHP); IR (KBr)

m

(cm^ꢁ1): 3344.4 (N–H,

yellow crystals; Yield 65%; ¹H NMR (DMSO-d₆) d (ppm) 11.65

(br s, 1H, NH-amide), 7.86 (d, J = 7.6 Hz, 1H, C4⁰-benzothiazole),

7.68 (d, J = 8.0 Hz, 1H, C7⁰H-benzothiazole), 7.35 (t, J = 8.4 Hz, 1H,

C5⁰H-benzothiazole), 7.20 (t, J = 8.0 Hz, 1H, C6⁰H-benzothiazole),

DHP), 2924.0 (C–H, aliphatic), 1667.6 (C@O, amide), 1598.3 (C@C,

alkene); MS m/z (%): 455(35) [M⁺], 410(12), 399(50), 313(75),

228(79), 202(100), 115(85), 42(21); Anal. Calcd (%) for C₇H₈N₂O₂S:

C, 45.64; H, 4.38; N, 15.21; S, 17.41. Found: C, 45.78; H, 4.44; N,

15.26; S, 17.34.

3.73 (s, 2H, COCH₂CO), 2.23 (s, 3H, CH₃CO); IR (KBr)

m

(cm^ꢁ1):

3178.7 (N–H, amide), 3067.5 (C–H, aromatic), 2917.3 and 2859.2

(C–H, aliphatic), 1719.4 (C@O, ketone), 1661.3 (C@O, amide); MS

m/z (%): 234(25) [M⁺], 177(6), 150(100), 123(11), 43(32); Anal.

Calcd (%) for C₁₁H₁₀N₂O₂S: C, 56.39; H, 4.30; N, 11.96; S, 13.69.

Found: C, 56.20; H, 4.42; N, 12.11; S, 13.52.

4.2.1.2. N-(5-Methyl-3-isoxazolyl)-3-oxobutanamide (3b). Pale

yellow crystals; yield 91%; ¹H NMR (DMSO-d₆) d (ppm) 10.49 (br s,

1H, NH-amide), 6.43 (s, 1H, C4H-isoxazole), 3.59 (s, 2H, COCH₂CO),

2.37 (s, 3H, CH₃CO), 2.18 (s, 3H, CH₃-isoxazole); IR (KBr)

m

(cm^ꢁ1):

3265.1 (N–H, amide), 3089.5 (C–H, aromatic), 2979.0 and 2934.7

(CꢁH, aliphatic), 1729.3 (C@O, ketone), 1673.8 (C@O, amide); MS

m/z (%): 182(35) [M⁺], 140(12), 125(14), 98(100), 43(79); Anal.

Calcd (%) for C₈H₁₀N₂O₃: C, 52.74; H, 5.53; N, 15.38. Found: C,

52.69; H, 5.51; N, 15.46.

4.2.1.5. N-(6-Ethoxy-2-benzothiazolyl)-3-oxobutanamide (3e). Pale

yellow crystals; Yield 41%; ¹H NMR (DMSO-d₆) d (ppm) 11.88 (br s,

1H, NH-amide), 7.62 (d, J = 8.8 Hz, 1H, C4⁰H-benzothiazole), 7.54

(d, J = 2.4 Hz, 1H, C7⁰H-benzothiazole), 7.01 (d, J = 8.8 Hz, 1H, C5⁰H-

benzothiazole), 4.06 (q, J = 6.8 Hz, 2H, –OCH₂CH₃), 3.75 (s, 2H,

COCH₂CO), 2.23 (s, 3H, CH₃CO), 1.35 (t, J = 6.4 Hz, 3H, –OCH₂CH₃);

4.2.1.3. N-(4-Methyl-2-thiazolyl)-3-oxobutanamide (3c). Pale

yellow crystals; Yield 86%; ¹H NMR (DMSO-d₆) d (ppm) 11.65 (br s,

1H, NH-amide), 7.26 (s, 1H, C5H-thiazole), 3.76 (s, 2H, COCH₂CO),

IR (KBr) m

(cm^ꢁ1): 3190.2 (N–H, amide), 3085.8 (C–H, aromatic),

2962.5 and 2889.6 (C–H, aliphatic), 1720.6 (C@O, ketone), 1661.9

(C@O, amide); MS m/z (%): 278(58) [M⁺], 220(18), 194(100),

2.35 (s, 3H, CH₃CO), 2.21 (s, 3H, CH₃-thiazole); IR (KBr)

m

(cm^ꢁ1):

165(86), 43(41); Anal. Calcd (%) for C₁₃H₁₄N₂O₃S: C, 56.10;

3179.8 (N–H, amide), 3053.0 (C–H, aromatic), 2974.2 and 2899.2

(C–H, aliphatic), 1723.5 (C@O, ketone), 1672.9 (C@O, amide);

H, 5.07; N, 10.06; S, 11.52. Found: C, 56.23; H, 4.95; N, 10.18; S,

11.41.

Figure 8. Characterization of prepared nano-Fe₃O₄nano particles by (a) TEM, (b) FTIR and (c) particle size analyzing methods.

6906

N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

4.2.2. Preparation of superparamagnetic Fe₃O₄nanoparticles

Fe₃O₄nanoparticles were prepared using co-precipitation

method.⁴⁹The average particle size was found to be 39.9 nm. The

characterization of Nano Fe₃O₄particles was performed via TEM,

particle size analyzer and FTIR spectra⁵¹(Fig. 8).

4.2.2.1. 2,6-Dimethyl-3,5-bis-N-(4-methyl-2-thiazolyl) carbam-

oyl-4-(2-furyl)-1,4-dihydropyridine (6a).

Pale yellow precip-

itates; Yield 32%; ¹H NMR (DMSO-d₆) d (ppm) 11.32 (s, 2H,

NH-amide), 8.93 (s, 1H, NH-DHP), 7.46 (d, J = 4 Hz, 1H, CH-furyl),

6.71 (t, J = 3.6 Hz, 1H, CH-furyl), 6.40 (s, 2H, CH-thiazole), 5.90 (d,

J = 3.2 Hz, 1H, CH-furyl), 5.34 (s, 1H, C4H-DHP), 2.29 (s, 6H, CH₃-

4.2.3. General procedure for the synthesis of 2,6-dimethyl-3,5-

bis-N-(aryl) carbamoyl-4-aryl-1,4-dihydropyridines (6a–p)

Conventional method: A mixture of aldehyde 4 (1 mmol), corre-

sponding N-arylacetoacetamide 3a–e (2 mmol) and ammonium

acetate 5 (2 mmol) was reﬂuxed in 20 mL absolute ethanol in an

oil bath for 24–48 h. The progress of the reaction was monitored

by TLC. On completion of the reaction, the solvent was removed

to some extent under reduced pressure. The product was ﬁltered,

washed with small portions of cold methanol and then dried. Fur-

ther puriﬁcation by column chromatography or/and preparative

TLC using ethylacetate/petroleum ether as eluent followed by re-

crystallization from appropriate solvent(s) gave pure compounds

6a–p.

Nano-Fe₃O₄catalyzed: To a stirred suspension of nano-iron oxide

(20 mol %) in absolute ethanol (25 mL) were added aldehyde 4

(1 mmol), N-arylacetoacetamide 3a–e (2 mmol) and ammonium

acetate 5 (2 mmol). The mixture was stirred and heated for desired

period while the completion of the reaction was monitored by TLC.

After completion of the reaction, nano iron oxides were separated

thiazole), 2.25 (s, 6H, CH₃-DHP); IR (KBr) m

(cm^ꢁ1): 3344.4 (N–H,

DHP), 2924.0 (C–H, aliphatic), 1667.6 (C@O, amide), 1598.3 (C@C,

alkene); MS m/z (%): 455(35) [M⁺], 410(12), 399(50), 313(75),

228(79), 202(100), 115(85), 42(21); Anal. Calcd (%) for C₂₁H₂₁N₅O₃S₂:

C, 55.37; H, 4.65; N, 15.37, S, 14.08. Found: C, 55.26; H, 4.51; N, 15.28;

S, 14.17.

4.2.2.2. 2,6-Dimethyl-3,5-bis-N-(2-thiazolyl) carbamoyl-4-(4-

nitrophenyl)-1,4-dihydropyridine (6b).

Yellow crystals;

Yield 32%; ¹H NMR (DMSO-d₆) d (ppm) 11.74 (s, 2H, NH-amide),

8.92 (s, 1H, NH-DHP), 8.14 (d, J = 8.8 Hz, 2H, CH-phenyl), 7.49 (d,

J = 3.2 Hz, 2H, C4⁰H-thiazole), 7.38 (d, J = 8.8 Hz, 2H, CH-phenyl),

7.17 (d, J = 3.6, 2H, Hz, C5⁰H-thiazole), 5.43 (s, 1H, C4H-DHP), 2.23

(s, 6H, CH₃-DHP); IR (KBr) m

(cm^ꢁ1): 3345.9 (N–H, DHP), 2921.3

(C–H, aliphatic), 1676.2 (C@O, amide), 1647.8 (C@C, alkene); MS

m/z (%): 482(12) [M⁺], 465 (100), 381(18), 339(11), 255(31),

101(33), 91(42), 55(12); Anal. Calcd (%) for C₂₁H₁₈N₆O₄S₂: C,

52.27; H, 3.76; N, 17.42; S, 13.29. Found: C, 52.10; H, 4.91; N,

17.41; S, 13.46.

using

a

super-magnet. Preparative TLC in ethyl acetate/

petroleum ether eluent gave the pure DHP compounds. Further

puriﬁcation process was done via re-crystallization in appropriate

solvent(s).

4.2.2.3. 2,6-Dimethyl-3,5-bis-N-(5-methyl-3-isoxazolyl) carbam-

oyl-4-(4-nitrophenyl)-1,4-dihydropyridine

(6c).

Yellow

crystals; Yield 16%; ¹H NMR (CDCl₃) d (ppm) 8.17 (d, J = 8.5 Hz,

2H, CH-phenyl), 7.85 (s, 2H, NH-amide), 7.50 (d, J = 8.5 Hz, 2H,

CH-phenyl), 6.64 (s, 2H, C4⁰H-isoxazole), 5.62 (s, 1H, NH-DHP),

5.03 (s, 1H, C4H-DHP), 2.38 (s, 6H, CH₃-isoxazole), 2.33 (s, 6H,

Obtained DHP compounds were characterized by IR, MS and ¹H

NMR spectra along with CHN/CHNS analysis. Physical and charac-

teristic data of the ﬁnal compounds are summarized in Table 8.

Characteristic data for synthesized DHP derivatives are as

follows:

CH₃-DHP); IR (KBr)

amide), 2920.9 (C–H, aliphatic), 1692.1 (C@O, amide), 1643.8,

m

(cm^ꢁ1): 3244.1 (N–H, DHP), 3107.9 (N–H,

Table 8

Physical and analytical data of 1,4-DHPs (6a–p)

Compd.

Ar’

Ar⁰

Mol. formula (Mol. wt)

mp (°C)

Conventional

method^ayield^b

(%)/period (h)

Nano-Fe₃O₄catalyzed

method^cyield^d

(%)/period (h)

6a

6b

6c

6d

6e

6f

6g

6h

6i

6j

6k

6l

6m

6n

6o

6p

4-Methyl-2-thiazolyl

2-Thiazolyl

5-Methyl-3-isoxazolyl

4-Methyl-2-thiazolyl

2-Benzothiazolyl

5-Methyl-3-isoxazolyl

4-Methyl-2-thiazolyl

2-Benzothiazolyl

6-Ethoxy-2-benzothiazolyl

5-Methyl-3-isoxazolyl

2-Benzothiazolyl

5-Methyl-3-isoxazolyl

2-Benzothiazolyl

4-Methyl-2-thiazolyl

2-Furyl

C

₂₁H₂₁N₅O₃S₂455.11

₂₁H₁₈N₆O₄S₂482.08

₂₃H₂₂N₆O₆478.16

₂₃H₂₂N₆O₄S₂510.11

₂₉H₂₂N₆O₄S₂582.11

₂₃H₂₂N₆O₆478.16

₂₃H₂₂N₆O₄S₂510.11

₂₉H₂₂N₆O₄S₂582.11

₃₃H₃₀N₆O₆S₂670.17

₂₃H₂₂N₆O₆478.16

₂₉H₂₂N₆O₄S₂582.11

₂₁H₂₂N₆O₄422.17

₂₉H₂₃N₅O₂S₂537.13

₂₃H₂₂N₆O₄S₂510.11

₂₄H₂₅N₅O₃S₂495.14

₂₃H₂₄N₆O₂S₂480.14

194–196

159–161

240–242

206–208

209–211

243–245

162–164

202–204

160–162

209–211

214–216

136–137

182–184

177–180

151–153

230–232

32 (48)

26 (24)

16 (24)

33 (28)

35 (24)

17 (24)

31 (24)

37 (36)

24 (40)

11 (48)

31 (30)

16 (48)

21 (24)

28 (24)

23 (48)

15 (48)

79 (4)

81 (1)

69 (8)

73 (7)

83 (1)

61 (9)

65 (5)

77 (6)

68 (4)

66 (6)

81 (4)

62 (6)

76 (3)

78 (4)

71 (5)

69 (3)

4-Nitrophenyl

3-Nitrophenyl

2-Nitrophenyl

2-Pyrrolyl

Phenyl

2-Nitrophenyl

4-Methoxyphenyl

6-Methyl-2-pyridyl

^ePE: petroleum ether.

a

Reﬂuxing in EtOH for 24-48 h.

Isolated yield.

Reﬂuxing in EtOH in the presence of nano-Fe₃O₄.

Isolated yield.

b

c

d

N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

6907

1621.5, (C@C, alkene), 1519.7 and 1344.6 (C–NO₂aromatic); MS m/

z (%): 478(3) [M⁺], 463 (7), 379(25), 356(19), 255(56), 228(50),

182(37), 98(33), 43(100); Anal. Calcd (%) for C₂₃H₂₂N₆O₆: C,

57.74; H, 4.63; N, 17.56. Found: C, 57.88; H, 4.84; N, 17.41.

1H, NH-DHP), 5.29 (s, 1H, C4H-DHP), 2.39 (s, 6H, CH₃-DHP); IR

(KBr)

(cm^ꢁ1): 3366.5 (N-H, DHP), 2926 (C–H, aliphatic), 1660.6

m

(C@O, amide), 1595.7 (C@C, alkene), 1530.8 and 1344.1 (C–NO₂

aromatic), 685.8, 759.1, 863.8 (meta substituted benzene); MS m/

z (%): 582(5) [M⁺], 580(7), 431(10), 389(58), 255(78), 228(64),

176(58), 150(100); Anal. Calcd (%) for C₂₉H₂₂N₆O₄S₂: C, 59.78; H,

3.81; N, 14.42; S, 11.01. Found: C, 59.83; H, 3.68; N, 14.56, S, 10.94.

4.2.2.4. 2,6-Dimethyl-3,5-bis-N-(4-methyl-2-thiazolyl) carbamoyl-

4-(4-nitrophenyl)-1,4-dihydropyridine (6d).

Yellow precipi-

tates; Yield 33%; ¹H NMR (CDCl₃) d (ppm) 10.25 (s, 2H, NH-amide),

8.09 (d, J = 8.3 Hz, 2H, CH-phenyl), 7.43 (d, J = 8.3 Hz, 2H, CH-phenyl),

6.48 (s, 2H, CH-thiazole), 5.91 (br s, 1H, NH-DHP), 5.41 (s, 1H, C4H-

4.2.2.9.

2,6-Dimethyl-3,5-bis-N-(6-ethoxy-2-benzothiazolyl)

Yellow

carbamoyl-4-(3-nitrophenyl)-1,4-dihydropyridine (6i).

DHP), 2.43 (s, 6H, CH₃-thiazole), 2.34 (s, 6H, CH₃-DHP); IR (KBr)

m

precipitates; Yield 24%; ¹H NMR (CDCl₃) d (ppm) 10.66 (s, 2H,

NH-amide), 8.16 (s, 1H, CH-phenyl), 7.99 (d, J = 8.2 Hz, 1H, CH-phe-

nyl), 7.83 (d, J = 7.1 Hz, 2H, CH-phenyl), 7.60 (d, J = 8.9 Hz, 2H,

C4⁰H-benzothiazole), 7.37 (t, J = 8.0 Hz, 1H, CH-phenyl), 7.23 (t,

J = 2.4 Hz, 2H, C7⁰H-benzothiazole), 7.04 (d, J = 8.8, 2H, C5⁰H-benzo-

thiazole), 6.45 (br s, 1H, NH-DHP), 5.75 (s, 1H, C4H-DHP), 4.07 (q,

J = 6.9 Hz, 4H, –OCH₂CH₃), 2.46 (s, 6H, CH₃-DHP), 1.45 (t,

(cm^ꢁ1): 3311.1 (N–H, DHP), 3107.9 (N–H, amide), 2920.9 (C–H, ali-

phatic), 1670.5 (C@O, amide), 1604.7 (C@C, alkene), 1526.6 and

1348.6 (C–NO₂aromatic); MS m/z (%): 510(3) [M⁺], 508(22),

395(67), 281(54), 255(100), 114(63); Anal. Calcd (%) for C₂₃H₂₂N₆O_4-

S₂: C, 54.10; H, 4.34; N, 16.46; S, 12.56. Found: C, 53.98; H, 4.70; N,

16.61; S, 12.24.

J = 6.9 Hz, 6H, –OCH₂CH₃); IR (KBr)

m

(cm^ꢁ1): 3373.3 (N–H, DHP),

4.2.2.5. 2,6-Dimethyl-3,5-bis-N-(2-benzothiazolyl) carbamoyl-4-

2924.2 (C–H, aliphatic), 1662.2 (C@O, amide), 1605.5 (C@C, al-

kene); MS m/z (%): 670(9) [M⁺], 668(10), 475(19), 448(38),

255(78), 194(100), 165(94), 139(33); Anal. Calcd (%) for C₃₃H₃₀N_6-

O₆S₂: C, 59.09; H, 4.51; N, 12.53; S, 9.56. Found: C, 59.19; H, 4.56;

N, 12.44; S, 9.48.

(4-nitrophenyl)-1,4-dihydropyridine (6e).

Yellow crystals;

Yield 35%; ¹H NMR (CDCl₃) d (ppm) 10.21 (s, 2H, NH-amide),

8.11 (d, J = 8.1 Hz, 2H, CH-phenyl), 7.80 (d, J = 7.8 Hz, 2H, C4⁰H-ben-

zothiazole), 7.74 (d, J = 7.8 Hz, 2H, C7⁰H-benzothiazole), 7.53 (d,

J = 8.2 Hz, 2H, CH-phenyl), 7.46 (t, J = 7.4 Hz, 2H, C5⁰H-benzothia-

zole), 7.33 (t, J = 7.4 Hz, 2H, C6⁰H-benzothiazole), 5.97 (s, 1H, NH-

4.2.2.10. 2,6-Dimethyl-3,5-bis-N-(5-methyl-3-isoxazolyl) car-

DHP), 5.48 (s, 1H, C4H-DHP), 2.46 (s, 6H, CH₃-DHP); IR (KBr)

m

bamoyl-4-(2-nitrophenyl)-1,4-dihydropyridine (6j).

Yellow

(cm^ꢁ1): 3332.9 (N–H, DHP), 2924 (C–H, aliphatic), 1655.4 (C@O,

amide), 1598.2 (C@C, alkene), 1534.5 and 1345.9 (C–NO₂aro-

matic); MS m/z (%): 582 (3) [M⁺], 580 (9), 431(24), 389(37),

255(89), 150(100); Anal. Calcd (%) for C₂₉H₂₂N₆O₄S₂: C, 59.78; H,

3.81; N, 14.42; S, 11.01. Found: C, 59.68; H, 3.98; N, 14.61, S, 11.09.

precipitates; Yield 11%; ¹H NMR (CDCl₃) d (ppm) 8.69 (s, 2H, NH-

amide), 7.70 (d, J = 8.0 Hz, 1H, CH-phenyl), 7.62 (t, J = 7.3 Hz, 1H,

CH-phenyl), 7.55 (d, J = 7.2 Hz, 1H, CH-phenyl), 7.32 (t, J = 7.8 Hz,

1H, CH-phenyl), 6.59 (s, 2H, C4⁰H-isoxazole), 5.75 (s, 1H, NH-DHP),

5.64 (s, 1H, C4H-DHP), 2.36 (s, 6H, CH₃-isoxazole), 2.25 (s, 6H, CH₃-

DHP); IR (KBr) m

(cm^ꢁ1): 3348.4 (N–H, DHP), 2924.2 (C–H, aliphatic),

4.2.2.6. 2,6-Dimethyl-3,5-bis-N-(5-methyl-3-isoxazolyl) carbam-

1679.6 (C@O, amide), 1615.7 (C@C, alkene), 1527.4 and 1352.7 (C–

NO₂aromatic); MS m/z (%): 478(9) [M⁺], 461(100), 379(12),

334(29), 236(38), 182(8), 43(14); Anal. Calcd (%) for C₂₃H₂₂N₆O₆: C,

57.74; H, 4.63; N, 17.56; Found: C, 57.68; H, 4.78; N, 17.61.

oyl-4-(3-nitrophenyl)-1,4-dihydropyridine (6f).

Pale yellow

crystals; Yield 17%; ¹H NMR (DMSO-d₆) d (ppm) 10.48 (s, 2H, NH-

amide), 8.63 (br s, 1H, NH-DHP), 8.13 (s, 1H, CH-phenyl), 8.05 (d,

J = 9 Hz, 1H, CH-phenyl), 7.99 (d, J = 9 Hz, 1H, CH-phenyl), 7.56 (t,

J = 9.5 Hz, 1H, CH-phenyl), 6.57 (s, 2H, C4⁰H-isoxazole), 5.26 (s,

1H, C4H-DHP), 2.34 (s, 6H, CH₃-isoxazole), 2.15 (s, 6H, CH₃-DHP);

4.2.2.11. 2,6-Dimethyl-3,5-bis-N-(2-benzothiazolyl) carbamoyl-

4-(2-nitrophenyl)-1,4-dihydropyridine (6k).

Yellow crys-

IR (KBr)

m

(cm^ꢁ1): 3331.8 (N–H, DHP), 1672.2 (C@O, amide),

tals; Yield 31%; ¹H NMR (CDCl₃) d (ppm) 10.46 (s, 2H, NH-amide),

7.91 (d, J = 7.5 Hz, 1H, CH-phenyl), 7.66–7.75 (m, 6H, C4⁰H, C5⁰H

and C7⁰H-benzothiazole), 7.54 (t, J = 7.5 Hz, 1H, CH-phenyl), 7.39

(t, J = 7.5 Hz, 1H, CH-phenyl), 7.32 (m, 3H, C6⁰H-benzothiazole

and CH-phenyl), 6.08 (s, 1H, NH-DHP), 5.93 (s, 1H, C4H-DHP),

1622.1 (C@C, alkene), 1523.8 and 1346.1 (C–NO₂aromatic),

697.5, 788.2, 878 (meta substituted benzene); MS m/z (%):

478(11) [M⁺], 463 (100), 379(51), 356(95), 255(47), 232(77),

134(46), 43(100); Anal. Calcd (%) for C₂₃H₂₂N₆O₆: C, 57.74; H,

4.63; N, 17.56. Found: C, 57.77; H, 4.69; N, 17.41.

2.51 (s, 6H, CH₃-DHP); IR (KBr)

m

(cm^ꢁ1): 3300.4 (N–H, DHP),

2922.1 (C–H, aliphatic), 1663.5 (C@O, amide), 1598.8 (C@C,

alkene), 1525.9 and 1350.3 (C–NO₂aromatic); MS m/z (%):582 (5)

[M⁺], 580 (43), 567(28), 431(81), 389(73), 370(32), 267(68),

150(100); Anal. Calcd (%) for C₂₉H₂₂N₆O₄S₂: C, 59.78; H, 3.81; N,

14.42; S, 11.01. Found: C, 59.97; H, 3.88; N, 14.36, S, 11.12.

4.2.2.7. 2,6-Dimethyl-3,5-bis-N-(4-methyl-2-thiazolyl) carbam-

oyl-4-(3-nitrophenyl)-1,4-dihydropyridine (6g).

Pale yellow

precipitates; Yield 31%; ¹H NMR (CDCl₃) d (ppm) 10.11 (s, 2H, NH-

amide), 8.14 (s, 1H, CH-phenyl), 8.01 (d, J = 8.2 Hz, 1H, CH-phenyl),

7.97 (d, J = 8.2 Hz, 1H, CH-phenyl), 7.40 (t, J = 7.5 Hz, 1H, CH-

phenyl), 6.48 (s, 2H, CH-thiazole), 6.29 (br s, 1H, NH-DHP), 5.74

(s, 1H, C4H-DHP), 2.48 (s, 6H, CH₃- thiazole), 2.37 (s, 6H,

4.2.2.12. 2,6-Dimethyl-3,5-bis-N-(5-methyl-3-isoxazolyl) car-

bamoyl-4-(2-pyrrolyl)-1,4-dihydropyridine (6l).

Pale yellow

CH₃-DHP); IR (KBr)

m

(cm^ꢁ1): 3211.6 (N–H, DHP), 2923.9 (C–H,

precipitates; Yield 16%; ¹H NMR (CDCl₃) d (ppm) 9.50 (br s, 1H, NH-

pyrrol), 8.29 (s, 2H, NH-amide), 6.78 (Apparent br s, 1H, CH-pyrrol),

6.68 (s, 2H, C4⁰H-isoxazole), 6.09 (br s, 1H, NH-DHP), 6.00 (Appar-

ent br s, 1H, CH-pyrrol), 5.83 (Apparent s, 1H, CH-pyrrol), 4.66 (s,

1H, C4H-DHP), 2.39 (s, 6H, CH₃-isoxazole), 2.29 (s, 6H, CH₃-DHP);

aliphatic), 1660.5 (C@O, amide), 1523.5, 1349.3 (C–NO₂, aromatic);

MS m/z (%): 510(8) [M⁺], 508(19), 493(64), 395(72), 255(100),

141(27), 114(67); Anal. Calcd (%) for C₂₃H₂₂N₆O₄S₂: C, 54.10; H,

4.34; N, 16.46; S, 12.56. Found: C, 54.23; H, 4.41; N, 16.39; S, 12.50.

IR (KBr)

m

(cm^ꢁ1): 3318.01 (N–H, DHP), 2924.2 (C–H, aliphatic),

4.2.2.8. 2,6-Dimethyl-3,5-bis-N-(2-benzothiazolyl) carbamoyl-4-

1669.7 (C@O, amide), 1615.4 (C@C, alkene); MS m/z (%): 422(5)

[M⁺], 407(25), 340(67), 258(100), 225(28), 16(18), 43(22); Anal.

Calcd (%) for C₂₁H₂₂N₆O₄: C, 59.71; H, 5.25; N, 19.89. Found: C,

59.63; H, 5.28; N, 19.69.

(3-nitrophenyl)-1,4-dihydropyridine (6h).

Yellow crystals;

Yield 37%; ¹H NMR (CDCl₃) d (ppm) 9.91 (s, 2H, NH-amide), 8.20

(s, 1H, CH-phenyl), 7.99 (d, J = 9 Hz, 1H, CH-phenyl), 7.78 (d,

J = 7.45 Hz, 2H, C4⁰H-benzothiazole), 7.71 (br s, 3H, C7⁰H-benzothi-

azole and CH-phenyl), 7.45 (t, J = 7.5, 2H, C5⁰H-benzothiazole), 7.32

(t, J = 7.2 Hz, 3H, C6⁰H-benzothiazole and CH-phenyl), 6.00 (br s,

4.2.2.13. 2,6-Dimethyl-3,5-bis-N-(2-benzothiazolyl) carbamoyl-

4-phenyl-1,4-dihydropyridine (6m).

Pale yellow precipitates;

6908

N. Razzaghi-Asl et al. / Bioorg. Med. Chem. 21 (2013) 6893–6909

Yield 16%; ¹H NMR (CDCl₃) d (ppm) 9.77 (s, 2H, NH-amide), 7.76 (2H,

d, J = 9 Hz, C4⁰H-benzothiazole), 7.66 (2H, d, J = 9 Hz, C7⁰H-benzothi-

azole), 7.48 (2H, d, J = 7.4 Hz, CH-phenyl), 7.31–7.42 (7H, m, CH-phe-

nyl and C5⁰H and C6⁰H-benzothiazole), 5.85 (1H, s, NH-DHP), 5.06

The assay procedure was as follows; the provided BACE-1 (puri-

ﬁed baclovirus-expressed) and the peptide substrate (Rh-EVNL-

DAEFK-Quencher) were diluted in BACE-1 assay buffer (50 mM

sodium acetate, pH 4.5) to obtain 3X solutions. Stock solutions of

inhibitors in DMSO were diluted in assay buffer to provide 3X solu-

tion of test compounds at different concentrations. The ﬁnal con-

centration of DMSO was kept under 4%. BACE-1 enzyme and test

(1H, s, C4H-DHP), 2.40 (6H, s, CH₃-DHP); IR (KBr)

m

(cm^ꢁ1): 3338.5

(N–H, DHP), 2923.7 (C–H, aliphatic), 1666.7 (C@O, amide), 1598.5

(C@C, alkene); MS m/z (%): 537(5) [M⁺], 535(8), 389(10), 361(27),

236(24), 212(71), 183(100), 150(83), 115(23); Anal. Calcd (%) for C_29-

H₂₃N₅O₂S₂: C, 64.78; H, 4.31; N, 13.03; S, 11.93. Found: C, 64.67; H,

4.40; N, 13.31, S, 11.79.

samples (10

ll of each) were introduced into the 96-well plate

while gently mixing. Then, BACE-1 substrate (10

l

l) was added

to the mixture and the plates were incubated at 25 °C for 90 min

in the dark and then the reaction was stopped with 2.5 M sodium

acetate (stop solution). Fluorescence was recorded at 544 nm

(excitation) and 590 nm (emission) wavelengths to monitor the

4.2.2.14. 2,6-Dimethyl-3,5-bis-N-(4-methyl-2-thiazolyl) carbam-

oyl-4-(2-nitrophenyl)-1,4-dihydropyridine

(6n).

Yellow

crystals; Yield 28%; ¹H NMR (CDCl₃) d (ppm) 10.09 (s, 2H, NH-

amide), 7.67 (d, J = 8.0 Hz, 2H, CH-phenyl), 7.63 (d, J = 7.9 Hz, 1H,

CH-phenyl), 7.52 (t, J = 7.6 Hz, 1H, CH-phenyl), 7.30 (t, J = 7.6 Hz,

1H, CH-phenyl), 6.47 (s, 2H, CH-thiazole), 6.12 (br s, 1H, NH-

DHP), 5.82 (s, 1H, C4H-DHP), 2.52 (s, 6H, CH₃-thiazole), 2.36 (s,

hydrolysis of the substrate. OM99-2 (Glu-Val-Asn-Leu-

W-Ala-Ala-

Glu-Phe, Calbiochem) was used as a reference inhibitor.

The percentages of enzyme inhibition for each concentration of

tested compounds were calculated regarding the maximum en-

zyme activity wells (containing substrate plus enzyme) and base-

line wells (containing substrate). IC₅₀values were calculated

with CurveExpert software version 1.34 for Windows. Each exper-

iment was repeated for three to four times. All data were presented

as mean S.E.

6H, CH₃-DHP); IR (KBr)

m

(cm^ꢁ1): 3360.6 (N–H, DHP), 2923.3

(C–H, aliphatic), 1665.5 (C@O, amide), 1607.9 (C@C, alkene),

1525.2 and 1354.6 (C-NO₂aromatic); MS m/z (%): 510(9) [M⁺],

493(70), 395(44), 353(90), 267(55), 210(86), 141(54), 114(100);

Anal. Calcd (%) for C₂₃H₂₂N₆O₄S₂: C, 54.10; H, 4.34; N, 16.46.

Found: C, 53.99; H, 4.99; N, 16.31.

4.3.2. Calcium channel blocking activity

Male albino guinea pigs (300–450 g) were purchased from ani-

mal house department of Shiraz University of Medical Sciences.

They had free access to standard rodent chow and tap water at

all times. The animals were housed at 23 2 °C temperature,

55 10% humidity, and on a 12 h light/dark cycle. The feeding

was disrupted 1 day before starting in vitro tests. The animals were

sacriﬁced by a blow to the head. The intestine was removed above

the ileocecal junction and longitudinal smooth muscle segments of

2 cm length were mounted under a resting tension of 0.5 g. The

segments were maintained at 37 °C in a 20 ml jacketed organ bath

containing oxygenated physiological saline solution with the fol-

lowing composition (mM): NaCl 137, CaCl₂1.8, KCl 2.7, MgSO₄

1.1, NaH₂PO₄0.4, NaHCO₃12 and glucose 5. The muscle was equil-

ibrated for 1 h with a solution varying every 15 min. The contrac-

tions were recorded with a force displacement transducer (F-50)

on a Narco Physiograph (Narco Biosystems, Houston, TX, USA). Test

agents were prepared as 2–10 M stock solutions in DMSO and

stored away from light. Dilutions were made with DMSO. The con-

tractile response was taken as the 100% value for the tonic (slow)

component of the response. The contraction was elicited with

40 mM KCl. Test compounds were cumulatively added and com-

pound-induced relaxation of contracted muscle was expressed as

percentage of control. Nifedipine was used as the reference com-

pound. The IC₃₀values (concentration needed to produce 30%

relaxation on contracted ileal smooth muscle) were graphically

determined using Curve Expert 1.34 program. Data were expressed

as means SE. The one-way analysis of variance (ANOVA) followed

by Tukey–Kramer multiple comparisons was used to analyze the

data. A value of P < 0.05 was considered as the signiﬁcance level

between the groups.

4.2.2.15. 2,6-Dimethyl-3,5-bis-N-(4-methyl-2-thiazolyl) carbam-

oyl-4-(4-methoxyphenyl)-1,4-dihydropyridine

(6o).

Pale

yellow precipitates; Yield 23%; ¹H NMR (DMSO-d₆) d (ppm) 11.54

(s, 2H, NH-amide), 8.74 (s, 1H, NH-DHP), 7.02 (d, J = 8.8 Hz, 2H,

CH-phenyl), 6.79 (d, J = 8.8 Hz, 2H, CH-phenyl), 6.69 (s, 2H, CH-

thiazole), 5.18 (s, 1H, C4H-DHP), 3.64 (s, 3H, –OCH₃), 2.29 (s, 6H,

N-CH₃), 2.21 (s, 6H, CH₃-DHP); IR (KBr)

m

(cm^ꢁ1): 3356.7 (N–H,

DHP), 2924.3 (C–H, aliphatic), 1667.1 (C@O, amide), 1608.9 (C@C,

alkene); MS m/z (%): 495(12) [M⁺], 493 (17), 380(46), 354(20),

266(100), 240(45), 115(31); Anal. Calcd (%) for C₂₄H₂₅N₅O₃S₂: C,

58.16; H, 5.08; N, 14.13; S, 12.94. Found: C, 58.27; H, 4.99; N,

14.31; S, 12.79.

4.2.2.16. 2,6-Dimethyl-3,5-bis-N-(4-methyl-2-thiazolyl) carbam-

oyl-4-(6-methyl-2-pyridyl)-1,4-dihydropyridine (6p).

Pale

yellow crystals; Yield 15%; ¹H NMR (CDCl₃) d (ppm) 10.26 (br s,

2H, NH-amide), 7.67 (t, J = 7.5 Hz, 1H, CH-pyridyl), 7.11 (d,

J = 7.6 Hz, 1H, CH-pyridyl), 7.07 (d, J = 7.4 Hz, 1H, CH-pyridyl), 6.63

(s, 2H, CH-thiazole), 6.21 (br s, 1H, NH-DHP), 4.91 (s, 1H, C4H-

DHP), 2.75 (s, 3H, CH₃-pyridyl), 2.39 (s, 6H, CH₃-thiazole), 2.36 (s,

6H, CH₃-DHP); IR (KBr)

m

(cm^ꢁ1): 3355.2 (N–H, DHP), 2922.9 (C–H,

aliphatic), 1665.8 (C@O, amide), 1594.3 (C@C, alkene); MS m/z (%):

480(5) [M⁺], 388(39), 366(100), 274(15), 225(85), 115(23), 42(7);

Anal. Calcd (%) for C₂₃H₂₄N₆O₂S₂: C, 57.48; H, 5.03; N, 17.49; S,

13.34. Found: C, 57.27; H, 4.96; N, 17.41; S, 13.28.

4.3. Biological assessment

4.3.1. BACE-1 inhibition assay

All synthesized compounds (6a-p) were evaluated for their

BACE-1 inhibitory activity using a ﬂuorescence resonance energy

transfer (FRET) assay with recombinant human BACE-1 and

quenched ﬂuorescent peptide substrate based on the Swedish mu-

tant APP sequence (SEVNLDAEFK). FRET-based enzymatic kit was

purchased from Invitrogen (former Pan Vera corporation, Madison,

WI) and the assay was carried out according to the manufacturers’

instructions (Invitrogen. http://tools.invitrogen.com/content/sfs/

manuals/L0724.pdf) using a multiplate spectroﬂuorometer instru-

ment (Recording ﬂuorescence at 544 nm excitation and 590 nm

emission wavelengths) (BMG LABTECH, Polar star, Germany).

Acknowledgments

Financial support of this project by Iranian National Science

Foundation (INSF) is acknowledged. Authors also thank the sup-

port of the Vice-chancellor of Research of Shiraz University of

Medical Sciences (Grant number: 5001).

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Article Doi

Design and synthesis of novel 3,5-bis-N-(aryl/heteroaryl) carbamoyl-4-aryl-1,4-dihydropyridines as small molecule BACE-1 inhibitors

DOI: 10.1016/j.bmc.2013.09.033

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Full text of DOI:10.1016/j.bmc.2013.09.033

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