Synthesis and evaluation of 6-heteroarylamino-2,4,5-trimethylpyridin-3-ols as inhibitors of TNF-α-induced cell adhesion and inflammatory bowel disease

Article abstract of DOI:10.1039/c8md00156a

Inflammatory bowel disease (IBD) is an inflammatory disease of the gastrointestinal tract with complex pathogenesis. Here, we synthesized 6-heteroarylamino analogues to inhibit TNF-α-induced adhesion of monocytes to colon epithelial cells which are implicated in the initial inflammation process of IBD. The best analogue, 16a, showed IC₅₀ = 0.29 μM, which is about five orders of magnitude better than that of 5-aminosalicylic acid (5-ASA), a positive control. Oral administration of 6f and 16a dramatically ameliorated 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colon inflammation in rat. The ameliorating effects were accompanied by a high level of recovery in colon and body weights and in the myeloperoxidase (MPO) level. Consistently, the compounds suppressed the expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein 1 (MCP-1). Moreover, they significantly suppressed the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 while increasing the level of IL-10, an anti-inflammatory cytokine.

Full text of DOI:10.1039/c8md00156a

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Synthesis and evaluation of 6-heteroarylamino-2,4,5-
trimethylpyridin-3-ols as inhibitors of TNF-α-induced cell adhesion
and inflammatory bowel disease
Received 00th January 20xx,
Accepted 00th January 20xx
Sang Won Park,†a Suhrid Banskota,†b Pallavi Gurung,b You Jin Jin,a Han-eol Kang,a Chhabi Lal
Chaudhary,b Sang Yeul Lee,a Byeong-Seon Jeong,*b Jung-Ae Kim,*b and Tae-gyu Nam*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Inflammatory bowel disease (IBD) is an inflammatory disease in gastrointestinal tract with complex pathogenesis. Here, we
synthesized 6-heteroarylamino analogues to inhibit TNF-α-induced adhesion of monocyte to colon epithelial cells which is
implicated in initial inflammation process of IBD. The best analogue, 16a, showed IC50 = 0.29 μM, which is about five orders
of magnitude better than that of 5-aminosalicylic acid (5-ASA), a positive control. Oral administration of 6f and 16a
dramatically ameliorated 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colon inflammation in rat. The ameliorating
effects were accompanied by high level of recovery in colon and body weights and in myeloperoxidase (MPO) level.
Consistently, the compounds suppressed the expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte
chemoattractant protein 1 (MCP-1). Moreover, they significantly suppressed the expression of pro-inflammatory cytokines
such as TNF-α, IL-1β, and IL-6 while increasing the level of IL-10, an anti-inflammatory cytokine.
them, tumor necrosis factor-α (TNF-α) is secreted from monocytes
and colon epithelial cells. TNF-α, in turn, induces colon epithelial
cells to express other inflammatory cytokines and adhesion
molecules in higher level. It recruits more inflammatory cells,
including immune cells, into the damaged intestinal epithelium. This
process is one of the critical steps in the initiation of inflammation
and tissue injury in IBD.6,7
Introduction
Inflammatory bowel disease (IBD) is
a chronic inflammatory
pathology occurring in gastrointestinal tract with major subtypes
being Crohn’s disease and ulcerative colitis.1 Although it is
considered a low-mortality disease, it can severely devastate quality
of life of patients. It is spreading all around the world with over 1
million people in USA and 2.5 million in Europe being estimated to
have IBD.2 Recently, unprecedented growing rates are observed
even in traditionally low-incidence regions including developing
countries. Several drugs are currently used in clinic to treat
symptoms of IBD.2-4
Beside TNF-α, other pro-inflammatory cytokines also contribute
to the aggravation of colitis. Interleukin-6 (IL-6) plays a crucial role
in the uncontrolled intestinal inflammatory process by enhancing
STAT-3 nuclear translocation where it induces anti-apoptotic genes
such as Bcl-xL. The development of T cells’ resistance to apoptosis
further aggravates intestinal inflammation.8 IL-1β level is increased
in colonic tissues of IBD patients,9 and its levels are positively linked
with the disease severity of IBD.10 In contrast to the enhanced pro-
inflammatory cytokines, decrease in IL-10, an anti-inflammatory
cytokine mainly produced by innate immune cells such as
monocytes and regulatory T cells, also aggravates IBD11 by losing its
ability to inhibit bacterial product-mediated induction of pro-
inflammatory cytokines, such as TNF-α and IL-1β.12 Considering
these network and interaction between pro- and anti-inflammatory
cytokines, a compound that corrects or restores the altered balance
between the pro- and anti-inflammatory cytokines in IBD would be
the most desirable therapeutic drug.
Although the causes of IBD have not been clearly understood, it
is believed that complex association of genetic, environmental, and
immune factors underlies the disease.1,5 In gut, mucosal layer
performs a first-line defense conducting a number of innate
immune functions. Under harmful conditions such as disturbance of
microbiotic and cytokine balance, intestinal epithelial cells
participate in the initiation and dissemination of inflammation by
secreting pro-inflammatory cytokines and chemokines.5 Among
Although several molecular targets have been proposed for
IBD,13-16 TNF-α currently seems to be the most successful target
mainly because of the high efficacy of anti-TNF-α monoclonal
antibodies, such as infliximab and adalimumab. As virtually no small
molecule has been approved for IBD since the anti-TNF-α antibodies,
there are huge medical unmet needs for chemical version of anti-
IBD agents. A few small molecule anti-IBD agents have been
reported in literatures or in clinical trials.14,17-20
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We have previously reported the inhibitory activity of 6-alkyl-
Journal Name
OH
and 6-phenyl-amino-2,4,5-trimethylpyridin-3-ols against colitis.21
Some of them inhibited TNF-α-induced monocyte adhesion to colon
epithelial cell better than 5-aminosalicylic acid (5-ASA a.k.a.
mesalazine), a positive control and the active metabolite of
sulfasalazine (SSZ) which is a medication used for the treatment of
IBD. Oral administration of the best compound showed about 30-
times better efficacy than sulfasalazine, a prodrug of 5-ASA, in a rat
model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis,25
a widely used animal model of IBD.
HO
3
HO
BnO
d
OH
a,b,c
6
N
HCl
N
Br
N
Br
1
2
3
BnO
HO
e
f
HetAr
HetAr
N
N
N
N
H
H
5
6
In this study, we further expanded the scope of side chain (HetAr
group in Scheme 1) by installing heteroarylamino groups on C(6)-
position to render more polarity. In fact, cLogP values of heteroaryl
compounds shown here are smaller than those of the
corresponding phenyl analogues by about one unit. Compounds
shown here inhibited TNF-α-induced adhesion of monocytes to HT-
29 human colonic epithelial cells.22-24 In order to demonstrate in
vivo efficacy, selected compounds were tested in the rat model of
TNBS-induced colitis. Mode of action of the anti-colitis activities
were further characterized by the relevant molecules in vivo.
HetAr =
CF3
CF3
Cl
CF3
Cl
N
N
N
N
N
N
6e
6a
6b
6c
6d
6f
N
N
N
N
N
N
6g
6h
6i
6j
6k
6l
NH
Results and Discussion
CO2Et
N
S
HN
N
S
N
CO2Me
6n
O
Synthesis
6m
6o
6p
6q
6-Heteroarylamino-2,4,5-trimethylpyridin-3-ols (6a
synthesized using a synthetic route we have developed
(Scheme 1)26 but with novel heteroarylamines
. In short,
SOCl2 with catalytic amount of DMF converted two primary
hydroxy groups of pyridoxine∙HCl ( ) to chlorides which were
–6q) were
Scheme 1. Synthetic scheme and structure of 6-heteroarylamino-2,4,5-
trimethylpyridin-3-ols (6). 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) H2N-HetAr (4), Pd2(dba)3, BINAP,
NaOtBu, toluene, reflux or H2NHetAr (4), Pd(OAc)2, Xantphos, Cs2CO3,
toluene, reflux; (f) H2, Pd/C, MeOH, r.t. or BCl3, CH2Cl2, r.t.
4
1
then removed reductively using Zn and acetic acid under
refluxing condition to give 2,4,5-trimethylpyridin-3-ol. C(6)-
position was brominated by electrophilic aromatic
bromination
(DBDMH) to afford compound
then protected with benzyl group to give compound
Bromide in the compound was replaced with various
heteroarylamines under Buchwald-Hartwig amination
reaction conditions to afford compounds . Lastly, benzyl
with
1,3-dibromo-5,5-dimethylhydantoin
2
and phenolic OH group was
In cases of 6-(1,3,4-oxadiazol-2-yl)amino analogues 15 and 6-(1,3,4-
thiadiazol-2-yl)amino analogues 16, the oxadiazole and thiadiazole
groups did not survive debenzylation conditions used in the final
step of Scheme 1. Therefore, we introduced tert-butyldiphenylsilyl
(TBDPS) group instead of benzyl group for the protection of
3.
3
4
5
protective group was removed either by catalytic
hydrogenolysis or by BCl3 depending on the substituents to
give 6-heteroarylamino-2,4,5-trimethylpyridin-3-ols (6a–6q).
phenolic OH of compound
2 (Scheme 2). TBDPS-protected
compound 7 was coupled with benzophenone imine, a synthetic
equivalent for ammonia, under Buchwald-Hartwig amination
reaction condition to give 8. Free primary amino group was
liberated from the imine of 8 by acidic methanolysis to afford
compound 9 which was then treated with thiophosgene and
Hünig's base to give isothiocyanate 10. Treatment of 10 with
various N-acylhydrazides 11 leads to the corresponding N-acyl
thiosemicarbazides 12 which serve as key intermediate for the
preparation of both 2-amino-1,3,4-oxadiazoles (13) and 2-amino-
1,3,4-thiadiazoles (14).27 A desulfurative cyclization method using 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC)
was
successfully employed for preparation of 6-(1,3,4-oxadiazol-2-
yl)amino analogues 13. On the other hand, an efficient reaction
system (i.e., p-toluenesulfonyl chloride (p-TsCl) with triethylamine
in a polar solvent, N-methyl-2-pyrrolidine (NMP)) for dehydrative
cyclization of 12 gave 6-(1,3,4-thiadiazol-2-yl)amino compounds 14.
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Finally, deprotection of TBDPS of the cyclized compounds 13 and 14 (50.5%) even at 20 mM against the adhesion (Table 1).
with tetra-n-butylammonium fluoride afforded Meanwhile, 6-heteroarylamino-2,4,5-trimethylpyridin-3-ols (
oxadiazole/thiadiazole-containing pyridinol compounds 15 and 16, 15 and 16) exerted up to 78% inhibition at 1 μM, equivalent to
6,
respectively.
three to five orders of magnitude stronger activities than that
of 5-ASA. Of note, 1 μM concentration of the compounds 6f
6k, and 16a showed superior activity to 20 mM of 5-ASA.
,
Table 1. Inhibitory activity against TNF-α-induced monocyte
adhesion to colon epithelial cells.
Inhibition
Comp.
Inhibition (%)†
Comp.
(%)†
5-ASA (1 ꢀM)
3.5 ± 2.4 *
6m
6n
31.9 ± 6.4 *
23.7 ± 2.4 *
23.7 ± 6.1 *
38.5 ± 1.3 *
59.9 ± 4.7 *
26.2 ± 4.6 *
35.8 ± 5.0 *
1.4 ± 3.0
5-ASA (20 mM) 50.5 ± 1.8 *
6a
6b
6c
6d
6e
6f
8.5 ± 1.2 *
22.8 ± 10.9
34.8 ± 16.3
50.6 ± 10.6 *
59.0 ± 8.4 *
75.4 ± 3.4 *
60.2 ± 5.8 *
55.4 ± 4.5 *
10.4 ± 5.9
6o
6p
6q
15a
15b
15c
15d
16a
16b
16c
16d
16e
6g
6h
6i
55.7 ± 4.9 *
78.7 ± 2.5 *
13.7 ± 2.3 *
13.2 ± 1.9 *
16.6 ± 3.1 *
52.3 ± 1.1 *
6j
52.8 ± 6.9 *
63.4 ± 1.6 *
56.6 ± 1.1 *
6k
6l
†
Data are represented as inhibition % at 1 μM concentration of
compound and as mean ± SEM. All experiments independently carried
out at least three times. * P < 0.05 compared to vehicle-treated
control group.
2-Pyridyl analogue 6a showed little activity (8.5%) compared to its
phenyl analogue (73.6%) published by us21 in the inhibition of TNF-
α-induced adhesion. However, installation of -CF3 group on m-
position of 2-pyridyl group (6b) increased the activity by 2.7-fold
from 6a, while the same group on para-position increased the
activity by 4.1-fold (6c). Interestingly, with the identical p-CF3
substituent, 3-pyridyl analogue (6d) showed 6.0-fold higher activity
than 6a. Replacement of p-CF3 group of 6c with weaker electron-
withdrawing p-Cl increased the activity to 59.0% (6e). Upon change
of 2-pyridyl substitution of 6e to 3-pyridyl position with the same p-
Cl (6f), the activity reached the highest point (75.4%). Among
pyridine and quinoline analogues (6a – 6l), analogues that can assist
a cyclic hydrogen bond network with or without H2O between C(6)-
amino group and heteroaryl group (e.g., 6c and 6e) generally
showed lower activity than their counterparts that have
unfavorable structure for the cyclic network (e.g., 6d and 6f).
Quinoline analogues, 6g–6l, showed the same trend; 6i being the
only one that possibly forms the cyclic hydrogen bond showed
marginal activity (10.4%) while the rest of quinolones analogues
showed several fold higher activity (52.8–63.4%).
Scheme 2. Synthesis of 6-(1,3,4-oxadiazol-2-yl)amino analogues (15) and 6-
(1,3,4-thiadiazol-2-yl)amino analogues (16). Reagents and conditions: (a)
TBDPSCl, imidazole, DMF, r.t., 24 h, 87%; (b) Ph2C=NH, Pd2(dba)3, BINAP,
NaOtBu, toluene, reflux, 5 h, 89%; (c) HCl-MeOH, THF-MeOH, r.t. 24 h, 93%;
i
(d) CSCl2, Pr2NEt, r.t., 1 h, 98%; (e) CH2Cl2, r.t.; (f) EDCI, DMSO, 60 °C; (g) n-
Bu4NF, THF, 0 °C; (h) p-TsCl, Et3N, NMP, r.t.
Inhibition of TNF-α-induced cell adhesion
TNF-α-induced attachment and infiltration of immune cells to
colon epithelium is one of the hallmark events leading to
IBD.6,7 To quantitatively assess inhibitory activity of the
compounds against this adhesion process, we set up a cell-
based screening system in which monocytes (U937 cells) and
colon epithelial cells (HT-29 cells) were co-cultured in the
presence of TNF-α.26 Treatment of TNF-α indeed induced the
adhesion of monocytic cells to colonic epithelial cells, indicated
by increased fluorescence that was pre-loaded in U937 cells
(Supporting Information). 5-ASA showed very weak inhibition
(3.5%) at 1 μM concentration and only moderate inhibition
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best activity in Table 1 for this assay, 6f and 16a, which represent
distinctive heterocyclic scaffolds. As a macroscopic marker to
represent a disease phenotype, we measured recovery level on
colon and body weights upon drug treatment. Upon rectal
administration of TNBS, rats showed colitis signs, such as bloody
diarrhea, and wasting conditions with sluggish and weak
movement.27 Compared to rats in the sham-operated group, TNBS-
treated rats showed a significant reduction in body weight followed
by stagnated increase. In contrast, rats receiving oral administration
of 6f or 16a (1 mg/kg) showed significant recovery of the TNBS-
induced decrease in body weight (Fig. 2A). Besides, 16a showed 79%
recovery in colon weight at 1 mg/kg dose, which is superior
macroscopic activity to SSZ (300 mg/kg), 6f and our previous
compound21 which showed about 72% recovery in colon weight at 1
mg/kg dose, demonstrating significant efficacy of new scaffold (Fig.
2C). Colon tissues in the TNBS-treated lesion site showed significant
inflammation, as revealed by edema and adhesion in gross
morphology examination (Fig. 2B), and increased wet weight per
colon length (Fig. 2C). In addition, myeloperoxidase (MPO) level in
colon tissues is directly related to neutrophil infiltration into tissues,
serving as a biochemical marker of inflammation.28,29 TNBS
treatment induced a tremendous increase in MPO activity (Fig. 2D),
and the increased MPO activity by TNBS (456.67 ± 41.78 ng/mL)
was significantly suppressed by treatment with 6f (127.02 ± 19.24
ng/mL) and 16a (107.37 ± 14.09 ng/mL). The inhibitory effect of
these compounds at 1 mg/kg was better than that of 300 mg/kg SSZ
(MPO level of 209.65 ± 4.13) (Fig. 2D).
HO
3
HO
X
6
N
N
H
N
N
N
H
N
H
6c, 6e
6i
O
H
Indole (6m), thiophene (6n), thiazole (6o) and oxazole (6p
)
analogues showed modest range of activity while
benzimidazole analogue (6q) showed high activity (59.9%).
Next, we decided to explore 1,3,4-oxadiazole (15) and 1,3,4-
thiadiazole (16) series which have three heteroatoms in the
cycles. Among oxadiazole series (15), p-trifluoromethylphenyl
analogue (15d) showed the highest activity (55.7%) while the
rest showed low to modest activity. Of thiadiazoles (16), the
simplest phenyl substitution (16a) showed tremendously high
activity (78.7%). There seems to be no clear structure-activity
relationship (SAR) among this series in terms of electronic
effect of the substitution. Although electron-withdrawing
substitution (16e) is more active than electron-donating
substitution (16c), neither compounds is more active than the
phenyl substitution (16a). We then measured half maximal
inhibitory concentration (IC50, μM) of three representative
compounds: 6f, 6k, and 16a, whose activities are aligned in a
narrow range in a high activity group in Table 1. As shown in
Figure 1, their IC50 values are turned out 0.36, 0.54 and 0.29
μM, respectively. The order of their IC50’s is exactly consistent
with that of
a single concentration cell-based assay,
Although fundamental molecular targets of the compounds
demonstrating high degree of correlation between the assays
and validity of our methodology. The best IC50 is up to about responsible for the phenotype are to be investigated, we further
7×104-fold better than that of 5-ASA (IC50 ≈ 20 mM).
examined in this study the inhibitory effects of the compounds on
the expression levels of cytokines responsible for inflammatory
response. It will provide more consistency to our mechanism based
on the assay against TNF-α-induced cell adhesion. In ELISA (Fig. 2E)
and western blotting (Fig. 2F−2I) analyses, TNBS treatment
dramatically increased expression of monocyte chemoattractant-1
(MCP-1), intercellular adhesion molecule-1 (ICAM-1), and pro-
inflammatory cytokines, TNF-α, IL-1β and IL-6, whereas anti-
inflammatory cytokine, IL-10, was significantly decreased in the
inflamed colon tissues (Fig. 2F, 2I). However, oral administration of
6f or 16a significantly restored the TNBS-altered protein
expressions. All those changes consistently and strongly support the
anti-IBD activity of our compounds. Indeed, the inhibitory effect of
1 mg/kg 6f or 16a was much stronger than that of 300 mg/kg SSZ.
We believe that data in this report also confirmed the validity of our
phenotypic screening where inhibitory activity of compounds
against pathological role of TNF-α can be monitored.
Figure 1. IC50 values of the selected compounds, 6f
,
6k and 16a. Each
data represents mean
performed in triplicate.
± S.E.M for three independent experiments
In vivo activity against TNBS-induced colitis model and mode of
action
Next, we conducted a TNBS-induced rat colitis model to assess anti-
colitis activity in vivo. We chose two compounds that showed the
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group. #P < 0.05 compared to the TNBS-treated group. (
appearance of the large intestine. ( ) Body weight was recorded daily
from day 0 to day 5. ( ) Colon weight per unit length of colon (distal 5–
6 cm segment). ( ) MPO activity of colon tissue measured with an
MPO assay kit. ( ) Expression of MCP-1 of colon tissue. ( ) Inhibitory
A) Macroscopic
B
C
D
E
F–I
effects of compounds 6f and 16a on TNBS-altered protein expressions
of TNF-α, IL-1β, IL-6, IL-10, and ICAM-1 in rat colon tissues. The bar
diagrams,
G,
H
and
I, represent the quantitative change in the
expressions of the proteins.
*
P < 0.05 vs. the vehicle-treated controls.
#P < 0.05 vs. TNBS-treated group.
Conclusions
We synthesized 6-heteroarylamino-2,4,5,-trimethylpyridin-3-ols and
tested them as anti-colitis agents in vitro and in vivo. Most
analogues showed superior inhibition to 5-ASA against TNF-α-
induced adhesion of monocytes to colon epithelial cells at >103-fold
lower concentration. In TNBS-induced in vivo rat colitis model, oral
administration of 1 mg/kg 16a showed the most active recovery
profiles in body and colon weight among aminopyridinol series
including 6-alkylamino and 6-phenylamino analogues we previously
reported. The activity was also demonstrated by significant
decrease of MPO activity. In addition, 16a significantly decreased
the expression of inflammatory molecules and pro-inflammatory
cytokines while increasing anti-inflammatory cytokine. The effects
of 1 mg/kg dose of 6f and 16a were much stronger than 300 mg/kg
SSZ in those profiles, demonstrating their remarkable potency.
Although the detailed mechanism of action should be studied
further, it is strongly suggested that 6-heteroaylamino-2,4,5-
trimethylpyridin-3-ol can be an excellent anti-IBD scaffold.
Conflicts of interest
There authors declare no competing interest.
Acknowledgements
This work was supported by grant of the Korean Health
Technology R&D Project, Ministry of Health & Welfare,
Republic of Korea (HI15C0542).
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