In vitro and in vivo inhibitory activity of 6-amino-2,4,5-trimethylpyridin-3-ols against inflammatory bowel disease

Article abstract of DOI:10.1016/j.bmcl.2016.08.075

Although the pathogenesis of inflammatory bowel disease (IBD) is complex, attachment and infiltration of leukocytes to gut epithelium induced by pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) represents the initial step of inflammation in IBD. Previously, we have reported that some 6-amino-2,4,5-trimethylpyridin-3-ols have significant levels of antiangiogenic activity via PI3K inhibition. Based on the reports that angiogenesis is involved in the aggravation of IBD and that PI3K is a potential target for IBD therapy, we investigated whether the scaffold has inhibitory activity against in vitro and in vivo models of colitis. Many analogues showed >80% inhibition against TNF-α-induced monocyte adhesion to colon epithelial cells at 1?μM. Compound 8m showed IC₅₀?=?0.19?μM, which is about five orders of magnitude better than that of 5-aminosalicylic acid (5-ASA, IC₅₀?=?18.1?mM), a positive control. In a rat model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis, orally administered 8m dramatically ameliorated TNBS-induced colon inflammation. It was demonstrated by a high level of suppression in myeloperoxidase (MPO), a surrogate marker of colitis, as well as almost perfect recovery of colon and body weights in a dose-dependent manner. Compared to sulfasalazine, a prodrug of 5-ASA, compound 8m showed >300-fold better efficacy in those parameters. Taken together, 6-amino-2,4,5-trimethylpyridin-3-ols can provide a novel platform for anti-IBD drug discovery.

Full text of DOI:10.1016/j.bmcl.2016.08.075

Accepted Manuscript
In vitro and in vivo inhibitory activity of 6-amino-2,4,5-trimethylpyridin-3-ols
against inflammatory bowel disease
Suhrid Banskota, Han-eol Kang, Dong-Guk Kim, Sang Won Park, Hyeonjin
Jang, Ujjwala Karmacharya, Byeong-Seon Jeong, Jung-Ae Kim, Tae-gyu Nam
PII:
S0960-894X(16)30907-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.08.075
BMCL 24201
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
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17 August 2016
24 August 2016
Please cite this article as: Banskota, S., Kang, H-e., Kim, D-G., Park, S.W., Jang, H., Karmacharya, U., Jeong, B-
S., Kim, J-A., Nam, T-g., In vitro and in vivo inhibitory activity of 6-amino-2,4,5-trimethylpyridin-3-ols against
inflammatory bowel disease, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/
j.bmcl.2016.08.075
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In vitro and in vivo inhibitory activity of 6-
amino-2,4,5-trimethylpyridin-3-ols against
inflammatory bowel disease
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Suhrid Banskotaa,† , Han-eol Kangb,† , Dong-Guk Kima , Sang Won Parkb , Hyeonjin Janga , Ujjwala
Karmacharyaa , Byeong-Seon Jeonga, , Jung-Ae Kima, and Tae-gyu Namb,

Bioorganic & Medicinal Chemistry Letters
In vitro and in vivo inhibitory activity of 6-amino-2,4,5-trimethylpyridin-3-ols
against inflammatory bowel disease
Suhrid Banskotaa,† , Han-eol Kangb,† , Dong-Guk Kima , Sang Won Parkb , Hyeonjin Janga , Ujjwala
Karmacharyaa , Byeong-Seon Jeonga,* , Jung-Ae Kima,* and Tae-gyu Namb,
aCollege of Pharmacy and Institute for Drug Research, Yeungnam University, Gyeongsan 38541, Republic of Korea
bDepartment of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do 15588, Republic of Korea
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Although the pathogenesis of inflammatory bowel disease (IBD) is complex, attachment and
infiltration of leukocytes to gut epithelium induced by pro-inflammatory cytokines such as
tumor necrosis factor- (TNF-) represents the initial step of inflammation in IBD. Previously,
we have reported that some 6-amino-2,4,5-trimethylpyridin-3-ols have significant levels of
antiangiogenic activity via PI3K inhibition. Based on the reports that angiogenesis is involved in
the aggravation of IBD and that PI3K is a potential target for IBD therapy, we investigated
whether the scaffold has inhibitory activity against in vitro and in vivo models of colitis. Many
analogues showed >80% inhibition against TNF--induced monocyte adhesion to colon
epithelial cells at 1 M. Compound 8m showed IC50 = 0.19 M, which is about five orders of
magnitude better than that of 5-aminosalicylic acid (5-ASA, IC50 = 18.1 mM), a positive control.
In a rat model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis, orally administered
8m dramatically ameliorated TNBS-induced colon inflammation. It was demonstrated by a high
level of suppression in myeloperoxidase (MPO), a surrogate marker of colitis, as well as almost
perfect recovery of colon and body weights in a dose-dependent manner. Compared to
sulfasalazine, a prodrug of 5-ASA, compound 8m showed > 300-fold better efficacy in those
parameters. Taken together, 6-amino-2,4,5-trimethylpyridin-3-ols can provide a novel platform
for anti-IBD drug discovery.
Keywords:
Inflammatory bowel disease
TNF-
6-Amino-2,4,5-trimethylpyridin-3-ol
Cell adhesion
TNBS-induced colitis
2009 Elsevier Ltd. All rights reserved.
———
 Corresponding author. Tel.: +82-53-810-2814; fax: +82-53-810-4654; e-mail: jeongb@ynu.ac.kr (B. -S. J); Tel.: +82-53-810-2816; fax: +82-
53-810-4654; e-mail: jakim@yu.ac.kr (J. -A. K.); Tel.: +82-31-400-5807; fax: +82-31-400-5958; e-mail: tnam@hanyang.ac.kr (T. -G. N.)
† Equally contributed as first author

Introduction
Results and discussion
Synthesis
Inflammatory bowel disease (IBD) refers to a set of chronic
inflammatory pathologies occurring in gastrointestinal (GI) tract
and is spreading rapidly all around the world, causing over
50,000 deaths globally in 2013.1 Major types of the pathology are
Crohn’s disease (CD) and ulcerative colitis (UC) although
several other types are also present.1,2 With no specific molecular
markers for each type, the location and nature of the lesion are
quite different. CD usually affects any location throughout GI
tract with transmural injuries in the intestinal wall, while UC is
more likely to occur in lower tract such as colon and rectum with
more limited damage confined to epithelial layer of intestine.2,3.
Although the cause of IBD is not clearly understood, it is
believed to be the result of complex association of genetic,
environmental, and immune factors.4 Gut mucosa plays a primary
role as a first line defense and performs a number of innate
immune functions, intestinal epithelial cells, however, upon
harmful stimuli such as gut microbiota disturbance and
imbalance of cytokine homeostasis, participate in the initiation
and perpetuation of inflammation by secreting pro-inflammatory
cytokines and chemokines.5 Among the various cytokines, tumor
necrosis factor- (TNF-) secreted from monocytes and colon
epithelial cells induces more expression of other inflammatory
cytokines and adhesion molecules in colon epithelial cells, which
recruits more inflammatory cells into the damaged intestinal
epithelium. It is one of the critical steps in inflammation and
tissue injury in IBD.6,7 Although several molecular targets have
been proposed for IBD,8-11 TNF- seems to be the most
successful thus far mainly because of anti-TNF- monoclonal
antibodies, such as infliximab and adalimumab. Since the
introduction of the antibodies, virtually no small molecule has
been approved for IBD. As a result, there are enormous medical
unmet needs for chemical version of anti-IBD agents. A few
small molecule anti-IBD agents have been reported in
literatures12-14 or in clinical trials.15-17
Scheme 1. General synthetic scheme for 6-amino-2,4,5-
trimethylpyridin-3-ols (8 and 9).
6-Amino-2,4,5-trimethylpyridin-3-ols (8 and 9) were
synthesized via the synthetic route we reported (Scheme 1).18
Briefly, pyridoxine HCl (1) was treated with SOCl2 and catalytic
amount of DMF to convert two primary hydroxy groups to
chlorides, which were removed by reductive cleavage using Zn
dust in refluxing acetic acid to afford 2,4,5-trimethylpyridin-3-ol
(2). C(6)-Position was then brominated with 1,3-dibromo-5,5-
dimethylhydantoin (DBDMH) and the phenolic OH group was
protected with benzyl group to give the key intermediate 3.
Previously, we showed that 6-amino-2,4,5-trimethyl-
pyridin-3-ols significantly inhibited angiogenesis induced by
VEGF18 and serotonin.19 The compounds showed structure-
activity relationship and dose-dependency in antiangiogenic
activity, clearly suggesting some kind of specific interaction
with common target molecules involved in both VEGF and
serotonin signaling such as phosphoinositide-3-kinase
(PI3K)/Akt.19 The importance of the PI3K/Akt pathway in
the pathogenesis of IBD is highlighted and suggested as a
target for IBD therapy.20,21 In conjunction with our previous
findings on 6-amino-2,4,5-trimethyl-pyridin-3-ols and
increasing amounts of evidence that support the crosstalk
between angiogenesis and IBD,22-24 we examined in the
current study whether 6-amino-2,4,5-trimethyl-pyridin-3-ols
suppress intestinal inflammation.
Here, we report the synthesis, anti-colitis activity, and
structure-activity relationship (SAR) of novel 6-amino-2,4,5-
trimethylpyridin-3-ol derivatives using
a cell-based anti-
inflammatory assay and in vivo model of IBD. Based on the
notion that IBD is of complex etiology, we have tried a
phenotype-based primary screening to inhibit the pathological
action of TNF-, instead of conducting a specific target-based
approach to directly target TNF- molecule. The anti-
inflammatory activity in vitro was measured as an inhibitory
activity against TNF--induced adhesion of monocytes to HT-29
human colonic epithelial cells.25-27 In order to demonstrate in vivo
efficacy, some compounds were tested in the rat model of 2,4,6-
trinitrobenzenesulfonic acid (TNBS)-induced colitis,28 a widely
used animal model of IBD.

Although IBD is of complex etiology, one of the hallmark
events leading to IBD is the attachment and infiltration of
immune cells to colon epithelium, which is induced mainly by
TNF-α.6,7 To quantitatively assess the inhibitory activity of the
compounds against TNF-α-induced adhesion process, we set up
and used a cell-based assay system where monocytes (U937 cells)
and colon epithelial cells (HT-29 cells) were co-cultured in the
presence of TNF-α.27 The level of cell adhesion was measured as
fluorescence signal after wash-out of unattached cells.
Table 1. Inhibitory activity of aminopyridinols against TNF-α-
induced monocyte adhesion to colon epithelial cells [1 μM
compound concentrations except for 5-ASA (20 mM)].
Inhibition
(%)
Inhibition
(%)
Compound
Compound
5-ASAa
10
8b
8d
8f
8h
8j
8l
52.7 ± 7.2*a
22.9 ± 7.2
26.9 ± 10.0
35.7 ± 8.9*
49.2 ± 6.4*
36.0 ± 9.2*
26.5 ± 10.6
84.4 ± 4.8*
76.6 ± 3.8*
74.0 ± 3.7*
14.0 ± 6.5
24.9 ± 14.1
28.4 ± 5.7*
16.3 ± 3.4*
20.5 ± 1.4*
18.5 ± 1.2*
67.4 ± 2.5*
77.2 ± 5.2*
41.3 ± 8.7*
47.9 ± 2.7*
47.6 ± 1.2*
36.7 ± 9.0
LENb
8a
8c
8e
8g
8i
8k
8m
8o
8q
8s
8u
8w
8y
8aa
9a
64.6 ± 6.0*
28.4 ± 2.4
19.0 ± 21.3
1.3 ± 31.0
73.3 ± 6.4*
73.6 ± 13.6*
52.4 ± 11.7*
77.9 ± 7.7*
27.3 ± 28.3
37.8 ± 4.3*
17.3 ± 4.7
71.5 ± 2.5*
47.2 ± 14.9
42.5 ± 4.1
39.0 ± 5.5
58.1 ± 5.3*
63.2 ± 4.1*
65.2 ± 3.6*
13.4 ± 9.6
67.7 ± 7.0*
–7.0 ± 15.7
56.1 ± 3.2*
8n
8p
8r
8t
8v
8x
8z
8ab
9b
9d
9f
9h
9j
9l
9c
9e
9g
9i
9k
9m
a20 mM concentration of 5-ASA. At 1 μM concentration, 5-ASA
showed 3.8% inhibition. bLenalidomide (1 μM).
*P<0.05 compared to vehicle-treated control group.
As shown in Table 1 and Figure 2, TNF-α induced a
remarkable increase in the adhesion of monocytic cells to colonic
epithelial cells, and such adhesion was significantly suppressed
by lenalidomide (LEN, 64.6% inhibition at 1 μM), a positive
control drug that is well-known for its anti-angiogenic
activity.29,30 5-Aminosalicylic acid (5-ASA), the active
metabolite of sulfasalazine (SSZ), a clinical drug for the
treatment of IBD, showed weak inhibitory effects at the same
concentration (3.8% inhibition at 1 μM) with the 50% inhibitory
concentration (IC50) being 18.1 mM. In contrast, majority of
aminopyridinols shown in Table 1 exerted three to four orders of
magnitude stronger activities than 5-ASA, and about a third of
them showed comparable to superior inhibitory activity to LEN
(Table 1). Indeed, aminopyridinols with monosubstitutions on
C(6)-nitrogen (8), significantly inhibited TNF-α-induced
adhesion. Of them, several N-(alkyl-substituted)phenyl analogues
(8l–8n and 8p) showed the best activities (74.0–84.4%). N-
Aliphatic alkyl analogues (8a–8c) as well as N-hydrogen
compound (10) in general showed rather low inhibitions
regardless of the length of aliphatic chain and tethering functional
group.
Figure 1. 6-Amino-2,4,5-trimethylpyridin-3-ols.
Then, a variety of primary, secondary and heterocyclic amines
were reacted with 3 under one of the Buchwald-Hartwig
amination reaction conditions. After the amination, benzyl
protective group was removed either by catalytic hydrogenolysis
or by BCl3 to afford the final product, 6-amino-2,4,5-
trimethylpyridin-3-ols (8, 9). The simplest derivative 10 with
C(6)-NH2 group was also prepared with benzophenone imine, an
ammonia equivalent reagent, by a known procedure developed by
us.18 Structures of all aminopyridinols prepared for this study are
shown in Figure 1.
Inhibitory effect of 6-amino-2,4,5-trimethylpyridin-3-ols on
the TNF-α-induced adhesion of monocytes to colon epithelial
cells

However, the electron-withdrawing group must be located at
para-position to the C(6)-nitrogen in order to preserve the
activity (8u vs. 8x, and 8aa vs. 8ab).
Next, investigation of additional N-substitution effect on
inhibitory activity was carried out. Additional N-methyl or
N-phenyl, or N-2-naphthyl group was substituted on five N-
phenyl derivatives (8i: N-phenyl, 8l: N-p-methylphenyl, 8p
:
N-m-methylphenyl, 8u: N-p-fluorophenyl, and 8v: N-p-
chlorophenyl), under Buchwald-Hartwig reaction conditions,
to afford 9a
inhibitory activity were observed in the cases of N-phenyl
substitution (8i vs. 9a 9f, and 9g) and the degree of the
–9g. Significant to partial decreases in the
,
decrease seemed to be proportional to the size of the second
substituent. The activity of 8i (73.6%) decreased to 58.1%
(9a, additional N-methyl on 8i), 41.3% (9f, additional N-
phenyl on 8i), and 13.4% (9g, additional N-naphthalen-2-yl
on 8i), respectively. This tendency was also observed in N-
methylphenyl analogues, but the degree of the decline was
smaller than that in the N-phenyl series. The activity of 8l
(84.4%) decreased to 67.4% (9b: additional N-methyl on 8l),
and that of 8p (74.0%) decreased to 63.2% (9c: additional
N-methyl on 8p). However, the compounds with N-
halophenyl substituents showed the opposite trend. N-
Hydrogen-N-halophenyl derivatives [8u, (71.5%) and 8v
(28.4%)] showed slight to significantly less activity than N-
methyl-N-halophenyl analogues [9d (77.2%) and 9e (65.2%),
respectively]. Non-aromatic heterocyclic amine-containing
Figure 2. Inhibitory effects of 5-aminosalicylic acid (5-ASA),
lenalidomide (LEN), and an aminopyridinol 8m on TNF-α-induced
adhesion of U937 cells to HT-29 cells. Confluent monolayers of HT-29
cells were pretreated with drugs for 1 h, and then stimulated by 10
ng/mL of TNF-α. After 3 h, HT-29 cells were co-cultured with U937
cells that were already labeled with BCECF-AM (10 μg/mL). Images of
the adhesion of BCECF fluorescence-labeled U937 cells to HT-29 colon
epithelial cells were captured by light microscopy (first column) and
fluorescent microscopy (middle column). Fluorescent images were then
merged over the corresponding light microscopy images to show the
adhering position of U937 cells (third column) (magnification, 200×).
derivatives (9h–9l) generally showed moderate to good
levels of inhibition except for 9k which contains a bulky
substituent. Compared to its saturated analogue, the
pyrrolidine derivative (9l, 36.7%), the activity increased in
the aromatic pyrrole analogue (9m, 56.1%)
.
Suppression of TNBS-induced colitis in rats by compound 8m
Next, we examined the effect of aminopyridinol on colon
inflammation in vivo using a TNBS-induced rat colitis
model, and compared it to that of SSZ. We chose 8m, which
has strong inhibitory activity against the cell adhesion (IC50
= 0.19 μM, data not shown) and represents the highly active
N-alkylphenyl series. N-phenyl series to which 8m belongs
also showed the best antiangiogenic activity (>70% at 0.01
nmol/CAM) in chick chorioallantoic membrane (CAM)
assay.18 N-morpholine analogue (9i) showed considerable
anti-adhesion (67.7%, Table 1) and antiangiogenic activity
(59% at 0.01 nmol/CAM)18 as well. All other compounds
whose antiangiogenic activities were already published18
only showed low to medium level anti-adhesion activity (19%
- 48%, Table 1), implying an somewhat rough agreement
between two activities.
Rats treated with rectal administration of TNBS
showed colitis signs, including bloody diarrhea and wasting
conditions with sluggish and weak movement.28 Compared
to rats in the sham-operated group, TNBS-treated rats
showed a significant reduction followed by stagnated
increase in body weight (Figure 3A). In contrast, rats
receiving oral administration of 8m (0.1, 1, and 10 mg/kg)
clearly showed significant recovery of the TNBS-induced
decrease in body weight in a dose-dependent manner (Figure
3A). Surprisingly, 0.1 mg/kg dose of compound 8m showed
almost comparable body weight recovery than 300 mg/kg
dose of SSZ (Figure 3E). Among other important clinical
sign, colon tissues in the TNBS-treated lesion site showed a
significant level of inflammation, as revealed by edema and
adhesion in gross morphology examination (Figure 3B), and
increased wet weight (Figure 3C). Such damage was
N-Benzyl pyridinols (8d
N-Cycloalkyl analogues (8g and 8h) revealed a high level of
sensitivity to the ring size; the N-cyclopentyl group (8g
showed almost two-fold better inhibitory activity than the N-
cyclohexyl group (8h). Replacement of the cyclohexyl
group of 8h with a phenyl group (8i) restored the activity
–8f) also showed weak activities.
)
(73.6%) to the same level as cyclopentyl analogue (8g
,
73.3%). However, the activity decreased in analogues with
fused benzene substituents (8j and 8k). Of the analogues
with alkyl- and alkoxy-phenyl substituents (8l
alkylphenyl analogues (8l 8n) exerted especially high levels
of inhibition. With the N-p-methylphenyl analogue (8l
–8t), N-p-n-
–
)
being the most active compound, it seems that the size of the
n-alkyl chain slightly changed the activity, except that the
bulky t-butyl substituent (8o) significantly decreased the
activity. o-Isopropyl substitution of phenyl ring also reduced
the activity (8q) while m-methyl substitution retained the
activity (8p). All N-alkoxyphenyl analogues (8r
only marginal activities and all N-halophenyl analogues
8u 8z) showed low to medium activities (16.3–47.2%)
–8t) showed
(
–
except for N-p-fluorophenyl substitution (8u, 71.5%). The
position of the halogen substitution did not show special
SAR. N-nitrophenyl pyridinols (8aa, 8ab) also showed only
marginal activities. It seems that the electron density of the
N-phenyl ring affects the activity. Electron-withdrawing
substituents such as 4-nitrophenyl (8aa) and 4-fluorophenyl
groups (8u) afforded several times higher activity (39.0%
and 71.5%, respectively) than an electron-donating
substituent, such as p-methoxyphenyl
(
8r
)
and p-
phenoxyphenyl group (8s) (14.0% and 17.3%, respectively).

significantly recovered in rats orally treated with compound
8m in a dose-dependent manner. As low as 1 mg/kg 8m
showed better colon weight recovery than 300 mg/kg SSZ,
while 10 mg/kg 8m recovered colon weight almost to the
same level as control (Figure 3C and 3E). High level of
body and colon weight recoveries, in turn, might imply low
possibility of systemic toxicity caused by 8m. In addition, to
measure the degree of colon inflammation, we measured
myeloperoxidase (MPO) level in colon tissue. TNBS
treatment induced a tremendous increase in MPO (Figure
3D) which is directly related to neutrophil infiltration into
tissues with inflammation on the basis that MPO is
abundantly expressed in neutrophils, and that neutrophils in
the circulation are highly recruited to tissues where
inflammation occurs.31,32 The increased MPO level was
significantly suppressed by the treatment with 8m. The
inhibitory effect of 8m at 0.1 mg/kg was almost comparable
to that of 300 mg/kg SSZ, while the efficacy of 1 and 10
mg/kg 8m treatment far exceeded that of 300 mg/kg SSZ
(Figure 3D).
Conclusions
In this study, we synthesized 6-amino-2,4,5,-trimethylpyridin-
3-ols and tested them in colitis models in vitro and in vivo as
surrogate conditions of IBD. Many analogues, especially N-p-
alkylphenyl analogues, showed superior inhibition to 5-ASA
against TNF--induced adhesion of monocytes to colon
epithelial cells at three orders of magnitude lower concentration.
In TNBS-induced in vivo rat colitis model, compound 8m
showed especially excellent recovery profiles in body and colon
weight and inhibitory activity against MPO level. As low as 1
mg/kg 8m showed comparable recovery activities to 300 mg/kg
SSZ in those profiles, demonstrating its remarkable potency.
Although the detailed mechanism of action should be studied
further, it is strongly suggested that 6-amino-2,4,5-
trimethylpyridin-3-ol can be an excellent anti-IBD scaffold.
Acknowledgments
Taken together, remarkable potency and a mechanistic
evidence of 6-amino-2,4,5-trimethylpyridin-3-ols were identified
in cell-based phenotypic assay, in vivo rat colitis model, and
myeloperoxidase assay to reveal their potential as anti-IBD
agents. In addition, the high level of activity and dose-
dependency of compound 8m shown in TNBS-induced rat colitis
model demonstrated, in turn, the validity of our in vitro assay
using TNF--induced adhesion as a functional phenotypic
screening to discover anti-IBD agents.
This study was supported by grant of the Korean Health
Technology R7D Project, Ministry of Health & Welfare,
Republic of Korea (HI15C0542).

Figure 3. 6-Amino-2,4,5-trimethylpyridin-3-ol (8m) ameliorates the clinical features of TNBS-induced colitis in rats. Colitis was induced by rectal
administration of TNBS (2,4,6-trinitrobenzenesulfonic acid). The control group received 50% ethanol as a vehicle. Data represent the mean ± S.E.M. for
five rats per group. *P < 0.05 compared to the vehicle-treated control group. #P < 0.05 compared to the TNBS-treated group. (A) Body weight was recorded
daily from day 0 to day 5. (B) Macroscopic appearance of the large intestine. (C) Wet weight of the colon (distal 5–6 cm segment). (D) MPO level in colon
tissue measured with an MPO assay kit. (E) Quantified summary of the results.
13. Hayakawa, N.; Noguchi, M.; Takeshita, S. et al. Bioorg. Med.
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