Synthesis, biological activity screening and molecular modeling study of acylaminoacetamide derivatives

MEDICINAL

CHEMISTRY

RESEARCH

Med Chem Res

DOI 10.1007/s00044-015-1419-4

ORIGINAL RESEARCH

Synthesis, biological activity screening and molecular modeling

study of acylaminoacetamide derivatives

Gunes Coban¹Fadime Aydin Kose²Petek Ballar Kirmizibayrak²

Varol Pabuccuoglu¹

•

Received: 19 December 2014 / Accepted: 15 July 2015

Ó Springer Science+Business Media New York 2015

Abstract In this study, non-rigid analogs of thalidomide

have been designed in order to develop potentially active,

more effective and safer lead molecules for disorders

caused or contributed by inﬂammation. Five different ser-

ies of acylaminoacetamide compounds were synthesized,

and the biological inhibitory potency of the title com-

pounds has been determined by evaluating their effects on

COX-2 isoenzyme expression and PGE₂production in

A549 (human lung adenocarcinoma) cell lines. Among the

studied series, N-[2-(isopropylamino)-2-oxoethyl]isoni-

cotinamide is the most active inhibitory compound on

COX-2 isoenzyme expression, and N-[2-oxo-2-(pyroly-

dine-1-yl)etyl]isonicotinamide is the most active inhibitory

compound on the biosynthesis of PGE₂. Molecular docking

studies and molecular dynamics simulations were also

applied to investigate non-covalent interactions of the most

active compounds inside the active side of the crystal

Abbreviations

ATCC

BCA

American Type Culture Collection

Bicinchoninic acid

COX-1

COX-2

COX-3

DMEM

DMSO

EDTA

EIA

Cyclooxygenase-1

Cyclooxygenase-2

Cyclooxygenase-3

Dulbecco’s modiﬁed Eagle’s medium

Dimethyl sulfoxide

Ethylenediaminetetraacetic acid

Enzyme immunoassay

Electrospray ionization

United States Food and Drug Administration

Generalized Born

ESI

FDA

GB

IL-1b

mCOX-2

Interleukin-1b

Murine cyclooxygenase 2

MD

Molecular dynamics

MM-GBSA Molecular mechanics–Generalized Born

structure of murine cyclooxygenase

isoenzyme.

2

(mCOX-2)

surface area

MM-PBSA Molecular mechanics–Poisson–Boltzmann

surface area

Keywords Cyclooxygenase-2 Á Prostaglandin E₂Á

Synthesis Á Acylaminoacetamide Á Western blot analysis Á

Enzyme immuno assay Á Molecular modeling

NMR

PBS

Nuclear magnetic resonance

Phosphate-buffered saline

Prostaglandin E₂

PGE₂

PME

RIPA

RMSD

Particle mesh Ewald

Radioimmunoprecipitation assay

Root-mean-square deviation

Electronic supplementary material The online version of this

article (doi:10.1007/s00044-015-1419-4) contains supplementary

material, which is available to authorized users.

SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis

& Varol Pabuccuoglu

varol.pabuccuoglu@ege.edu.tr

Introduction

1

Department of Pharmaceutical Chemistry, Faculty of

Pharmacy, Ege University, 35100 Izmir, Turkey

Inﬂammation, a protective tissue response to the patho-

genic, traumatic, or toxic factors, has variable conse-

quences depending on the potency period (Costa et al.,

2

Department of Biochemistry, Faculty of Pharmacy, Ege

University, 35100 Izmir, Turkey

123

Med Chem Res

2007). In acute state, inﬂammation can be accepted as a

part of the defense response of the body and has therapeutic

implications (Aggarwal et al., 2006). However, chronic

inﬂammation is associated with a wide variety of chronic

diseases such as cancer, diabetes, arthritis, pulmonary,

auto-immune and Alzheimer’s diseases (Costa et al., 2007;

Aggarwal et al., 2006; Gaspirini et al., 2003; Ku and Ilson,

2010; Mao et al., 2005; Smith et al., 2008, 2012; Sethi

et al., 2012; Prins et al., 2012; Hyde and Missailidis, 2009;

`

Maccio and Madeddu, 2012; Al-Maghrabi et al., 2012;

Salvado et al., 2012; Heravi et al., 2011; Phillis et al.,

2006; Luo et al., 2011). Therefore, the inﬂammation pro-

cess is a preferential therapeutic target (Aggarwal et al.,

2006; Mao et al., 2005; Hyde and Missailidis, 2009).

Among the components of the process such as proinﬂam-

matory cytokines, chemokines, eicosanoids and inﬂam-

matory enzymes, eicosanoids and cyclooxygenase enzymes

in arachidonic acid cascade have been taken great attention

as medicinal targets (Aggarwal et al., 2006; Hyde and

Missailidis, 2009).

Fig. 1 Design of the title compounds

thalidomide is known to possess ‘‘moderate non-selective or

weakly COX-2-selective COX-inhibiting activity’’ (Nogu-

chi et al., 2002).

The literature survey described above prompted us to

design non-rigid analogs of thalidomide in order to eval-

uate their effects on COX-2 isoenzyme expression and

prostaglandin E₂(PGE₂) biosynthesis. For this purpose,

thalidomide structure was converted to open-chain analogs

to yield 2-acylaminoacetamide derivatives (Fig. 1). The

synthesis of the compounds, their effects on COX-2

isoenzyme expression and PGE₂biosynthesis and their

non-covalent interactions inside the active side of the

crystal structure of murine cyclooxygenase 2 (mCOX-2)

isoenzyme (PDB id: 4llz) using molecular docking meth-

ods are reported in this article.

Cyclooxygenase (COX) enzymes responsible for the

prostanoid synthesis in arachidonic acid cascade have three

isoforms, COX-1, COX-2 and COX-3 (Hyde and Missai-

lidis, 2009). Recently, it has been reported that COX-2

isoform is involved in several cancer types including eso-

phageal adenocarcinoma, breast, colon, prostate and lung

cancers and also in several cancer-related processes such as

apoptosis, proliferation, angiogenesis and metastasis dis-

eases (Costa et al., 2007; Aggarwal et al., 2006; Gaspirini

et al., 2003; Ku and Ilson, 2010; Mao et al., 2005; Smith

et al., 2008; Sethi et al., 2012; Prins et al., 2012; Hyde and

Missailidis, 2009; Al-Maghrabi et al., 2012; Salvado et al.,

2012). Moreover, COX-2 contributes neuroinﬂammation in

neurodegenerative disorders by inducing inﬂammation and

oxidative stress (Phillis et al., 2006). Despite the with-

drawn of rofecoxib and some other COX-2 inhibitors due

to their cardiovascular side effects, it became a challenge to

explore alternative templates with COX-2 inhibitory

activity (Heravi et al., 2011).

Materials and methods

Chemistry

Melting points were determined with Barnstead Elec-

trothermal 9100 melting point apparatus (Electrothermal,

Essex, U.K.). The UV spectra of compounds were taken on

a Shimadzu UV-160 in methanol solution. The IR spectra

of the compounds were monitored as potassium bromide

pellets on Jasco FT/IR-430 spectrometer (Jasco, Tokyo,

Thalidomide, a sedative-hypnotic drug which had been

withdrawn from market in 1960s because of the severe ter-

atogenicity, is accepted as a ‘‘multi-template’’ for develop-

ment of biologically active compounds (DeCicco et al.,

2004; Hashimoto, 2008; Onn et al., 2001; Sano et al., 2005;

Sampaio et al., 2006). It has been approved for Hansen’s

disease and multiple myeloma by United States Food and

Drug Administration (FDA) and also reported to have vari-

ous beneﬁcial pharmacological effects such as antiangio-

1

Japan). The NMR spectra (400 MHz for H and 100 MHz

for ¹³C) were recorded on AS 400 Mercury Plus NMR

Varian (Varian Inc., Palo Alto, CA, USA) using DMSO-d₆

and CDCl₃. Chemical shifts were measured in parts per

million (d). The J values were given in Hz. Mass spectra

were taken on a Waters Micromass ZQ connected with

Waters Alliance HPLC (Waters Corp., Milford, Mas-

sachusetts, USA), using ESI (?) method, with C-18 col-

umn. Elemental analyses were performed by Leco CHNS-

932 (Leco-932, St. Joseph, MI, USA) and were within

genic,

anticachexia,

anti-inﬂammatory,

antitumor

promoting, antiviral, tumor cell invasion inhibiting activity

and hypoglycemic effect (Hashimoto, 2008; Onn et al.,

2001; Sano et al., 2005; Sampaio et al., 2006; Noguchi et al.,

2002; Hashimoto, 2002; Harousseau, 2010). Racemic

123

Med Chem Res

0.4 % of the theoretical values. Compounds G1, G4, G5,

G11, G17, G19, G24 and G29 are reported with fractional

moles of water. Analytical thin-layer chromatography

(TLC) was run on Merck silica gel plates (Kieselgel

60F₂₅₄) with detection by UV light; column chromatogra-

phy was performed using Merck silica gel 60 (63–200 mm

diameter), and the synthesized compounds were eluted

with chloroform–methanol (11:1). All starting materials

and reagents were of high-grade commercial products.

methyl ester and 0.03 mol of appropriate amine in 5 mL of

methanol were reﬂuxed for 4 h at 80 °C. After the reaction

was completed, the excess of solvent was evaporated. The

compounds were puriﬁed by column chromatography

[CHCl₃:MeOH (11:1)] and then crystallized with an

appropriate solvent.

General procedure for the synthesis of the 2-(2-

picolinoylamino)acetamide, 2-nicotinoylaminoacetamide

and 2-isonicotinoylaminoacetamide derivatives

General procedure for the synthesis of the 2-

benzoylaminoacetamide and 2-(2-thienoylamino)acetamide

derivatives

The preparation of the 2-(2-picolinoylamino)acetamides,

2-nicotinoylaminoacetamides and 2-isonicotinoylaminoac-

etamides is outlined in Scheme 1. 0.01 mol of glycine was

dissolved in 4 mL of methanol and 2 mL of thionyl chloride

was added dropwise at 0–5 °C. This mixture was reﬂuxed

for 2 h at 80 °C, and the excess of thionyl chloride and the

methanol were evaporated. The crude methyl glycinate was

obtained in 90 % yield. 0.01 mol of methyl glycinate was

dissolved in 25 mL of tetrahydrofuran and 0.01 mol of tri-

ethylamine was added at 0–5 °C. The mixture was stirred

for 30 min at 0–5 °C. 0.012 mol of acid chloride

hydrochloride (prepared by the reaction of thionyl chloride

and 2-picolinic acid, nicotinic acid and isonicotinic acid) and

additional 0.024 mol of triethylamine were added. The

mixture was stirred for 3 h at room temperature and ﬁltered.

Tetrahydrofuran was evaporated to dryness. To the oily

residue, 25 mL of saturated sodium bicarbonate solution

was added, and then the mixture was extracted by ethyl

acetate (3 9 25 mL). The ethyl acetate phase was dried over

anhydrous sodium sulfate and evaporated. Methyl 2-(2-pi-

colinoylamino)acetate (31 % yield), 2-nicotinoylaminoac-

etate (56 %) and 2-isonicotinoylaminoacetate (62 % yield)

were obtained. 0.005 mol of ester derivative and 0.03 mol of

appropriate amine in 5 mL of methanol were reﬂuxed for

4 h at 80 °C, and the excess of amine and methanol were

evaporated. The compounds were puriﬁed by column

chromatography [chloroform–methanol (11:1)] and then

crystallized with an appropriate solvent.

The preparation of the 2-benzoylaminoacetamides and

2-(2-thienoylamino)acetamides is outlined in Scheme 1.

0.02 mol of acid (benzoic acid and 2-thienoic acid) in

5 mL of thionyl chloride were reﬂuxed for 1 h at 80 °C and

thionyl chloride was evaporated. The crude acid chloride

was used in the next step without puriﬁcation. 0.02 mol of

glycine and 0.02 mol of acid chloride were separately

dissolved in 15 mL of 2 M sodium hydroxide solution at

0–5 °C. Acid chloride in sodium hydroxide solution was

added to glycine solution in portions for 30 min by stirring

and cooling. The mixture was stirred for additional 30 min

at room temperature. The mixture was acidiﬁed by con-

centrated hydrochloride acid. The precipitated product was

ﬁltered and recrystallized from water to obtain hippuric

acid (81 % yield) and 2-(2-thienoylamino)acetic acid

(80 % yield). 0.01 mol of 2-acylaminoacetic acid and

0.25 mL of concentrated sulfuric acid in 15 mL of

methanol were reﬂuxed for 2 h at 80 °C and the excess of

methanol was evaporated. The residue was extracted by

chloroform (3 9 30 mL). The chloroform phase was

washed by 20 mL of 1 M sodium hydroxide solution and

dried over anhydrous sodium sulfate. Chloroform was

evaporated, and the residue was recrystallized from ethyl

acetate to obtain methyl hippurate (84 % yield) and methyl

2-(2-thienoylamino)acetate (70 % yield). 0.005 mol of

Scheme 1 Synthesis of the title

compounds

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Med Chem Res

Structural data of the 2-benzoylaminoacetamide

derivatives

C-5), 131.72 (CH, C-4), 134.27 (C, C-1), 166.32 (C, C=O),

167.32 (C, PhC=O); LC/MS (ESI?) m/z 247 (M ? H, 60),

269 (M ? Na, 100), 310 (M ? Na ? CH₃CN, 100). Anal.

Calcd for: C₁₄H₁₈N₂O₂: C, 68.27; H, 7.37; N, 11.37.

Found: C, 68.17; H, 7.39; N, 11.38.

N-[2-(phenylamino)-2-oxoethyl]benzamide (G1)

N-(2-morpholino-2-oxoethyl)benzamide(G3): White crys-

tals (EtOAc). This compound (G3) was prepared from

methyl benzoylglycinate (5 mmol, 0.97 g) and morpholine

(30 mmol, 2.61 g) according to the general procedure. The

product obtained as white solid was puriﬁed from ethyl

acetate. 37 % yield (0.46 g); mp 163 °C (150 °C, Ref.

Haworth et al., 1952); UV (Methanol) k_max(loge) 224.5

(4.13), 205.0 (4.24) nm; IR (KBr) 3338, 2981, 2921, 2859,

White crystals (EtOH-H₂O). This compound (G1) was

prepared from methyl benzoylglycinate (5 mmol, 0.97 g)

and aniline (30 mmol, 2.79 g) according to the general

procedure. The product obtained as white solid was puri-

ﬁed from ethanol–water (1:9) mixture. 40 % yield (0.51 g);

mp 221 °C (215 °C, Ref. Schwyzer et al., 1955); UV

(Methanol) k_max(loge) 240.5 (4.36), 205.0 (4.40) nm; IR

(KBr) 3309, 3137, 3060, 1681, 1639, 1602, 1540, 1492,

1

1664, 1643, 1577, 1515, 1471, 1434, 709, 688 cm^-1; H

NMR (CDCl₃, 400 MHz) d 3.47–3.50 (m, 2H, H-3⁰or H-5⁰),

3.68–3.74 (m, 6H, H-3⁰or H-5⁰, H-2⁰, H-6⁰), 4.25 (d, 2H,

J = 3.9 Hz, H-a), 7.29 (brs, 1H, NH), 7.42–7.46 (m, 2H,

H-3, H-5), 7.50–7.53 (m, 1H, H-4), 7.83–7.85 (m, 2H, H-2,

H-6); ¹³C NMR (CDCl₃, 100 MHz) d 41.77 (CH₂, C-a),

42.53 (CH₂, C-3⁰or C-5⁰), 45.05 (CH₂, C-3⁰or C-5⁰), 66.53

(CH₂, C-2⁰or C-6⁰), 66.84 (CH₂, C-2⁰or C-6⁰), 127.30 (CH,

C-2, C-6), 128.74 (CH, C-3, C-5), 131.85 (CH, C-4), 134.09

(C, C-1), 167.07 (C, C=O), 167.40 (C, PhC=O); LC/MS

(ESI?) m/z 249 (M ? H, 95), 271 (M ? Na, 100). Anal.

Calcd for: C₁₃H₁₆N₂O₃: C, 62.89; H, 6.50; N, 11.28. Found:

C, 62.73; H, 6.33; N, 11.20.

1

1442, 748, 690 cm^-1; H NMR (DMSO-d₆, 400 MHz) d

4.09 (d, 2H, J = 5.6 Hz, H-a), 7.03 (t, 1H, J_o= 7.2 Hz,

H-4⁰), 7.30 (t, 2H, J_o= 8.0 Hz, H-3, H-5), 7.47 (t, 2H,

J_o= 7.2 Hz, H-3⁰, H-5⁰), 7.53 (t, 1H, J_o= 6.8 Hz, H-4),

7.61 (d, 2H, J_o= 8.4 Hz, H-2⁰, H-6⁰), 7.92 (d, 2H,

J_o= 7.4 Hz, H-2, H-6), 8.81 (t, 1H, J = 5.6 Hz, NH),

10.03 (brs, 1H, NH–Ph); ¹³C NMR (DMSO-d₆, 100 MHz)

d 44.01 (CH₂, C-a), 119.88 (CH, C-2⁰, C-6⁰), 123.93 (CH,

C-4⁰), 128.03 (CH, C-2, C-6), 128.99 (CH, C-3, C-5),

129.42 (CH, C-3⁰, C-5⁰), 132.04 (CH, C-4), 134.69 (C,

C-1), 139.66 (C-1⁰), 167.35 (C, PhC=O), 168.52 (C, C=O);

LC/MS (ESI?) m/z 162 (C₉H₈NO₂^?, 100), 255 (M ? H,

60), 277 (M ? Na, 97). Anal. Calcd for C₁₅H₁₄N₂O₂

0.2H₂O: C, 69.86; H, 5.63; N, 10.86. Found: C, 69.73; H,

5.57; N, 10.54.

N-[2-oxo-2-(pyrrolidin-1-yl) ethyl]benzamide(G4) White

crystals (EtOAc). This compound (G4) was prepared from

methyl benzoylglycinate (5 mmol, 0.97 g) and pyrrolidine

(30 mmol, 2.13 g) according to the general procedure. The

product obtained as white solid was puriﬁed from ethyl

acetate. 35 % yield (0.41 g); mp 109 °C; UV (Methanol)

k_max(loge) 224.5 (4.05), 203.5 (4.21) nm; IR (KBr) 3390,

1

3056, 2975, 2877, 1637, 1515, 1454, 709 cm^-1; H NMR

N-[2-oxo-2-(piperidin-1-yl) ethyl]benzamide(G2) White

crystals (EtOAc). This compound (G2) was prepared from

methyl benzoylglycinate (5 mmol, 0.97 g) and piperidine

(30 mmol, 2.55 g) according to the general procedure. The

product obtained as white solid was puriﬁed from ethyl

acetate. 35 % yield (0.43 g); mp 97 °C (91 °C, Ref.

Thomsen et al., 1983); UV (Methanol) k_max(loge) 224.0

(4.15), 204.0 (4.29) nm; IR (KBr) 3338, 3060, 3006, 2935,

(CDCl₃, 400 MHz) d 1.89–1.96 (m, 2H, H-3⁰or H-4⁰),

2.01–2.07 (m, 2H, H-3⁰or H-4⁰), 3.47 (t, 2H, J = 6.8 Hz,

H-2⁰or H-5⁰), 3.55 (t, 2H, J = 6.8 Hz, H-2⁰or H-5⁰), 4.16

(d, 2H, J = 4.3 Hz, H-a), 7.27 (brs, 1H, NH), 7.41–7.46

(m, 2H, H-3, H-5), 7.47–7.51 (m, 1H, H-4), 7.82–7.86 (m,

2H, H-2, H-6); ¹³C NMR (CDCl₃, 100 MHz) d 24.30 (CH₂,

C-3⁰or C-4⁰), 26.08 (CH₂, C-3⁰or C-4⁰), 42.69 (CH₂, C-a),

45.65 (CH₂, C-2⁰or C-5⁰), 46.16 (CH₂, C-2⁰or C-5⁰),

127.29 (CH, C-2, C-6), 128.63 (CH, C-3, C-5), 131.67

(CH, C-4), 134.17 (C, C-1), 166.74 (C, C=O), 167.33 (C,

PhC=O); LC/MS (ESI?) m/z 233 (M ? H, 100), 255

(M ? Na, 100). Anal. Calcd for: C₁₃H₁₆N₂O₂Á0.4H₂O: C,

65.20; H, 7.07; N, 11.70. Found: C, 65.39; H, 6.84; N,

11.65.

2852, 1660, 1633, 1579, 1540, 1444, 775, 713, 692 cm^-1

;

¹H NMR (CDCl₃, 400 MHz) d 1.54–1.62 (m, 4H, H-3⁰,

H-5⁰), 1.66–1.71 (m, 2H, H-4⁰), 3.40 (t, 2H, J = 5.5 Hz,

H-2⁰or H-6⁰), 3.62 (t, 2H, J = 5.5 Hz, H-2⁰or H-6⁰), 4.24

(d, 2H, J = 3.5 Hz, H-a), 7.38 (brs, 1H, NH), 7.42–7.46

(m, 2H, H-3, H-5), 7.48–7.52 (m, 1H, H-4), 7.84–7.86 (m,

2H, H-2, H-6); ¹³C NMR (CDCl₃, 100 MHz) d 24.54 (CH₂,

C-4⁰), 25.63 (CH₂, C-3⁰or C-5⁰), 26.37 (CH₂, C-3⁰or C-5⁰),

41.89 (CH₂, C-a), 43.40 (CH₂, C-2⁰or C-6⁰), 45.65 (CH₂,

C-2⁰or C-6⁰), 127.28 (CH, C-2, C-6), 128.70 (CH, C-3,

N-[2-(isopropylamino)-2-oxoethyl]benzamide (G5) White

crystals (EtOH-H₂O). This compound (G5) was prepared

from methyl benzoylglycinate (5 mmol, 0.97 g) and

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Med Chem Res

White crystals (EtOH-H₂O). This compound (G7) was

prepared from methyl (thiophene-2-carbonyl)glycinate

(5 mmol, 1.00 g) and aniline (30 mmol, 2.79 g) according

to the general procedure. The product obtained as white

solid was puriﬁed from ethanol–water (1:9) mixture. 48 %

yield (0.55 g); mp 251 °C; UV (Methanol) k_max(loge)

246.5 (4.34), 204.5 (4.30) nm; IR (KBr) 3307, 3278, 3077,

isopropylamine (30 mmol, 1.77 g) according to the general

procedure. The product obtained as white solid was puri-

ﬁed from ethanol–water (1:9) mixture. 50 % yield (0.55 g);

mp 194 °C (186 °C, Ref. Schwyzer et al., 1955); UV

(Methanol) k_max(loge) 227.5 (4.05), 203.0 (4.18) nm; IR

(KBr) 3289, 3064, 2973, 2933, 2875, 1658, 1643, 1548,

1

1492, 1465, 692 cm^-1; H NMR (DMSO-d₆, 400 MHz) d

1

1681, 1633, 1550, 1442, 755, 719, 692 cm^-1; H NMR

1.05 (d, 6H, J = 6.4 Hz, H-2⁰, H-3⁰), 3.84 (d, 2H,

J = 6.0 Hz, H-a), 3.86 (q, 1H, J = 6.8 Hz, H-1⁰), 7.45 (t,

2H, J_o= 7.2 Hz, H-3, H-5), 7.51 (t, 1H, J_o= 7.4 Hz,

H-4), 7.73 (d, 1H, J = 8.0 Hz, NHPrⁱ), 7.80 (d, 2H,

(DMSO-d₆, 400 MHz) d 4.04 (d, 2H, J 5.9 Hz, H-a), 7.05

(1H, t, J_o= 7.4 Hz, H-4⁰), 7.17 (dd, 1H, J = 3.9/5.1 Hz,

H-4), 7.31 (t, 2H, J_o= 7.8 Hz, H-3⁰, H-5⁰), 7.60 (d, 2H,

J_o= 7.8 Hz, H-2⁰, H-6⁰), 7.68–7.78 (m, 1H, H-5),

7.82–7.83 (m, 1H, H-3), 8.83 (t, 1H, J = 5.9 Hz, NH),

10.03 (brs, 1H, PhNH); ¹³C NMR (DMSO-d₆, 100 MHz) d

43.76 (CH₂, C-a), 119.88 (CH, C-2⁰, C-6⁰), 123.96 (CH,

C-4⁰), 128.62 (CH, C-4), 129.22 (CH, C-3), 129.43 (CH,

C-3⁰, C-5⁰), 131.61 (CH, C-5), 139.61 (C, C-1⁰), 140.18 (C,

C-2), 162.34 (C, ThC=O), 168.38 (C, C=O); LC/MS

(ESI?) m/z 261 (M ? H, 19), 283 (M ? Na, 100), 324

(M ? Na ? CH₃CN, 100). Anal. Calcd for: C₁₃H₁₂N₂O₂S:

C, 59.98; H, 4.65; N, 10.76; S, 12.32. Found: C, 59.64; H,

4.39; N, 10.76, S, 12.30.

J_o= 7.2 Hz, H-2, H-6), 8.60 (t, 1H, J = 5.8 Hz, NH); ¹³

C

NMR (DMSO-d₆, 100 MHz) d 23.04 (CH₃, C-2⁰, C-3⁰),

41.17 (CH, C-1⁰), 43.25 (CH₂, C-a), 127.99 (CH, C-2,

C-6), 128.91 (CH, C-3, C-5), 131.91 (CH, C-4), 134.82 (C,

C-1), 167.07 (C, PhC=O), 168.49 (C, C=O); LC/MS

(ESI?) m/z 221 (M ? H, 100), 243 (M ? Na, 100). Anal.

Calcd for: C₁₂H₁₆N₂O₂Á0.1H₂O: C, 64.90; H, 7.35; N,

12.61. Found: C, 64.68; H, 6.99; N, 12.44.

N-[2-(dimethylamino)-2-oxoethyl]benzamide (G6) White

crystals (EtOAc). This compound (G6) was prepared from

methyl benzoylglycinate (5 mmol, 0.97 g) and dimethy-

lamine (30 mmol, 1.35 g) according to the general proce-

dure. The product obtained as white solid was puriﬁed from

ethyl acetate. 35 % yield (0.36 g); mp 122 °C (113 °C,

Ref. (Haworth et al., 1952); UV (Methanol) k_max(loge)

225.5 (4.09), 205.0 (4.19) nm; IR (KBr) 3289, 3060, 2925,

N-[2-oxo-2-(piperidin-1-yl) ethyl]thiopene-2-carboxamide

(G8) White crystals (EtOAc). This compound (G8) was

prepared from methyl (thiophene-2-carbonyl)glycinate

(5 mmol, 1.00 g) and piperidine (30 mmol, 2.55 g)

according to the general procedure. The product obtained

as white solid was puriﬁed from ethyl acetate. 42 % yield

(0.53 g); mp 138 °C; UV (Methanol) k_max(loge) 249.0

(4.04), 203.5 (4.11) nm; IR (KBr) 3276, 3073, 2933, 2856,

2867, 1668, 1646, 1513, 1475, 709 cm^-^{1 1}H NMR

;

(CDCl₃, 400 MHz) d 3.04 (d, 3H, J = 5,2 Hz, H-1⁰or

H-2⁰), 3.06 (d, 3H, J = 4,8 Hz, H-1⁰or H-2⁰), 4.22 (d, 2H,

J = 3.5 Hz, H-a), 7.31 (brs, 1H, NH), 7.41–7.46 (m, 2H,

H-3, H-5), 7.48–7.52 (m, 1H, H-4), 7.83–7.86 (m, 2H, H-2,

1635, 1554, 1459, 746, 718 cm^-^{1 1}H NMR (CDCl₃,

;

400 MHz) d 1.56–1.72 (m, 6H, H-3⁰, H-4⁰, H-5⁰), 3.39 (t,

2H, J = 5.5 Hz, H-2⁰or H-6⁰), 3.60–3.63 (m, 2H, H-2⁰or

H-6⁰), 4.21 (d, 2H, J = 3.9 Hz, H-a), 7.08 (dd, 1H,

J = 3.9/5.1 Hz, H-4), 7.23 (brs, 1H, NH), 7.48 (dd, 1H,

J = 1.2/5.1 Hz, H-5), 7.58 (dd, 1H, J = 1.2/3.9 Hz, H-3);

¹³C NMR (CDCl₃, 100 MHz) d 24.54 (CH₂, C-4⁰), 25.63

(CH₂, C-3⁰or C-5⁰), 26.37 (CH₂, C-3⁰or C-5⁰), 41.73 (CH₂,

C-a), 43.45 (CH₂, C-2⁰or C-6⁰), 45.69 (CH₂, C-2⁰or C-6⁰),

127.81 (CH, C-4), 128.52 (CH, C-3), 130.24 (CH, C-5),

138.84 (C, C-2), 161.89 (C, ThC=O), 166.15 (C, C=O);

LC/MS (ESI?) m/z 253 (M ? H, 53), 275 (M ? Na, 100),

316 (M ? Na ? CH₃CN, 100). Anal. Calcd for: C₁₂H_16-

N₂O₂S: C, 57.12; H, 6.39; N, 11.10, S, 12.71. Found: C,

57.16; H, 6.41; N, 11.07; S, 12.78.

13

H-6); C NMR (CDCl₃, 100 MHz) d 35.69 (CH₃, C-1⁰or

C-2⁰), 36.05 (CH₃, C-1⁰or C-2⁰), 41.87 (CH₂, C-a), 127.26

(CH, C-2, C-6), 128.65 (CH, C-3, C-5), 131.70 (CH, C-4),

134.17 (C, C-1), 167.27 (C, PhC=O), 168.24 (C, C=O);

LC/MS (ESI?) m/z 207 (M ? H, 32), 229 (M ? Na, 100),

270 (M ? Na ? CH₃CN, 100). Anal. Calcd for:

C₁₁H₁₄N₂O₂: C, 64.06; H, 6.84; N, 13.58. Found: C, 63.79;

H, 6.71; N, 13.39.

Structural data of the 2-(2-thienoylamino)acetamide

derivatives

N-[2-(phenylamino)-2-oxoethyl]thiopene-2-carboxamide

(G7)

N-(2-morpholino-2-oxoethyl)thiopene-2-carboxamide

(G9) White crystals (EtOAc). This compound (G9) was

prepared from methyl (thiophene-2-carbonyl)glycinate

(5 mmol, 1.00 g) and morpholine (30 mmol, 2.61 g)

according to the general procedure. The product obtained

as white solid was puriﬁed from ethyl acetate. 32 % yield

(0.41 g); mp 156 °C; UV (Methanol) k_max(loge) 249.0

123

Med Chem Res

(3.95), 204.0 (4.01) nm; IR (KBr) 3396, 3374, 3089, 2967,

2933, 2856, 1641, 1554, 1459, 738 cm^-^{1 1}H NMR

(CH, C-1⁰), 42.97 (CH₂, C-a), 128.53 (CH, C-4), 129.03

(CH, C-3), 131.44 (CH, C-5), 140.37 (C, C-2), 162.05 (C,

ThC=O), 168.33 (C, C=O); LC/MS (ESI?) m/z 227

(M ? H, 100), 249 (M ? Na, 100). Anal. Calcd for: C₁₀

H₁₄N₂O₂S 0.1H₂O: C, 52.66; H, 6.27; N, 12.28, S, 14.06.

Found: C, 52.50; H, 5.99; N, 12.15; S, 14.11.

;

(CDCl₃, 400 MHz) d 3.46–3.48 (m, 2H, H-2⁰or H-6⁰),

3.68–3.73 (m, 6H, H-3⁰, H-5⁰, H-2⁰or H-6⁰), 4.23 (d, 2H,

J = 4.3 Hz, H-a), 7.09 (dd, 1H, J = 4.3/5.1 Hz, H-4), 7.13

(brs, 1H, NH), 7.49 (d, 1H, J = 5.1 Hz, H-5), 7.58 (d, 1H,

J = 3.9 Hz, H-3); ¹³C NMR (CDCl₃, 100 MHz) d 41.59

(CH₂, C-a), 42.55 (CH₂, C-2⁰or C-6⁰), 45.10 (CH₂, C-2⁰or

C-6⁰), 66.55 (CH₂, C-3⁰or C-5⁰), 66.82 (CH₂, C-3⁰or C-5⁰),

127.88 (CH, C-4), 128.64 (CH, C-3), 130.46 (CH, C-5),

138.66 (C, C-2), 161.99 (C, ThC=O), 167.01 (C, C=O);

LC/MS (ESI?) m/z 255 (M ? H, 63), 277 (M ? Na, 100).

Anal. Calcd for: C₁₁H₁₄N₂O₃S: C, 51.95; H, 5.55; N,

11.02, S, 12.61. Found: C, 51.99; H, 5.35; N, 10.96; S,

12.59.

N-[2-(dimethylamino)-2-oxoethyl]thiopene-2-carboxamide

(G12) White crystals (EtOAc). This compound (G12)

was prepared from methyl (thiophene-2-carbonyl)glycinate

(5 mmol, 1.00 g) and dimethylamine (30 mmol, 1.35 g)

according to the general procedure. The product obtained

as white solid was puriﬁed from ethyl acetate. 27 % yield

(0.29 g); mp 121 °C; UV (Methanol) k_max(loge) 249.0

(4.30), 203.0 (4.34) nm; IR (KBr) 3367, 3112, 3068, 2923,

1

2873, 1660, 1623, 1533, 1486, 754, 717 cm^-1; H NMR

N-[2-oxo-2-(pyrrolidin-1-yl) ethyl]thiopene-2-carboxam-

ide (G10) White crystals (EtOAc). This compound (G10)

was prepared from methyl (thiophene-2-carbonyl)glycinate

(5 mmol, 1.00 g) and pyrrolidine (30 mmol, 2.13 g)

according to the general procedure. The product obtained

as white solid was puriﬁed from ethyl acetate. 37 % yield

(0.44 g); mp 156 °C; UV (Methanol) k_max(loge) 249.5

(4.01), 203.5 (4.05) nm; IR (KBr) 3295, 3085, 2933, 2873,

(CDCl₃, 400 MHz) d 3.03 (s, 6H, H-1⁰, H-2⁰), 4.21 (d, 2H,

J = 3.9 Hz, H-a), 7.08 (dd, 1H, J = 3.9/5.1 Hz, H-4), 7.17

(brs, 1H, NH), 7.47–7.49 (m, 1H, H-5), 7.58 (dd, 1H,

J = 1.2/3.9 Hz, H-3); ¹³C NMR (CDCl₃, 100 MHz) d

35.79 (CH₃, C-1⁰or C-2⁰), 36.16 (CH₃, C-1⁰or C-2⁰), 41.73

(CH₂, C-a), 127.83 (CH, C-4), 128.54 (CH, C-3), 130.29

(CH, C-5), 138.80 (C, C-2), 161.93 (C, ThC=O), 168.18

(C, C=O); LC/MS (ESI?) m/z 213 (M ? H, 46), 235

(M ? Na, 100). Anal. Calcd for: C₉H₁₂N₂O₂S: C, 50.92;

H, 5.70; N, 13.20, S, 15.11. Found: C, 51.01; H, 5.52; N,

13.09; S, 15.07.

1656, 1633, 1552, 1454, 742 cm^-^{1 1}H NMR (CDCl₃,

;

400 MHz) d 1.88–1.95 (m, 2H, H-3⁰or H-4⁰), 1.98–2.06

(m, 2H, H-3⁰or H-4⁰), 3.42–3.46 (m, 2H, H-2⁰or H-5⁰),

3.54 (t, 2H, J = 7.0 Hz, H-2⁰or H-5⁰), 4.15 (d, 2H,

J = 3.9 Hz, H-a), 7.08 (dd, 1H, J = 3.9/5.1 Hz, H-4), 7.14

(brs, 1H, NH), 7.47–7.48 (m, 1H, H-5), 7.58 (dd, 1H,

J = 1.2/3.9 Hz, H-3); ¹³C NMR (CDCl₃, 100 MHz) d

24.33 (CH₂, C-3⁰or C-4⁰), 26.12 (CH₂, C-3⁰or C-4⁰), 42.53

(CH₂, C-a), 45.74 (CH₂, C-2⁰or C-5⁰), 46.23(CH₂, C-2⁰or

C-5⁰), 127.80 (CH, C-4), 128.55 (CH, C-3), 130.26 (CH,

C-5), 138.84 (C, C-2), 162.01 (C, ThC=O), 166.74 (C,

C=O); LC/MS (ESI?) m/z 239 (M ? H, 75), 261

(M ? Na, 100), 302 (M ? Na ? CH₃CN, 100). Anal.

Calcd for: C₁₁H₁₄N₂O₂S: C, 55.44; H, 5.92; N, 11.76, S,

13.46. Found: C, 55.32; H, 5.93; N, 11.75; S, 13.55.

Structural data of the 2-nicotinoylaminoacetamide

derivatives

N-[2-(phenylamino)-2-oxoethyl]nicotinamide (G13)

White crystals (EtOH-H₂O). This compound (G13) was

prepared from methyl nicotinoylglycinate (5 mmol, 0.97 g)

and aniline (30 mmol, 2.79 g) according to the general

procedure. The product obtained as white solid was puri-

ﬁed from ethanol–water (1:9) mixture. 45 % yield (0.52 g);

mp 231 °C (224–226 °C, Ref. Farese et al., 1996); UV

(Methanol) k_max(loge) 242.5 (4.30), 207.0 (4.34) nm; IR

(KBr) 3344, 3096, 1675, 1655, 1606, 1556, 1474, 1449,

N-[2-(isopropylamino)-2-oxoethyl]thiopene-2-carboxamide

(G11) White crystals (EtOAc). This compound (G11)

was prepared from methyl (thiophene-2-carbonyl)glycinate

(5 mmol, 1.00 g) and isopropylamine (30 mmol, 1.77 g)

according to the general procedure. The product obtained

as white solid was puriﬁed from ethyl acetate. 38 % yield

(0.43 g); mp 189 °C; UV (Methanol) k_max(loge) 249.5

(4.09) nm. IR (KBr) 3284, 3075, 2973, 2931, 1658, 1633,

1

1415, 728, 703 cm^-1; H NMR (DMSO-d₆, 400 MHz) d

1548, 1463, 752, 709 cm^-1

;

¹H NMR (DMSO-d₆,

4.08 (d, 2H, J = 5.9 Hz, H-a), 7.01–7.05 (m, 1H, H-4⁰),

7.27–7.31 (m, 2H, H-3⁰, H-5⁰), 7.50–7.53 (m, 1H, H-5),

7.57–7.59 (m, 2H, H-2⁰, H-6⁰), 8.20–8.24 (m, 1H, H-4),

8.70–8.72 (m, 1H, H-6), 9.02 (d, 1H, J = 5.9 Hz, NH),

9.04 (t, 1H, J = 1.2 Hz, H-2), 10.02 (brs, 1H, PhNH); ¹³C

400 MHz) d 1.05 (d, 6H, J = 6.6 Hz, H-2⁰, H-3⁰), 3.77 (d,

2H, J = 6.2 Hz, H-a), 3.81–3.87 (m, 1H, H-1⁰), 7.12–7.14

(m, 1H, H-4), 7.73–7.74 (m, 2H, H-5, NHPrⁱ), 7.76–7.78

(m, 1H, H-3), 8.62 (t, 1H, J = 5.9 Hz, NH); ¹³C NMR

(DMSO-d₆, 100 MHz) d 23.04 (CH₃, C-2⁰, C-3⁰), 41.17

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Med Chem Res

N-[2-oxo-2-(pyrrolidin-1-yl) ethyl]nicotinamide (G16)

White crystals (EtOAc). This compound (G16) was pre-

pared from methyl nicotinoylglycinate (5 mmol, 0.97 g)

and isopropylamine (30 mmol, 1.77 g) according to the

general procedure. The product obtained as white solid was

puriﬁed from ethyl acetate. 32 % yield (0.37 g); mp

120 °C; UV (Methanol) k_max(loge) 262.5 (3.65), 257.0

(3.68), 205.0 (4.27) nm; IR (KBr) 3276, 3064, 2973, 2883,

NMR (DMSO-d₆, 100 MHz) d 43.94 (CH₂, C-a), 119.95

(CH, C-2⁰, C-6⁰), 123.99 (CH, C-4⁰), 124.14 (CH, C-5),

129.42 (CH, C-3⁰, C-5⁰), 130.18 (C, C-3), 135.75 (CH,

C-4), 139.57 (C, C-1⁰), 149.24 (CH, C-2), 152.70 (CH,

C-6), 166.00 (C, NicC=O), 168.25 (C, C=O); LC/MS

(ESI?) m/z 256 (M ? H, 100), 297 (M ? H?CH₃CN,

100). Anal. Calcd for: C₁₄H₁₃N₃O₂: C, 65.87; H, 5.13; N,

16.46. Found: C, 66.06; H, 5.16; N, 16.33.

1

1662, 1635, 1594, 1546, 1452, 736, 701 cm^-1; H NMR

N-[2-oxo-2-(piperidin-1-yl) ethyl]nicotinamide (G14)

White crystals (EtOAc-PE). This compound (G14) was

prepared from methyl nicotinoylglycinate (5 mmol, 0.97 g)

and piperidine (30 mmol, 2.55 g) according to the general

procedure. The product obtained as white solid was puri-

ﬁed from ethyl acetate-petrolium ether (7:3) mixture. 30 %

yield (0.37 g); mp 80 °C; UV (Methanol) k_max(loge) 262.5

(3.64), 257.0 (3.64), 205.0 (4.27) nm; IR (KBr) 3347, 3056,

(CDCl₃, 400 MHz) d 1.88–1.95 (m, 2H, H-3⁰or H-4⁰),

2.00–2.07 (m, 2H, H-3⁰or H-4⁰), 3.44–3.47 (m, 2H, H-2⁰or

H-5⁰), 3.53–3.56 (m, 2H, H-2⁰or H-5⁰), 4.18 (d, 2H,

J = 3.5 Hz, H-a), 7.40–7.44 (m, 1H, H-5), 7.46 (brs, 1H,

NH), 8.18 (d, 1H, J = 8.2 Hz, H-4), 8.75 (brs, 1H, H-6),

9.10 (brs, 1H, H-2); ¹³C NMR (CDCl₃, 100 MHz) d 24.26

(CH₂, C-3⁰or C-4⁰), 26.06 (CH₂, C-3⁰or C-4⁰), 42.54 (CH₂,

C-a), 45.76 (CH₂, C-2⁰or C-5⁰), 46.22 (CH₂, C-2⁰or C-5⁰),

123.39 (CH, C-5), 129.68 (C, C-3), 135.07 (CH, C-4),

148.72 (CH, C-2), 152.30 (CH, C-6), 165.60 (C, NicC=O),

166.70 (C, C=O); LC/MS (ESI?) m/z 234 (M ? H, 100).

Anal. Calcd for: C₁₂H₁₅N₃O₂: C, 61.79; H, 6.48; N, 18.01.

Found: C, 62.09; H, 6.26; N, 17.93.

1

2938, 2856, 1639, 1546, 1444, 1419, 740, 701 cm^-1; H

NMR (CDCl₃, 400 MHz) d 1.61 (t, 4H, J = 5.1 Hz, H-3⁰,

H-5⁰), 1.69–1.70 (m, 2H, H-4⁰), 3.39–3.42 (m, 2H, H-2⁰or

H-6⁰), 3.62 (t, 2H, J = 5.5 Hz, H-2⁰or H-6⁰), 4.24 (d, 2H,

J = 3.5 Hz, H-a), 7.40 (dd, 1H, J = 4.7/7.8 Hz, H-5), 7.46

(brs, 1H, NH), 8.15 (d, 1H, J = 7.8 Hz, H-4), 8.74 (d, 1H,

J = 4.7 Hz, H-6), 9.09 (s, 1H, H-2); ¹³C NMR (CDCl₃,

100 MHz) d 24.44 (CH₂, C-4⁰), 25.54 (CH₂, C-3⁰or C-5⁰),

26.31 (CH₂, C-3⁰or C-5⁰), 41.74 (CH₂, C-a), 43.38 (CH₂,

C-2⁰or C-6⁰), 45.67 (CH₂, C-2⁰or C-6⁰), 123.42 (CH, C-5),

129.83 (C, C-3), 135.02 (CH, C-4), 148.64 (CH, C-2),

152.38 (CH, C-6), 165.48 (C, NicC=O), 166.05 (C, C=O);

LC/MS (ESI?) m/z 248 (M ? H, 100). Anal. Calcd for:

C₁₃H₁₇N₃O₂: C, 63.14; H, 6.93; N, 16.99. Found: C, 62.94;

H, 6.97; N, 16.85.

N-[2-(isopropylamino)-2-oxoethyl]nicotinamide (G17)

White crystals (EtOAc). This compound (G17) was pre-

pared from methyl nicotinoylglycinate (5 mmol, 0.97 g)

and isopropylamine (30 mmol, 1.77 g) according to the

general procedure. The product obtained as white solid was

puriﬁed from ethyl acetate. 30 % yield (0.33 g); mp

188 °C (182 °C, Ref. Blanco et al., 2005); UV (Methanol)

k_max(loge) 256.5 (3.67), 203.0 (4.15) nm. IR (KBr) 3313,

3087, 2971, 2933, 2875, 1654, 1641, 1594, 1548, 1471,

744, 703 cm^-1; ¹H NMR (CDCl₃, 400 MHz) d 1.20 (d, 6H,

J = 6.6 Hz, H-2⁰, H-3⁰), 4.09–4.14 (m, 3H, H-1⁰, H-a),

5.94 (brs, 1H, NHPrⁱ), 7.43–7.46 (m, 2H, NH, H-5),

8.21–8.23 (m, 1H, H-4), 8.74–8.76 (m, 1H, H-6), 9.14 (d,

1H, J = 2 Hz, H-2); ¹³C NMR (CDCl₃, 100 MHz) d 22.69

(CH₃, C-2⁰, C-3⁰), 41.97 (CH, C-1⁰), 44.05 (CH₂, C-a),

123.53 (CH, C-5), 129.45 (C, C-3), 135.25 (CH, C-4),

148.85 (CH, C-2), 152.48 (CH, C-6), 166.31 (C, NicC=O),

168.35 (C, C=O); LC/MS (ESI?) m/z 222 (M ? H, 100).

Anal. Calcd for: C₁₁H₁₅N₃O₂0.1H₂O: C, 59.23; H, 6.87;

N, 18.84. Found: C, 59.17; H, 6.58; N, 18.79.

N-(2-morpholino-2-oxoethyl)nicotinamide (G15) White

crystals (EtOAc). This compound (G15) was prepared from

methyl nicotinoylglycinate (5 mmol, 0.97 g) and morpho-

line (30 mmol, 2.61 g) according to the general procedure.

The product obtained as white solid was puriﬁed from ethyl

acetate. 27 % yield (0.34 g); mp 157 °C; UV (Methanol)

k

_max(loge) 262.5 (3.60), 257.0 (3.61), 205.0 (4.23) nm; IR

(KBr) 3345, 3064, 2958, 2913, 2869, 1656, 1633, 1589,

1531, 1467, 746, 707 cm^-1; ¹H NMR (CDCl₃, 400 MHz) d

3.49 (t, 2H, J = 4.7 Hz, H-2⁰or H-6⁰), 3.68–3.72 (m, 6H,

H-2⁰or H-6⁰, H-3⁰, H-5⁰), 4.24 (d, 2H, J = 3.9 Hz, H-a),

7.36 (brs, 1H, NH), 7.37–7.41 (m, 1H, H-5), 8.12–8.15 (m,

1H, H-4), 8.74–8.75 (m, 1H, H-6), 9.06 (d, 1H,

J = 2.3 Hz, H-2); ¹³C NMR (CDCl₃, 100 MHz) d 41.68

(CH₂, C-a), 42.57 (CH₂, C-2⁰or C-6⁰), 45.09 (CH₂, C-2⁰or

C-6⁰), 66.50 (CH₂, C-3⁰or C-5⁰), 66.80 (CH₂, C-3⁰or C-5⁰),

123.53 (CH, C-5), 129.71 (C, C-3), 135.13 (CH, C-4),

148.59 (CH, C-2), 152.56 (CH, C-6), 165.63 (C, NicC=O),

166.80 (C, C=O); LC/MS (ESI?) m/z 250 (M ? H, 100).

Anal. Calcd for: C₁₂H₁₅N₃O₃: C, 57.82; H, 6.07; N, 16.86.

Found: C, 57.80; H, 6.14; N, 16.60.

N-[2-(dimethylamino)-2-oxoethyl]nicotinamide

(G18)

White crystals (EtOAc). This compound (G18) was pre-

pared from methyl nicotinoylglycinate (5 mmol, 0.97 g)

and dimethylamine (30 mmol, 1.35 g) according to the

general procedure. The product obtained as white solid was

puriﬁed from ethyl acetate. 28 % yield (0.29 g); mp 91 °C;

UV (Methanol) k_max(loge) 262.5 (3.59), 257.0 (3.59),

206.5 (4.17) nm; IR (KBr) 3332, 3054, 2927, 1643, 1542,

1

1419, 707 cm^-1; H NMR (CDCl₃, 400 MHz) d 3.04 (d,

6H, J = 3.9 Hz, H-1⁰, H-2⁰), 4.25 (d, 2H, J = 3.9 Hz,

123

Med Chem Res

H-a), 7.39 (dd, 1H, J = 4.7/7.8 Hz, H-5), 7.42 (brs, 1H,

NH), 8.13–8.16 (m, 1H, H-4), 8.74 (dd, 1H, J = 1.6/

4.7 Hz, H-6), 9.08 (d, 1H, J = 2 Hz, H-2); ¹³C NMR

(CDCl₃, 100 MHz) d 35.79 (CH₃, C-1⁰or C-2⁰), 36.14

(CH₃, C-1⁰or C-2⁰), 41.83 (CH₂, C-a), 123.48 (CH, C-5),

129.77 (C, C-3), 135.05 (CH, C-4), 148.64 (CH, C-2),

152.47 (CH, C-6), 165.52 (C, NicC=O), 168.00 (C, C=O);

LC/MS (ESI?) m/z 208 (M ? H, 100). Anal. Calcd for:

C₁₀H₁₃N₃O₂: C, 57.96; H, 6.32; N, 20.28. Found: C, 57.68;

H, 6.45; N, 19.98.

400 MHz) d 1.57–1.64 (m, 4H, H-3⁰, H-5⁰), 1.68–1.72 (m,

2H, H-4⁰), 3.38–3.41 (m, 2H, H-2⁰or H-6⁰), 3.61–3.63 (m,

2H, H-2⁰or H-6⁰), 4.22 (d, 2H, J = 3.9 Hz, H-a), 7.52 (brs,

1H, NH), 7.67–7.68 (m, 2H, H-3, H-5), 8.74–8.76 (m, 2H,

H-2, H-6); ¹³C NMR (CDCl₃, 100 MHz) d 24.49 (CH₂,

C-4⁰), 25.60 (CH₂, C-3⁰or C-5⁰), 26.36 (CH₂, C-3⁰or C-5⁰),

41.83 (CH₂, C-2*), 43.50 (CH₂, C-2⁰or C-6⁰), 45.71 (CH₂,

C-2⁰or C-6⁰), 121.15 (CH, C-3, C-5), 141.23 (C, C-4),

150.75 (CH, C-2, C-6), 165.33 (C, C-1*), 165.84 (C, C-3*);

LC/MS (ESI?) m/z 248 (M ? H, 100). Anal. Calcd for:

C₁₃H₁₇N₃O₂: C, 63.14; H, 6.93; N, 16.99. Found: C, 63.13;

H, 6.80; N, 16.93.

Structural data of the 2-isonicotinoylaminoacetamide

derivatives

N-(2-morpholino-2-oxoethyl)isonicotinamide (G21) White

crystals (EtOAc). This compound (G21) was prepared from

methyl isonicotinoylglycinate (5 mmol, 0.97 g) and mor-

pholine (30 mmol, 2.61 g) according to the general pro-

cedure. The product obtained as white solid was puriﬁed

from ethyl acetate. 23 % yield (0.29 g); mp 182 °C; UV

(Methanol) k_max(loge) 237.0 3.71), 205.0 (4.23) nm; IR

(KBr) 3403, 2985, 2917, 2857, 1646, 1552, 1515, 1475,

N-[2-(phenylamino)-2-oxoethyl]isonicotinamide (G19)

1

1430, 752, 707, 674 cm^-1; H NMR (CDCl₃, 400 MHz) d

3.47–3.49 (m, 2H, H-2⁰or H-6⁰), 3.69–3.75 (m, 6H, H-2⁰or

H-6⁰, H-3⁰, H-5⁰), 4.25 (d, 2H, J = 3.9 Hz, H-2*), 7.43

(brs, 1H, NH-1⁰⁰), 7.67 (dd, 2H, J = 1.6/4.3 Hz, H-3, H-5),

8.76 (dd, 2H, J = 1.6/4.7 Hz, H-2, H-6); ¹³C NMR

(CDCl₃, 100 MHz) d 41.73 (CH₂, C-2*), 42.63 (CH₂, C-2⁰

or C-6⁰), 45.07 (CH₂, C-2⁰or C-6⁰), 66.52 (CH₂, C-3⁰or

C-5⁰), 66.84 (CH₂, C-3⁰or C-5⁰), 121.13 (CH, C-3, C-5),

141.03 (C, C-4), 150.81 (CH, C-2, C-6), 165.45 (C, C-1*),

166.56 (C, C-3*); LC/MS (ESI?) m/z 250 (M ? H, 100).

Anal. Calcd for: C₁₂H₁₅N₃O₃: C, 57.82; H, 6.07; N, 16.86.

Found: C, 57.61; H, 5.80; N, 16.58.

White crystals (EtOH-H₂O). This compound (G19) was

prepared from methyl isonicotinoylglycinate (5 mmol,

0.97 g) and aniline (30 mmol, 2.79 g) according to the

general procedure. The product obtained as white solid was

puriﬁed from ethanol–water (1:9) mixture. 34 % yield

(0.43 g); mp 191 °C (191–194 °C, Ref. Farese et al.,

1996); UV (Methanol) k_max(loge) 242.5 (4.30), 206.0

(4.38) nm; IR (KBr) 3301, 3073, 1677, 1646, 1600, 1535,

1

1490, 1442, 746 cm^-1; H NMR (DMSO-d₆, 400 MHz) d

4.08 (d, 2H, J = 5.9 Hz, H-a), 7.04 (t, 1H, J_o= 7.4 Hz,

H-4⁰), 7.27–7.31 (m, 2H, H-3⁰, H-5⁰), 7.58 (d, 2H,

J_o= 7.8 Hz, H-2⁰, H-6⁰), 7.78 (dd, 2H, J = 1.6/4.3 Hz,

H-3, H-5), 8.73–8.74 (m, 2H, H-2, H-6), 9.09 (d, 1H,

J = 5.9 Hz, NH), 10.04 (brs, 1H, PhNH); ¹³C NMR

(DMSO-d₆, 100 MHz) d 43.98 (CH₂, C-a), 119.94 (CH,

C-2⁰, C-6⁰), 122.00 (CH, C-3, C-5), 124.02 (CH, C-4⁰),

129.43 (CH, C-3⁰, C-5⁰), 139.54 (C, C-1⁰), 141.06 (C, C-4),

150.96 (CH, C-2, C-6), 165.87 (C, NicⁱC=O), 168.06 (C,

C=O); LC/MS (ESI?) m/z 256 (M ? H, 100). Anal. Calcd

for: C₁₄H₁₃N₃O₂0,25H₂O: C, 64.73; H, 5.24; N, 16.18.

Found: C, 64.52; H, 5.14; N, 16.09.

N-[2-oxo-2-(pyrrolidin-1-yl) ethyl]isonicotinamide (G22)

White crystals (EtOAc). This compound (G22) was pre-

pared from methyl isonicotinoylglycinate (5 mmol, 0.97 g)

and pyrrolidine (30 mmol, 1.77 g) according to the general

procedure. The product obtained as white solid was puri-

ﬁed from ethyl acetate. 42 % yield (0.49 g); mp 157 °C;

UV (Methanol) k_max(loge) 237.0 (3.71), 205.5 (4.23) nm;

IR (KBr) 3272, 3237, 3043, 2962, 2884, 1668, 1627, 1548,

1

1452, 754, 717, 676 cm^-1; H NMR (CDCl₃, 400 MHz) d

1.91–1.96 (m, 2H, H-3⁰or H-4⁰), 2.01–2.06 (m, 2H, H-3⁰or

H-4⁰), 3.44–3.47 (m, 2H, H-2⁰or H-5⁰), 3.53–3.57 (m, 2H,

H-2⁰or H-5⁰), 4.17 (d, 2H, J = 3.9 Hz, H-2*), 7.47 (brs,

1H, NH-1⁰⁰), 7.69 (dd, 2H, J = 1.6/4.7 Hz, H-3, H-5),

8.75–8.76 (m, 2H, H-2, H-6); ¹³C NMR (CDCl₃,

100 MHz) d 24.28 (CH₂, C-3⁰or C-4⁰), 26.08 (CH₂, C-3⁰or

C-4⁰), 42.56 (CH₂, C-2*), 45.78 (CH₂, C-2⁰or C-5⁰), 46.27

(CH₂, C-2⁰or C-5⁰), 121.19 (CH, C-3, C-5), 141.01 (C,

C-4), 150.59 (CH, C-2, C-6), 165.39 (C, C-1*), 166.58 (C,

C-3*); LC/MS (ESI?) m/z 234 (M ? H, 100). Anal. Calcd

N-[2-oxo-2-(piperidin-1-yl) ethyl]isonicotinamide (G20)

White crystals (EtOAc-PE). This compound (G20) was

prepared from methyl isonicotinoylglycinate (5 mmol,

0.97 g) and piperidine (30 mmol, 2.55 g) according to the

general procedure. The product obtained as white solid was

puriﬁed from ethyl acetate–petroleum ether (7:3) mixture.

20 % yield (0.25 g); mp 125 °C; UV (Methanol) k_max

(loge) 237.0 (3.73), 205.5 (4.20) nm; IR (KBr) 3392, 2935,

2857, 1641, 1552, 1446, 754, 707 cm^-1; ¹H NMR (CDCl₃,

123

Med Chem Res

for: C₁₂H₁₅N₃O₂: C, 61.79; H, 6.48; N, 18.01. Found: C,

61.73; H, 6.56; N, 17.93.

and aniline (30 mmol, 2.79 g) according to the general

procedure. The product obtained as white solid was puri-

ﬁed from ethanol–water (1:9) mixture. 24 % yield (0.31 g);

mp 201 °C (204–206 °C, Ref. Farese et al., 1996); UV

(Methanol) k_max(loge) 242.5 (4.27), 206.0 (4.33) nm; IR

(KBr) 3367, 3322, 3149, 1675, 1608, 1558, 1531, 1463,

750, 694 cm^-1; ¹H NMR (DMSO-d₆, 400 MHz) d 4.15 (d,

2H, J = 5.9 Hz, H-2*), 7.03–7.06 (m, 1H, H-4⁰), 7.30–7.34

(m, 2H, H-3⁰, H-5⁰), 7.58–7.60 (m, 2H, H-2⁰, H-6⁰),

7.62–7.66 (m, 1H, H-5), 8.00–8.04 (m, 1H, H-4), 8.05–8.08

(m, 1H, H-3), 8.68–8.70 (m, 1H, H-6), 8.94–8.97 (m, 1H,

NH-1⁰⁰), 10.07 (brs, 1H, NH-2⁰⁰); ¹³C NMR (DMSO-d₆,

100 MHz) d 43.63 (CH₂, C-2*), 119.85 (CH, C-2⁰, C-6⁰),

122.56 (CH, C-3), 123.98 (CH, C-4⁰), 127.34 (CH, C-5),

129.44 (CH, C-3⁰, C-5⁰), 138.49 (CH, C-4), 139.54 (C,

C-1⁰), 149.20 (CH, C-6), 150.26 (C, C-2), 164.78 (C,

C-1*), 168.03 (C, C-3*); LC/MS (ESI?) m/z 163

(C₈H₇N₂O₂^?, 100), 256 (M ? H, 100), 278 (M ? Na,

100). Anal. Calcd for: C₁₄H₁₃N₃O₂: C, 65.87; H, 5.13; N,

16.46. Found: C, 65.62; H, 5.14; N, 16.28.

N-[2-(isopropylamino)-2-oxoethyl]isonicotinamide (G23)

White crystals (EtOAc). This compound (G23) was prepared

from methyl isonicotinoylglycinate (5 mmol, 0.97 g) and

isopropylamine (30 mmol, 1.77 g) according to the general

procedure. The product obtained as white solid was puriﬁed

from ethyl acetate. 20 % yield (0.22 g); mp 208 °C; UV

(Methanol) k_max(loge) 236.5 (3.72), 204.0 (4.13) nm; IR

(KBr) 3288, 3068, 2975, 2937, 1654, 1645, 1550, 1463, 754,

1

674 cm^-1; H NMR (DMSO-d₆, 400 MHz) d 1.05 (d, 6H,

J = 6.6 Hz, H-2⁰, H-3⁰), 3.82–3.87 (m, 3H, H-1⁰, H-2*), 7.76

(2H, dd, J = 1.6/4.3 Hz, H-3, H-5), 7.78 (brs, 1H, NH-2⁰⁰),

8.71 (dd, 2H, J = 1.6/4.3 Hz, H-2, H-6), 8.90 (t, 1H,

J = 5.7 Hz, NH-1⁰⁰); ¹³C NMR (CDCl₃, 100 MHz) d 23.03

(CH₃, C-2⁰, C-3⁰), 40.84 (CH, C-1⁰), 43.19 (CH₂, C-2*),

121.98 (CH, C-3, C-5), 141.72 (C, C-4), 150.88 (CH, C-2,

C-6), 165.54 (C, C-1*), 168.02 (C, C-3*); LC/MS (ESI?) m/z

222(M ? H,100). Anal. Calcdfor:C₁₁H₁₅N₃O₂:C, 59.71;H,

6.83; N, 18.99. Found: C, 59.62; H, 6.64; N, 18.85.

N-[2-oxo-2-(piperidin-1-yl) ethyl]picolinamide (G26)

White crystals (EtOAc). This compound (G26) was pre-

pared from methyl picolinoylglycinate (5 mmol, 0.97 g)

and piperidine (30 mmol, 2.55 g) according to the general

procedure. The product obtained as white solid was puri-

ﬁed from ethyl acetate. 30 % yield (0.37 g); mp 84 °C; UV

(Methanol) k_max(loge) 265.5 (3.74), 206.5 (4.21) nm; IR

(KBr) 3378, 3056, 3008, 2946, 2859, 1671, 1641, 1567,

1509, 1434, 758, 694 cm^-1; ¹H NMR (CDCl₃, 400 MHz) d

1.56–1.62 (m, 4H, H-3⁰, H-5⁰), 1.67–1.70 (m, 2H, H-4⁰),

3.40–3.43 (m, 2H, H-2⁰or H-6⁰), 3.63 (t, 2H, J = 5.5 Hz,

H-2⁰or H-6⁰), 4.27 (d, 2H, J = 4.7 Hz, H-2*), 7.40–7.43

(m, 1H, H-5), 7.80–7.85 (m, 1H, H-4), 8.17 (ddd, 1H,

J = 1.2/2/7.8 Hz, H-3), 8.59–8.62 (m, 1H, H-6), 8.93 (brs,

1H, NH-1⁰⁰); ¹³C NMR (CDCl₃, 100 MHz) d 24.54 (CH₂,

C-4⁰), 25.58 (CH₂, C-3⁰or C-5⁰), 26.38 (CH₂, C-3⁰or C-5⁰),

41.37 (CH₂, C-2*), 43.28 (CH₂, C-2⁰or C-6⁰), 45.69 (CH₂,

C-2⁰or C-6⁰), 122.11 (CH, C-3), 126.30 (CH, C-5), 137.22

(CH, C-4), 148.58 (CH, C-6), 149.88 (C, C-2), 164.59 (C,

C-1*), 166.11 (C, C-3*); LC/MS (ESI?) m/z 248 (M ? H,

100), 270 (M ? Na, 100), 312 (M ? H?Na ? CH₃CN,

100). Anal. Calcd for: C₁₃H₁₇N₃O₂: C, 63.14; H, 6.93; N,

16.99. Found: C, 63.01; H, 7.22; N, 16.71.

N-[2-(dimethylamino)-2-oxoethyl]isonicotinamide (G24)

White crystals (EtOAc). This compound (G24) was pre-

pared from methyl isonicotinoylglycinate (5 mmol, 0.97 g)

and dimethylamine (30 mmol, 1.35 g) according to the

general procedure. The product obtained as white solid was

puriﬁed from ethyl acetate. 33 % yield (0.34 g); mp

147 °C; UV (Methanol) k_max(loge) 237.0 (3.69), 205.5

(4.18) nm; IR (KBr) 3345, 3070, 2923, 1677, 1637, 1546,

1

1428, 752, 707, 674 cm^-1; H NMR (CDCl₃, 400 MHz) d

3.05 (d, 6H, J = 1.2 Hz, H-1⁰, H-2⁰), 4.23 (d, 2H,

J = 3.9 Hz, H-2*), 7.46 (brs, 1H, NH-1⁰⁰), 7.67–7.68 (m,

2H, H-3, H-5), 8.75–8.76 (m, 2H, H-2, H-6); ¹³C NMR

(CDCl₃, 100 MHz) d 35.73 (CH₃, C-1⁰or C-2⁰), 36.08

(CH₃, C-1⁰or C-2⁰), 41.75 (CH₂, C-2*), 121.15 (CH, C-3,

C-5), 141.05 (C, C-4), 150.61 (CH, C-2, C-6), 165.33 (C,

C-1*), 167.97 (C, C-3*); LC/MS (ESI?) m/z 208 (M ? H,

100). Anal. Calcd for: C₁₀H₁₃N₃O₂0.1H₂O: C, 57.46; H,

6.37; N, 20.10. Found: C, 57.42; H, 5.97; N, 19.89.

Structural data of the 2-picolinoylaminoacetamide

derivatives

N-[2-(phenylamino)-2-oxoethyl]picolinamide (G25)

N-(2-morpholino-2-oxoethyl)picolinamide (G27) White

crystals (EtOAc). This compound (G27) was prepared from

methyl picolinoylglycinate (5 mmol, 0.97 g) and morpho-

line (30 mmol, 2.61 g) according to the general procedure.

The product obtained as white solid was puriﬁed from ethyl

acetate. 33 % yield (0.41 g); mp 209 °C; UV (Methanol)

k_max(loge) 265.5 (3.77), 206.5 (4.22) nm; IR (KBr) 3380,

2977, 2929, 2863, 1675, 1633, 1511, 1432, 748, 694 cm^-1

;

White crystals (EtOH-H₂O). This compound (G25) was

prepared from methyl picolinoylglycinate (5 mmol, 0.97 g)

¹H NMR (DMSO-d₆, 400 MHz) d 3.48 (t, 4H, J = 5.0 Hz,

123

Med Chem Res

H-2⁰, H-6⁰), 3.60 (t, 4H, J = 4.8 Hz, H-3⁰, H-5⁰), 4.20 (d,

2H, J = 4.8 Hz, H-2*), 7.56–7.59 (m, 1H, H-5), 7.97 (dt,

1H, J = 1.6/7.8 Hz, H-4), 8.04 (dd, 1H, J = 0.8/7.8 Hz,

H-3), 8.64 (dd, 1H, J = 1.0/5.7 Hz, H-6), 8.66 (brs, 1H,

NH-1⁰⁰); ¹³C NMR (DMSO-d₆, 100 MHz) d 41.42 (CH₂,

C-2*), 42.50 (CH₂, C-2⁰or C-6⁰), 45.11 (CH₂, C-2⁰or

C-6⁰), 66.66 (CH₂, C-3⁰,C-5⁰), 122.44 (CH, C-3), 127.39

(CH, C-5), 138.57 (CH, C-4), 149.29 (CH, C-6), 150.18 (C,

C-2), 164.24 (C, C-1*), 167.45 (C, C-3*); LC/MS (ESI?)

m/z 163 (C₈H₇N₂O₂^?, 100), 250 (M ? H, 100), 272

(M ? Na, 100), 313 (M ? Na ? CH₃CN, 100). Anal.

Calcd for: C₁₂H₁₅N₃O₃: C, 57.82; H, 6.07; N, 16.86.

Found: C, 57.53; H, 6.10; N, 16.65.

148.59 (CH, C-6), 149.58 (C, C-2), 165.10 (C, C-1*),

167.93 (C, C-3*); LC/MS (ESI?) m/z 222 (M ? H, 99),

244 (M ? Na, 100). Anal. Calcd for: C₁₁H₁₅N₃O₂

0,15H₂O: C, 58.99; H, 6.89; N, 18.76. Found: C, 58.93; H,

6.70; N, 18.63.

N-[2-(dimethylamino)-2-oxoethyl]picolinamide

(G30)

White crystals (EtOAc). This compound (G30) was pre-

pared from methyl picolinoylglycinate (5 mmol, 0.97 g)

and dimethylamine (30 mmol, 1.35 g) according to the

general procedure. The product obtained as white solid was

puriﬁed from ethyl acetate. 33 % yield (0.34 g); mp

134 °C; UV (Methanol) k_max(loge) 265.5 (3.71), 206.5

(4.12) nm; IR (KBr) 3369, 2927, 1678, 1652, 1513, 1459,

1

N-[2-oxo-2-(pyrrolidin-1-yl) ethyl]picolinamide (G28)

White crystals (EtOAc). This compound (G28) was pre-

pared from methyl picolinoylglycinate (5 mmol, 0.97 g)

and pyrrolidine (30 mmol, 1.77 g) according to the general

procedure. The product obtained as white solid was puri-

ﬁed from ethyl acetate. 42 % yield (0.49 g); mp 155 °C;

UV (Methanol) k_max(loge) 265.5 (3.69), 206.5 (4.13) nm;

IR (KBr) 3378, 3056, 2971, 2886, 1675, 1633, 1565, 1513,

1432, 755, 642 cm^-1; H NMR (CDCl₃, 400 MHz) d 3.05

(d, 6H, J = 8.2 Hz, H-1⁰, H-2⁰), 4.27 (d, 2H, J = 4.7 Hz,

H-2*), 7.41 (ddd, 1H, J = 1.2/4.7/7.4 Hz, H-5), 7.83 (td,

1H, J = 1.6/7.8 Hz, H-4), 8.16–8.18 (m, 1H, H-3),

8.59–8.61 (m, 1H, H-6), 8.89 (brs, 1H, NH-1⁰⁰); ¹³C NMR

(CDCl₃, 100 MHz) d 35.68 (CH₃, C-1⁰or C-2⁰), 36.13

(CH₃, C-1⁰or C-2⁰), 41.43 (CH₂, C-2*), 122.13 (CH, C-3),

126.34 (CH, C-5), 137.26 (CH, C-4), 148.58 (CH, C-6),

149.82 (C, C-2), 164.61 (C, C-1*), 167.99 (C, C-3*); LC/

MS (ESI?) m/z 163 (C₈H₇N₂O₂^?, 100), 208 (M ? H,

100), 230 (M ? Na, 100), 271 (M ? Na ? CH₃CN, 100).

Anal. Calcd for: C₁₀H₁₃N₃O₂: C, 57.96; H, 6.32; N, 20.28.

Found: C, 57.91; H, 6.28; N, 20.23.

1432, 750, 701 cm^-^{1 1}H NMR (CDCl₃, 400 MHz) d

;

1.87–1.93 (m, 2H, H-3⁰or H-4⁰), 2.00–2.06 (m, 2H, H-3⁰or

H-4⁰), 3.46–3.49 (m, 2H, H-2⁰or H-5⁰), 3.55 (t, 2H,

J = 7.0 Hz, H-2⁰or H-5⁰), 4.21 (d, 2H, J = 4.7 Hz, H-2*),

7.40–7.43 (m, 1H, H-5), 7.80–7.85 (m, 1H, H-4), 8.17 (d,

1H, J = 7.7 Hz, H-3), 8.60 (d, 1H, J = 5.7 Hz, H-6), 8.86

(brs, 1H, NH-1⁰⁰); ¹³C NMR (CDCl₃, 100 MHz) d 24.30

(CH₂, C-3⁰or C-4⁰), 26.15 (CH₂, C-3⁰or C-4⁰), 42.25 (CH₂,

C-2*), 45.69 (CH₂, C-2⁰or C-5⁰), 46.13 (CH₂, C-2⁰or

C-5⁰), 122.15 (CH, C-3), 126.33 (CH, C-5), 137.24 (CH,

C-4), 148.57 (CH, C-6), 149.85 (C, C-2), 164.65 (C, C-1*),

166.46 (C, C-3*); LC/MS (ESI?) m/z 234 (M ? H, 100),

256 (M ? Na, 100). Anal. Calcd for: C₁₂H₁₅N₃O₂: C,

61.79; H, 6.48; N, 18.01. Found: C, 61.79; H, 6.50; N,

17.91.

Cell lines and cell culture

The A549 cancer cell line was purchased from American

Type Culture Collection (ATCC) and cultured at 37 °C in a

humidiﬁed atmosphere of 5 % CO₂in Dulbecco’s modiﬁed

Eagle’s medium (DMEM) supplemented with 10 % heat-

inactivated fetal bovine serum. Cells were grown to 90 %

conﬂuence and trypsinized with 0.25 % trypsin–EDTA.

Western blot analysis

N-[2-(isopropylamino)-2-oxoethyl]picolinamide (G29)

White crystals (EtOAc). This compound (G29) was pre-

pared from methyl picolinoylglycinate (5 mmol, 0.97 g)

and isopropylamine (30 mmol, 1.77 g) according to the

general procedure. The product obtained as white solid was

puriﬁed from ethyl acetate. 20 % yield (0.22 g); mp

169 °C; UV (Methanol) k_max(loge) 265.5 (3.75), 217.0

(4.02), 203.5 (4.06) nm; IR (KBr) 3336, 3293, 3068, 2971,

2927, 2875, 1679, 1644, 1544, 1463, 1411, 744, 719,

A549 cells (2 9 10⁵) were seeded on 24-well plates and

grown to 80 % conﬂuent. The cells were treated for 6 h

with IL-1b (2 ng/mL) alone, followed by the incubation

with 50 lM of samples or DMSO (as control) for 24 h.

After treatment, A549 cells were washed with phosphate-

buffered saline (PBS) and then lysed at 4 °C for 20 min in

radioimmunoprecipitation assay (RIPA) buffer. The pro-

tein concentration of the lysate was measured by using the

bicinchoninic acid (BCA) reagents (Pierce Biotechnology

Inc., Rockford, IL, USA). Equal amounts of protein

(25 lg) were loaded for sodium dodecyl sulfate-polyacry-

lamide gel electrophoresis (SDS-PAGE), and the gel was

transferred to the polyvinylidene ﬂuoride membrane (EMD

Millipore, Billerica, Massachusetts, USA). The transblotted

1

674 cm^-1; H NMR (CDCl₃, 400 MHz) d 1.17 (d, 6H,

J = 6.6 Hz, H-2⁰, H-3⁰), 4.06–4.13 (m, 3H, H-1⁰, H-2*),

5.96 (brs, 1H, NH-2⁰⁰), 7.44–7.47 (m, 1H, H-5), 7.84–7.89

(m, 1H, H-4), 8.18 (d, 1H, J = 7.8 Hz, H-3), 8.58–8.60 (m,

2H, NH-1⁰⁰, H-6); ¹³C NMR (CDCl₃, 100 MHz) d 22.77

(CH₃, C-2⁰, C-3⁰), 41.77 (CH, C-1⁰), 43.63 (CH₂, C-2*),

122.29 (CH, C-3), 126.60 (CH, C-5), 137.45 (CH, C-4),

123

Med Chem Res

membranes were blotted with monoclonal anti-COX-2

antibody (Santa Cruz Biotechnology Inc., Dallas, TX,

USA) in order to determine the COX-2 protein level. Actin

levels were used as loading control by blotting with mon-

oclonal anti-actin antibody (Sigma-Aldrich Corp., St.

Louis, MO, USA). The detection was performed by using

SuperSignal West Pico Chemiluminescent Substrate

(Pierce Biotechnology Inc., Rockford, IL, USA) and X-ray

ﬁlm (CL-Xposure Film, Pierce Biotechnology Inc., Rock-

ford, IL, USA). The relative expression levels of COX-2

protein were quantiﬁed by analyzing the total intensity of

each band as arbitrary units using ImageJ software (Sch-

neider et al., 2012). The effect of tested compounds was

calculated by comparison of COX-2 levels to the control

samples.

conformations of the ligands was performed by using GOLD

5.2.1 program with default generic algorithm parameters:

population size = 100; selection pressure = 1.1; # opera-

tions = 100.000; # islands = 5; niche size = 2; migra-

tion = 10; mutation = 95; crossover = 95 (Genetic

Optimization for Ligand Docking, Jones et al., 1995, 1997).

˚

The ligands were docked within a radius of 10 A around the

binding site from a single solvent-accessible point approxi-

mately at the center of the active site in the protein and its

orthogonal coordinates: x = -16.3552; y = 41.307;

z = 26.3551; that deﬁned by HERMES 1.6 using cavity

residues (Hermes Visualiser, http://www.ccdc.cam.ac.uk/

Solutions/GoldSuite/Pages/Hermes.aspx). Ten conforma-

tions were allowed per structure. GoldScore and ChemScore

ﬁtness functions are used as scoring functions (Jones et al.,

1995, 1997; Eldridge et al., 1997). Figure 2 is setup with

MOE2013.08 program.

PGE₂assay

A549 cells (2 9 10⁵) were seeded on 24-well plates and

grown to 80–90 % conﬂuent. The cells were treated for 6 h

with IL-1b (2 ng/mL) alone. After IL-1b treatment, the

cells were exposed to the samples (50 lM) or DMSO for

24 h. After the treatment, the conditioned medium was

collected and the PGE₂accumulation (pg/mL) in media

was evaluated by Prostaglandin E₂Enzyme Immuno Assay

kit (Cayman), according to the manufacturer’s instructions.

The effect of tested compounds was calculated by the

comparison of PGE₂accumulation to the control samples.

Concentration of PGE₂value is 216.2187 pg/mL for the

control (DMSO).

Molecular dynamics simulations

Molecular dynamics simulations were performed for pro-

tein and for three protein–ligand complexes (G21, G22,

G27) using AMBER v 12.0 package (Case et al., 2012).

The best-ranked docking solution of the three ligands

obtained by using GOLD 5.2.1 program was selected to

prepare the initial protein–ligand complexes. Hydrogen

atoms were added to the protein. The protein and complex

systems were neutralized with appropriate number of

sodium counter ions and were solvated in an octahedral

˚

box with TIP3P water molecules with 10-A distance

between the protein surface and the box distance between

the protein surface and the box boundary (Jorgensen et al.,

1983). The partial atomic charges for each ligand were

gained with AM1-BCC charge model using antechamber

module of AMBER v.12 package (Jakalian et al., 2000).

Protein and ligand’s topology and parameter ﬁles were

constituted using AMBERff99 and general AMBER force

ﬁeld (gaff), respectively (Cornell et al., 1995; Wang et al.,

2004). The energy minimizations and MD simulations of

protein and protein–ligand complexes were executed with

SANDER.MPI and PMEMD.CUDA modules of AMBER

v 12.0 package, respectively. At ﬁrst, the simulated protein

and binding complexes were exposed to 15,000-step min-

imization using steepest descent algorithm succeeded by

10,000-step minimization using conjugate gradient to avoid

bad steric contacts. After the minimization steps, protein–

ligand complex and protein systems including the involved

water molecule and sodium ions were slowly heated from 0

Molecular docking study

Ligand docking

The chemical structures of the studied compounds were built

with builder panel of MOE2013.08, protonated using the

protonate 3D protocol and subjected to an energy mini-

mization with MOE.2013.08 using MMFF94x force ﬁeld

(Molecular Operating Environment Chemical Computing

Group Inc., Canada; Halgren, 1996). Then, all of the con-

formations of the studied compounds were generated using

the systematic search algorithm available in MOE2013.08

(Chen and Foloppe, 2008). The crystal structure of lumira-

coxib in complex with mCOX-2 (PDB id: 4llz resolved at

˚

2.35 A) was obtained from the RCSB Protein Data Bank

(http://www.rcsb.org/pdb). Chain A was chosen for docking

structure and chain B, heteroatoms; water molecules (ex-

cluded the water molecules included in the binding site) in

the 4llz pdb ﬁle were deleted, and hydrogen atoms were

added to the protein using MOE2013.08. Chain A of the

enzyme was subjected to an energy minimization using

MMFF94x force ﬁeld in MOE2013.08. Docking of all

˚

to 300 K with 10 kcal/mol/A restraint force during the

term of 0.2 ns permitting water molecules and ions to move

freely. The SHAKE algorithm was carried out to constrain

band vibrations involving hydrogen atoms (Ryckaert et al.,

˚

1977). 10-A cutoff for non-bonded interactions and 2 fs for

123

Med Chem Res

Fig. 2 Comparison of the ﬁrst-ranked solutions of the studied com-

pounds in mCOX-2 (PDB id: 4llz). The ranked solutions of the studied

compounds are based on Goldscore. Green, yellow and cyan sticks

represent compounds G21, G22 and G27, respectively. The active side

residues are named and represented as gray sticks in mCOX2. For

clarity, all hydrogen atoms have been removed except for those

involved in hydrogen bonding. H bonds and CH–p interactions are

represented black dashed lines (Color ﬁgure online)

a time step were used. The particle mesh Ewald (PME)

method was executed for long-range electrostatic interac-

tions (Essmann et al., 1995). The entire systems were

equilibrated at 300 K for 1 ns and were exposed to free

MD simulation for 20 ns. Xmgrace program was used for

visualization of the trajectories (Grace Development Team,

http://plasma-gate.weizmann.ac.il/Grace/). Free binding

energies of compounds were calculated with MMPBSA.py

module of AMBER v 12.0 package using the Generalized

Born (GB) model from 200 spaced snapshots of the unre-

strained MD simulations (Miller III et al., 2012). The

calculated free energies are not real binding free energies

and are only used to compare relative to one another. Ptraj

module of AMBER v.12 package were used to determine

the hydrogen bonding using the maximum donor–acceptor

Those chlorides were reacted with glycine (in this case, it is

followed by esteriﬁcation) or methyl glycinate to yield

2-acylaminoacetic acid methyl esters, which were then

converted to 2-acylaminoacetamide by treating with appro-

priated amines (Scheme 1). According to literature survey,

compounds G1, G2, G3, G5, G6, G13, G17, G19 and G25

are reported derivatives and G4, G7, G8, G10, G11, G16,

G22, G26 and G28 are listed in the literature with chemical

abstracts service registry number 95204-52-7, 932130-00-2,

1002499-52-6, 705955-02-8, 705954-02-5, 119755-00-2,

1197607-87-6, 1223618-35-6 and 1211129-24-6, respec-

tively, but corresponding scientiﬁc reference data are not

available (Schwyzer et al., 1955; Thomsen et al., 1983;

Haworth et al., 1952; Farese et al., 1996; Blanco et al.,

2005).

˚

distance of 3.5 A and a donor–hydrogen–acceptor angle of

The structures of title compounds were conﬁrmed by

1

180° 60 (Roe and Cheatham III, 2013; Fabiola et al.,

2002). Figure 4 is setup with MOE2013.08 program.

spectral (UV, IR, H NMR, ¹³C NMR and ESI-Mass) and

elemental analysis. In the IR spectra of the title compounds,

carbonyl and amide N–H stretching frequencies were

observed between 1623 and 1681 and 3237 and 3403 cm^-1

respectively, indicating the existence of amidoamine func-

Results and discussion

Chemistry

1

tional group (Hesse et al., 1997). H NMR spectra of the

compounds veriﬁed the existence of the expected numbers of

protons, and the chemical shifts, multiplicities and coupling

constants were also used to prove the integrity and

intramolecular vicinity of the synthesized molecular struc-

tures. ¹³C NMR spectra also supported the structural conﬁr-

mation (see experimental part). The mass spectra of the title

compounds were recorded according to the electrospray ion-

ization (ESI) technique and interpreted for positive ionization.

The [M ? H]^?ions of title compounds were in complete

agreement with the calculated molecular weights.

2-Acylaminoacetamide derivatives were synthesized by

modiﬁcation of the reported methods in the literature (Singh

et al., 1994; Kurita et al., 2002; Gros et al., 2006; Naumova

et al., 1969). Thus, aromatic carboxylic acids [benzoic acid,

thiophen-2-carboxylic acid (2-thienoic acid), pyridin-2-car-

boxylic acid (2-picolinoic acid), pyridin-3-carboxylic acid

(nicotinic acid) and pyridin-4-carboxylic acid (isonicotinic

acid)] were ﬁrst converted to corresponding acyl chlorides.

123

Med Chem Res

Biological activity

PGE₂level in A549 cell line. Among the compounds which

increased the PGE₂level, compound G23 is the unique

one; while reducing the expression of COX-2 as fourfold

according to the control, it did not cause any signiﬁcant

shift on the PGE₂level.

The biological activities of the title compounds have

been determined by evaluating their effects on COX-2

isoenzyme expression and PGE₂production in A549

(human lung adenocarcinoma) cell lines. A549 cell line

is a powerful tool to study the effect of different con-

ditions on COX-2 expression due to its low or unde-

tectable level of COX-2 isoenzyme expression (Mitchell

et al., 1994; Sievers et al., 2005). Moreover, it is also

known that the inﬂammatory cytokine, interleukin-1b

(IL-1b), strongly upregulates COX-2 expression and

PGE₂synthesis in A549 cells (Ling et al., 2010). Since

IL-1b-stimulated A549 cells do not express COX-1, it

has been suggested that the upregulation of the PGE₂is

mainly dependent on COX-2 enzyme activity (Koeberle

et al., 2008). Therefore, in our experimental model, the

expression of COX-2 has been triggered in A549 cells

with IL-1b treatment, and the effect of the title com-

pounds on upregulated COX-2 protein expression was

determined by Western Blotting experiment (Ling et al.,

2010; Jiang et al., 2004). Similarly, inhibitory effects of

title compounds on PGE₂production (pg/mL) in IL-1b-

stimulated A549 cell lines were performed by enzyme

immunoassay procedure (EIA) (Ling et al., 2010). In this

method, the elevated PGE₂level in IL-1b-activated

A549 cell lines has been linked to the induced COX-2

isoenzyme expression (Huang et al., 1998). Meanwhile,

the inhibitory effect of DUP-697, selective COX-2

inhibitor, has also been determined as the positive con-

trol. The effect of tested compounds on COX-2 protein

expression levels and PGE₂production was evaluated by

comparison with the negative control (100 %).

The general structure of the designed derivatives is

2-acylamino-N-substituted acetamide. The structural mod-

iﬁcations made on the general structure are limited to the

replacement of the acyl group and the N-substituent. In our

study, ﬁve different acyl groups (namely benzoyl, 2-thie-

noyl, nicotinoyl, isonicotinoyl and 2-picolinoyl) and six

different N-substituents on amide nitrogen (N-phenyl

derivative, N-alkyl derivatives and the derivatives of which

the amide nitrogen belongs to a heterocyclic ring such as

piperidine, morpholine and pyrrolidine) have been used to

build the target molecules.

Under the frame of the biological data summarized in

Table 1, the contribution of the acyl groups independent

from N-substituent on the 2-acylamino-N-substituted acet-

amide general structure could be stated in the order

of isonicotinoyl [ 2-thienoyl, 2-picolinoyl [ nicotinoyl [

benzoyl for the inhibition of COX-2 expression. Benzoyl

group as acyl moiety generally afforded compounds,

which are either expression inducer (compounds G1, G3,

G4 and G6) or mild expression reducer (compounds G2,

G5).

The contribution of the acyl moiety on inhibitor poten-

cies of the title compounds on PGE₂biosynthesis was not

clear-cut to make general assumptions. It was observed that

isonicotinoyl, 2-thienoyl, 2-picolinoyl, nicotinoyl afforded

compounds with diverse inhibitor potency. According to

the data summarized in Table 1, the most active com-

pounds (compounds G21 and G22) belong to isonicoti-

namidoacetamide derivatives. On the contrary, benzoyl as

the acyl moiety in compounds G1, G3, G4 and G6

increased the PGE₂level in A549 cell line.

In the light of the results summarized in Table 1, com-

pounds G9, G23, G24, G27 and G29 have decreased the

induced COX-2 isoenzyme expression in A549 cell line

more than 50 % compared with the control. The inhibitor

potencies of these compounds can be ranged as

G23 [ G24 [ G27 [ G29, G9 in descending order

(Table 1). Surprisingly, compounds G3, G4, G16, G25 and

DUP-697, COX-2 selective inhibitor standard, have

increased the COX-2 isoenzyme expression in A549 cell

line signiﬁcantly compared to the control. The inhibitor

potencies of the title compounds on the level of PGE₂in

IL-1b-induced A549 cells have also varied. Compounds

G21 and G22 were the most active derivatives with inhi-

bitor potency over 40 % in comparison with control. The

inhibitor potencies of these two derivatives were compa-

rable with standard drug DUP-697. The inhibitory ratio of

compounds G29, G27, G11 and G9 were between 30 and

40 %. However, this ratio was between 20 and 30 % for

compounds G26, G19, G18, G17 and G15. Interestingly,

compounds G3, G4, G6, G23 and G25 have increased the

Among the six different N-substituents on amide nitrogen

(N-phenyl derivative, N-alkyl derivatives and the derivatives

of which the amide nitrogen belongs to a heterocyclic ring

such as piperidine, morpholine and pyrrolidine), morpholine

ring lead to more potent inhibitors on COX-2 expression

in the series of 2-thienoylaminoacetamides, nicotinoy-

laminoacetamides and 2-picolinoylaminoacetamides. In the

case of isonicotinoylaminoacetamide derivatives, N,N-

dimethyl and N-isopropyl substituents yielded the most

active compounds. When the overall contribution of N-

substituents to inhibitor effects of the title compounds on

COX-2 expression were reviewed, N-isopropyl and N,N-

dimethyl stand out as the most promising substituents in the

studied series.

Regarding the contribution of N-substituent on

inhibitory activity of the title compounds on PGE₂

biosynthesis, the compounds bearing isopropyl or

123

Med Chem Res

Table 1 In vitro biological activity data summarizing the effect of synthesized compounds on the level of COX-2 expression and PGE₂

biosynthesis in A549 cell line

Compound

Ar

NRR^ı

% COX-2^a

% PGE^b₂

Control

G1

–

100 1.5

107 1.2

87 0.9

151 2.4

147 3.0

99 2.9

116 0.9

72 0.6

101 3.0

46 3.1

54 1.8

59 1.5

106 1.5

87 0.6

108 0.9

85 0.3

135 1.8

85 1.2

101 1.5

61 0.6

81 1.8

97 1.6

87 0.7

18 0.9

21 1.0

134 0.9

63 1.2

36 0.6

70 1.8

46 0.9

105 1.2

188 1.5

100.0

114.8

0

Phenyl

Phenylamino

Piperidin-1-yl

Morpholino

G2

Phenyl

93.3 3.0

144.03 3.7

163.2 1.5

98.3 6.5

126.6 4.8

na

G3

Phenyl

G4

Phenyl

Pyrrolidin-1-yl

Isopropylamino

Dimethylamino

Phenylamino

Piperidin-1-yl

Morpholino

G5

Phenyl

G6

Phenyl

G7

2-Thienyl

3-Pyridinyl

4-Pyridinyl

2-Pyridinyl

–

G8

97.2 1.3

64.6 3.5

87.7 6.5

67.1 1.5

100.9 2.7

97.4 6.4

87.3 4.3

71.1 0.4

97.0 7.0

65.6 2.1

74.4 2.6

70.3 2.3

88.8 1.1

59.0 1.2

50.0 1.6

110.2 4.3

83.3 2.7

113.6 1.6

71.2 2.0

61.5 2.3

87.0 4.8

69.8 3.9

na

G9

G10

G11

G12

G13

G14

G15

G16

G17

G18

G19

G20

G21

G22

G23

G24

G25

G26

G27

G28

G29

G30

DUP-697

Pyrrolidin-1-yl

Isopropylamino

Dimethylamino

Phenylamino

Piperidin-1-yl

Morpholino

Pyrrolidin-1-yl

Isopropylamino

Dimethylamino

Phenylamino

Piperidin-1-yl

Morpholino

Pyrrolidin-1-yl

Isopropylamino

Dimethylamino

Phenylamino

Piperidin-1-yl

Morpholino

Pyrrolidin-1-yl

Isopropylamino

Dimethylamino

–

46.3 2.3

na not available

a

Data are indicated as percentage of COX-2 level in A549 cells (n = 3) compared to the control sample

Data are indicated as percentage of PGE₂level in conditioned media (n = 3) compared to the control sample

b

Molecular modeling studies

morpholine moieties as N-substituent were appeared

to be most active ones in each series with the exception

of isonicotinoylaminoacetamide derivatives. In the case

of isonicotinoylaminoacetamide derivatives, pyrro-

lidine seemed to have priority in comparison with

morpholine.

Molecular docking studies

In this study, molecular docking was practiced to detect the

interaction of the studied compounds for mCOX-2 using

123

Med Chem Res

Table 2 Docking score for compounds G21, G22 and G27, inside mCOX2 (PDB id: 4llz), using Goldscore and Chemscore

Compounds

4LLZ

GoldScore

ChemScore

G21

G22

G27

45.7594 (1)

45.9231 (1)

45.1932 (1)

13.4045 (117)

15.3856 (64)

16.5502 (37)

The absolute ranking positions for the suggested binding poses were given inside brackets

Fig. 3 Plots of root-mean-

square deviation (RMSD) of

mCOX2 (PDB id: 4llz) and

mCOX2-ligand complexes.

RMSDs were calculated using

initial structures as templates.

The initial pdb structure is the

reference for mCOX2 (red) and

the initial docked structures are

references for the complexes

(black). The trajectories were

captured every 1 ps until the

simulations end (Color ﬁgure

online)

GOLD 5.2.1 (Jones et al., 1995, 1997). The crystal struc-

ture of lumiracoxib in complex with mCOX-2 (PDB id:

4llz) was selected as the initial model of COX-2. The most

active compounds (G21, G22 and G27) were docked in

order to propose the non-covalent interactions of the syn-

thesized compounds inside the active side of mCOX-2.

According to the docking results, morpholine and

pyrrolidine rings of the studied compounds (G21, G22 and

G27) are oriented toward the mouth of the COX-2 channel

in the vicinity of amino acid residues Met113, Val116,

Arg120, Val349, Tyr355, Leu359 and Leu531. In addition,

pyridine ring of the most active compounds substituted at 2

123

Med Chem Res

or 4 positions are oriented toward the hydrophobic pocket

formed by Leu352, Phe381, Tyr385, Trp387, Phe518,

Met522, Val523, Gly526, Ala527 and Ser530 (Fig. 2). In

this pocket, the hydrophobic interactions such as CH–p and

aromatic interactions such as CH–p appear between the

ligands and amino acid residues as Leu352, Phe 518,

Val523 and Gly526 (Takahashi et al., 2000; Brandl et al.,

2001). The CH–p interactions are observed between the

pyridine ring of the studied compounds and the alkyl chain

of Leu352, Gly526 and Val523 residues whereas aromatic

CH–p interactions occur between the pyridine ring of the

studied compounds and the phenyl ring of Phe518 residue.

The scoring values and the absolute ranking position

(inside brackets) of the selected docking poses of the

compounds are reported in Table 2.

Molecular dynamics simulations

Molecular dynamics simulation is a powerful molecular

modeling method that enables the dynamic behavior of

protein–ligand complexes, in opposition to docking

which mostly ensures a single conformation. In our

study, MD simulations were used to analyze the sta-

bility and hydrogen bonding of the selected compounds

in the active side of mCOX-2 and to calculate the

average free binding energies of the studied compounds.

Fig. 4 Main interactions between compounds G21 (a), G22 (b), G27

(c) and mCOX2 during a 20-ns MD simulation. The percentages

represent the time when a hydrogen bond was present during the

dynamics. Green, yellow and cyan sticks represent compounds G21, G22

and G27, respectively. The active side residues are named and

represented as gray sticks in mCOX2. For clarity, all hydrogen atoms

have been removed except for those involved in hydrogen bonding. H

bonds and CH–p interactions are represented black dashed lines (Color

ﬁgure online)

Table 3 Hydrogen bond analysis with MD simulations for the studied compounds inside the active site of mCOX2

˚

Distance (A)

Compounds

G21

Acceptor

Donor-H

Donor

H bond occupancy (%)

Angle (°)

G21:O1

G22:O1

G27:O1

Arg120:HH21

Arg120:HE

Ser530:HG

Arg120:NH2

Arg120:NE

Ser530:OG

73.83

18.35

85.87

63.77

2.815

2.899

2.706

2.709

26.79

34.71

28.33

22.91

G22

G27

The combination of donor D, hydrogen H, and acceptor A atom with a D–HÁÁÁA conﬁguration was regarded as a hydrogen bond when the

˚

distance between donor D and acceptor A was shorter than R_max(3.5 A) and the angle H–D–A was smaller than h_max(60.0 °). The percentage of

hydrogen bond was occurring during the process of the MD simulation

123

Med Chem Res

Table 4 Calculated MM-GBSA free binding energies and the van der

Waals energies of compound G21, G22 and G27 with standard errors

of the mean, and in vitro biological activity data summarizing the

effect of studied compounds on the level of PGE₂biosynthesis with

standard errors of the mean in A549 cell line

Compounds

MM-GBSA free binding energy (kcal/mol)

Van der Waals Energy (kcal/mol)

% PGE₂inhibition

G21

G22

G27

-29.4446 0.1613

-30.5273 0.1674

-31.3873 0.1833

-41.8946 0.1343

-40.6072 0.1601

-40.9803 0.1851

59.0 1.2

50.0 1.6

61.5 2.3

Table 5 Best docking scores for all 30 compounds, DUP-697 and

lumiracoxib, inside mCOX2 (PDB id: 4llz), for each scoring functions

molecular dynamics (MD) simulations. These three

protein–ligand complexes of the most active compounds

were then subjected to 20-ns simulations after the

equilibrium step.

Compounds

4LLZ

Goldscore

Chemscore

G1

48.5322

45.6771

46.5456

46.6042

45.7700

43.2603

48.9115

48.8905

47.6948

47.2328

45.6634

43.2201

48.2853

46.9592

46.2284

45.5160

45.6810

43.2680

48.2826

44.5805

45.7594

45.9231

46.3652

42.8652

49.4017

47.4551

45.1932

44.2938

45.5826

42.1806

63.5386

50.5532

28.6891

24.8408

22.1398

22.6276

21.9693

19.1647

26.9644

22.5798

20.2616

20.9577

20.3185

17.7727

26.7782

22.3785

20.3180

21.4686

21.9874

17.4862

26.5454

22.3293

19.9770

20.3602

20.7145

17.2617

27.1024

22.5748

20.3180

21.0730

20.1880

16.5030

36.0067

29.2209

Structural stability analysis

G2

G3

It is appraised that the MD simulations for the stability of

compound G21, G22 and G27 in complex with mCOX-2,

we have them observed stable during 20 ns. Firstly, the

average root-mean-square deviation (RMSD) values of

ligand–protein complexes gradually increase from *1.7 to

G4

G5

G6

G7

˚

G8

*2.5 A for G21 and from *1.8 to *3.5 A for G27 at the

time slot between 0 and 20 ns, respectively. However, the

key interactions between protein and ligands are still con-

served, and they are also stable till the end of the simulation

in spite of the ﬂexibility of the compounds in the active side

of mCOX-2. In addition, average RMSD value of G22-

G9

G10

G11

G12

G13

G14

G15

G16

G17

G18

G19

G20

G21

G22

G23

G24

G25

G26

G27

G28

G29

G30

Dup-697

Lumiracoxib

˚

protein complex is *1.6 A and is stable at whole MD

simulation. Lastly, in the MD plot of the receptor protein,

average RMSD value ﬂuctuatingly increases from *1.0 to

˚

*3.2 A and is stable throughout the MD simulation because

of the fact that the protein is ﬂexible in its apo form (Fig. 3).

Binding mode analysis

In terms of the interactions between the protein–ligand

complexes, hydrogen bonds play an important role in sta-

bilizing protein–ligand complexes (Chou, 2004). In this

study, the hydrogen bonds were examined on the basis of

the trajectories of the MD simulations, and the geometry

and stability of the hydrogen bond network formed by the

ligand were investigated in the active side of the mCOX-2.

As shown in Fig. 4, putative binding mode obtained from

MD simulation presents the hydrogen bonds between the

amide oxygen (O1) of the morpholine ring of compound

G21 and Arg120 with 73.83 and 18.35 % occupancies. On

the other hand, amide oxygen (O1) which also belongs to

pyrrolidine ring of compound G22 and morpholine ring of

compound G27 create a hydrogen bond with different

amino acid residue as Ser530, with 85.87 and 63.77 %

occupancy for G22 and G27, respectively. The hydrogen

For this purpose, the best-ranked solutions of the most

active compounds inside mCOX-2 (PDB id: 4llz)

obtained by using GOLD 5.2.1 program were chosen for

123

Med Chem Res

Fig. 5 Plots of root-mean-square deviation (RMSD) of mCOX2

(PDB id: 4llz) and mCOX2-DUP-697 complex. RMSDs were

calculated using initial structures as templates. The initial pdb

structure is the reference for mCOX2 (red) and the initial docked

structure is the reference for the complex (black). The trajectory was

captured every 1 ps until the simulation end (Color ﬁgure online)

bonding analysis results of the studied compounds are

presented in Table 3.

Conclusion

According to the putative binding modes of the studied

compounds obtained from MD snapshots, the CH–p

interactions occur in the hydrophobic pocket of active side

of mCOX-2 as well as hydrogen bonds. The CH–p inter-

actions are seen between pyridine ring of the selected

compounds and Leu352 and Gly526 residues. Additionally,

the aromatic CH–p interaction appears between Tyr385

and pyridine ring of compound G27 (Fig. 4).

In this study, molecular simpliﬁcation technique has been

applied to the thalidomide molecule to design the title

compounds since thalidomide is known to possess ‘‘mod-

erate non-selective or weakly COX-2-selective COX-in-

hibiting activity.’’ The synthesized compounds are the

derivatives of acylaminoacetamide structure, namely,

2-benzoylaminoacetamide, 2-(2-thienoylamino)acetamide,

2-nicotinoylaminoacetamide,

2-isonicotinoylaminoac-

etamide and 2-picolinoylaminoacetamide. The inhibitory

potencies of the title compounds on COX-2 expression and

PGE₂biosynthesis were determined in A549 cells.

Molecular docking, molecular dynamics simulations and

relative free binding energy calculations were applied to

determine the binding mechanism of the studied com-

pounds to COX-2 isoenzyme.

MM-GBSA binding free energy calculations

The MM-GBSA binding free energies were calculated for

compounds G21, G22 and G27 from MD simulations

(Massova and Kollman, 2000). However, these should not

be expected to be true free binding energies; they were

used to compare binding afﬁnity of ligands to mCOX-2

(Miller and Roitberg, 2013). The average free binding

energies of compounds are very close when compared to

each other (Table 4). The van der Waals energy (spanning

to -40 to -42 kcal/mol) is the dominant factor for the

binding of the three inhibitors among the binding free

energy components. This dominant factor also shows the

contribution of CH–p interactions and hydrogen bonds

which are favorable for binding. However, the average free

binding energy of compound G27 yielded from MD sim-

ulation is slightly bigger than the MM-GBSA free binding

energy of compound G21 and G22, but the average free

binding energies of the three compound are close by

-30 kcal/mol, and they are not signiﬁcantly different from

each other, so the binding afﬁnities of the compounds to

mCOX-2 display similar behavior according to acquired

results from MD simulations.

In conclusion, acylaminoacetamide template bearing

heterocyclic acyl group seems as a promising lead to derive

for further studies on medicinal targets of arachidonic acid

cascade. Regarding molecular modeling studies, hydrogen

bonds and hydrophobic and aromatic interactions such as

CH–p and CH–p, respectively, play an important role for

binding of the studied compounds to COX-2.

Supporting information

Docking results

Docking studies were carried out GOLD 5.2.1 program using

Goldscore and Chemscore as the scoring functions inside the

crystal structure of mCOX2 (PDB id: 4llz). The scoring

values of the best ranking positions of all title compounds,

DUP-697 and lumiracoxib were reported in Table 5.

123

Med Chem Res

Eldridge MD, Murray CW, Auton TR, Paolini GV, Mee RP (1997)

Empirical scoring functions—I: the development of a fast

empirical scoring function to estimate the binding afﬁnity of

ligands in receptor complexes. J Comput Aided Mol Des

11:425–445

Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG

(1995) A smooth particle mesh Ewald method. J Chem Phys

103:8577–8593

Molecular dynamics of DUP-697 inside mCOX2

The initial structure of mCOX2-DUP-697 complex was

prepared with chain A of the crystal structure of mCOX2

(PDB id: 4llz) and the best-ranked solutions of the ligand

obtained using GOLD 5.2.1 program. The MD plot of DUP-

697-protein complex showed that the MD simulation is

Fabiola F, Bertram R, Korostelev A, Chapman MS (2002) An

improved hydrogen bond potential: impact on medium resolution

protein structures. Protein Sci 11:1415–1423

˚

stable with *1.8 A value of the average RMSD in the ﬁrst

10 ns. In the second part of the MD simulation, the average

Farese A, Peytou V, Condom R, Sinet M, Patino N, Kirn A, Moog C,

Aubertin AM, Guedj R (1996) Synthesis of analogues of the

benzodiazepine Ro-5-3335, antagonist of Tat HIV-1: biological

evaluation by luciferase transactivation and anti-viral assay.

J Med Chem 31:497–505

˚

RMSD value increases to *2.2 A and keeps it until 17.5 ns.

In the rest of the MD simulation, the average RMSD value

˚

gradually increases to *3.2 A (Fig. 5). The MM-GBSA

free binding energy of DUP-697 gained from unrestrained

Grace Development Team, Weizmann Institute of Science, Israel.

http://plasma-gate.weizmann.ac.il/Grace/

MD simulation is calculated -36.1591 kcal/mol.

Gasparini G, Longo R, Sarmiento R, Morabito A (2003) Inhibitors of

cyclo-oxygenase 2: a new class of anticancer agents? Lancet

Oncol 4:605–615

Gros L, Lorente Jimenez SO, Yardley V, Rattray L, Wharton H, Little

S, Croft SL, Ruiz-Perez LM, Gonzalez-Pacanowska D, Gilbert

IH (2006) Evaluation of azasterols as anti-parasitics. J Med

Chem 49:6094–6103

Acknowledgments This study was supported by research grants

from Ege University (Project Number: 2009/Ecz/015). We also thank

Pharmaceutical Sciences Research Centre (FABAL) of Ege Univer-

sity Faculty of Pharmacy for equipmental support.

Hermes Visualiser. http://www.ccdc.cam.ac.uk/Solutions/GoldSuite/

Pages/Hermes.aspx

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Synthesis, biological activity screening and molecular modeling study of acylaminoacetamide derivatives

DOI: 10.1007/s00044-015-1419-4

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