Synthesis, activity and mechanism of alkoxy-, carbamato-, sulfonamido-, thioureido-, and ureido-derivatives of 2,4,5-trimethylpyridin-3-ol against inflammatory bowel disease

Journal of Enzyme Inhibition and Medicinal Chemistry

ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20

Synthesis, activity and mechanism of alkoxy-,

carbamato-, sulfonamido-, thioureido-, and ureido-

derivatives of 2,4,5-trimethylpyridin-3-ol against

inflammatory bowel disease

Chhabi Lal Chaudhary, Pallavi Gurung, Seoul Jang, Suhrid Banskota, Tae-Gyu

Nam, Jung-Ae Kim & Byeong-Seon Jeong

To cite this article: Chhabi Lal Chaudhary, Pallavi Gurung, Seoul Jang, Suhrid Banskota, Tae-Gyu

Nam, Jung-Ae Kim & Byeong-Seon Jeong (2020) Synthesis, activity and mechanism of alkoxy-,

carbamato-, sulfonamido-, thioureido-, and ureido-derivatives of 2,4,5-trimethylpyridin-3-ol against

inflammatory bowel disease, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 1-20,

DOI: 10.1080/14756366.2019.1677637

To link to this article: https://doi.org/10.1080/14756366.2019.1677637

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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

2019, VOL. 35, NO. 1, 1–20

https://doi.org/10.1080/14756366.2019.1677637

ORIGINAL ARTICLE

Synthesis, activity and mechanism of alkoxy-, carbamato-, sulfonamido-,

thioureido-, and ureido-derivatives of 2,4,5-trimethylpyridin-3-ol against

inflammatory bowel disease

a

b

a

b

a

ꢀ

Chhabi Lal Chaudhary , Pallavi Gurung , Seoul Jang , Suhrid Banskota , Tae-Gyu Nam , Jung-Ae Kim and

Byeong-Seon Jeong^a

b

^aCollege of Pharmacy and Institute for Drug Research, Yeungnam University, Gyeongsan, Republic of Korea; Department of Pharmacy and

Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Republic of Korea

ABSTRACT

ARTICLE HISTORY

Received 22 July 2019

Revised 25 September 2019

Accepted 2 October 2019

Inflammatory bowel disease (IBD) is a chronic immuno-inflammation in gastrointestinal tract. We have

evaluated the activity of the compounds to inhibit the adhesion of monocytes to colon epithelial cells is

triggered by a pro-inflammatory cytokine, tumour necrosis factor (TNF)-a. The in vitro activity of the com-

pounds, 13b (an ureido-derivative), 14c, 14j, 14k, 14n (thioureido-), 18c and 18d (sulfonamido-), was in

correlation with in vivo anti-colitis activity revealed as significant recovery in body- and colon-weights and

colon myeloperoxidase level, a biochemical marker of inflammation reflecting neutrophil infiltration.

In vivo, TNBS-induced changes in the expression of inflammatory cytokines (TNF-a, IL-6, IL-1b, IL-10, and

TGF-b), NLRP3 inflammasome components (NLRP-3, Caspase-1, and IL-18), and epithelial junction mole-

cules (E-cadherin, claudin2/3, and ZO-1) were blocked and recovered by oral administration of the com-

pounds (1 mg/kg). Compound 14n which showed the best efficacy can be a promising lead for orally

available therapeutics for pathology of IBD.

KEYWORDS

Pyridin-3-ols; inflammatory

bowel disease; TNF-a;

adhesion; epithelial junction

1. Introduction

IL-6, IL-1b, and anti-inflammatory cytokines like IL-10 and TGF-b^3,4

.

TNF-a is a master cytokine which induces the production of IL-1b

Inflammatory bowel disease (IBD) refers to a chronic inflammation

occurring in the gastrointestinal tract with increasing worldwide

incidence and prevalence. Two major subtypes of IBD include

ulcerative colitis (UC) and Crohn’s disease (CD) with distinct injury

site and disease types. The exact cause of IBD is not fully under-

stood, but mounting evidences suggest that its pathogenesis is

associated with close interactions between genetic, immune, and

environmental factors, resulting in impairment of intestinal epithe-

lial barrier and dysregulation of inflammatory cytokine homeostasis.

While a couple of molecular targets have been studied for treat-

ment of IBD, only a few target-based approaches are successful

and there are no well-validated molecular targets known so far. The

best examples of successful anti-IBD treatment would be anti-

tumour necrosis factor-a (anti-TNF-a) antibody therapies such as

infliximab and adalimumab used in clinics, showing mucosal heal-

ing in CD¹. However, some patients do not respond to the drugs,

which highlights the necessity of new pharmacological interven-

tions for of IBD. Examples of small molecule inhibitors with good

therapeutic outcome are even scarcer. A Janus kinase (JAK) inhibi-

tor, tofacitinib has recently been included as a therapeutic option

in the treatment of UC and possibly of CD². Therefore, medical

unmet need for small molecule IBD treatment still remains huge.

Uncontrolled inflammatory response results from an imbalance

and IL-6 through activation of NF-kB, leading to a vicious cycle of

persistent inflammation and deterioration of epithelial function^5,6

.

IL-10 and TGF-b which downregulate proinflammatory cytokine

(TNF-a and IL-6) production and Th1 immune response, also play a

crucial role in maintenance of colon mucosa integrity during inflam-

mation^7–9. The effects of IL-10 depend on the timing and sources

of its secretion. In low to moderate inflammatory condition, IL-10

from dendritic cells or macrophages drives the production of IL-10

by regulatory T cells (Treg), whereas in case of challenging with

strong proinflammatory response condition, a large amount of IL-

10 is produced from larger populations of cells to minimise path-

ology during the resolution phase of the challenge¹⁰.

During cellular stress or infection, an innate immune response

occurs through pattern recognition receptors (PRRs) like toll-like

receptors (TLR) and NOD-like receptors. NOD-like receptor protein

3 (NLRP3), one of the most characterised PRRs, forms a multipro-

tein complex called inflammasome, leading to caspase-1 activation

and release of IL-1b and IL-18¹¹. NLRP3 further triggers pyroptosis,

an inflammatory programmed cell death, and thus intestinal bar-

rier damage¹². Abnormal activation of NLRP3 inflammasome has

been linked to IBD¹³. Based on the findings that inhibition of

NLRP3 blocks not only cytokine release but also p’yroptotic cell

death, NLRP3 inflammasome is proposed to be a potential target

between the levels of proinflammatory cytokines such as TNF-a, in IBD drug discovery efforts¹³.

CONTACT Tae-Gyu Nam

Republic of Korea; Jung-Ae Kim

tnam@hanyang.ac.kr

Department of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan,

jeongb@ynu.ac.kr College of Pharmacy and Institute for Drug Research, Yeungnam

jakim@yu.ac.kr; Byeong-Seon Jeong

University, Gyeongsan, Republic of Korea

ꢀ

These authors equally contributed to this work.

ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

2

C. L. CHAUDHARY ET AL.

or 400 MHz for ¹H-NMR and 62.5 MHz or 100 MHz for ¹³C-NMR.

Chemical shifts (d) were expressed in ppm using solvent as

an internal standard and coupling constant (J) in hertz. Low-

resolution mass spectra (LRMS) were obtained using an Advion

Expression CMS, and recorded in a positive ion mode with an

electrospray (ESI) source. Described below are detailed methods

for synthesising the new compounds introduced in this paper.

Other compounds were prepared using the synthetic methods we

had previously reported²⁰.

Previous studies

This study

HO

3

HO

R¹

6

N

G

R²

R¹

R

²= alkyl, aryl, heteroaryl

,

O

S

R

G =

O

R

S

R

N

H

HO

N

O

R

N

R

N

H

O

X

N

2.1.1. 5-(Benzyloxy)-3,4,6-trimethylpyridin-2-ol (6)

H

To a solution of 5-benzyloxy-3,4,6-trimethylpyridin-6-amine (5, 2 g,

8.25 mmol) in a mixed solvent of THF (3 ml) and water (100 ml)

was added 10% H₂SO₄(1.1 ml, 20.63 mmol) followed by addition

of NaNO₂(285 mg, 4.13 mmol) at 0 ^ꢁC. The reaction temperature

was gradually increased to room temperature and stirred for 2 h.

The reaction mixture was diluted with CH₂Cl₂; washed with sat.

NaHCO₃. The aqueous layer separated was extracted with CH₂Cl₂.

The combined CH₂Cl₂solution was washed with brine, dried over

MgSO₄, filtered, and concentrated to give 6 (1.95 g, 98%). Yellow

solid; TLC R_f0.20 (CH₂Cl₂/MeOH ¼ 20/1); m.p. 198 ^ꢁC; MS (ESI) m/z

244 [M þ H]^þ; ¹H-NMR (CDCl₃) d 13.04 (s, 1H), 7.48–7.32 (m, 5H),

4.70 (s, 2H), 2.28 (s, 3H), 2.17 (s, 3H), 2.10 (s, 3H); ¹³C-NMR (CDCl₃)

d 163.20, 145.35, 139.06, 136.95, 133.43, 128.75 (2 C), 128.42,

128.20 (2 C), 123.53, 75.83, 13.98, 13.85, 12.49.

X = O, S ; R = alkyl, aryl

Figure 1. 2,4,5-Trimethylpyridin-3-ol as anti-IBD scaffold.

For the last several years, we have conducted a phenotype-

based approach to identify small molecule inhibitors^14–19. Based

on the notion that immune cells such as monocytes infiltrate and

adhere to colon epithelium in the presence of TNF-a, we have

developed a cell-based screening assay to monitor the cell adhe-

sion phenotype in the presence of compounds. We recently pub-

lished a couple of papers on the discovery of 6-aminopyridin-3-ols

to inhibit IBD in vitro and in vivo (Figure 1). Among the many

compounds identified as effective in the previous studies, the ana-

logues having (4-propylphenyl)amino, (6-chloropyridin-3-yl)amino,

and (5-phenyl-1,3,4-thiadiazol-2-yl)amino substructures at C(6)-

position could be of particular excellence. These three compounds

showed >75% inhibitory effect against TNF-a-induced cell adhe-

2.1.2. 2,5-Bis(benzyloxy)-3,4,6-trimethylpyridine (7a)

sion between monocyte and colon epithelial cells at 1 mM concen- To a solution of 5-(benzyloxy)-3,4,6-trimethylpyridin-2-ol (6, 50 mg,

tration. Considering that 5-aminosalicylic acid (5-ASA, mesalazine), 0.21 mmol) in a mixed solvent of DMF (1 ml) and THF (1 ml) was

an active metabolite of sulfasalazine (SSZ) which is widely used to added Ag₂CO₃(69 mg, 0.25 mmol) followed by addition of 3-

treat IBD in the clinical field, has only 3.5% inhibitory activity at bromo-1-propylbenzeze (38 mL, 0.32 mmol). The mixture was

the same drug concentration (1 mM), the in vitro activity of our stirred at room temperature for 24 h. After filtration of the reaction

three compounds can be quite marvellous. Moreover, in vivo effi- mixture through a Celite pad, the filtrate was diluted with CH₂Cl₂

cacy studies using rats with severe colon inflammation induced by and water. The aqueous layer separated was extracted with

2,4,6-trinitrobenzenesulfonic acid (TNBS) have confirmed that our CH₂Cl₂, and the combined CH₂Cl₂solution was dried over MgSO₄,

compounds are certainly effective against IBD. When orally admin- filtered and concentrated. The residue was purified by silica gel

istered at the dose of 1 mg/kg, those compounds showed very column chromatography (Hexanes/EtOAc ¼ 20/1) to give 7a

good efficacy demonstrated by ameliorating disease parameters

such as % of the recovery in colon- and body-weights (up to 79%

(60 mg, 88%). White solid; TLC R_f0.61 (Hexanes/EtOAc ¼ 10/1);

1

m.p. 64 ^ꢁC; MS (ESI) m/z 334 [M þ H]^þ; H-NMR (CDCl₃) d 7.54–7.29

and 59%, respectively) and myeloperoxidase (MPO) level.

In this study, we made some changes in the functional group

at C(6)-position of the pyridin-3-ol ring and examined how these

structural changes affect on efficacy against IBD. We considered

five types of functional groups (alkoxy-, ureido-, thioureido-, carba-

mato-, and sulfonamido-group) to replace 6-amino groups, and

synthesised several to a dozen new derivatives for each family

(Figure 1).

(m, 10H), 5.39 (s, 2H), 4.76 (s, 2H), 2.44 (s, 3H), 2.22 (s, 3H), 2.16 (s,

3H); ¹³C-NMR (CDCl₃) d 156.88, 146.70, 144.54, 141.09, 138.43,

137.33, 128.57 (2 C), 128.28 (2 C), 128.10, 127.92 (2 C), 127.68 (2 C),

127.39, 117.07, 74.97, 67.26, 18.97, 12.78, 11.83.

2.1.3. 3,4,6-Trimethylpyridine-2,5-diol (8a)

To a suspension of 10% palladium on activated carbon (5 mg) in

MeOH (2 ml) was added 7a (20 mg, 0.06 mmol). The mixture was

stirred with hydrogen balloon at room temperature for 1 h. After

filtration of the reaction mixture through a Celite pad, the filtrate

was concentrated. The residue was purified by silica gel column

chromatography (CH₂Cl₂/MeOH ¼ 20/1) to give 8a (9 mg, 98%).

White solid; TLC R_f0.27 (CH₂Cl₂/MeOH ¼ 9/1); m.p. 176 ^ꢁC; MS

2. Materials and methods

2.1. Chemistry

Unless noted otherwise, materials were purchased from

commercial suppliers and used without further purification. Air- or

moisture-sensitive reactions were carried out under an inert

gas atmosphere. Reaction progress was monitored by thin-

layer-chromatography (TLC) using silica gel F₂₅₄plates. The prod-

ucts were purified by flash column chromatography using silica

gel 60 (70–230 mesh) or by Biotage ‘Isolera One’ system with

indicated solvents. Melting points were determined using a To

1

(ESI) m/z 154 [M þ H]^þ; H-NMR (DMSO-d₆) d 10.96 (s, 1H), 7.51 (s,

1H), 2.07 (s, 3H), 2.03 (s, 3H), 1.90 (s, 3H); ¹³C-NMR (DMSO-d₆) d

160.04, 141.97, 134.45, 126.76, 121.30, 13.70, 13.55, 12.27.

2.1.4. 3-(Benzyloxy)-6-butoxy-2,4,5-trimethylpyridine (7 b)

a

solution of 5-(benzyloxy)-3,4,6-trimethylpyridin-2-ol (6,

Fischer-Jones melting point apparatus and were not corrected. 100 mg, 0.41 mmol) in DMF (4 ml) was added Ag₂CO₃(136 mg,

NMR spectra were obtained using Bruker spectrometers 250 MHz 0.49 mmol) followed by addition of 1-iodobutane (70 mL,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

3

0.62 mmol). The mixture was stirred at 40 ^ꢁC for 2 h. After filtration 2.1.8. 2,4,5-Trimethyl-6-(octyloxy)pyridin-3-ol (7d)

of the reaction mixture through a Celite pad, the filtrate was con- To

a solution of 5-(benzyloxy)-3,4,6-trimethylpyridin-2-ol (6,

centrated then the residue was diluted with EtOAc and washed 100 mg, 0.41 mmol) in DMF (4 ml) was added Ag₂CO₃(136 mg,

0.49 mmol) followed by addition of 1-iodo-3-methylbutane (71 mL,

0.62 mmol). The mixture was stirred at 50 ^ꢁC for 20 h. After filtra-

tion of the reaction mixture through a Celite pad, the filtrate was

concentrated. The residue was diluted with EtOAc and washed

with water and brine. The EtOAc solution was dried over MgSO₄,

filtered and concentrated. The residue was purified by silica gel

column chromatography (Hexanes/EtOAc ¼ 30/1) to give 7d

(98 mg, 76%). Colourless liquid; TLC R_f0.30 (Hexanes/EtOAc ¼ 20/

with water and brine. The EtOAc solution was dried over MgSO₄,

filtered and concentrated. The residue was further purified by sil-

ica gel column chromatography (Hexanes/EtOAc ¼ 30/1) to give

7 b (87 mg, 71%). Pale yellow solid; TLC R_f0.29 (Hexanes/EtOAc ¼

20/1); m.p. 33 ^ꢁC; MS (ESI) m/z 300 [M þ H]^þ; ¹H-NMR (CDCl₃) d

7.55–7.31 (m, 5H), 4.74 (s, 2H), 4.28 (t, J ¼ 6.5 Hz, 2H), 2.41 (s, 3H),

2.20 (s, 3H), 2.11 (s, 3H), 1.76 (dt, J ¼ 14.6, 6.5 Hz, 2H), 1.51 (dq,

J ¼ 14.2, 7.3 Hz, 2H), 0.99 (t, J ¼ 7.3 Hz, 3H); ¹³C-NMR (CDCl₃) d

157.57, 146.47, 144.59, 140.90, 137.53, 128.69 (2 C), 128.21, 128.06

(2 C), 117.08, 75.09, 65.57, 31.58, 19.62, 19.12, 14.12, 12.87, 11.87.

1

1); MS (ESI) m/z 514 [M þ H]^þ; H-NMR (CDCl₃) d 7.52–7.33 (m, 5H),

4.72 (s, 2H), 4.29 (t, J ¼ 6.6 Hz, 2H), 2.40 (s, 3H), 2.19 (s, 3H), 2.09 (s,

3H), 1.82 (td, J ¼ 13.3, 6.6 Hz, 1H), 1.65 (q, J ¼ 6.7 Hz, 2H), 0.97 (d,

J ¼ 6.6 Hz, 6H); ¹³C-NMR (CDCl₃) d 157.52, 146.42, 144.55, 142.42,

137.46, 128.72 (2 C), 128.24, 128.07 (2 C), 117.11, 75.09, 64.33,

38.26, 25.42, 22.88 (2 C), 19.10, 12.91, 11.92.

2.1.5. 6-Butoxy-2,4,5-trimethylpyridin-3-ol (8 b)

To a suspension of 10% palladium on activated carbon (11 mg) in

MeOH (2 ml) was added 7 b (57 mg, 0.19 mmol). The mixture was

stirred with hydrogen balloon at room temperature for 1 h. After

filtration of the reaction mixture through a Celite pad, the filtrate

was concentrated. The residue was dissolved in MeOH and filtered

through a membrane filter. The filtrate was concentrated to give

8 b (28 mg, 70%). Yellow solid; TLC R_f0.41 (CH₂Cl₂/MeOH ¼ 20/1);

m.p. 63 ^ꢁC; MS (ESI) m/z 210 [M þ H]^þ; ¹H-NMR (CDCl₃) d 4.23 (t,

J ¼ 6.5 Hz, 2H), 4.07 (s, 1H), 2.35 (s, 3H), 2.16 (s, 3H), 2.10 (s, 3H),

1.79–1.65 (m, 2H), 1.47 (dd, J ¼ 15.0, 7.5 Hz, 2H), 0.97 (t, J ¼ 7.3 Hz,

3H); ¹³C-NMR (CDCl₃) d 155.76, 143.03, 136.41, 134.63, 117.05,

65.54, 31.62, 19.61, 18.61, 14.12, 12.25, 11.86.

2.1.9. 6-(Isopentyloxy)-2,4,5-trimethylpyridin-3-ol (8d)

To a suspension of 10% palladium on activated carbon (14 mg) in

MeOH (2 ml) was added 7d (72 mg, 0.23 mmol). The mixture was

stirred with hydrogen balloon at room temperature for 1 h. After

filtration of the reaction mixture through a Celite pad, the filtrate

was concentrated. The residue was dissolved in MeOH and filtered

through a membrane filter. The filtrate was concentrated to give

8d (40 mg, 78%). Pale yellow liquid; TLC R_f0.41 (CH₂Cl₂/MeOH ¼

20/1); MS (ESI) m/z 224 [M þ H]^þ; ¹H-NMR (CDCl₃) d 4.26 (t,

J ¼ 6.6 Hz, 2H), 4.10 (s, 1H), 2.36 (s, 3H), 2.16 (s, 3H), 2.09 (s, 3H),

1.81 (dt, J ¼ 13.3, 6.7 Hz, 1H), 1.63 (q, J ¼ 6.7 Hz, 2H), 0.96 (d,

J ¼ 6.6 Hz, 6H); ¹³C-NMR (CDCl₃) d 155.72, 143.05, 136.40, 134.75,

117.08, 64.33, 38.34, 25.46, 22.87 (2 C), 18.58, 12.27, 11.88.

2.1.6. 3-(Benzyloxy)-2,4,5-trimethyl-6-(octyloxy)pyridine (7c)

To

a solution of 5-(benzyloxy)-3,4,6-trimethylpyridin-2-ol (6,

100 mg, 0.41 mmol) in DMF (4 ml) was added Ag₂CO₃(136 mg,

0.49 mmol) followed by addition of 1-iodooctane (111 mL,

0.62 mmol). The mixture was stirred at 40 ^ꢁC for 4 h. After filtration

of the reaction mixture through a Celite pad, the filtrate was con-

centrated. The residue was diluted with EtOAc and washed with

water and brine. The EtOAc solution was dried over MgSO₄, fil-

tered and concentrated. The residue was purified by silica gel col-

umn chromatography (Hexanes/EtOAc ¼ 30/1) to give 7c (131 mg,

90%). Pale yellow solid; TLC R_f0.28 (Hexanes/EtOAc ¼ 20/1); m.p.

31 ^ꢁC; MS (ESI) m/z 356 [M þ H]^þ; ¹H-NMR (CDCl₃) d 7.52–7.32 (m,

5H), 4.73 (s, 2H), 4.27 (t, J ¼ 6.6 Hz, 2H), 2.40 (s, 3H), 2.19 (s, 3H),

2.10 (s, 3H), 1.75 (dd, J ¼ 14.3, 6.6 Hz, 2H), 1.32 (m, 10H), 0.89 (t,

J ¼ 6.5 Hz, 3H); ¹³C-NMR (CDCl₃) d 157.58, 146.54, 144.60, 140.99,

137.58, 128.71 (2 C), 128.22, 128.08 (2 C), 117.15, 75.14, 65.99,

32.02, 29.58, 29.45 (2 C), 26.42, 22.83, 19.10, 14.24, 12.90, 11.89.

2.1.10. 6-(Isopentyloxy)-2,4,5-trimethylpyridin-3-ol (7e)

To

a solution of 5-(benzyloxy)-3,4,6-trimethylpyridin-2-ol (6,

100 mg, 0.41 mmol) in DMF (4 ml) was added Ag₂CO₃(136 mg,

0.49 mmol) followed by addition of 1-iodocyclopentane (71 mL,

0.62 mmol). The mixture was stirred at 50 ^ꢁC for 7 h. After filtration

of the reaction mixture through a Celite pad, the filtrate was con-

centrated. The residue was diluted with EtOAc and washed with

water and brine. The EtOAc solution was dried over MgSO₄, fil-

tered and concentrated. The residue was purified by silica gel col-

umn chromatography (Hexanes/EtOAc ¼ 50/1) to give 7e (88 mg,

69%). Colourless liquid; TLC R_f0.29 (Hexanes/EtOAc ¼ 20/1); MS

(ESI) m/z 312 [M þ H]^þ; ¹H-NMR (CDCl₃) d 7.52–7.31 (m, 5H),

5.54–5.39 (m, 1H), 4.74 (s, 2H), 2.39 (s, 3H), 2.18 (s, 3H), 2.06 (s,

3H), 1.93 (dd, J ¼ 10.8, 6.3 Hz, 2H), 1.77 (dd, J ¼ 20.6, 5.4 Hz, 4H),

1.63 (dd, J ¼ 10.9, 4.1 Hz, 2H); ¹³C-NMR (CDCl₃) d 157.18, 146.30,

144.66, 140.72, 137.60, 128.69 (2 C), 128.19, 128.05 (2 C), 117.49,

75.07, 33.09 (2 C), 29.86, 24.07 (2 C), 19.26, 12.89, 11.9.

2.1.7. 2,4,5-Trimethyl-6-(octyloxy)pyridin-3-ol (8c)

To a suspension of 10% palladium on activated carbon (20 mg) in

MeOH (2 ml) was added 7c (99 mg, 0.28 mmol). The mixture was

stirred with hydrogen balloon at room temperature for 1 h. After

filtration of the reaction mixture through a Celite pad, the filtrate

was concentrated. The residue was dissolved in MeOH and filtered

through a membrane filter. The filtrate was concentrated to give

8c (70 mg, 95%). Pale yellow solid; TLC R_f0.48 (CH₂Cl₂/MeOH ¼

20/1); m.p. 52 ^ꢁC; MS (ESI) m/z 266 [M þ H]^þ; ¹H-NMR (CDCl₃) d

4.22 (t, J ¼ 6.6 Hz, 2H), 4.14 (s, 1H), 2.35 (s, 3H), 2.16 (s, 3H), 2.10 (s,

3H), 1.72 (dd, J ¼ 14.2, 6.7 Hz, 2H), 1.51–1.18 (m, 10H), 0.89 (dd,

J ¼ 8.9, 4.4 Hz, 3H); ¹³C-NMR (CDCl₃) d 155.75, 143.05, 136.47,

134.77, 117.09, 65.93, 32.00, 29.57, 29.46, 29.44, 26.40, 22.83,

18.58, 14.25, 12.27, 11.87.

2.1.11. 6-(Cyclopentyloxy)-2,4,5-trimethylpyridin-3-ol (8e)

To a suspension of 10% palladium on activated carbon (13 mg) in

MeOH (2 ml) was added 7e (63 mg, 0.20 mmol). The mixture was

stirred with hydrogen balloon at room temperature for 1 h. After

filtration of the reaction mixture through a Celite pad, the filtrate

was concentrated. The residue was dissolved in MeOH and filtered

through a membrane filter. The filtrate was concentrated to give

8e (39 mg, 87%). Pale yellow solid; TLC R_f0.41 (CH₂Cl₂/MeOH ¼

20/1); m.p. 79 ^ꢁC; MS (ESI) m/z 222 [M þ H]^þ; ¹H-NMR (CDCl₃) d

5.38 (dt, J ¼ 8.2, 3.0 Hz, 1H), 4.18 (s, 1H), 2.34 (s, 3H), 2.14 (s, 3H),

2.06 (s, 3H), 1.95–1.82 (m, 2H), 1.82–1.67 (m, 4H), 1.66–1.51 (m,

4

C. L. CHAUDHARY ET AL.

2H); ¹³C-NMR (CDCl₃) d 155.33, 142.90, 136.71, 134.56, 117.61, (s, 3H), 2.05 (s, 3H), 1.56–1.41 (m, 2H), 0.91 (t, J ¼ 7.4 Hz, 3H); ¹³C-

77.46, 33.03 (2 C), 24.03 (2 C), 18.71, 12.26, 11.92.

NMR (DMSO-d₆) d 155.50, 144.40, 143.36, 138.73, 135.53, 118.36,

40.75, 22.78, 19.13, 13.08, 12.66, 11.39.

2.1.12. 3-(Benzyloxy)-2,4,5-trimethyl-6–(3-phenylpropoxy)pyridine (7f)

To a solution of 5-(benzyloxy)-3,4,6-trimethylpyridin-2-ol (6, 30 mg,

0.12 mmol) in DMF (1 ml) was added Ag₂CO₃(41 mg, 0.15 mmol)

followed by addition of 3-bromo-1-propylbenzeze (28 mL,

0.19 mmol). The mixture was stirred at 80 ^ꢁC for 12 h. After filtra-

tion of the reaction mixture through a Celite pad, the filtrate was

concentrated. The residue was diluted with EtOAc and washed

with water and brine. The EtOAc solution was dried over MgSO₄,

filtered and concentrated. The residue was purified by silica gel

column chromatography (Hexanes/EtOAc ¼ 30/1) to give 7f

(37 mg, 83%). Colourless liquid; TLC R_f0.31 (Hexanes/EtOAc ¼ 15/

2.1.16. 1–(5-(Benzyloxy)-3,4,6-trimethylpyridin-2-yl)-3–(4-nitrophe-

nyl)urea (11k)

To a solution of 5-(benzyloxy)-3,4,6-trimethylpyridin-6-amine (5,

50 mg, 0.21 mmol) in CH₂Cl₂(2 ml) was added 4-nitrophenyl iso-

cyanate (41 mg, 0.25 mmol). The mixture was stirred at room tem-

perature for 4 h. The reaction mixture was diluted Et₂O and the

precipitate was filtered. The filter cake was washed with Et₂O and

acetone to give 11k (73 mg, 87%). White solid; TLC R_f0.33

(CH₂Cl₂/MeOH ¼ 40/1); m.p. 227 ^ꢁC; MS (ESI) m/z 429 [M þ Na]^þ;

¹H-NMR (DMSO-d₆) d 11.77 (s, 1H), 8.72 (s, 1H), 8.21 (d, J ¼ 8.4 Hz,

2H), 7.77 (d, J ¼ 8.5 Hz, 2H), 7.60–7.32 (m, 5H), 4.81 (s, 2H), 2.44 (s,

3H), 2.22 (s, 3H), 2.16 (s, 3H); ¹³C-NMR (DMSO-d₆) d 152.21, 147.72,

145.66, 145.12, 144.96, 141.49, 141.38, 136.80, 128.22 (2 C), 127.98

(2 C), 127.90, 124.87 (2 C), 120.88, 118.06 (2 C), 74.34, 18.59,

13.18, 12.75.

1

1); MS (ESI) m/z 362 [M þ H]^þ; H-NMR (CDCl₃) d 7.54–7.36 (m, 5H),

7.36–7.17 (m, 5H), 4.76 (s, 2H), 4.35 (t, J ¼ 6.3 Hz, 2H), 2.84 (dd,

J ¼ 8.6, 6.9 Hz, 2H), 2.42 (s, 3H), 2.23 (s, 3H), 2.15 (s, 3H), 2.14–2.05

(m, 2H); ¹³C-NMR (CDCl₃) d 157.35, 146.57, 144.65, 142.21, 140.96,

137.51, 128.69 (2 C), 128.62 (2 C), 128.46 (2 C), 128.21, 128.05 (2 C),

125.88, 117.04, 75.08, 64.95, 32.66, 31.09, 19.12, 12.88, 11.89.

2.1.17. 1–(4-Aminophenyl)-3–(5-hydroxy-3,4,6-trimethylpyridin-2-

yl)urea (13k)

2.1.13. 2,4,5-Trimethyl-6–(3-phenylpropoxy)pyridin-3-ol (8f)

To a suspension of 10% palladium on activated carbon (15 mg) in

MeOH (2 ml) was added 7f (77 mg, 0.21 mmol). The mixture was

stirred with hydrogen balloon at room temperature for 1 h. After

filtration of the reaction mixture through a Celite pad, the filtrate

was concentrated. The residue was dissolved in MeOH and filtered

through a membrane filter. The filtrate was concentrated to give

8f (50 mg, 87%). White solid; TLC R_f0.30 (CH₂Cl₂/MeOH ¼ 20/1);

To a suspension of 10% palladium on activated carbon (15 mg) in

MeOH (3 ml) was added 11k (75 mg, 0.18 mmol). The mixture was

stirred with hydrogen balloon at room temperature for 12 h. After

filtration of the reaction mixture through a Celite pad, the filtrate

was concentrated. The residue was purified by silica gel column

chromatography (CH₂Cl₂/MeOH ¼ 20/1) to give 13k (14 mg, 26%).

White solid; TLC R_f0.35 (CH₂Cl₂/MeOH ¼ 20/1); m.p. 200 ^ꢁC

1

m.p. 73 ^ꢁC; MS (ESI) m/z 272 [M þ H]^þ; H-NMR (CDCl₃) d 7.35–7.16

1

(decomp.); MS (ESI) m/z 309 [M þ Na]^þ; H-NMR (DMSO-d₆) d 11.03

(m, 5H), 4.29 (t, J ¼ 6.3 Hz, 3H), 2.89–2.71 (m, 2H), 2.36 (s, 3H), 2.18

(s, 3H), 2.14 (s, 3H), 2.13–2.02 (m, 2H); ¹³C-NMR (CDCl₃) d 155.55,

143.15, 142.24, 136.65, 134.92, 128.61 (2 C), 128.44 (2 C), 125.86,

117.04, 65.00, 32.64, 31.14, 18.55, 12.28, 11.87.

(s, 1H), 8.34 (s, 1H), 7.94 (s, 1H), 7.15 (d, J ¼ 8.7 Hz, 2H), 6.52 (d,

J ¼ 8.7 Hz, 2H), 4.77 (s, 2H), 2.36 (s, 3H), 2.14 (s, 3H), 2.10 (s, 3H);

¹³C-NMR (DMSO-d₆) d 153.01, 144.74, 144.09, 142.99, 138.81,

135.83, 128.50, 120.61 (2 C), 118.97, 114.17 (2 C), 19.20,

13.19, 12.76.

2.1.14. 1–(5-(Benzyloxy)-3,4,6-trimethylpyridin-2-yl)-3-propylurea (11a)

To a solution of 5-(benzyloxy)-3,4,6-trimethylpyridin-6-amine (5,

100 mg, 0.41 mmol) in CH₂Cl₂(5 ml) was added propyl isocyanate

(46 mL, 0.50 mmol). The mixture was stirred at room temperature

for 40 h. After concentration of the reaction mixture, the residue

was purified by silica gel column chromatography (CHCl₃/MeOH ¼

50/1 to 20/1) to give 13a (125 mg, 92%). White solid; TLC R_f0.34

(CH₂Cl₂/MeOH ¼ 30/1); m.p. 175 ^ꢁC; MS (ESI) m/z 328 [M þ H]^þ;

¹H-NMR (CDCl₃) d 9.79 (s, 1H), 7.49–7.33 (m, 5H), 6.69 (s, 1H), 4.74

(s, 2H), 3.35 (td, J ¼ 6.9, 5.6 Hz, 2H), 2.40 (s, 3H), 2.22 (s, 3H), 2.10

(s, 3H), 1.64 (dd, J ¼ 14.3, 7.1 Hz, 2H), 1.02 (t, J ¼ 7.4 Hz, 3H); ¹³C-

NMR (CDCl₃) d 155.95, 146.87, 146.75, 144.92, 141.54, 137.01,

128.77 (2 C), 128.43, 128.10 (2 C), 115.89, 75.38, 41.73, 23.20, 19.11,

13.31, 12.84, 11.75.

2.1.18. 1–(5-(Benzyloxy)-3,4,6-trimethylpyridin-2-yl)thiourea (12a)

To a solution of N-((5-(benzyloxy)-3,4,6-trimethylpyridin-2-yl)carba-

mothioyl)benzamide (12n, 100 mg, 0.25 mmol) in a mixed solvent

of THF (1 ml) and water (1 ml) was added NaOH (20 mg,

0.49 mmol). The mixture was refluxed for 17 h. The reaction mix-

ture was cooled to room temperature and diluted with water. The

pH of the reaction mixture was adjusted to around 7 using 2 M

HCl. The precipitate formed was collected by filtration and the fil-

ter cake was washed with water to give 12a (43 mg, 58%). Ivory

solid; TLC R_f0.22 (Hex/EtOAc ¼ 2/1); m.p. 165 ^ꢁC; MS (ESI) m/z 302

[M þ H]^þ; ¹H-NMR (CDCl₃) d 11.26 (s, 1H), 7.96 (s, 1H), 7.46 – 7.35

(m, 5H), 6.84 (s, 1H), 4.76 (s, 2H), 2.40 (s, 3H), 2.24 (s, 3H), 2.18 (s,

3H); ¹³C-NMR (CDCl₃) d 148.10, 148.00, 146.49, 145.99, 142.17,

136.69, 128.83 (2 C), 128.58, 128.14 (2 C), 116.92, 75.41, 19.25,

13.43, 12.99.

2.1.15. 1–(5-Hydroxy-3,4,6-trimethylpyridin-2-yl)-3-propylurea (13a)

To a suspension of 10% palladium on activated carbon (21 mg) in

CH₂Cl₂(4 ml) was added 11a (105 mg, 0.32 mmol). The mixture 2.1.19. 1–(5-Hydroxy-3,4,6-trimethylpyridin-2-yl)thiourea (14a)

was stirred with hydrogen balloon at room temperature for 2 h.

After filtration of the reaction mixture through a Celite pad, the fil-

trate was concentrated. The residue was purified by silica gel col-

To a solution of 12a (34 mg, 0.11 mmol) in CH₂Cl₂(1 ml), was

added pentamethylbenzene (50 mg, 0.34 mmol) followed by drop-

wise addition of boron trichloride (1 M in CH₂Cl₂, 0.2 ml) at 0 ^ꢁC.

umn chromatography (CH₂Cl₂/MeOH ¼ 20/1) to give 13a (58 mg, The reaction mixture was stirred at 0 ^ꢁC for 15 min then quenched

75%). White solid; TLC R_f0.26 (CH₂Cl₂/MeOH ¼ 20/1); m.p. 176 ^ꢁC; with CHCl₃/MeOH solution (9/1, 1.5 ml) and stirred at room tem-

1

MS (ESI) m/z 239 [M þ H]^þ; H-NMR (DMSO-d₆) d 8.88 (s, 1H), 8.20 perature for 30 min. After concentration of the reaction mixture,

(s, 1H), 7.71 (s, 1H), 3.14 (dd, J ¼ 12.4, 6.7 Hz, 2H), 2.29 (s, 3H), 2.12 2 ml of a mixed solvent (CH₂Cl₂/MeOH ¼ 40/1) was added to the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

5

residue and precipitate formed was filtered, washed with CH₂Cl₂2.1.23.

and collected to give 14a (23.6 mg, 99%). White solid; TLC R_þf 0.35

NMR (CD₃OD) d 2.35 (s, 3H), 2.22 (s, 3H), 2.18 (s, 3H); ¹³C-NMR

(CD₃OD) d 147.61, 144.33, 141.39, 137.55, 129.24, 128.91, 19.08,

12.98, 12.77.

Butyl

(5-hydroxy-3,4,6-trimethylpyridin-2-yl)carba-

mate (16 b)

1

(CH₂Cl₂/MeOH ¼ 9/1); m.p. 189 ^ꢁC; MS (ESI) m/z 212 [M þ H] ; H-

To a suspension of 10% palladium on activated carbon (16 mg) in

MeOH (5 ml) was added 15 b (79 mg, 0.23 mmol). The mixture was

stirred with hydrogen balloon at room temperature for 1 h. After

filtration of the reaction mixture through a Celite pad, the filtrate

was concentrated. The residue was dissolved in MeOH and filtered

through a membrane filter and concentrated to give 16 b (54 mg,

93%). White solid; TLC R_f0.24 (CH₂Cl₂/MeOH ¼ 15/1); m.p. 166 ^ꢁC;

2.1.20.

mate (15a)

Methyl

(5-benzyloxy-3,4,6-trimethylpyridin-2-yl)carba-

MS (ESI) m/z 253 [M þ H]^þ; H-NMR (DMSO-d₆) d 8.95 (s, 1H), 3.98

1

To a solution of 5-benzyloxy-3,4,6-trimethylpyridin-6-amine (5,

70 mg, 0.29 mmol) in acetone (1 ml) was added K₂CO₃(120 mg,

0.87 mmol) followed by dropwise addition of methyl chlorofor-

mate (112 mL, 1.45 mmol) at 0 ^ꢁC. The mixture was stirred at room 140.33, 133.76, 126.26, 63.68, 30.80, 19.32, 18.65, 14.30,

(t, J ¼ 6.5 Hz, 2H), 2.27 (s, 3H), 2.11 (s, 3H), 1.99 (s, 3H), 1.55 (dt,

J ¼ 14.5, 6.6 Hz, 2H), 1.34 (dq, J ¼ 14.1, 7.1 Hz, 2H), 0.89 (t,

J ¼ 7.3 Hz, 3H); ¹³C-NMR (DMSO-d₆) d 154.94, 147.82, 141.42,

13.70, 12.65.

temperature for 24 h. The reaction mixture was concentrated and

then diluted with CH₂Cl₂and water. The aqueous layer separated

was extracted with CH₂Cl₂. The combined CH₂Cl₂solution was

washed with brine and dried over MgSO₄, filtered and concen-

trated. The residue was purified by silica gel column chromatog-

raphy (CH₂Cl₂/MeOH ¼ 30/1 to 20/1) to give 15a (63 mg, 73%).

Pale yellow solid; TLC R_f0.29 (CH₂Cl₂/MeOH ¼ 15/1); m.p. 117 ^ꢁC;

MS (ESI) m/z 301 [M þ H]^þ; ¹H-NMR (CDCl₃) d 7.50–7.32 (m, 5H),

6.96 (s, 1H), 4.77 (s, 2H), 3.76 (s, 3H), 2.43 (s, 3H), 2.25 (s, 3H), 2.16

(s, 3H); ¹³C-NMR (CDCl₃) d 155.16, 150.60, 148.10, 143.55, 141.69,

136.96, 128.78 (2 C), 128.43, 128.05 (2 C), 126.58, 75.01, 52.71,

19.19, 14.98, 13.32.

2.1.24. Isobutyl (5-(benzyloxy)-3,4,6-trimethylpyridin-2-yl)carba-

mate (15c)

To a solution of 5-benzyloxy-3,4,6-trimethylpyridin-6-amine (5,

30 mg, 0.12 mmol) in acetone (1 ml) was added K₂CO₃(86 mg,

0.62 mmol) followed by dropwise addition of isobutyl chlorofor-

mate (24 mL, 0.19 mmol) at 0 ^ꢁC. The mixture was stirred at room

temperature for 24 h. The reaction mixture was concentrated and

then diluted with CH₂Cl₂. It was washed with sat. NaHCO₃and

brine. The CH₂Cl₂solution was dried over MgSO₄, filtered and con-

centrated. The residue was purified by silica gel column chroma-

tography (CH₂Cl₂/MeOH ¼ 40/1) to give 15c (40 mg, 87%). White

solid; TLC R_f0.29 (CH₂Cl₂/MeOH ¼ 15/1); m.p. 70 ^ꢁC; MS (ESI) m/z

343 [M þ H]^þ; ¹H-NMR (CDCl₃) d 7.52–7.34 (m, 5H), 7.28 (s, 1H),

4.77 (s, 2H), 3.93 (d, J ¼ 6.6 Hz, 2H), 2.44 (s, 3H), 2.24 (s, 3H), 2.17

(s, 3H), 2.04–1.87 (m, 1H), 0.94 (d, J ¼ 6.7 Hz, 6H); ¹³C-NMR (CDCl₃)

d 154.89, 150.50, 147.89, 143.87, 141.61, 137.01, 128.73 (2 C),

128.37, 128.02 (2 C), 126.53, 75.00, 71.63, 29.80, 28.09, 19.11 (d,

2 C), 15.00, 13.27.

2.1.21.

Methyl

(5-hydroxy-3,4,6-trimethylpyridin-2-yl)carba-

mate (16a)

To a suspension of 10% palladium on activated carbon (12 mg) in

a mixed solvent of CHCl₃(1.5 ml) and MeOH (1.5 ml) was added

15a (55 mg, 0.18 mmol). The mixture was stirred with hydrogen

balloon at room temperature for 1 h. After filtration of the reaction

mixture through a Celite pad, the filtrate was concentrated. The

residue was purified by silica gel column chromatography (CH₂Cl₂/

MeOH ¼ 20/1 to 5/1) to give 16a (37 mg, 96%). White solid; TLC

R_f0.19 (CH₂Cl₂/MeOH ¼ 15/1); m.p. 210 ^ꢁC; MS (ESI) m/z 233

[M þ Na]^þ; ¹H-NMR (DMSO-d₆) d 9.02 (s, 1H), 8.53 (s, 1H), 3.57 (s,

3H), 2.27 (s, 3H), 2.11 (s, 3H), 2.00 (s, 3H); ¹³C-NMR (DMSO-d₆) d

155.20, 147.63, 141.39, 140.32, 133.75, 126.18, 51.47, 19.15,

14.14, 12.51.

2.1.25. Isobutyl (5-(benzyloxy)-3,4,6-trimethylpyridin-2-yl)carba-

mate (16c)

To a suspension of 10% palladium on activated carbon (18 mg) in

MeOH (5 ml) was added 15c (91 mg, 0.27 mmol). The mixture was

stirred with hydrogen balloon at room temperature for 6 h. After

filtration of the reaction mixture through a Celite pad, the filtrate

was concentrated. The residue was purified by silica gel column

chromatography (CH₂Cl₂/MeOH ¼ 30/1 to 20/1) to give 16c

(34 mg, 51%). White solid; TLC R_f0.35 (CH₂Cl₂/MeOH ¼ 15/1); m.p.

166 ^ꢁC; MS (ESI) m/z 253 [M þ H]^þ; ¹H-NMR (DMSO-d₆) d 8.89 (s,

1H), 8.44 (s, 1H), 3.78 (d, J ¼ 6.6 Hz, 2H), 2.28 (s, 3H), 2.12 (s, 3H),

2.01 (s, 3H), 1.86 (dt, J ¼ 13.3, 6.6 Hz, 1H), 0.89 (d, J ¼ 6.7 Hz, 6H);

¹³C-NMR (DMSO-d₆) d 154.85, 147.54, 141.30, 140.38, 133.66,

126.12, 69.84, 27.59, 19.10, 18.80 (2 C), 14.11, 12.47.

2.1.22.

Methyl

(5-hydroxy-3,4,6-trimethylpyridin-2-yl)carba-

mate (15 b)

To a solution of 5-benzyloxy-3,4,6-trimethylpyridin-6-amine (5,

70 mg, 0.29 mmol) in acetone (1 ml) was added K₂CO₃(200 mg,

1.45 mmol) followed by dropwise addition of butyl chloroformate

(184 mL, 1.45 mmol) at 0 ^ꢁC. The mixture was stirred at room tem-

perature for 24 h. The reaction mixture was concentrated then

diluted with CH₂Cl₂and washed with sat. NaHCO₃and brine. The

CH₂Cl₂layer was dried over MgSO₄, filtered and concentrated. The

residue was purified by silica gel column chromatography (CH₂Cl₂/

MeOH ¼ 30/1 to 20/1) to give 15 b (90 mg, 91%). White solid; TLC

R_f0.33 (CH₂Cl₂/MeOH ¼ 15/1); m.p. 81 ^ꢁC; MS (ESI) m/z 399

2.1.26. 2-Chloroethyl (5-(benzyloxy)-3,4,6-trimethylpyridin-2-yl)car-

bamate (15d)

To a solution of 5-benzyloxy-3,4,6-trimethylpyridin-6-amine (5,

70 mg, 0.29 mmol) in acetone (1 ml) was added K₂CO₃(200 mg,

1.45 mmol) followed by dropwise addition of 2-chloroethyl chloro-

formate (149 mL, 1.45 mmol) at 0 ^ꢁC. The mixture was stirred at

room temperature for 9 h. The reaction mixture was concentrated.

The residue was diluted with CH₂Cl₂and washed with sat.

NaHCO₃and brine. The CH₂Cl₂layer was dried over MgSO₄, fil-

tered and concentrated. The residue was purified by silica gel

1

[M þ H]^þ; H-NMR (CDCl₃) d 7.51–7.33 (m, 5H), 7.08 (s, 1H), 4.77 (s,

2H), 4.14 (t, J ¼ 6.6 Hz, 2H), 2.43 (s, 3H), 2.24 (s, 3H), 2.16 (s, 3H),

1.65 (dt, J ¼ 14.8, 6.9 Hz, 2H), 1.40 (dq, J ¼ 14.2, 7.2 Hz, 2H), 0.94 (t,

J ¼ 7.3 Hz, 3H); ¹³C-NMR (CDCl₃) d 154.83, 150.52, 147.90, 143.80,

141.66, 137.01, 128.75 (2 C), 128.38, 128.03 (2 C), 126.53, 75.02,

65.43, 31.11, 19.18, 19.08, 15.01, 13.85, 13.28.

6

C. L. CHAUDHARY ET AL.

column chromatography (CH₂Cl₂/MeOH ¼ 50/1 to 30/1) to give 2.1.30. N-(5-(benzyloxy)-3,4,6-trimethylpyridin-2-yl)methanesulfo-

15d (84 mg, 83%). White solid; TLC R_f0.21 (CH₂Cl₂/MeOH ¼ 30/1);

namide (17a)

m.p. 128 ^ꢁC; MS (ESI) m/z 349 [M þ H]^þ; ¹H-NMR (CDCl₃) d

To a solution of 5-benzyloxy-3,4,6-trimethylpyridin-6-amine (5,

7.51–7.34 (m, 5H), 4.78 (s, 2H), 4.44–4.34 (m, 2H), 3.78–3.66 (m,

2H), 2.44 (s, 3H), 2.25 (s, 3H), 2.17 (s, 3H); ¹³C-NMR (CDCl₃) d

154.05, 150.70, 148.22, 143.22, 141.80, 136.90, 128.79 (2 C), 128.45,

128.05 (2 C), 126.62, 75.02, 65.05, 42.06, 19.14, 14.96, 13.33.

50 mg, 0.21 mmol) in pyridine (2 ml) was added methanesulfonyl

chloride (48 mL, 0.62 mmol). The mixture was stirred at room tem-

perature for 24 h and then concentrated. The residue was diluted

with CH₂Cl₂and washed with brine. The CH₂Cl₂layer was dried

over MgSO₄, filtered and concentrated. The residue was purified

by silica gel column chromatography (CH₂Cl₂/MeOH ¼ 40/1 to

20/1) to give 17a (34 mg, 52%). White solid; TLC R_f0.35 (Hexanes/

EtOAc ¼ 1/1); m.p. 122 ^ꢁC; MS (ESI) m/z 321 [M þ H]^þ; ¹H-NMR

(CDCl₃) d 9.23 (br, s, 1H), 7.52–7.29 (m, 5H), 4.75 (s, 2H), 3.23 (s,

3H), 2.33 (s, 3H), 2.23 (s, 3H), 2.15 (s, 3H); ¹³C-NMR (CDCl₃) d

147.81, 145.88, 144.54, 140.41, 136.48, 128.86 (2 C), 128.66, 128.23

(2 C), 123.99, 75.68, 42.92, 17.18, 13.77, 13.74.

2.1.27. 2-Chloroethyl (5-(benzyloxy)-3,4,6-trimethylpyridin-2-yl)car-

bamate (16d)

To a suspension of 15d (75 mg, 0.22 mmol) in CH₂Cl₂(2 ml), was

added pentamethylbenzene (96 mg, 0.65 mmol) followed by drop-

wise addition of boron trichloride (1 M in CH₂Cl₂, 0.32 ml) at 0 ^ꢁC.

The reaction mixture was stirred at 0 ^ꢁC for 30 min, The mixture

was then quenched with a mixed solvent of CHCl₃/MeOH (9/1,

1.5 ml) and stirred at room temperature for 30 min. After concen- 2.1.31. N-(5-Hydroxy-3,4,6-trimethylpyridin-2-yl)methanesulfona-

tration of the reaction mixture, the residue was purified by silica

gel column chromatography (CHCl₃/MeOH ¼ 30/1 to 10/1) to give

16d (51 mg, 92%). White solid; TLC R_f0.18 (CH₂Cl₂/MeOH ¼ 30/1);

mide (18a)

To a suspension of 10% palladium on activated carbon (5 mg) in a

mixed solvent of MeOH (2.5 ml) and CH₂Cl₂(2.5 ml) was added

17a (26 mg, 0.08 mmol). The mixture was stirred with hydrogen

balloon at room temperature for 1 h. After filtration of the reaction

mixture through a Celite pad, the filtrate was concentrated. The

residue was dissolved in MeOH and filtered through a membrane

filter and concentrated to give 18a (19 mg, 100%). Pale yellow

solid; TLC R_f0.2 (CH₂Cl₂/MeOH ¼ 20/1); m.p. 142 ^ꢁC; MS (ESI) m/z

231 [M þ H]^þ; ¹H-NMR (DMSO-d₆) d 9.00 (s, 2H), 3.20 (s, 3H), 2.30

(s, 3H), 2.11 (s, 3H), 2.10 (s, 3H); ¹³C-NMR (DMSO-d₆) d 147.22,

141.11, 140.84, 134.54, 124.95, 42.20, 19.48, 14.07, 12.69.

1

m.p. 163 ^ꢁC; MS (ESI) m/z 259 [M þ H]^þ; H-NMR (DMSO-d₆) d 9.11

(s, 1H), 8.48 (s, 1H), 4.30–4.22 (m, 2H), 3.85–3.78 (m, 2H), 2.28 (s,

3H), 2.12 (s, 3H), 2.02 (s, 3H); ¹³C-NMR (DMSO-d₆) d 154.28, 147.70,

141.39, 140.01, 133.74, 126.29, 64.06, 42.99, 19.11, 14.08, 12.48.

2.1.28. 2-Methoxyethyl (5-(benzyloxy)-3,4,6-trimethylpyridin-2-

yl)carbamate (15e)

To a solution of 5-benzyloxy-3,4,6-trimethylpyridin-6-amine (5,

70 mg, 0.29 mmol) in acetone (2 ml) was added K₂CO₃(200 mg,

1.45 mmol) followed by dropwise addition of 2-methoxyethyl

chloroformate (168 mL, 1.45 mmol) at 0 ^ꢁC. The mixture was stirred

at room temperature for 17 h and then concentrated. The residue

was diluted with CH₂Cl₂and washed with sat. NaHCO₃and brine.

The CH₂Cl₂layer was dried over MgSO₄, filtered and concentrated.

The residue was purified by silica gel column chromatography

(CH₂Cl₂/MeOH ¼ 40/1) to give 15e (72 mg, 72%). White solid; TLC

R_f0.22 (CH₂Cl₂/MeOH ¼ 30/1); m.p. 98 ^ꢁC; MS (ESI) m/z 345

2.1.32. N-(5-(benzyloxy)-3,4,6-trimethylpyridin-2-yl)-4-methylbenze-

nesulfonamide (17 b)

To a solution of 5-benzyloxy-3,4,6-trimethylpyridin-6-amine (5,

100 mg, 0.41 mmol) in pyridine (2 ml) was added p-toluenesulfonyl

chloride (87 mg, 0.45 mmol). The mixture was stirred at room tem-

perature for 2 h and then concentrated. The residue was diluted

with CH₂Cl₂and washed with brine. The CH₂Cl₂layer was dried

over MgSO₄, filtered and concentrated. The residue was purified

by silica gel column chromatography (Hexanes/EtOAc ¼ 3/1) to

give 17 b (82 mg, 50%). White solid; TLC R_f0.34 (Hexanes/EtOAc ¼

2/1); m.p. 198 ^ꢁC; MS (ESI) m/z 397 [M þ H]^þ; ¹H-NMR (CDCl₃) d

1

[M þ H]^þ; H-NMR (CDCl₃) d 7.50–7.35 (m, 5H), 6.94 (s, 1H), 4.77 (s,

2H), 4.35–4.27 (m, 2H), 3.67–3.58 (m, 2H), 3.41 (s, 3H), 2.44 (s, 3H),

2.24 (s, 3H), 2.16 (s, 3H); ¹³C-NMR (CDCl₃) d 154.41, 150.65, 148.01,

143.46, 141.81, 136.98, 128.78 (2 C), 128.43, 128.06 (2 C), 126.51, 7.83 (d, J ¼ 8.3 Hz, 2H), 7.43–7.33 (m, 5H), 7.24 (d, J ¼ 8.0 Hz, 2H),

75.06, 70.90, 64.57, 59.11, 19.15, 14.91, 13.32.

4.72 (s, 2H), 2.39 (s, 3H), 2.25 (s, 3H), 2.21 (s, 3H), 2.16 (s, 3H); ¹³C-

NMR (CDCl₃) d 149.33, 146.34, 144.21, 142.36, 140.62, 136.70,

136.15, 129.26 (2 C), 128.88 (2 C), 128.76, 128.29 (2 C), 126.54 (2 C),

125.87, 75.93, 21.62, 15.88, 14.06, 13.85.

2.1.29. 2-Methoxyethyl (5-hydroxy-3,4,6-trimethylpyridin-2-yl)car-

bamate (16e)

To a suspension of 10% palladium on activated carbon (19 mg) in

MeOH (5 ml) was added 15e (95 mg, 0.28 mmol). The mixture was

stirred with hydrogen balloon at room temperature for 1 h. After

filtration of the reaction mixture through a Celite pad, the filtrate

was concentrated. The residue was dissolved in MeOH and filtered

through a membrane filter. The filtrate was concentrated to give

16e (70 mg, 99%). White solid; TLC R_f0.16 (CH₂Cl₂/MeOH ¼ 30/1);

2.1.33. N-(5-Hydroxy-3,4,6-trimethylpyridin-2-yl)-4-methylbenzene-

sulfonamide (18 b)

To a suspension of 10% palladium on activated carbon (26 mg) in

MeOH (10 ml) was added 17 b (130 mg, 0.33 mmol). The mixture

was stirred with hydrogen balloon at room temperature for 2 h.

After filtration of the reaction mixture through a Celite pad, the fil-

trate was concentrated. The residue was dissolved in MeOH and

filtered through a membrane filter and concentrated to give 18 b

(86 mg, 86%). White solid; TLC R_f0.21 (Hexanes/EtOAc ¼ 1/1); m.p.

135 ^ꢁC; MS (ESI) m/z 307 [M þ H]^þ; ¹H-NMR (DMSO-d₆) d 9.13 (br,

1

m.p. 147 ^ꢁC; MS (ESI) m/z 255 [M þ H]^þ; H-NMR (DMSO-d₆) d 9.01

(s, 1H), 8.52 (br s, 1H), 4.11 (dd, J ¼ 5.4, 3.9 Hz, 2H), 3.52 (dd,

J ¼ 5.4, 4.0 Hz, 2H), 3.27 (s, 3H), 2.28 (s, 3H), 2.12 (s, 3H), 2.00 (s,

3H); ¹³C-NMR (DMSO-d₆) d 154.74, 147.70, 141.40, 140.31, 133.77, s, 2H), 7.72 (d, J ¼ 8.3 Hz, 2H), 7.32 (d, J ¼ 8.2 Hz, 2H), 2.36 (s, 3H),

126.35, 70.29, 63.20, 58.05, 19.31, 14.25, 12.63.

2.12 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H); ¹³C-NMR (DMSO-d₆) d 146.78,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

7

141.98, 140.59, 140.41, 139.53, 134.66, 128.71 (2 C), 127.32 (2 C), 2.1.37. N-(5-Hydroxy-3,4,6-trimethylpyridin-2-yl)-4-(trifluoromethyl)-

124.58, 20.95, 18.72, 14.05, 12.61.

benzenesulfonamide (18d)

To a suspension of 10% palladium on activated carbon (27 mg) in

MeOH (5 ml) was added 17d (146 mg, 0.32 mmol). The mixture

was stirred with hydrogen balloon at room temperature for 4 h.

After filtration of the reaction mixture through a Celite pad, the fil-

trate was concentrated. The residue was dissolved in MeOH and

filtered through a membrane filter and concentrated to give 18d

(82 mg, 70%). White solid; TLC R_f0.32 (CH₂Cl₂/MeOH ¼ 15/1); m.p.

2.1.34. N-(5-(benzyloxy)-3,4,6-trimethylpyridin-2-yl)naphthalene-1-

sulphonamide (17c)

To a solution of 5-benzyloxy-3,4,6-trimethylpyridin-6-amine (5,

50 mg, 0.21 mmol) in CH₂Cl₂(1 ml) was added 1-napthalenesul-

fonyl chloride (70 mg, 0.31 mmol) followed by addition of triethyl-

amine (57 mL, 0.41 mmol). The mixture was stirred at room 165 ^ꢁC; MS (ESI) m/z 361 [M þ H]^þ; ¹H-NMR (DMSO-d₆) d 10.17 (s,

temperature for 70 h. The reaction mixture was diluted with 1H), 8.55 (s, 1H), 8.03 (d, J ¼ 8.3 Hz, 2H), 7.93 (d, J ¼ 8.4 Hz, 2H),

CH₂Cl₂and washed with sat. NaHCO₃and brine. The CH₂Cl₂layer 2.12 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H); ¹³C-NMR (DMSO-d₆) d 146.89,

was dried over MgSO₄, filtered and concentrated. The residue was 146.42, 140.31, 131.62 (dd, J ¼ 64.2, 32.1 Hz), 128.17 (2 C), 125.83,

purified by silica gel column chromatography (Hexanes/EtOAc ¼ 125.58 (q, J ¼ 3.8 Hz), 124.84 (2 C), 121.50, 18.40, 13.93, 12.66.

15/1 to 2/1) to give 17c (54 mg, 61%). Pale yellow solid; TLC R_f

0.31 (Hexanes/EtOAc ¼ 2/1);) ; m.p. 153 ^ꢁC; MS (ESI) m/z 433

[M þ H]^þ; ¹H-NMR (CDCl₃) d 11.85 (br s, 1H), 9.03 (d, J ¼ 8.5 Hz,

2.1.38. N-(5-(benzyloxy)-3,4,6-trimethylpyridin-2-yl)-4-nitrobenzene-

sulfonamide (17e)

To a solution of 5-benzyloxy-3,4,6-trimethylpyridin-6-amine (5,

100 mg, 0.41 mmol) in pyridine (2 ml) was added 4-nitrobenzene-

sulfonyl chloride (110 mg, 0.50 mmol). The mixture was stirred at

room temperature for 4 h and then concentrated. The residue was

1H), 8.26 (dd, J ¼ 7.3, 1.0 Hz, 1H), 7.96 (d, J ¼ 8.2 Hz, 1H), 7.88 (d,

J ¼ 7.9 Hz, 1H), 7.65 (ddd, J ¼ 8.5, 6.9, 1.4 Hz, 1H), 7.56 (dd, J ¼ 11.0,

4.0 Hz, 1H), 7.50–7.43 (m, 1H), 7.43–7.29 (m, 5H), 4.69 (s, 2H), 2.23

(s, 3H), 2.18 (s, 3H), 2.12 (s, 3H); ¹³C-NMR (CDCl₃) d 160.04, 150.54,

147.41, 143.15, 139.48, 136.05, 134.45, 132.93, 128.90 (2 C), 128.82,

diluted with CH₂Cl₂and washed with sat. NaHCO₃and brine. The

128.75, 128.47, 128.31 (2 C), 127.54, 126.78, 126.70, 126.63, 126.43,

CH₂Cl₂layer was dried over MgSO₄, filtered and concentrated. The

124.06, 76.11, 15.30, 14.19, 13.68.

residue was purified by silica gel column chromatography

(Hexanes/EtOAc ¼ 4/1) to give 17e (126 mg, 71%). Yellow solid;

TLC R_f0.25 (Hexanes/EtOAc ¼ 2/1); m.p. 62 ^ꢁC; MS (ESI) m/z 428

2.1.35. N-(5-Hydroxy-3,4,6-trimethylpyridin-2-yl)naphthalene-1-sul-

[M þ H]^þ; ¹H-NMR (CDCl₃) d 11.86 (br s, 1H), 8.25 (d, J ¼ 8.7 Hz,

phonamide (18c)

To a suspension of 10% palladium on activated carbon (22 mg) in

MeOH (5 ml) was added 17c (109 mg, 0.25 mmol). The mixture

2H), 8.10 (d, J ¼ 8.8 Hz, 2H), 7.49–7.31 (m, 5H), 4.74 (s, 2H), 2.28 (s,

3H), 2.24 (s, 3H), 2.14 (s, 3H); ¹³C-NMR (CDCl₃) d 150.09, 149.30,

148.24, 143.85, 135.85, 135.09, 135.07, 128.86 (2 C), 128.83, 128.27

was stirred with hydrogen balloon at room temperature for 8 h.

(2 C), 127.28 (2 C), 126.36, 123.97 (2 C), 76.10, 15.44, 14.27, 13.63.

After filtration of the reaction mixture through a Celite pad, the fil-

trate was concentrated. The residue was purified by silica gel col-

umn chromatography (Hexanes/EtOAc ¼ 2/1 to 1/1) to give 18c

2.1.39. N-(5-Hydroxy-3,4,6-trimethylpyridin-2-yl)-4-nitrobenzenesul-

(77 mg, 89%). Pale yellow solid; TLC R_f0.32 (CH₂Cl₂/MeOH ¼ 15/

fonamide (18e)

1); m.p. 95 ^ꢁC; MS (ESI) m/z 343 [M þ H]^þ; ¹H-NMR (CDCl₃) d 8.92

To a suspension of 17e (74 mg, 0.17 mmol) in CH₂Cl₂(2 ml), was

(d, J ¼ 8.5 Hz, 1H), 8.17 (dd, J ¼ 7.3, 0.9 Hz, 1H), 7.90 (d, J ¼ 8.2 Hz,

added pentamethylbenzene (77 mg, 0.52 mmol) followed by drop-

1H), 7.83 (d, J ¼ 7.9 Hz, 1H), 7.68–7.56 (m, 1H), 7.52 (dd, J ¼ 11.0,

4.0 Hz, 1H), 7.47–7.35 (m, 1H), 2.27 (s, 3H), 2.14 (s, 3H), 2.04 (s, 3H);

¹³C-NMR (CDCl₃) d 148.37, 144.31, 141.04, 139.26, 134.40, 132.99,

129.12, 128.60, 128.56, 127.52, 126.63, 126.46, 126.42, 125.67,

124.04, 15.50, 13.93, 13.59.

wise addition of boron trichloride (1 M in CH₂Cl₂, 0.35 ml) at 0 ^ꢁC.

The reaction mixture was stirred at 0 ^ꢁC for 15 min then quenched

with a mixed solvent of CHCl₃/MeOH (9/1, 1.5 ml) and stirred at

room temperature for 30 min. After concentration of the reaction

mixture, the residue was purified by silica gel column chromatog-

raphy (CH₂Cl₂/MeOH ¼ 20/1) to give 18e (47 mg, 81%). Yellow

solid; TLC R_f0.29 (Hexanes/EtOAc ¼ 2/1); m.p. 199 ^ꢁC; MS (ESI) m/z

388 [M þ H]^þ; ¹H-NMR (CD₃OD) d 8.36 ꢂ 8.27 (m, 2H), 8.09 ꢂ 8.00

(m, 2H), 2.28 (s, 3H), 2.19 (s, 3H), 2.10 (s, 3H); ¹³C-NMR (CD₃OD) d

150.91, 150.53, 147.15, 144.33, 141.49, 137.52, 129.19 (2 C), 127.54,

124.85 (2 C), 16.80, 14.01, 13.35.

2.1.36. N-(5-(benzyloxy)-3,4,6-trimethylpyridin-2-yl)-4-(trifluorome-

thyl)benzenesulfonamide (17d)

To a solution of 5-benzyloxy-3,4,6-trimethylpyridin-6-amine (5,

150 mg, 0.62 mmol) in pyridine (3 ml) was added 4-(trifluorome-

thyl)benzenesulfonyl chloride (227 mg, 0.93 mmol). The mixture

was stirred at room temperature for 24 h and then concentrated.

The residue was diluted with CH₂Cl₂and washed with sat.

NaHCO₃and brine. The CH₂Cl₂layer was dried over MgSO₄, fil-

tered and concentrated. The residue was purified by silica gel col-

2.1.40. 2-Bromo-5-methoxy-3,4,6-trimethylpyridine (19)

To a solution of 6-bromo-2,4,5-trimethylpyridin-3-ol (3, 216 mg,

1.0 mmol) of CH₃CN (10 ml) were added K₂CO₃(207 mg, 1.5 mmol)

and iodomethane (0.12 ml, 2.0 mmol). The mixture was stirred at

50 ^ꢁC for 16 h and then cooled to room temperature. It was

diluted with EtOAc and washed with 1 M HCl, water and brine

successively. The EtOAc solution was dried over MgSO₄, filtered

and concentrated. The residue was purified by silica gel column

chromatography (Hexanes/EtOAc ¼ 9/1) to give 19 (154 mg, 67%).

umn chromatography (Hexanes/EtOAc

¼

3/1) to give 17d

(231 mg, 83%). White solid; TLC R_f0.33 (Hexanes/EtOAc ¼ 3/1);

1

m.p. 131 ^ꢁC; MS (ESI) m/z 451 [M þ H]^þ; H-NMR (CDCl₃) d 11.72 (s,

1H), 8.07 (d, J ¼ 8.2 Hz, 2H), 7.69 (d, J ¼ 8.3 Hz, 2H), 7.45–7.32 (m,

5H), 4.74 (s, 2H), 2.27 (s, 3H), 2.24 (s, 3H), 2.16 (s, 3H); ¹³C-NMR

(CDCl₃)

d 150.14, 147.69, 143.80, 135.98, 135.23, 133.21 (q,

J ¼ 32.9 Hz), 128.90 (2 C), 128.84, 128.31 (2 C), 126.70 (4 C), 126.27, Yellow oil; TLC R_f0.37 (Hexanes/EtOAc ¼ 9/1); MS (ESI) m/z 230

125.81 (q, J ¼ 3.7 Hz), 121.47, 76.10, 15.52, 14.23, 13.69.

[M þ H]^þ; ¹H-NMR (CDCl₃) d 3.67 (s, 3H), 2.44 (s, 3H), 2.30 (s, 3H),

8

C. L. CHAUDHARY ET AL.

2.24 (s, 3H); ¹³C-NMR (CDCl₃) d 152.76, 150.07, 141.29, 137.88, (s, 1H), 8.22 (s, 1H), 7.81 (d, J ¼ 8.0 Hz, 1H), 7.59 (t, J ¼ 7.6 Hz, 1H),

131.95, 60.33, 18.88, 18.77, 13.50.

7.55 ꢂ 7.47 (m, 1H), 3.66 (s, 3H), 2.41 (s, 3H), 2.21 (s, 3H), 2.19 (s,

3H); ¹³C-NMR (DMSO-d₆) d 178.52, 149.47, 145.59, 145.06, 141.90,

139.91, 129.67, 128.98 (q, J ¼ 31.6 Hz), 127.27, 125.37, 122.67,

2.1.41. N-(diphenylmethylene)-5-methoxy-3,4,6-trimethylpyridin-2-

amine (20)

121.27, 119.61, 60.20, 18.42, 13.20, 12.66.

To a suspension of 19 (155 mg, 0.67 mmol) in toluene (5 ml) were

added BINAP (42 mg, 0.07 mmol), Pd₂(dba)₃(31 mg, 0.03 mmol),

NaOBu^t(71 mg, 0.74 mmol), and benzophenone imine (0.11 ml,

0.67 mmol). The mixture was stirred at 115 ^ꢁC for 4 h and then

cooled to room temperature. It was diluted with EtOAc and

washed with water and brine. The EtOAc layer was dried over

MgSO₄, filtered and concentrated. The residue was purified by sil-

ica gel column chromatography (Hexanes/EtOAc ¼ 4/1) to give 20

(170 mg, 77%). Yellow solid; TLC R_f0.15 (Hexanes/EtOAc ¼ 4/1);

2.2. Materials for biology

RPMI-1640, foetal bovine serum (FBS), penicillin/streptomycin and

trizol reagent were obtained from Invitrogen Life Technologies

(Carlsbad, CA, USA). Trypsin/EDTA was purchased from Clonetics,

Inc. (Walkersville, MD, USA). Recombinant human TNF-a was pro-

cured from R & D system Inc, (Minneapolis, MN, USA). BCECF/AM

(acetoxymethyl ester) was received from Molecular probes

(Eugene, Oregon, USA). Antibodies directed against TNF-a, IL-1b,

Claudin-2, NLRP3, Claudin-3 and Caspase-1 were purchased from

Abcam (Cambridge, MA, USA). Antibodies against IL-6 and IL-10

were obtained from Abbiotec (San Diego, CA, USA). E-cadherin,

ZO-1, IL-18 and I-jB antibodies were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA, USA) while NF-jB, TGFb and phos-

pho-I-jB antibodies were purchased from Cell Signalling

Technology Inc. (Beverly, MA, USA).

m.p. 95 ^ꢁC; MS (ESI) m/z 331 [M þ H]^þ; ¹H-NMR (CDCl₃)

d

7.80 ꢂ 7.36 (m, 10H), 3.66 (s, 3H), 2.52 (s, 3H), 2.29 (s, 3H), 2.10 (s,

3H); ¹³C-NMR (CDCl₃) d 169.36, 156.96, 149.30, 146.80, 139.56,

139.23, 136.89, 130.80, 129.60, 128.89, 128.56, 127.98, 127.53,

119.56, 60.36, 18.74, 13.79, 12.37.

2.1.42. 5-Methoxy-3,4,6-trimethylpyridin-2-amine (21)

A solution of 20 (168 mg, 0.51 mmol) in a mixed solvent of THF/

MeOH (1/10, 5.5 ml) was cooled to 0 ^ꢁC. A mixed solution of

CH₃COCl/MeOH (1/10, 1 ml) was slowly added to the mixture. The

reaction mixture was stirred at room temperature for 18 h and

then concentrated. The residue was diluted with EtOAc and water.

2.3. Cell culture

HT-29 human colonic epithelial cell line and U937 human pre-

monocytic cell line were obtained from the American Type

The EtOAc layer separated was extracted with water. To the com- Culture Collection (Manassas, VA, USA). HT-29 cells and U937 cells

bined aqueous solution was added 1 M NaOH until the pH of the were cultured in RPMI-1640 media containing 10% FBS, 100 IU/ml

solution reached 10. After extraction of the mixture with EtOAc,

the EtOAc solution was dried over MgSO₄, filtered and concen-

trated. The residue was purified by silica gel column chromatog-

raphy (CH₂Cl₂/MeOH ¼ 30/1) to give 21 (54 mg, 64%). Grey solid;

TLC R_f0.15 (CH₂Cl₂/MeOH ¼ 20/1); m.p. 84 ^ꢁC; MS (ESI) m/z 167

[M þ H]^þ; ¹H-NMR (DMSO-d₆) d 5.22 (s, 2H), 3.52 (s, 3H), 2.17 (s,

3H), 2.06 (s, 3H), 1.91 (s, 3H); ¹³C-NMR (CDCl₃) d 152.25, 146.62,

145.79, 139.72, 113.51, 60.53, 18.45, 13.05, 12.42.

of penicillin and 100 mg/mL of streptomycin. Cells were main-

tained at 37 ^ꢁC in 5% CO₂in humidifier incubator.

2.4. Measurements of monocyte adhesion to epithelial cell layer

Adhesion of monocytes to colon epithelial cell layer was measured

by using U937 pre-monocytic cells prelabeled with 2⁰,7⁰-bis(2-car-

boxyethyl)-5(6)-carboxylfluorescein acetoxymethyl ester (BCECF/

AM, 10 mg/ml) as previously reported^21,22with slight modification.

HT-29 cells (2 ꢃ 10⁵cells/well) cultured in 48-well plates were pre-

treated with compounds for 1 h. The prelabeled U937 cells were

added on the monolayer of HT-29 cells, and they were treated

with TNF-a (10 ng/mL) for 3 h at 37 ^ꢁC. Non adhering U937 cells

were removed by washing thrice with PBS. Cells were lysed with

0.1% Triton X-100 in Tris (0.1 M), and fluorescence intensity was

measured using Fluostar Optima microplate reader (BMG LABTECH

GmbH, Germany) at an excitation and emission wavelengths of

485 and 520 nm, respectively.

2.1.43. 1–(3,4-Dichlorophenyl)-3–(5-methoxy-3,4,6-trimethylpyridin-

2-yl)thiourea (22a)

To a solution of 21 (25 mg, 0.15 mmol) in EtOH (2 ml) was added

3,4-dichlorophenyl isothiocyanate (24 mL, 0.16 mmol). The reaction

mixture was stirred at room temperature for 48 h and then con-

centrated. The residue was purified by recrystallization over EtOH

to give 22a (39 mg, 70%). White solid; TLC R_f0.63 (Hexanes/EtOAc

1

¼ 4/1); m.p. 176 ^ꢁC; MS (ESI) m/z 370 [M þ H]^þ; H-NMR (DMSO-d₆)

d 13.05 (s, 1H), 9.54 (s, 1H), 8.22 (s, 1H), 7.81 (d, J ¼ 8.0 Hz, 1H),

7.59 (t, J ¼ 7.6 Hz, 1H), 7.55 ꢂ 7.47 (m, 1H), 3.66 (s, 3H), 2.41 (s, 3H),

2.21 (s, 3H), 2.19 (s, 3H); ¹³C-NMR (DMSO-d₆) d 178.41, 149.52,

145.53, 145.11, 141.92, 139.20, 130.49, 130.22, 126.54, 124.63,

123.60, 121.50, 60.21, 18.43, 13.23, 12.67.

2.5. TNBS-induced colitis in rats

Sprague-Dawley female rats (seven weeks old) were purchased

from Orient Bio Inc (Gyeonggi, South Korea) and were acclima-

tised to the lab condition for 1 week by giving food and water

2.1.44. 1–(5-Methoxy-3,4,6-trimethylpyridin-2-yl)-3–(3-(trifluorome- provided ad libitum. Rats were fasted for 24 h before TNBS injec-

tion (0.8 ml of 5% TNBS in 50% ethanol) into the lumen of the

colon (8 cm proximal to the anus through the rectum) through an

polyethylene catheter fitted onto a 1 ml syringe. Right after TNBS

injection, rats were kept in vertical position for 1 min before being

returned to the cage. Next day, sulfasalazine (300 mg/kg) or com-

pounds (1 mg/kg) in corn oil was administered for up to 5 days.

The doses of the drug were selected based on the previous stud-

thyl)phenyl)thiourea (22 b)

To a solution of 21 (25 mg, 0.15 mmol) in EtOH (2 ml) was added

3-(trifluoromethyl)phenyl isothiocyanate (24 mL, 0.16 mmol).

Reaction mixture was stirred at room temperature for 48 h and

then concentrated. The residue was purified with silica flash col-

umn chromatography (Hexanes/EtOAc ¼ 4/1) to give 22 b (30 mg,

54%). White solid; TLC R_f0.46 (Hexanes/EtOAc ¼ 4/1); m.p. 140 ^ꢁC;

MS (ESI) m/z 370 [M þ H]^þ; H-NMR (DMSO-d₆) d 13.05 (s, 1H), 9.54 ies^18,19. On the sixth day, all the rats were sacrificed and

1

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

9

macroscopic observations were evaluated for ulceration and sever-

ity of colitis. The colon tissues from 5 to 7 cm proximal to rectum

were excised. The colon tissues were stored at ꢂ80 ^ꢁC to examine

the protein expression of inflammatory mediators, junctional mol-

ecules, and transcription factors.

The study protocol of the animal experiment was reviewed

and approved beforehand by the Institutional Animal Care and

Use Committee of Yeungnam University and were performed fol-

lowing the institutional guidelines of the Institute of Laboratory

Animal Resources (1996), and of Yeungnam University for the care

and use of animals (2009).

3. Results

3.1. Design and synthesis

At first, we were curious about the effect of heteroatom at C(6)-

position on activity and replaced nitrogen with oxygen to give

6-alkoxy analogues 8. Synthesis of 8 was conducted via intermedi-

ate 5 which has been reported by us (Scheme 1)¹⁵. Briefly, two

primary hydroxy groups of pyridoxineꢄHCl (1) was converted to

chlorides using SOCl₂and catalytic amount of DMF. Then, two

benzyl chloride groups were reductively removed with Zn

and acetic acid under refluxing conditions to give 2,4,5-

trimethylpyridin-3-ol (2). The C(6)-position was brominated using

1,3-dibromo-5,5-dimethylhydantoin (DBDMH) and the phenolic

OH group of 3 was protected as benzyloxy group. Bromide on

C(6)-position was replaced with benzophenone imine under

Buchwald–Hartwig amination conditions to afford 4. The imine

group was then methanolyzed to give the free amino compound

(5). The amino group of 5 was converted to hydroxy group via

diazonium ion to give 6, which was then treated with various alkyl

halides to give alkoxy compounds 7. Lastly, the benzyl protective

group was removed by catalytic hydrogenolysis to give 6-

alkoxy-2,4,5-trimethylpyridin-3-ol analogues 8. The dihydroxy com-

pound 8a was obtained from the dibenzyloxy compound 7a. A

2.6. Measurement of myeloperoxidase level

Colon myeloperoxidase (MPO) level was measured by using MPO

Rat ELISA kit (HK105-01, Hycult biotechnology, Netherland). Colon

tissues were weighed, washed in cold 1ꢃ PBS (pH: 7.4), and

homogenised in 500 mL of ice-cold lysis buffer (pH 7.4, 200 mM

NaCl, 5 mM EDTA, 10 mM Tris, 10% glycerol, 1 mM PMSF, 1 mg/mL

leupeptide, 28 mg/mL aprotinin) by using

(Benchmark scientific, Edison, NJ, USA). MPO amount in the super-

natant was measured by detecting the absorbance at 450 nm in direct approach to obtain 8a from 6 under the standard catalytic

a

Bead Blaster

hydrogenolysis reaction conditions gave poor yield.

microplate reader (Spectrostar Nano, BMG LABTECH, Ortenberg,

Germany).

Unfortunately, all alkoxy analogues (8a–8f) showed modest

activity (29–67%) in our cell adhesion assay (Figure 2). Compound

8a (67% inhibition) and 8f (56% inhibition) showed 2.9- and 2.1-

fold higher activity than the corresponding nitrogen counterparts

whose inhibition level was 23% and 27%, respectively. Contrarily,

compound 8e (29% inhibition) showed 2.5-fold less activity than

the corresponding nitrogen counterpart (73% inhibition)¹⁸. The

rest of alkoxy analogues with alkyl groups (8 b, 8c, 8d) did not

show distinct activity from 6-alkylamino analogues. It seems that

nitrogen atom does not necessarily be replaced and might be bet-

ter to remain on C(6)-position for the better topological polar sur-

face area (tPSA) and calculated partition coefficient (cLogP). By

locating nitrogen back to C(6)-position, about 10% of both tPSA

and cLogP values can be restored compared to the alkoxy ana-

logues. Therefore, we decided to explore structural diversity with

nitrogen atom on the position.

2.7. Protein extraction and Western blotting

Total protein from the colon was extracted by using radioimmu-

noprecipitation assay (RIPA) buffer (150 mM Sodium chloride, 1%

Triton X-100, 0.5% Sodium deoxycholate, 0.1% SDS and 50 mM

Tris adjusted to pH 8.0) containing 1X proteases and phosphatase

inhibitor (Thermo Scientific, Rockford, USA). Cytosolic and nuclear

proteins were extracted by using manufacturer’s instruction given

by NE-PER nuclear and cytoplasmic extraction reagent kit (#78833,

Thermo Scientific, Rockford, USA). Protein concentrations were

measured using a BCA protein assay kit (Pierce, Rockford, IL, USA).

Equal amounts of total proteins were then separated by SDS-

PAGE and transferred onto Hybond ECL nitrocellulose membranes

(Amersham Life Science, Buckinghamshire, UK) at 200 mA for 1 h.

Blocking was done using 5% BSA in Tris-buffered saline (TBS)-

Tween 20 (TBS-T) at 20 ^ꢁC for 1 h. The membranes were incubated

for 16 h at 4 ^ꢁC with primary antibody diluted in TBS containing

3% BSA. After incubation, the membranes were washed three

times with TBS-T. Next, the membrane was incubated for 1 h at

20 ^ꢁC with horseradish peroxidase-conjugated secondary antibody

in TBS. Immunoreactive proteins were visualised using an

enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL, USA)

and digitally processed using an LAS-4000 mini (Fuji, Japan). The

membranes were stripped and re-probed with an actin antibody

as a loading control.

HO

O

C

S

C

O

C

O

R

S

N

O

N

R

H

urea

thiourea

carbamate

sulfonamide

Now that we have reported that 6-(substituted)amino-2,4,5-

trimethylpyridin-3-ols have high activity against colitis in vitro and

in vivo, structural exploration with nitrogen on C(6)-position was

designed to install ureido-, thioureido-, carbamato- and sulfona-

mido-linkage between pyridine ring and R group.

Described in Scheme 2 is the general scheme for the prepar-

ation of urea and thiourea analogues, 13 and 14. Isocyanates

(R ꢂ N ¼ C ¼ O)/isothiocyanates (R ꢂ N ¼ C ¼ S) were employed for

the construction of urea/thiourea moieties. Two key intermediates,

5 and 10, were used for the coupling reactions and the TBDPS-

protected intermediate (10) was prepared by the synthetic pro-

cedure which we reported before²⁰. In short, compound 10 was

prepared from 2,4,5-trimethylpyridin-3-ol (2) by the following

sequence; bromination of C(6)-position, protection of ꢂ OH with

TBDPS group, palladium-catalyzed amination with benzophenone

imine, and acidic methanolysis of the imine group. The coupled

products with isocyanates and isothiocyanates, 11 and 12, were

2.8. Statistical analysis

Data are presented as the mean SEM, and were analysed by one

way ANOVA followed by the Newman–Keuls comparison method

using GraphPad Prism software (version 5.0, San Diego, CA, USA).

P values less than 0.05 were considered statistically significant.

10

C. L. CHAUDHARY ET AL.

Scheme 1. Synthesis of alkoxy-analogues 8.

then transformed the final compounds, 13 and 14, by deprotec- 0.23 mM (18c). The order of these values is quite lined with the

tion of O-benzyl or O-silyl group. For 14a compound with ꢂ NH₂order of % inhibition measured at the fixed single concentra-

group, the precursor, O-benzyl protected compound 12a, was tion (1 mM).

obtained from N-benzoyl thiourea compound 12n by a debenzoy-

Next, we tried to investigate the role –OH group on C(3)-

lation under basic hydrolysis conditions.

position on the activity. The hydroxy group is protected as

For the synthesis of sulfonamido- (16) and carbamato-ana- methoxy group as shown in structure 22 to study if the OH group

logues (18), the key intermediate 5 was treated with either chloro- is essential (Scheme 4). Compounds 22a and 22 b are 3-methoxy

formates or sulphonyl chlorides to give 15 and 17. They were analogues of 14j and 14k, respectively, which showed excellent

then debenzylated to afford 16 and 18, respectively (Scheme 3).

activity in our in vitro assay as shown in Figure 1. The intermedi-

ate 3 is methylated with iodomethane and K₂CO₃to give

3-methoxy compound 19. Then, benzophenone imine was

coupled under Buchwald-Hartwig amination conditions to afford

imino compound 20 which in turn was hydrolysed to free amino

compound 21. The amino group of 21 was coupled with isothio-

cyanates to give 22a and 22 b. As shown in Figure 2, unfortu-

nately, 3-methoxy analogues 22a and 22 b showed more than

2-fold less activity than corresponding 3-hydroxy analogues, 14j

and 14k, respectively. It might imply that free hydroxyl group at

3-position is essential to the activity by contributing some sort of

hydrogen bonding to a binding partner which is responsible for

the activity. Further detail of the role of hydroxyl group will be

investigated in subsequent studies.

3.2. Inhibitory effects of 2,4,5-trimethylpyridin-3-ol derivatives

on TNF-a-induced adhesion of monocytes to colonic

epithelial cells

In vitro activity data of the alkoxy- (8), ureido- (13), thioureido-

(14), carbamato- (16) and sulfonamido-analogues (18) are listed in

Figure 2. As stated above, alkoxy analogues (8) showed activity in

a somewhat similar range to the nitrogen counterparts. The best

activity (67%) was seen in compound 8a which has no substituent

on C(6)-oxygen. Activity of urea (13) and thiourea (14) analogues

are also in a similar range. Among the compounds with the same

R group, the phenyl group is more active in urea analogue (13 b:

76.3%) than in thiourea analogue (14e: 49.7%). Both p-tolyl and

p-methoxyphenyl groups showed similar and low activity both in

urea analogues (13d: 20.1%, 13j: 12.6%, respectively) and thiourea

analogues (14f: 19.2%, 14 g: 13.3%, respectively). IC₅₀values of

3.3. Anti-inflammatory effects of seven 2,4,5-trimethylpyridin-3-

ol derivatives on TNBS-induced rat colitis

some compounds of high % inhibition were measured to be Next, we evaluated in vivo anti-inflammatory activity using TNBS-

0.75 mM (8f), 0.41 mM (13 b), 0.40 mM (14n), 0.32 mM (18d), and induced rat colitis model. Seven pyridin-3-ol compounds, 13 b,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

11

Figure 2. Inhibitory activity of the compounds against TNF-a-induced monocyte adhesion to colon epithelial cells. Data are shown as mean SEM of at least three

ꢀ

independent experiments. p < 0.05 compared to TNF-a-induced HT-29 cells.

14c, 14j, 14k, 14n, 18c, and 18d, were selected for the in vivo disease phenotype, we measured the recovery level in body- and

efficacy evaluation along with sulfasalazine (SSZ) as the positive colon-weights upon drug treatment (Figure 3). Oral administration

control drug. Colitis was induced by rectal administration of TNBS. of rats with compounds, 13 b, 14c, 14j, 14k, 14n, 18c, or 18d

Our seven pyridin-3-ol compounds along with SSZ were orally (1 mg/kg) significantly restored TNBS-induced body weight loss

administered once a day for five days starting one-day after (Figure 3(A)). The recovery rate by the compounds (1 mg/kg)

administration of TNBS at a dose of 1 mg/kg (for seven pyridin-3- ranged from 25% to 98.9%, among which compound 14n was the

ols) and 300 mg/kg (for SSZ), respectively. After treatment with most excellent. The recovery rate of SSZ (300 mg/kg), a positive

TNBS, the rats showed signs of colitis, such as body weight control, was 48.8%. Colon weight recovery was between 50.3%

decrease, bloody diarrhoea, and sluggish and weak movement. and 94.9%, which was better than the body weight recovery in

Compared to the sham-operated control group, the body weight most cases of oral administration of the compounds including

of the TNBS-treated rats was significantly reduced and then sulfasalazine (Figure 3(A)). Compound 14n (1 mg/kg) was the most

increased gradually. Colon tissues in the TNBS-treated lesion site effective in colon weight recovery as in the case of body weight

showed significant inflammation, as revealed by oedema and recovery. In addition, the myeloperoxidase (MPO) level in colon

adhesion in gross morphology examination and increased wet tissues, which is directly related to neutrophil infiltration into tis-

weight per colon length. As a macroscopic marker to represent a sues and serves as a biochemical marker of inflammation²³, was

12

C. L. CHAUDHARY ET AL.

Scheme 2. Synthesis of ureido- and thioureido-analogues, 13 and 14.

examined. TNBS treatment induced a tremendous increase in MPO IL-10 and TGF-b which are known to be secreted by M2 macro-

phages for termination of inflammation and tissue repair^24,25

,

activity (Figure 4), and the increase was significantly suppressed

by oral administration with compounds (1 mg/kg). The order of

the compounds having excellent effects was 14n, 14k, 14c, 14j,

13 b, 18c, and 18d, demonstrating the significant efficacy of the

new scaffold.

were also significantly increased in the inflamed colon tissues

(Figure 4(A,B)). In contrast, level of colon epithelial junction mole-

cules, E-cadherin, Claudin-2, Claudin-3 and ZO-1 was significantly

reduced by TNBS (Figure 4(A,C)). However, oral administration of

compounds, 13b, 14c, 14j, 14k, 14n, 18c, or 18d (1 mg/kg) sig-

nificantly restored the TNBS-altered protein expressions of junc-

tional molecules. All these changes consistently and strongly

support the anti-IBD activity of our compounds. Indeed, the inhibi-

tory effects of compounds, 14c, 14j, 14k, and 14n (1 mg/kg) was

much stronger than that of 300 mg/kg SSZ.

3.4. Suppressive effects of seven 2,4,5-trimethylpyridin-3-ol

derivatives on TNBS-induced expression changes of

inflammatory cytokines, junction molecules, inflammasome, and

nuclear transcription factor-jB

Next, we also examined whether NLRP3 inflammasome, which

plays an important role in IL-1b production²⁶, was suppressed by

the compounds. TNBS-induced alteration of NLRP3 inflammasome

components, NLRP3, pro-caspase-1²⁷, and cleaved and active cas-

pase-1, were blocked by compounds, 13b, 14c, 14j, 14k, 14n,

18c, or 18d (Figure 5). Similar to the effects of the compounds on

cytokine expressions, 14n exerted the strongest effect, which was

Although the fundamental molecular targets of the compounds

responsible for the phenotype are yet to be investigated, we fur-

ther examined in this study the inhibitory effects of the com-

pounds on the expression levels of cytokines responsible for the

inflammatory response. It will provide more consistency to our

mechanism based on the assay against TNF-a-induced cell adhe-

sion. In rat colon tissues, TNBS treatment significantly increased

the expression of TNF-a, IL-6 and IL-1b. The expression level of followed by 14c, 14j, 14k, 13b, 18c, and 18d. Consistent to the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

13

Scheme 2. (Continued).

result of NLRP3 inflammasome activation, IL-18 level was signifi- various compounds against TNF-a-induced monocyte adhesion to

colonic epithelial cells, an in vitro model of initial phase of

colitis. In the previous reports, we found that 6-amino-2,4,5-

trimethylpyridin-3-ol scaffold was effective against TNF-a-induced

cell adhesion between monocyte and colon epithelial cells and

against severe inflammation of colon induced by TNBS. Among

the 6-aminopyridin-3-ols prepared, compounds A ꢂ C showed

cantly increased in the colon of TNBS-treated rats, and com-

pounds, 13b, 14c, 14j, 14k, 14n, 18c, and 18d significantly

inhibited the TNBS-induced IL-18 expression.

In the colon of TNBS-treated rats, nuclear factor-kappa B (NF-

jB), a transcription factor associated with expression of inflamma-

tory cytokines and NLRP3 inflammasome was increased, while

cytosolic NF-jB level was decreased in along with inhibitory-jB

(I-jB) phosphorylation. However, the compounds significantly sup-

pressed the nuclear translocation of NF-jB (Figure 6).

excellent efficacy in in vivo experiments^18,19

.

HO

3

HO

Cl HO

N

S

N

6

N

H

N

H

N

H

sp²

C

sp²C

A

B

C

4. Discussion

Structurally speaking, these strong analogues (A ꢂ C) contain

some sort of sp²-carbon (indicated with arrows) attached to C(6)-

nitrogen. In this study, we decided to further explore the chemical

TNF-a is a critical player in the pathogenesis of IBD, which is sup-

ported by the most efficacious effects of anti-TNF-a biologics in

the clinic²⁸. Based on the notion, we examined anti-IBD activity of space of the substituents on C(6)-position. First, we wondered if

14

C. L. CHAUDHARY ET AL.

Scheme 3. Synthesis of carbamato- and sulfonamido- analogues, 16 and 18.

Scheme 4. Synthesis of 3-methoxy analogues, 22.

nitrogen is essential on C(6)-position for activity and decided to based on the activity of the alkoxy-analogues is to keep nitrogen

replace it with another heteroatom, oxygen. The resulting alkoxy on C(6)-position and instead introduce carbonyl-like functional

analogues (8a–8f) showed low to mediocre range activity in our groups attached to C(6)-nitrogen as an alteration of sp²-carbon

in vitro assay and it seems that nitrogen is quite necessary for the shown in previous papers. This type of analogues include ureido

position. Therefore, the next approach we decided to conduct (11), thioureido (12), carbamato (16) and sulfonamido (18)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

15

Figure 3. Recovery effects of compounds on TNBS-induced rat colitis, body and colon weights, and MPO activity. Colitis was induced by rectal administration of TNBS,

and sham group received vehicle (50% ethanol) in the same route. Compounds (1 mg/kg) and SSZ (300mg/kg) were given orally 1 day after TNBS treatment. Data rep-

resent the mean SEM for five rats per group. (A) Body weight was recorded daily from day 0 to day 5, and body weight recovery was calculated based on the last

day measurement. Colon wet weight (distal 5–6 cm segment) was measured right after dissection of the colon. ^#p < 0.05 compared to TNBS-treated group. ^$p < 0.05

#

ꢀ

compared to SSZ-treated group. (B) MPO level of colon tissues p < 0.05 compared to sham-operated control group. p < 0.05 compared to TNBS-treated group.

analogues. As a result, many compounds shown in this paper increase in the expressions of TNF-a, IL-6, and IL-1b, referred to as

were found to have very strong activity. In vitro, some compounds troika of pro-inflammatory cytokines in colon tissues of TNBS-

including thiourea and sulphonamide analogues showed up to treated rats, was consistent with many previous studies in IBD

93% inhibition against TNF-a-induced cell adhesion at 1 mM con- patients as well as animal models of IBD^30,31. TNBS treatment also

centration. It is the highest in vitro inhibitory activity measured significantly increased colonic levels of IL-10 and TGF-b, known as

with pyridin-3-ol analogues we synthesised so far. The order of anti-inflammatory cytokines. Although the expression level of IL-

the compounds having excellent in vitro effects was 14n, 14k, 10 in experimental colitis is contradictory³², IL-10 level may be

14c, 14j, 13b, 18c, and 18d. Such in vitro activity of the com- related to the inflammatory phase, increased at the initial phase

pounds was consistent with in vivo anti-colitis efficacy (1 mg/kg) and returned to baseline level in the resolution phase of inflam-

on colitis signs and biochemical inflammatory markers, which was mation^33,34. Oral treatments with the compounds significantly sup-

pressed TNBS-induced expressions of IL-10, in addition to TNF-a,

IL-6, and IL-1b in colon tissues, indicating that an anti-colitis activ-

ity of the compounds led to the resolution phase of TNBS-induced

inflammation. In the case of TGF-b, its production is upregulated

by bacteria, viruses, cytokines, and dying cells, in an autocrine or

much better than SSZ (300 mg/kg). Among high activity com-

pounds, 14n, a thiourea analogue, showed the best in vivo activity

represented by macroscopic parameters such as almost complete

recovery of colon and body weights (95% and 99%, respectively)

in TNBS-induced colitis rat model.

Although the exact aetiology of IBD has not yet been clarified, paracrine manner^35,36. TGF-b suppresses inflammatory responses

a defective innate immune response seems to play a critical role similar to IL-10, although previous reports on TGF-b expression in

in the chronic gut inflammation²⁹. Our current study showing an IBD have shown contradictory results depending on active or

16

C. L. CHAUDHARY ET AL.

Figure 4. Inhibitory effects of compounds on expressions of inflammatory cytokines and epithelial junction molecules. Total protein extracts from colon tissues homo-

genates were used for detecting the protein expression of inflammatory mediators, TNFa, IL-6, and IL-1b, and junction molecules, E-cadherin, claudin-2, claudin-3, and

ꢀ

ZO-1. The bar graphs represent the mean SEM of protein expression quantified in three independent experiments. p < 0.05, compared with vehicle-treated control

group. p < 0.05, compared with the TNBS-treated group.

#

inactive IBD^37,38, TGF-b levels are elevated in inflamed area of gut receptors (TLRs) and the cytosolic sensory protein complexes such

tissues in IBD³⁹. Consistently, our current results also showed that as NOD-like receptors (NLR)⁴³. NLRP3, an NLR family member, has

TNBS induced TGF-b increase in the colon, and oral administration been highlighted as a contributing factor in the pathogenesis of

of the compounds significantly reduced the level of TGF-b with IBD⁴⁴. NLRP3 mediates the assembly and activation of the inflam-

masome,

a multi-protein complex, which recognises cellular

the best effects of 14n, 14k, 14c, and 14j.

The innate immune system is activated by the pattern recogni-

tion receptors (PRRs) which recognise pathogen-associated

stresses and responds to them by activating caspase-1. Activated

caspase-1 converts pro-IL-1b and pro-IL-18 into their active forms,

molecular patterns (PAMPs) or endogenous danger-associated IL-1b and IL-18, and induces pyroptosis, an inflammatory form of

molecular patterns (DAMPs) generated during cellular injury or tis- cell death⁴⁵. In the present study, a significant increase in NLRP3

sue damage^40–42. PRRs comprise the membrane-bound toll-like and caspase-1 in colon tissues by TNBS treatment are correlated

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

17

Figure 4. (Continued).

Figure 5. NLRP3 inflammasome formation in TNBS-treated rat colon was suppressed by the compounds. Total protein extracts from colon tissue homogenates were

used for detection of NLRP3 inflammasome components. The bar graphs represent the mean SEM of protein expression quantified in three independent experiments.

ꢀ

p < 0.05, compared with vehicle-treated control group. #p < 0.05, compared with the TNBS-treated group.

18

C. L. CHAUDHARY ET AL.

Figure 6. Inhibitory effects of the compounds on the nuclear translocation of NF-jB in along with increase in cytosolic phospho-I-jB. Cytosolic and nuclear proteins

extracted from colon tissue homogenates were used for detection of phospho-I-jB, I-jB, and NF-jB. The bar graphs represent the mean SEM of protein expression

ꢀ

quantified in three independent experiments. p < 0.05, compared with vehicle-treated control group. #p < 0.05, compared with the TNBS-treated group.

to a high-level increase in IL-1b and IL-18. In addition, the correl- may be

a promising lead compound to develop anti-IBD

ation between casapase-1 increase and the significant reduction therapeutics.

in junction molecules, E-cadherin, claudins, and ZO-1 in TNBS-

treated colon implicates TNBS-induced mucosal damage through

pyroptosis. Inhibitory effects of compounds, 14n, 14k, 14c, 14j,

Conflict of interest: The authors declare no conflict of interest.

13 b, 18c, and 18d on TNBS-induced changes in inflammatory

cytokine expression, casapse-1 and junction molecules suggested

that anti-colitis effect of the compounds was associated with both

anti-inflammatory and mucosal healing activities.

Funding

This work was supported by a grant of the Korean Health

Technology R&D project, Republic of Korea [HI15C0542].

5. Conclusion

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Article Doi

Synthesis, activity and mechanism of alkoxy-, carbamato-, sulfonamido-, thioureido-, and ureido-derivatives of 2,4,5-trimethylpyridin-3-ol against inflammatory bowel disease

DOI: 10.1080/14756366.2019.1677637

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