Synthesis and activity of N-(5-hydroxy-3,4,6-trimethylpyridin-2-yl)acetamide analogues as anticolitis agents via dual inhibition of TNF-α- and IL-6-induced cell adhesions

Article abstract of DOI:10.1016/j.bmcl.2021.128059

Tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) are the critical pro-inflammatory cytokines involved in the pathogenesis of inflammatory bowel disease (IBD). Inhibition of these cytokines and related signaling pathways has been a target for the development of IBD therapeutics. In the current study, 6-acetamido-2,4,5-trimethylpyridin-3-ol (1) and various analogues with the amido scaffold were synthesized and examined for their inhibitory activities in in vitro and in vivo IBD models. The parent compound 1 (1 μM) showed an inhibitory activity against TNF-α- and IL-6-induced adhesion of monocytes to colon epithelial cells, which was similar to tofacitinib (1 μM), a JAK inhibitor, but much better than mesalazine (1,000 μM). All the analogues showed a positive relationship (R² = 0.8943 in a linear regression model) between the inhibitory activities against TNF-α-induced and those against IL-6-induced adhesion. Compound 2–19 turned out to be the best analogue and showed much better inhibitory activity against TNF-α- and IL-6-induced adhesion of the cells than tofacitinib. In addition, oral administration of compound 1 and 2–19 resulted in a significant suppression of clinical signs of TNBS-induced rat colitis, including weight loss, colon tissue edema, and myeloperoxidase activity, a marker for inflammatory cell infiltration in colon tissues. More importantly, compound 2–19 (1 mg/kg) was more efficacious in ameliorating colitis than compound 1 and sulfasalazine (300 mg/kg), the commonly prescribed oral IBD drug. Taken together, the results suggest that compound 2–19 can be a novel platform for dual-acting IBD drug discovery targeting both TNF-α and IL-6 signaling.

Full text of DOI:10.1016/j.bmcl.2021.128059

Bioorg. Med. Chem. Lett. 43 (2021) 128059
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and activity of N-(5-hydroxy-3,4,6-trimethylpyridin-2-yl)
acetamide analogues as anticolitis agents via dual inhibition of TNF-
IL-6-induced cell adhesions
α
- and
Ujjwala Karmacharyaa, Sushil Chandra Regmia, Bhuwan Prasad Awasthia,
,
,*
Prakash Chaudharya, Ye Eun Kimb, Iyn-Hyang Leea, Tae-gyu Namb *, Jung-Ae Kima
,
,
*
Byeong-Seon Jeonga
a College of Pharmacy and Institute for Drug Research, Yeungnam University, Gyeongsan 38541, Republic of Korea
b Department of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do 15588, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Tumor necrosis factor alpha (TNF-
α
) and interleukin-6 (IL-6) are the critical pro-inflammatory cytokines
Inflammatory bowel disease
involved in the pathogenesis of inflammatory bowel disease (IBD). Inhibition of these cytokines and related
signaling pathways has been a target for the development of IBD therapeutics. In the current study, 6-acetamido-
2,4,5-trimethylpyridin-3-ol (1) and various analogues with the amido scaffold were synthesized and examined
TNF-
IL-6
α
Amidopyridinol
for their inhibitory activities in in vitro and in vivo IBD models. The parent compound 1 (1
μ
M) showed an
TNBS-induced colitis
inhibitory activity against TNF-
α- and IL-6-induced adhesion of monocytes to colon epithelial cells, which was
similar to tofacitinib (1 μM), a JAK inhibitor, but much better than mesalazine (1,000 μM). All the analogues
showed a positive relationship (R2 = 0.8943 in a linear regression model) between the inhibitory activities
against TNF-α-induced and those against IL-6-induced adhesion. Compound 2–19 turned out to be the best
analogue and showed much better inhibitory activity against TNF-α- and IL-6-induced adhesion of the cells than
tofacitinib. In addition, oral administration of compound 1 and 2–19 resulted in a significant suppression of
clinical signs of TNBS-induced rat colitis, including weight loss, colon tissue edema, and myeloperoxidase ac-
tivity, a marker for inflammatory cell infiltration in colon tissues. More importantly, compound 2–19 (1 mg/kg)
was more efficacious in ameliorating colitis than compound 1 and sulfasalazine (300 mg/kg), the commonly
prescribed oral IBD drug. Taken together, the results suggest that compound 2–19 can be a novel platform for
dual-acting IBD drug discovery targeting both TNF-α and IL-6 signaling.
Inflammatory bowel disease (IBD) is the chronic, relapsing, and
abrogating inflammation of the gastrointestinal tract and is mainly
classified into Crohn’s disease (CD) and ulcerative colitis (UC) based on
pathologic features [1,2]. The etiology of IBD is complex, however, the
altered immune function of both innate and adaptive immunity is
regarded as a major player in the pathogenesis of IBD [3]. Although
different subtypes of adaptive immune T cells are involved in CD (a Th1/
Th17-associated) versus UC (a Th2-like disease) [4], abnormally high
level of expression of proinflammatory cytokines are common in CD and
epithelial cell death, and induction of other pro-inflammatory cytokines
[8,9]. Upon TNF- binding, TNF receptor type 1 (TNFR1) activates
α
signaling molecules, TNF-R1-associated death domain protein (TRADD),
receptor-interacting protein 1 (RIP1), and TNFR-associated factor 2
(TRAF2), which sequentially lead to activation of nuclear factor-κB (NF-
κB), a redox-sensitive transcription factor [10,11]. In the signaling
pathway, NADPH oxidase (NOX) is also activated. NOX-derived reactive
oxygen species (ROS) in phagocytic and non-phagocytic cells have been
shown to play an important role in TNF-α-mediated immune responses
UC, in particular, tumor necrosis factor-alpha (TNF-
α
) and interleukin-6
[12–15]. In TNF-α-treated cells, TNF-α–ROS–NF-κB pathway is also
(IL-6) [5–7].
involved in de novo synthesis of IL-6 and STAT3 [16].
TNF-
α
exerts a variety of detrimental effects on the intestinal mu-
IL-6 binds and activates a heterodimeric signaling complex consist-
cosa, such as disruption of barrier function, promotion of intestinal
ing of the IL-6 α-receptor (IL-6Rα) and the signal-transducing β-subunit
* Corresponding authors.
E-mail addresses: tnam@hanyang.ac.kr (T.-g. Nam), jakim@yu.ac.kr (J.-A. Kim), jeongb@ynu.ac.kr (B.-S. Jeong).
https://doi.org/10.1016/j.bmcl.2021.128059
Received 4 February 2021; Received in revised form 29 March 2021; Accepted 19 April 2021
Available online 23 April 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

U. Karmacharya et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128059
glycoprotein 130 (gp130) [17]. The expression of gp130 is found in
every tissue and cell types, whereas IL-6R is predominantly expressed in
certain types of cells, such as several leukocyte subpopulations, mega-
karyocytes, and hepatocytes. Heterodimeric association of gp130 with
study, we categorized the amido group into four classes (G1~G4), as
shown in the general structure 2 (Fig. 2) and examined if there is a
relationship between chemical structure and activity. We also investi-
gated a correlation by a statistical analysis between the inhibitory ac-
IL-6R
α
triggers phosphorylation of the Janus kinases, JAK1, JAK2, and
tivity of this series of compounds against TNF-
against IL-6-induced adhesion.
α-induced adhesion and
tyrosine kinase 2, which are constitutively associated with gp130,
leading to the activation (phosphorylation) of STAT3 [18]. Active
STAT3 subsequently translocates from the cytoplasm to the nucleus,
Described in Scheme 1 is the general synthetic method for amido
analogues starting from pyridoxine which has been well-established by
us [35]. Briefly, the two benzylic hydroxyl groups of pyridoxine were cut
off and converted into two methyl groups as in compounds 3, and then
benzyl protection followed by ring bromination afforded the substrate 4
for the introduction of nitrogen-containing functionality at C(6)-
position. The key compound 8 for the preparation of various amido
derivatives was obtained by palladium-catalyzed amination reaction
with benzophenone imine as the ammonia equivalent and successive
methanolysis. We used an alternative synthetic method for compound 8
which has also been developed by us and includes a nucleophilic aro-
matic substitution reaction of pyridine-N-oxide compound 6 with
phthalimide. Twenty-six acid chlorides which can be classified into
roughly four categories (G1 ~ G4) as shown in Fig. 2 were reacted with
the key intermediate compound 8 to give amido products 9. Subsequent
debenzylation toward the final compounds 2 was conducted using either
hydrogenolysis or BCl3 in the presence of pentamethylbenzene as a non-
Lewis-basic cation scavenger. The chemical structure of each compound
is shown in Table 1.
serving a critical role as a transcription factor [19]. TNF-α induction is
reported as one of the consequent gene expressions by IL-6–STAT3
pathway [20]. Also, IL-6 signaling activates NF-κB, which is critical for
induction of inflammatory molecules in colon epithelium [21]. TNF-
α
-induced upregulations of TNF-
TNF- and IL-6 [22].
Although anti-TNF-
and anti-IL-6 receptor antibody, such as tocilizumab, have been suc-
cessfully developed [23–26], anti-TNF antibody drugs become the
α and IL-6 suggest a crosstalk between
α
α
biologics, such as infliximab and adalimumab,
α
most effective therapeutics in the clinic among the biologics and orally
available drugs including tofacitinib, a JAK inhibitor [27–29]. In search
of small molecule version alternatives, we have reported several classes
of 2,4,5-trimethylpyridin-3-ol analogues (A~F in Fig. 1), derived from
pyridoxine⋅HCl (also known as vitamin B6⋅HCl) with anti-colitis activity
[30–32]. They mainly tether modifications on C(6)-position with
various functional group linkers such as amino-, ureido-, thioureido-,
sulfonamido-, carbamato-, and alkoxy-groups with trimethylpyridinol
moiety being retained. In a consecutive study, we have reported the
structural change of pyridinol ring moiety and its effect on anti-colitis
activity where 6-acetamido-2,4,5-trimethylpyridin-3-ol (1 in Fig. 2)
was used as a reference structure for modification [33]. Compound 1
was observed to be far superior to mesalazine/sulfasalazine and in a
similar level to tofacitinib in the activities against both in vitro TNF-
As shown in Table 1, high level of positive correlation was observed
between inhibitory activities against TNF-α-induced cell adhesion and
those against IL-6-induced one throughout the table, demonstrating the
crucial role of both cytokines in cell adhesion event. The adhesion of
monocytic cells to colonic epithelial cells can be initial pathological
characteristics of IBD. The parent compound, 1, showed 56% inhibition
α
-induced adhesion and in vivo DSS-induced colitis model. Mesalazine is
against TNF-α-induced adhesion at 1 μM concentration (Table 1). It also
an orally available IBD drug for patients with mild to moderate level of
severity. Because one of the action mechanism of mesalazine is sup-
pression of nuclear factor-κB [34], which is involved in the transcription
of a variety of inflammatory genes, it was used as a positive control to
compare anti-inflammatory action of compounds. Our efforts on the
discovery of effective anti-colitis agents continued. Various analogues
with the amido scaffold (1) were designed and synthesized in pursuit of
the diversity of side chains tethering on the C(6)-position. In the present
inhibited IL-6-induced adhesion with the activity slightly lower than
that against TNF-
of sulfasalazine (SSZ), showed very weak inhibition against both TNF-
and IL-6-induced adhesions even at 1000
ingly, tofacitinib showed quite an inhibitory activity against both TNF-
α
-induced adhesion. Mesalazine, the active metabolite
α
-
μ
M concentration. Interest-
α
-
and IL-6-induced adhesion (49.1% and 58.6%, respectively), indicating
JAK/STAT inhibition can result in cross activity against TNF-
α-induced
and IL-6-induced cell adhesions. Of the compound 1 analogues, six
Fig. 1. General structures of 2,4,5-trimethylpyridin-3-ol analogues (A ~ E) which were synthesized from pyridoxine⋅HCl and used for the discovery of anti-colitis
agents by us.
2

U. Karmacharya et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128059
3
HO
HO
Amido part derivatization
O
O
Me
G1~4
N
6 N
H
N
N
H
1
2
G1 : normal alkyl & related group
G2 : secondary alkyl & related group
G3 : benzyl & related group
G4 : phenethyl & related group
Fig. 2. Analogues of 6-acetamido-2,4,5-trimethylpyridin-3-ol (1) with diverse side chains for anti-colitis study.
HO
HO
OH
HO
BnO
BnO
a, b
c, d
e
N
N
Br
N
N
N
HCl
3
4
5
Pyridoxine•HCl
g, h
f
BnO
BnO
BnO
j
i
O
N
O
N
N
N
NH2
O
6
7
8
BnO
BnO
HO
k
l or m
O
O
N
NH2
N
N
G
N
N
G
H
H
8
9
2
Scheme 1. Synthetic method for derivatization of amino moiety of 6-amido-2,4,5-trimethylpyridinol. Reagents and conditions: (a) SOCl2, DMF, reflux, 30 min, 93%;
(b) Zn, AcOH, reflux, 3 h, 92%; (c) DBDMH, THF, r.t., 3 h, 80%; (d) PhCH2Cl, K2CO3, DMF, r.t., 12 h, 97%; (e) HN = CPh2, Pd2(dba)3, BINAP, NaOBut, PhMe, reflux,
12 h, 83%; (f) HCl in MeOH, THF, r.t., 12 h, 83%; (g) PhCH2Cl, K2CO3, DMF, r.t., 12 h, 71%; (h) m-CPBA, CH2Cl2, r.t., 1 h, 94%; (i) p-TsCl, i-Pr2NEt, phthalimide,
CH2Cl2, r.t., 12 h, 70%; (j) H2NNH2, THF-EtOH, r.t., 1 h, 90%; (k) G-COCl, Et3N, CH2Cl2; (l) H2, Pd/C, MeOH, r.t.; (m) BCl3, CHMe5, CH2Cl2, 0 ℃~r.t.
compounds (2–2, 2–10, 2–12, 2–14, 2–15, and 2–19) showed higher
proportional relationship (Fig. 3).
inhibition against TNF-
α
-induced adhesion than 1. Also, these com-
Of the analogues in G1 groups, there seems to be no simple struc-
ture–activity relationship (SAR) regarding the length of alkyl chain. C3
(normal-, chloro-, and unsaturated-C3 chain) and C15 side chains (2–1,
2–8, 2–9, and 2–4, respectively) showed low activity while C1, C7, and
C11 side chains showed high activity (1, 2–2, and 2–11, respectively).
Branched alkyl side chains in G1 group generally showed considerable
activity > 50% (2–5 and 2–6). Analogues in G2 group generally showed
high activity except for t-butyl side chain (2–11). More specifically, G2
group analogues with a methine carbon directly attached to amide
carbonyl showed high activity (2–10, 2–12, 2–13, and 2–14) while a
quaternary carbon-bearing analogue (2–11) showed low activity. Of the
G [3] group analogues, only a simple benzyl side chain (2–15) showed
considerable activity while all the halogenated analogues showed low
activity. Halogenated benzyl side chains (2–16 and 2–17) are especially
detrimental to the activity. G3 group has a methylene spacer between
pounds showed better activity than 1 in IL-6-induced adhesion.
All the compounds in Table 1 showed a positive correlation between
the inhibitory activities against TNF-α-induced and those against IL-6-
induced adhesion (Table 1 and Fig. 3). For the statistical analysis of
the correlation, a linear regression model was performed. The simple
linear regression equation with y (% inhibition against IL-6 induced
adhesion) as the response variable and × (% inhibition against TNF-
α
-induced adhesion) as the explanatory variable was
y = 0.82 x + 0.54 (R2 = 0.8943)
For every one-unit increase in x, the y increased by 0.82 on average.
In other words, increasing x by 10% is associated with an increase in the
y of 8.2% (P < 0.0001). That is, these series compounds inhibit
monocyte-epithelial cell adhesion induced by TNF-α or IL-6, and the
degree of inhibition against each cytokine-induced adhesion is a
3

U. Karmacharya et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128059
Table 1
Table 1 (continued)
Inhibitory activity of compounds at 1 μM concentration against TNF-α- and IL-6-
Compound
G
% inhibition in adhesion
assay (1
TNF-
μ
M)a
induced adhesion of human monocytic cells (U937) to human colonic epithelial
cells (HT-29).
Category
G4
Structure
IL-6-
α
-induced
induced
2–19
2–20
2–21
72.5 ±
69.6 ±
13.2*
9.8*
G4
G4
20.2 ± 1.4*
13.9 ± 6.6*
12.9 ±
4.5
18.0 ±
Compound
Mesalazine
Tofacitinib
G
% inhibition in adhesion
7.0*
assay (1
TNF-
μ
M)a
2–22
2–23
2–24
2–25
2–26
G4
G4
G4
G4
G4
30.6 ± 4.9*
35.3 ± 1.7*
41.5 ± 3.6*
20.8 ±
Category
Structure
IL-6-
4.6*
α-induced
induced
9.9 ± 14.7
(1,000 M)
1.7 ± 7.8
22.0 ±
μ
(1,000
5.5*
μM)
35.9 ±
5.1*
49.1 ± 0.1*
58.6 ±
2.3*
10.8 ±
11.4 ±
15.7
9.2*
25.2 ±
14.2 ±
14.7
5.7
1
–CH3
56.0 ± 6.3*
10.0 ± 6.1
61.5 ± 7.2*
55.8 ± 7.7*
47.2 ±
4.1*
*P < 0.05 versus vehicle-treated group.
G1
G1
G1
G1
G1
–CH2CH2CH3
–CH2(CH2)5CH3
–CH2(CH2)9CH3
–CH2(CH2)13CH3
17.5 ±
9.4*
a
2–1
2–2
2–3
2–4
2–5
Data are shown as ’mean ± SEM’ of at least three independent experiments
performed in triplicate.
54.8 ±
6.6*
44.3 ±
8.0*
31.7 ±
17.4 ±
7.1
10.5
52.3 ± 4.2*
48.0 ±
4.4*
2–6
2–7
G1
G1
54.5 ±
46.3 ±
10.4*
4.7*
6.8 ± 9.1
9.7 ±
5.7*
2–8
G1
G1
G2
24.7 ±
17.8 ±
4.2
18.0
2–9
31.9 ± 0.8*
21.3 ±
6.9
2–10
64.2 ± 1.4*
53.9 ±
4.4*
2–11
2–12
G2
G2
37.5 ±
21.1 ±
12.2
6.0*
60.6 ±
52.7 ±
12.4*
9.1*
2–13
2–14
G2
G2
42.8 ± 7.8*
34.8 ±
5.1*
64.5 ±
56.7 ±
12.5*
6.2*
2–15
2–16
G2
G2
56.6 ± 8.0*
7.2 ± 4.8*
50.2 ±
4.3*
Fig. 3. The regression of % inhibition against IL-6 induced adhesion (y) on %
18.1 ±
5.2*
inhibition against TNF-α-induced adhesion (x). Analyses were conducted using
SAS procedures (SAS version 9.4, SAS Institute, Cary, NC, USA) and significance
2–17
2–18
G3
G3
6.2 ± 10.7
36.6 ± 0.9*
9.2 ±
was defined at P < 0.05.
8.4*
amide carbonyl carbon and a phenyl ring, and the addition of one more
methylene spacer to that of G3 group rendered G4 group. Halogen sub-
stituents undermining the activity in G3 group displayed the same effects
on the analogues with phenethyl side chains in G4 group. Only a simple
phenethyl side chain (2–19) showed the best activity both against TNF-
22.5 ±
3.6
4

U. Karmacharya et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128059
α
- and IL-6-induced cell adhesion (72.5% and 69.6%, respectively) while
(Fig. 4B-1), colon morphology (Fig. 4B-2), and colon weight/ length
(Fig. 4B-3). Measurement of tissue myeloperoxidase (MPO) level, which
is directly related to neutrophil content and used for quantitation of
inflammation [36], also showed the superior activity of compound 2–19
to compound 1 or SSZ (Fig. 4C).
the other analogues within the group showed low to moderate activity.
We chose compounds 1 and 2–19 as the parent structure and the best
analogue, respectively, for the in vivo experiment using a TNBS-induced
rat colitis model.
Administration of TNBS to rat colon through rectum induced bloody
diarrhea, decreased activity, and a significant reduction followed by
stagnated increase in body weight (Fig. 4A-1). In contrast, oral admin-
istration of compound 1 showed a significant recovery of the TNBS-
induced decrease in body weight in a dose-dependent manner
(Fig. 4A-1). Also, compound 1 suppressed congestion, adhesion (Fig. 4A-
2), and increase in tissue weight/ length (Fig. 4A-3) in the colon treated
with TNBS. The effects of compound 1 (10 mg/kg) was better than SSZ
(300 mg/kg). Oral administration of compound 2–19 (1 mg/kg) showed
a better ameliorating effect on TNBS-induced changes in body weight
In this study, we synthesized 6-acetamido-2,4,5-trimethylpyridin-3-
ol analogues with the structural variation on amido-substituent and
examined their inhibitory activity in colitis models in vitro and in vivo.
High level of positive relationship was found in between the inhibitory
activity against TNF-α-induced and that against IL-6-induced adhesion
of monocytes to colon epithelial cells. Many compounds in this study
showed superior inhibition to mesalazine at three orders of magnitude
lower concentration. In TNBS-induced in vivo rat colitis model, oral
administration of compound 2–19 with phenethyl chain on amido part
at 1 mg/kg showed excellent recovery profiles in body and colon weight
Fig. 4. Inhibitory effect of compounds 1 and 2–19 on TNBS-induced rat colitis. TNBS was administered via rectum, and compounds 1 and 2–19 were orally
administered daily for 5 days. MPO level in colon tissues was determined by using MPO assay kit. *P < 0.05 vs control (Mock) group. #P < 0.05 vs vehicle-treated
TNBS colitis group. SSZ: sulfasalazine.
5

U. Karmacharya et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128059
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