A novel triazine-aryl-bis-indole derivative inhibits both phosphodiesterase IV and expression of cell adhesion molecules

Article abstract of DOI:10.1039/c5ra11495k

Asthma, like many inflammation related disorders, has a complex etiology. Drugs targeting multiple pathways may prove more efficacious in these complex disorders. Cyclic 3′,5′-adenosine monophosphate (cAMP) phosphodiesterase IV (PDE IV) is one of the vali

Full text of DOI:10.1039/c5ra11495k

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Bandyopadhyay, T. Banerjee, D. Kar, R. K. Palakodety, P. Sunchu, R. Nomula, V. S. Mallula, H.
Ravindranath, S. Gattu, A. Ramesh, G. Srikanth, M. Rajan, B. Ghosh, P. Jaisankar, R. Johri, D. Chakravarty,
V. Mishra, J. K. Chhabra, M. Shukla, B. N. Paul, S. Bandyopadhyay, S. Roy and G. V. Sharma, RSC Adv.,
2015, DOI: 10.1039/C5RA11495K.
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Biology & Bioinformatics
Sharma, Gangavaram; Indian Institute of Chemical Technology, Organic
Chemistry Division-III

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A Novel Triazine-Aryl-Bis-Indole Derivative Inhibits both
Phosphodiesterase IV and Expression of Cell Adhesion Molecules
Tanima Banerjeea, Dipak Kara, Palakodety Radha Krishnab, Sunchu Prabhakarb, Rajesh Nomulab,
Venkata Satyanarayana Mallulab, Hajari Ravindranathb, Gattu Sridharb, Ramesh Adepub,
Gourishetty Srikanthb, Mabali Rajanc, Balaram Ghoshc, Parasuraman Jaisankara, Rakesh Johrid,
Dolonchapa Chakrabortye, Vani Mishrae, Jasmeet Kaur Chhabrae, Mamta Shuklae, Bhola Nath
Paule, Santu Bandyopadhyaya, Siddhartha Roya, Gangavaram V M Sharmab* and Arun
Bandyopadhyaya*
Asthma, like many inflammation related disorders, has a complex etiology. Drugs targeting multiple pathways may prove
more efficacious in these complex disorders. Cyclic 3′,5′-adenosine monophosphate (cAMP) phosphodiesterase IV (PDE IV)
is one of the validated targets in bronchial asthma and despite availability of some therapeutic molecules targeting PDE-IV,
molecules with better properties are desired. Eosinophil/neutrophil infiltration into lung may also be an important
component of bronchial asthma in which increased expression of epithelial cell adhesion molecules may play an important
role. This study describes the synthesis of a novel class of compounds ‘triazine-aryl-bis-indoles’ having a catechol derived
structure constituting a part of ‘triazine’ and a part of ‘bis-indole’ moiety on it. This class of molecules potently inhibited
both phosphodiesterase IV and expression of cell adhesion molecules ICAM-1 and VCAM-1. The best molecule of this class
(Compound 11) inhibited PDE-IV activity in vitro, with an IC50 value of 14 µM compared to 12.7 µM for an existing drug
rolipram. The compound 11 not only stabilized the cAMP level in human lung epithelial cells (L132) following stimulation
with forskolin, but also inhibited TNF-α induced expression of cell adhesion molecules such as ICAM-1 and VCAM-1in
human umbilical vein epithelial cells (HUVECs). It also significantly inhibited the adhesion of human neutrophils to the
endothelial monolayer (IC50=17.86 µM) in a dose dependent manner. Its absolute bioavailability (in mice) was found to be
70% and its toxicity and pharmacokinetic profiles are excellent. The dual activity of this class of molecules suggests that
this class of molecules could have broad therapeutic applications in neutrophil dominant diseases such as severe asthma,
COPD and acute lung injury.
activity6. The elevation of cAMP levels leads to the suppression of
the synthesis and release of pro-inflammatory signals, cytokines
Introduction
The design and development of novel phosphodiesterase (PDE)
inhibitors in therapeutic applications have gained attention during
the last decade 1-5. The PDEs are a superfamily of enzymes that
catalyze the breakdown of cAMP and/or cyclic guanosine
monophosphate (GMP) to their inactive forms. The potential for
selective PDE inhibitors to be used as therapeutic agents for various
diseases was predicted earlier6 (). PDE4 is the main selective cAMP-
metabolizing enzyme in inflammatory and immune cells. The role of
cAMP as a second messenger is well established and it modulates
the response of immune cells to a variety of stimuli. Elevation of
cAMP has generally been associated with inhibition of lymphocyte
and inhibits the production of reactive oxygen species7-9
Indoles, bis-indoles and octahydro indoles are very common
structural skeletons of several biologically active compounds10,11
.
.
The indole ring system is a very important component in many
synthetic pharmaceuticals12,13 and it is worth to note that the World
Drug Index contains 74 indole derivatives as drug molecules.
Octahydro indole is a part structure of mesembrine, an alkaloid
with PDE-4 inhibitory activity. Furthermore, the indole 3-acetic acid
and its derivatives are commonly used as building blocks for the
synthesis of pharmaceutically important molecules14. Likewise, the
1,3,5-triazine skeleton is implicated in a variety of therapeutic
activities and some triazine derivates have shown anti-asthmatic
activity 15
.
a.CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata-
700032.
In the present study, we described the synthesis of a novel ‘triazine-
aryl-bis-indole’ derivative which inhibits phosphodiesterase activity
in vitro and stabilized cAMP in vivo and demonstrated the anti-
inflammatory activity of one of its potent derivative and thus these
b.CSIR-Indian Institute of Chemical Technology, Hyderabad.
c.CSIR-Institute of Genomics and Integrative Biology, New Delhi.
d.CSIR-Indian Institute of Integrative Medicine, Jammu, India.
e.CSIR-Indian Institute of Toxicology Research, Lucknow, India
*corresponsing authors Email: arunb@iicb.res.in; sharmagvm@gmail.com
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
R
R
derivatives might be useful for the development of potential
bronchodilator and/or anti-inflammatory agents for various airway
diseases.
NH
NH
N
N
ZrCl4, 600C, 8 h
N
N
OCH3
R'
OCH3
N
H
HN
R
N
O
HN
R
N
O
COOH
ZrCl4, 60 oC, 8 h
N
Results
Synthesis of triazine derivatives
CHO
H
N
4R =
R'
H
NH
The compounds described in this study were prepared following a
general synthetic scheme using trichloro triazine as the starting
material. Accordingly, trichloro triazine 1 on reacting with either
cycloproplyamine or aniline under basic conditions (1N
NaOH/acetone/ 0-40 oC/2h) furnished the corresponding
disubstituted chloro 1,3,5-triazine 2 (92%) and 3 (98%) in excellent
yields. Next, etherification (vanillin or isovanillin/K2CO3/DMF/70
oC/6h) of compound 2 with vanillin and isovanillin independently
led to triazine-aryl ethers 4 (74%) and 5 (82%) respectively, while
compound 3 under similar conditions gave 6 (87%) and 7 (87%)
(Scheme 1).
R' = H
8. R =
9. R =
R' = ꢀCH2COOH
R
NH
R'
H3CO
NHR
N
ZrCl4, 600C, 8 h
N
N
OCH3
CHO
N
N
H
N
O
HN
R
N
O
H
N
R'
COOH
ZrCl4, 60 oC, 8 h
NH
NHR
N
H
5 R =
R' = H
R' = ꢀCH2COOH
10 R =
11 R =
Scheme 2. Synthesis of triazine-aryl-bis indoles
Firstly, triazine-aryl ether 4 on reaction with indole or indole-3-
acetic acid in the presence of an acid at 60 ˚C for 8 h gave triazine-
aryl-bis-indoles 8 (76%) and 9 (52%), respectively. Similarly triazine-
aryl ether 5 upon reaction with indole or indole-3-acetic acid under
similar reaction conditions afforded triazine-aryl-bis-indoles 10
(67%) and 11 (61%), respectively (Scheme 2).
R
NH
N
N
R'
NH
OCH3
ZrCl4, 600C, 8 h
N
H
H CO
3
N
R
NH
N
HN
R
N
O
H
N
O
COOH
ZrC4, 60 oC, 8 h
CHO
6 R = Ph
R'
N
N
R
NH
H
12 R = Ph, R' = H
13 R = Ph, R' = ꢀCH2COOH
Next, the isomeric triazine-aryl ethers 6 and 7 on reaction with
indole or indole-3-acetic acid resulted in the corresponding triazine-
aryl-bis-indoles 12 (61%) and 13 (57%); 14 (66%) and 15 (64%) in
comparable yields (Scheme 3). All the compounds were thoroughly
characterized.
R
NH
R'
H3CO
N
N
R NH
OCH3
ZrCl4, 600C, 8 h
N
O
N
N
N
HN
N
O
N
H
H
R'
R
NH
R' NH
COOH
ZrCl4, 60 oC, 8 h
7 R = Ph
CHO
N
H
14 R = Ph, R' = H
15 R = Ph, R' = ꢀCH2COOH
CHO
R
N
NH
HO
N
Scheme 3. Synthesis of isomeric triazine-aryl-bis-indoles
OCH3
OCH3
R
N
HN
R
N
O
Cl
NH
K2CO3, DMF, 700C,
6
h
h
RNH2, acetone
N
N
N
CHO
1N NaOH, 0-400C, 2 h
4 R =
Triazine-aryl-bis-indole derivatives inhibit phosphodiesterase
activity in vitro
Cl
N
Cl
HN
N
Cl
H3CO
R
R
NH
1
HO
CHO
N
N
2 R =
3 R = Ph
PDE-4 enzyme preparations were purified from rat heart and
incubated in the absence or presence of the compounds (10 µg/ml)
and breakdown of cAMP was quantified. As shown in Table 1, all
the compounds inhibited PDE 4 activity which was comparable to
the known PDE 4 inhibitor, rolipram. Out of 12 compounds tested 2
of them (compounds 11 and 15) showed moderately higher
inhibition. We selected compound 11 for further investigation
because of easy synthesis and higher yield. Dose response (0.1-20
μg/ml) experiments showed that the IC50 value of compound 11
was higher than that of rolipram (13.03 ± 0.34 μM compound 11
compared to 12.7 ± 0.23 μM for rolipram) (Figure 1).
OCH3
CHO
K2CO3, DMF, 700C,
6
HN
R
N
O
CHO
5 R =
R
NH
HO
N
N
OCH3
OCH3
HN
R
N
O
R
K2CO3, DMF, 700C,
6 h
NH
CHO
6 R = Ph
N
N
H3CO
HO
K2CO3, DMF, 700C,
HN
R
N
Cl
R
NH
CHO
N
N
3 R = Ph
OCH3
CHO
HN
N
O
6
h
R
7 R = Ph
Isozyme specificity of compound 11
Scheme 1. Synthesis of Triazine aryl ethers
To check the specificity and selectivity on the inhibitory activity of
compound 11, screening of a panel of PDE isozymes was conducted
using high throughput analysis. Out of 13 isozymes screened,
compound 11 specifically inhibited PDE4 family. It also inhibited
the non selective PDE 5 and 6 isozymes. However, no inhibitory
activity was recorded with PDE isozymes -1B, -2A, -3A, -3B, -7A, -
8A1, -10A1 and -11A4 as shown in Table 2.
2 | J. Name., 2012, 00, 1-3
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Table 1. Effect of Triazine-aryl-bis-indole derivatives on cAMP Concentration vs. time curves for compound 11 after oral (Figure
dependent phosphodiesterase activity
5A) and i.v. (Figure 5B) administration in mice were established to
determine the pharmacokinetics and bioavailability of the
molecule. The results as summarized in Table 2 showed that after
oral administration (20 mg/kg) the half-life (t1/2 ) of compound 11
(IICTTA67) was 4.26 hrs; AUC = 502.75 ng. Hr/ml; Clearance (CL) =
663 ml/min, and volume of distribution (VD) = 474 L. After i.v. bolus
dosing of 10 mg/ kg, compound 11 showed AUC = 357.71 ng/hr.ml;
t1/2, = 0.93 hrs; CL = 466 ml/min; VD = 37.5 L. Absolute bioavailability
F (Oral) of compound 11 (in mice) was found to be 70.30%.
Inhibition
(%)
Compound no.
Name
Dose
2
IICT-TA42
IICT-TA57
IICT-TA43
IICT-TA58
IICT-TA44
IICT-TA45
IICT-TA66
IICT-TA67
IICT-TA59
IICT-TA60
IICT-TA77
IICT-TA78
10 μg/ml
10 μg/ml
10 μg/ml
10 μg/ml
10 μg/ml
10 μg/ml
10 μg/ml
10 μg/ml
10 μg/ml
10 μg/ml
10 μg/ml
10 μg/ml
10 μg/ml
44
53
29
30
16
42
34
60
34
31
45
73
69
3
4
5
8
9
Table 2: PDE Isozymes specificity of compound 11
10
Isozymes
IC50
Tissue Expression16,17
11
12
(µM)
13
14
PDE 1B
PDE 2A
PDE 3A
PDE 3B
PDE 4A
PDE4B1
PDE 4D2
non
ND
ND
ND
ND
9.81
10
Brain, Lymphocytes
15
Brain, Heart, Platelets, liver, thymocytes
Heart, blood vessels
Rolipram
Adipocytes, hepatocytes, lymphocytes
Lung, immune cells, brain, blood vessels
-do-
The enzyme preparations were incubated in the absence or presence of compounds 2-
14 and breakdown of cAMP was monitored by β counter. cAMP dependent PDE activity
was measured as described in the Methods.
10
-do-
Stabilization of cellular cAMP by compound 11
10
smooth muscles, platelets
When human lung epithelial cells (L 132) were stimulated with
forskolin, the level of cAMP increased significantly over time in the
presence of compound 11 compared to control treatment (Figure
2). These data confirm our previous results that compound 11
inhibited PDE-4 leading to accumulation of cellular cAMP.
Compound 11 inhibits TNF-α induced expression ICAM-1 and
VCAM-1 on HUVEC cells
selective
PDE 5
PDE 6 Non 5.34
selective
retinal photoreceptors
PDE 7A
ND
ND
ND
Brain, lymphocytes, kideney, heart
PDE 8A1
PDE 10A1
thyroid
testes, brain
PDE 11A4
ND
Prostate, Skeletal muscle, heart, testes
Since it has been earlier demonstrated that cAMP is crucial in
inducing various cell adhesion molecules via various mechanisms,
we wanted to determine the effect of compound 11 on TNF-α
induced expression of cell adhesion molecules, such as ICAM-1 and
VCAM-1. For this, HUVECs were pretreated with various
concentrations of the compound 11 for 2 hours followed by
induction with TNF-α for 16 hours. The cell surface expression of
cell adhesion molecules was measured by ELISA. As shown in Figure
3, compound 11 inhibited the adhesion of human neutrophils to
the endothelial monolayer in a dose-dependent way..
IC50 of each PDE isozyme was determined in the presence of increasing
concentrations of compound 11 (0.1 to 10 µg/ml) using high throughput platform as
described in the text. ND, non-detectable.
Table 3: Pharmacokinetics for compound 11 in rat
Oral
107
i.v.
Compound 11 inhibits the adhesion of Neutrophils on TNF-alpha
induced HUVEC cells
Cmax (ng/ml)
Tmax (hr)
T1/2 (hr)
----
2.0
----
The Cell adhesion molecules such as ICAM-1 and VCAM-1 are
critical in the adhesion of neutrophils to endothelial cells in most of
the neutrophil dominant diseases. To evaluate the functional
significance of inhibition of adhesion molecule by compound 11, we
next checked adhesion of neutrophils on TNF induced HUVEC cells.
As shown in Figure 4, compound 11 dose dependently inhibited the
adhesion of human neutrophils to the endothelial monolayer
(IC50=17.8 μM) (Figure 4). These data indicate that there is a strong
correlation between inhibition of adhesion molecule expression
and inhibition of neutrophil adhesion to the endothelial mono
layer by the compound
4.26
502.75
663
0.93
357.71
466
37.5
AUC (ng.hr/ml)
Cl (ml/min)
Vd (L)
474
No Cmax and Tmax in i.v. route
Pharmacokinetics of Compound 11
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Cytotoxicity of Compound 11
report, we describe 12 compounds having appreciable PDE4 activity
The 3T3 Neutral Red Dye Uptake (NRU) cytotoxicity assay was in vitro. Out of which 2 had better activity. In vitro inhibitory activity
conducted in the presence of increasing concentrations of of this compound on PDE4 is further supported by our data on
compound 11 (Figure 6). The derived IC50 of compound 11 was stabilization of intracellular cAMP in lung epithelial cells. For
found to be 48 µM indicating that a reasonably high concentration synthesis and better yield we had detailed investigation with
of the compound is necessary for inducing cell death in vitro.
isozyme specificity and other relevant studies with one compound
(Compound 11). This compound strongly inhibits PDE 4A, PDE 4B
and PDE 4D isozymes which are mainly expressed in lung and
immune cells 16,17. Compound 11 also inhibits non-selective PDE 5
and PDE 6 isozymes. However, these isozymes are not expressed
in lung or immune cells16,17 . Thus it appears that non selective
inhibitory activities of compound 11 may not have any influence in
asthma pathogenesis.. Interestingly, this compound does not
inhibit isozymes prevalent in brain (PDE 1A, PDE 1B and PDE 7) or
heart (PDE 2A, PDE3A, PDE 7A and PDE 11A4) which strengthens its
potential selective application against respiratory diseases.
Discussion
In this study, we demonstrate the synthesis of triazine-aryl-bis-
indole derivatives and their effect on phosphodiesterase enzyme,
an important target in the pathogenesis of asthma and related
respiratory disorders. The indole and its various derivatives such as
bis-indoles and octahydro indoles seem to be potent biologically
active compounds10, 11. The indole moiety has been found not only
in various natural molecules like lysergic acid, bufotenin, serotonin
but also important for drug discovery research.. These indole
derivatives have been found to have anti-cancer, anti-oxidant, anti-
inflammatory and antimicrobial activities18. Importantly, indole
derivatives have also shown to act as long acting β2-adrenoceptor
agonists similar to to salmeterol19. I Since, bis-indoles have been
The second messenger cAMP controls many cellular functions and it
is well established that an elevated cAMP level can inhibit various
inflammatory processes. Thus, inhibitors of enzymes that catalyze
cAMP hydrolysis would seem to be good candidates to treat
inflammatory conditions9. It is known that cAMP reduces
inflammatory signaling by inhibiting TNF-α induced cell adhesion
molecules through inhibiting either NF-KB or reactive oxygen
species32,33. On the other hand, cell adhesion molecules are crucial
in various inflammatory diseases. Recruitment of inflammatory
cells including eosinophils in the lung tissue plays an important role
in asthma pathogenesis34. The recruitment of various immune cells
into the site of the injury depends on the timely expression of cell
adhesion molecules on vascular endothelial cell surface and
adhesion of immune cells such as neutrophils. Migration of these
inflammatory cells is largely regulated by the expression of
adhesion molecules such as ICAM-1 and VCAM-1 either on the r
found to exist in various nutritional components such as cruciferous
18, 20, 21
vegetables
it is likely that bis-indoles may be highly
bioavailable and safer. The natural and unnatural analogs of
bisindole derivatives have potent anticancer activities18 . In addition
to these, triazine derivatives also have anti-eosinophilic and anti-
asthma activity22
.
Triazine aryl-bis-indole derivatives are
synthesized by the dialkylation of trizine followed by etherification
with vanallin isovallin. Furthermore, these triazine aryl ethers were
derivatised by the condensation of indole of indole acetic acid in
the presence of TCT under mild conditions for achieving the target
compounds. These compounds were evaluated for inhibitory
activity against various isoforms of PDE enzyme. These evidences
indicate that Traizine aryl-bis-indole derivatives could be potent in
various diseases.
epithelial or on the endothelial cell surface35-37
. Because of
antiinflammatory activities, PDE inhibitors are known to inhibit
cellular trafficking, production of oxygen species and cell adhesion
molecule expression8. Consistently, we also show significant
inhibition of adhesion molecules in HUVEC cells by our compound
suggesting its multipronged efficacy in inhibiting asthma
pathogenesis. These data indicate the potential of this compound
as a therapeutic molecule against bronchial asthma.
The cyclic nucleotide PDEs hydrolyze cAMP and cGMP to convert
into their inactive analogues. The potential for selective PDE
inhibitors as therapeutic agents was predicted earlier. Thus, it has
been focused to discover novel PDE4 inhibitors for using in various
diseases. In this context, indole derivatives also have been shown to
have PDE4 inhibitory activity23. In this study, we synthesize effective
indole derivatives and checked their PDE inhibitory activities.
Among various PDE isoenzymes known so far, PDE4 is crucial as it is
To determine whether the compound 11 is orally active, evaluation
of the in vivo pharmacokinetic parameters was performed. With
oral dosing maximum plasma concentration of compound 11
attains rapidly within 2 h. It also has a higher T1/2 value with
moderately higher clearance and distribution volume with oral
dosing compared to i.v. administration. Most of the compound was
eliminated by 24 h with elimination half life of about 4.25 h. Oral
bioavailability was found to be 70%. These data suggest that
compound 11 is orally bioavailable with good pharmacokinetic
profile.
predominantly present in various inflammatory cells23
.
It is
important to note that only PDE-4, 7 and 8 are associated with the
metabolism of cAMP. The role of cAMP has been implicated in
bronchial asthma pathogenesis24 . Because PDE inhibitors prevent
breakdown of cAMP they act as bronchodilators by maintaining
high level of this second messengers in bronchial cells25
.
Abundance of PDE 4 in lung tissue has been reported earlier26. PDE-
4 has four distinct genes, PDE-4A, PDE-4B, PDE-4C and PDE-4D27
with specificity to cAMP and thus have become potential
therapeutic targets. The importance of PDE 4 as a therapeutic
target for respiratory disorders has been supported further by the
recent approval of roflumilast, an inhibitor of PDE4, by FDA, for the
treatment of chronic obstructive pulmonary disease28-31. In this
The cytotoxic IC50 of compound 11 is approximately 5 -10 fold
greater than other anti-inflammatory compounds such as ursolic
acid38,39 and TiO240. However, the cytotoxic IC50 of several PDE4
inhibitors designed elsewhere41 were > 80 μM after a 24h assay.
4 | J. Name., 2012, 00, 1-3
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Notably, the duration of compound 11 exposure was 48h and this (ESI): m/z calculated for C15H13N5Cl [M+H]+: 298.0854; found:
298.0852.
prolonged exposure is likely to be associated with a pronounced
occurrence of a cumulative cytotoxic effect. Despite cumulative
cytotoxic effect, the IC50 of compound 11 offers ~2 fold margin in
comparison to our observed IC50 of Compound 11 inhibiting ICAM
(Fig 3a), VCAM (Fig 3b), neutrophil adhesion molecule (Fig 4), as
well as > 3 fold safe margin over PDE4 inhibition (14μM ). Hence,
the compound 11 is toxicologically safe enough to merit clinical
evaluation and use.
4-[4,6-Bis-cyclopropylamino-1,3,5-triazin-2-yl-oxy]-3-methoxy
benzaldehyde 4
A mixture of 2 (5.0 g, 22.1 mmol), vanillin (4.04 g, 26.6 mmol) and
K2CO3 (9.17 g, 66.48 mmol) in DMF (30 mL) was heated at 70 °C for
6 h. The reaction mixture was cooled to room temperature, diluted
with water (75 mL) and extracted with EtOAc (4 x 50 mL). The
combined organic layers were washed with water (50 mL), brine (50
mL) and dried (Na2SO4). Evaporation of the solvent under reduced
pressure and purification of the residue by column chromatography
(Silica gel, EtOAc in hexane 3:7) furnished 4 (5.58 g, 74%) as a white
Thus, triazine-aryl-bis-indole derivatives are novel structure with
ability to inhibit hydrolysis of cAMP in in vivo conditions in
association with inhibition of adhesion molecules (Fig 7). This
compound may potentiate bronchodilation due to sustenance of solid. mp 138-140 °C; IR (KBr): 3313, 3093, 3008, 2850, 1690, 1604,
1542, 1352, 1282, 809, 780 cm-1; 1H NMR (300 MHz, DMSO-d6): δ
intracellular cAMP. Furthermore, it may act as an anti-inflammatory
9.95 (s, 1H), 7.78 (s, 1H), 7.49 (s, 1H), 7.31 (d, 1H, J = 6.6 Hz), 6.98-
by inhibiting adhesion molecules (Fig 7).
6.55 (m, 2H), 3.87 (s, 3H), 3.07-2.62 (m, 2H), 0.75-0.39 (m, 8H); 13
C
Experimental Procedure
Synthesis of triazine-aryl-bis-indoles
NMR (75 MHz, DMSO-d6): δ 191.9, 168.3, 167.7, 167.2, 151.9,
146.1, 134.9, 123.6, 111.8, 55.8, 23.3, 23.2, 5.9, 5.7; HRMS (ESI):
m/z calculated for C17H20O3N5 (M+1)+ 342.1560; found: 342.1577.
Column chromatography was performed on silica gel, Acme grade
60-120 mesh. Unless stated otherwise, all reagents were purchased
from commercial sources (Sigma-Aldrich Chemicals, St. Louis, MO,
USA) and used without additional purification. 1H NMR and 13C NMR
spectra were recorded either on a Bruker 300 or Varian VXR 400 or
Varian VXR 500 in CDCl3 or CDCl3 + DMSO mixture as solvent with
TMS as reference unless otherwise indicated. Unless stated
otherwise, HRMS spectra were recorded on a QTOF analyser
(QSTAR XL, Applied Biosystems/MDS Sciex) at NCMS-IICT,
Hyderabad. Unless stated otherwise, elemental Analysis was carried
on a Vario Micro Cube Elementar at Analytical Chemistry Division
IICT, Hyderabad. The software ACD/Name Version 1.0, developed
by M/s Advanced Chemistry Development Inc., Toronto, Canada,
assisted nomenclature used in the experimental section. Unless
stated otherwise, all the reactions were performed under inert
atmosphere. Representative NMR and LCMS spectra of the
compounds are presented in supporting information file.
4-[4,6-Bis-cyclopropylamino-1,3,5-triazin-2-yl-oxy]-4-methoxy
benzaldehyde 5
A mixture of 2 (5.0 g, 22.1 mmol), isovanillin (4.04 g, 26.6 mmol)
and K2CO3 (9.17 g, 66.51 mmol) in DMF (30 mL) using the procedure
as described for 4 furnished 5 (6.2 g, 82%) as a white solid. mp 189-
191 °C; IR (KBr): 3297, 3255, 3008, 2847, 1605, 1575, 1506, 1363,
1
1280, 812, 729 cm-1; H NMR (500 MHz, CDCl3 + DMSO-d6 ): δ 9.88
(s, 1H), 7.75 (d, 1H, J = 8.3 Hz), 7.71-7.61 (m, 1H), 7.08 (d, 1H, J = 8.4
Hz), 5.97-5.58 (m, 2H), 3.89 (s, 3H), 2.94-2.48 (m, 2H), 0.81-0.39 (m,
8H); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 189.6, 169.6, 167.9,
167.3, 156.5, 141.3, 129.6, 128.6, 123.0, 111.3, 55.5, 22.7, 6.3;
HRMS (ESI): m/z calculated for C17H20N5O3: 342.1566(M+1)+; found
342.1574.
6-Chloro-N2,N4-dicyclopropyl-1,3,5-triazine-2,4-diamine 2
A fine slurry prepared from cyanuric chloride 1 (5.0 g, 27.17 mmol)
by the addition of acetone (25 mL) was stirred at 40 °C for 30 min.
4-[4,6-Bis-anilino-1,3,5-triazin-2-yl-oxy]-3-methoxy benzaldehyde
6
Using a mixture of 3 (5 g, 16.79 mmol), vanillin (3.06 g, 20.15 mmol)
and K2CO3 (9.17 g, 66.51 mmol) in DMF (30 mL) and adopting the
procedure as described for 4 gave 6 (6.05 g, 87%) as a white solid.
mp 178-180 °C. IR (KBr): 3292, 3110, 1684, 1623, 1581, 1525, 1443,
The reaction mixture was cooled to
0 °C and treated with
cyclopropyl amine (3.09 g, 54.34 mmol). It was stirred at the same
temperature for 1 h and neutralized with aq. 1N NaOH till the
reaction mixture becomes basic. It was then heated to 40 °C and
stirred at this temperature for 2 h. The reaction mixture was
filtered, washed with acetone (3 x 25 mL), evaporated solvent and
dried under vacuum to furnish 2 (5.6 g, 92%) as a white solid. mp
197-199 °C; IR (KBr): 3239, 3094, 3013, 2935, 1609, 1542, 1350,
1279, 1018, 807, 740 cm-1; 1H NMR (300 MHz, DMSO-d6): δ 7.08-
6.62 (m, 2H), 3.26-2.63 (m, 2H), 0.85-0.49 (m, 8H); 13C NMR (75
MHz, CDCl3): δ 167.9, 166.7, 166.5, 23.4, 23.3, 6.0, 5.8; HRMS (ESI):
m/z calculated for C9H13ClN5: 226.0854 (M+1)+; found: 226.0852.
6-Chloro-N2,N4-diphenyl-1,3,5-triazine-2,4-diamine 3
1
1400, 1355, 1287, 1156, 1112, 751; H NMR (75 MHz, DMSO-d6): δ
10.00 (s, 1H), 8.98 (br. s, 2H), 7.99 (s, 1H), 7.81-7.43 (m, 5H), 7.55-
6.9 (m, 7H), 3.85 (s, 3H); 13C NMR (75 MHz, DMSO-d6): δ 190.3,
164.5, 161.6, 151.5, 145.7, 137.9, 133.7, 127.4, 123.5, 122.0, 119.7,
110.1, 55.0; HRMS (ESI): m/z calculated for C23H20O3N5 [M+H]+:
414.1560; found: 414.1526.
4-[4,6-Bis-anilino-1,3,5-triazin-2-yl-oxy]-4-methoxy benzaldehyde
7
A mixture of 3 (5 g, 16.79 mm ol), isovanillin (3.06 g, 20.15 mmol)
and K2CO3 (6.95 g, 50.38 mmol) in DMF (30 mL) according to the
procedure as described for 4 gave 7 (6.01 g, 87%) as a white solid.
mp 148-150 oC; IR (KBr): 3405, 2925, 1698, 1612, 1574, 1505, 1440,
A fine slurry of cyanuric chloride 1 (5.0 g, 27.17 mmol) and acetone
(25 mL) was stirred at 40 °C for 30 min. The reaction mixture was
cooled to 0 °C and treated with aniline (5.05 g, 54.34 mmol) and
worked up as described for 2 to furnish 3 (7.45 g, 98%) as a white
solid. mp 210-212 oC; IR (KBr): 3291, 3053, 2924, 2850, 1687, 1609,
1512, 1442, 1276, 1124, 746 cm-1; 1H NMR (300 MHz, DMSO-d6): δ
9.30 (br. s, 1H), 7.56 (d, 2H, J = 7.5 Hz), 7.39 (s, 1H), 7.24-7.18 (m,
5H), 6.98 (t, 2H, J = 7.3 Hz), 163.4, 137.5, 127.8, 122.9, 120.4; HRMS
1
1391, 1348, 1210, 1125, 746 cm-1; H NMR (300 MHz, DMSO-d6): δ
9.89 (br. s, 1H), 8.75 (s, 2H), 7.70 (d, 1H, J = 2.0 Hz), 7.59-7.47 (m,
4H), 7.29-6.99 (m, 8H), 3.87 (s, 3H); 13C NMR (75 MHz, DMSO-d6): δ
190.3, 164.5, 161.6, 151.5, 145.7, 137.9, 137.7, 127.4, 123.5, 122.7,
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122.0, 119.7, 110.1, 55.0; HRMS (ESI): m/z calculated for C23H20O3N5 A mixture of 5 (1.0 g, 2.9 mmol) and indole 3-acetic acid (1.02 g,
[M+H]+: 414.1560; found: 414.1558.
5.85 mmol) in CH3CN (5 mL) using the procedure as described for 9
furnished 11 (1.2 g, 61%) as a pale brown solid. mp 204-206 °C; IR
(KBr): 3387, 2921, 2851, 1707, 1591, 1511, 1356, 1272, 1023, 810,
744 cm-1; 1H NMR (500 MHz, DMSO-d6): δ 10.4 (s, 2H), 7.77 (d, 2H, J
= 6.2 Hz), 7.48 (d, 2H, J = 7.5 Hz), 7.26 (d, 2H, J = 7.3 Hz), 7.13-6.91
(m, 6H), 6.8 (d, 1H, J = 8.8 Hz), 6.5 (br. s, 2H), 6.11 (s, 1H), 3.74 (s,
3H), 3.61 (s, 2H), 2.82-2.5 (m, 2H), 0.77-0.39 (m, 8H); 13C NMR (75
MHz, DMSO-d6): δ 173.2, 169.68, 168.33, 167.36,150.2, 140.9,
135.4, 127.7, 125.6, 122.5, 120.9, 118.2, 118.5, 110.9, 105.5,
55.7,38.72, 29.6, 23.4, 5.9; HRMS (ESI): m/z calculated for
C37H36N7O6:674.2721(M+1)+; found: 674.2716.
2,2’-[2,2’-4-(4,6-Bis-cyclopropylamino)-1,3,5-triazin-2-yl-oxy-3-
methoxyphenyl]-methy-lene-bis-1H-indole 8
To a stirred solution of 4 (1.0 g, 2.9 mmol) and indole (0.69 g, 5.8
mmol) in CH3CN (5 mL), catalytic amount of cyanuric chloride was
added and heated at 60 °C for 8 h. The reaction mixture was cooled
to room temperature, diluted with water (25 mL) and extracted
with EtOAc (4 x 50 mL). The combined organic layers were washed
with water (50 mL), brine (50 mL) and dried (Na2SO4). Evaporation
of the solvent under reduced pressure and purification of the
residue by column chromatography (Silica gel, EtOAc in hexane 1:4)
afforded 8 (1.25g, 76%) as a pale brown solid. mp 185-187 °C; IR
(KBr): 3414, 3267, 3008, 1586, 1505, 1356, 1207, 1147, 810, 749 cm-
2,2’-[2,2’-4-(4,6-Bis-anilino)-1,3,5-triazin-2-yl-oxy-3-
methoxyphenyl]-methylene-bis-1H-indole 12
1; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 10.28 (s, 2H), 7.73 (m, 2H), Using a stirred solution of 6 (1.0 g, 2.42 mmol) and indole (0.56 g,
7.33 (d, 4H, J = 7.9 Hz), 7.12-7.00 (m, 3H), 7.00-6.74 (m, 5H), 6.63 (s, 4.84 mmol) in CH3CN (5 mL) adopting the procedure as described
1H), 5.8 (s, 1H), 3.66 (s, 3H), 2.90-2.53 (m, 2H), 0.74-0.34 (m, 8H); for 8 furnished 12 (1.0 g, 61%) as a pale brown solid. mp180-182 °C.
13C NMR (75 MHz, CDCl3+DMSO-d6): δ 169.7, 167.9, 167.36, 150.7, IR (KBr) : 3396, 3052, 2927, 1609, 1576, 1503, 1441, 1391, 1346,
142.2, 139.1, 136.3, 126.2, 123.2, 121.8, 120.6, 118.8, 117.8, 112.6, 1200, 1121, 1031, 744; 1H NMR (75 MHz, DMSO-d6): 10.78 (m, 2H),
110.9, 55.25, 30.8, 22.9, 6.0; HRMS (ESI): m/z calculated for 9.30 (br. s, 2H), 7.92-6.63 (m, 23H), 5.86 (s, 1H), 3.37 (br. s, 3H); 13C
C33H32N7O2: 558.2612(M+1)+; found: 558.2587
NMR (75 MHz, DMSO-d6): δ 170.3, 165.1, 150.6, 143.5, 139.0,
136.4, 128.1, 126.5, 123.3, 122.3, 120.8, 120.2, 118.9, 118.1, 111.3,
99.6, 59.6, 55.5; HRMS (ESI): m/z calculated for C39H32O2N7 [M+H]+:
630.2612; found: 630.2563.
2,2’-[2,2’-4-(4,6-Bis-cyclopropylamino)-1,3,5-triazin-2-yl-oxy-3-
methoxyphenyl]-methyl-ene-bis-(1H-indole-3,3-diyl)-diacetic acid
9
2,2’-[2,2’-4-(4,6-Bis-anilino)-1,3,5-triazin-2-yl-oxy-3-
A mixture of 4 (1.0 g, 2.9 mmol) and indole 3-acetic acid (1.02 g,
5.86 mmol) in CH3CN (5 mL) was treated with a catalytic amount of
ZrCl4 and heated at 60 °C for 8 h. The reaction mixture was cooled
methoxyphenyl]-methylene-bis-(1H-indole-3,3-diyl)-diacetic acid
13
to room temperature, diluted with water (25 mL) and extracted A mixture of 6 (1.0 g, 2.41 mmol) and indole 3-acetic acid (0.84 g,
with EtOAc (4 x 50 mL). The combined organic layers were washed 4.83 mmol) in CH3CN (5 mL) using the procedure as described for 9
with water (50 mL), brine (50 mL) and dried (Na2SO4). Evaporation gave 13 (1.02 g, 57%) as a pale brown solid. mp 240-242 °C. IR (KBr):
of the solvent under reduced pressure and purification of the 3364, 2924, 1707, 1611, 1576, 1514, 1443, 1413, 1352, 1299, 1204,
residue by column chromatography (Silica gel, EtOAc in hexane 1:4) 1123, 1027, 747; 1H NMR (500 MHz, DMSO-d6): δ 9.78-9.53(m, 2H),
afforded 9 (1.02 g, 52%) as a pale brown solid. mp 254-256 °C; IR 8.46 (br. s, 2H), 7.61-7.28 (m, 10H), 7.16-6.86 (m, 13H), 6.73 (s, 1H),
(KBr): 3429, 3357, 3007, 2924, 1708, 1584, 1509, 1408, 1352, 1204, 3.60 (br. s, 7H); 13C NMR (75 MHz, DMSO-d6): δ 173.1, 171.8, 170.2,
1
809, 743 cm-1; H NMR (300 MHz, DMSO-d6): δ 10.5 (br. s, 2H), 7.8 165.1, 151.0, 139.6, 139.1, 139.0, 135.6, 135.2, 128.1, 127.6, 122.4,
(d, 3H, J = 6.4 Hz), 7.5 (d, 2H, J = 7.5 Hz), 7.2 (d, 2H, J = 7.7 Hz), 7.12- 120.9, 120.3, 118.5, 118.1, 113.4, 111.1, 105.4, 59.6, 55.6, 29.9;
6.9 (m, 7H), 6.8 (s, 1H), 6.1 (s, 1H), 3.63 (s, 3H), 3.62 (s, 2H), 3.3 (s, HRMS (ESI): m/z calculated for C43H36O6N7 [M+H]+: 746.2721; found:
2H), 2.92-2.5 (m, 2H), 0.62 (m, 8H); 13C NMR (75 MHz, DMSO-d6): δ 746.2721.
173.1,169.33, 167.2, 151.0, 139.6, 138.6, 135.5, 135.2, 127.7, 122.6,
2,2’-[2,2’-3-(4,6-Bis-anilino)-1,3,5-triazin-2-yl-oxy-4-
methoxyphenyl]-methylene-bis-1H-indole 14
120.9, 118.6, 118.1, 111.0, 105.5, 55.6, 30.0, 23.4, 5.9; HRMS (ESI):
m/z calculated for C37H36N7O6: 674.2721(M+1)+; found: 674.2716.
To a stirred solution of 7 (1.0 g, 2.4 mmol) and indole (0.56 g, 4.8
mmol) in CH3CN (5 mL) adopting the procedure as described for 8
furnished 14 (1.0 g, 66%) as a pale brown solid. mp 165-168 oC: IR
2,2’-[2,2’-3-(4,6-Bis-cyclopropylamino)-1,3,5-triazin-2-yl-oxy-4-
methoxyphenyl]-methyl-ene-bis-1H-indole 10
To a stirred solution of 5 (1.0 g, 2.9 mmol) and indole (0.68 g, 5.85 (KBr): 3379, 2924, 1614, 1579, 1513, 1443, 1399, 1268, 1129, 743;
mmol) in CH3CN (5 mL) adopting the procedure as described for 8 1H NMR (300 MHz, DMSO-d6): δ 9.84 (br. s, 2H), 8.74 (br. s, 2H),
gave 10 (1.1 g, 67%) as a pale brown solid. mp 176-178 °C; IR (KBr): 7.82-7.46 (m, 5H), 7.44-7.32 (m, 4H), 7.31-6.63 (m, 14H), 5.89 (s,
3410, 3007, 2923, 2852, 1586, 1506, 1354, 1270, 1129, 808, 747 cm- 1H), 3.71 (s, 3H); 13C NMR (75 MHz, DMSO-d6): δ 170.0, 164.7,
1
1; H NMR (500 MHz, DMSO-d6): δ 10.33 (s, 2H), 7.29-7.19 (m, 4H), 148.9, 140.3, 138.2, 136.9, 136.0, 127.5, 126.0, 125.2, 123.0, 122.1,
7.08 (d, 1H, J = 7.1 Hz), 7.01-6.92 (m, 3H), 6.84-6.66 (m, 7H), 5.67 (s, 120.3, 119.8, 118.6, 117.5, 115.0, 111.4, 110.5, 59.3, 55.0; HRMS
1H), 3.6 (s, 3H), 2.83-2.45 (m, 2H), 0.45 (m, 8H); 13C NMR (75 MHz, (ESI): m/z calculated for C39H32O2N7 [M+H]+: 630.2612; found:
DMSO-d6): δ 167.77, 168.31,149.3, 136.4, 126.4, 123.3, 120.7, 630.2604.
118.9, 118.08, 117.97, 111.29, 55.56, 38.5, 22.9, 5.9; HRMS (ESI):
m/z calculated for C33H32N7O2: 558.2612 (M+1)+; found: 558.2642.
2,2’-[2,2’-3-(4,6-Bis-anilino)-1,3,5-triazin-2-yl-oxy-4-
methoxyphenyl]-methylene-bis-(1H-indole-3,3-diyl)-diacetic acid
2,2’-[2,2’-3-(4,6-Bis-cyclopropylamino)-1,3,5-triazin-2-yl-oxy-4-
15
methoxyphenyl]-methy-lene-bis-(1H-indole-3,3-diyl)-diacetic acid
11
6 | J. Name., 2012, 00, 1-3
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ARTICLE
A mixture of 7 (1.0 g, 2.4 mmol) and indole 3-acetic acid (0.85 g, 4.8 terminated by the addition of ice-cold 95% ethanol at different time
mmol) in CH3CN (5 mL) using the procedure as described for 9 gave periods. The cells were harvested and centrifuged to remove cell
15 (1.15 g, 64%) as a pale brown solid. m.p. 188-190 oC: IR (KBr): debris. The experiment was repeated on at least three separate
3262, 3092, 1607, 1575, 1521, 1445, 1388, 1226, 990, 805, 754, occasions in duplicate wells for each time point. Measurement of
703, 599; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.93 (br, s, 2H), cAMP was conducted using enzyme immunoassay kit (GE
8.27 (br. s, 2H), 7.69-7.34 (m, 8H), 7.24-6.99 (m, 15H), 6.06 (s, 1H), healthcare) as described by the manufacturer.
3.63 (s, 3H), 3.25 (s, 4H); 13C NMR (75 MHz, CDCl3+DMSO-d6): δ Measurement of ICAM-1 and VCAM-1
188.7, 172.0, 168.8, 163.9, 155.4, 140.3, 137.7, 134.8. 134.1, 128.3, Cell-ELISA was used for measuring the expression of ICAM-1 and
126.7, 125.7, 122.1, 121.7, 121.1, 119.6, 119.0, 111.0, 109.9, 106.2, VCAM-1 on surface of endothelial cells (HUVEC) as described
104.0, 54.7, 54.3, 29.7; HRMS (ESI): m/z calculated for C43H36O6N7 earlier45. Briefly, HUVEC cells were incubated with or without
[M+H]+: 746.2721; found: 746.2711.
compound 11 at desired concentrations for the required period,
followed by treatment with TNF-α (10 ng/ml) for 16 h. The cells
were then fixed with 1.0% glutaraldehyde. Non-specific binding of
antibody was blocked by using skimmed milk (3.0% in PBS). Cells
were incubated overnight at 4 °C with anti-ICAM-1 mAb or anti-
VCAM-1 antibody, diluted in blocking buffer, the cells were further
washed with PBS and incubated with peroxidase-conjugated goat
anti-mouse secondary Abs. After washings, cells were exposed to
the peroxidase substrate (o-phenylenediamine dihydrochloride
40 mg/100 ml in citrate phosphate buffer, pH 4.5). Reaction was
stopped by the addition of 2 N sulfuric acid and absorbance at
490 nm was measured using microplate reader (Spectramax 190,
Molecular Devices, USA).
Animal : Healthy Swiss mice of both the sexes (n=25-30) were used
for pharmacokinetic study. The animals were kept under controlled
conditions (temp 26±2 oC; relative humidity 50±5%; 12h light/dark
cycle) and maintained on pelleted rodent diet (Ashirwad Industries
Ltd. Chandigarh, India). Water was provided ad libitum. Institutional
Animal Ethics committee approved the animal experiments.
Animals were fasted for 16 hours before use.
Phosphodiesterase (PDE-4) assay. PDE enzyme preparations were
performed from rat heart42 (. Heart was excised from anesthetized
rat and transfer into normal saline solution. Heart weighing 687 mg
was taken and homogenized in 2.5 volumes of homogenization
buffer containing 20 mM Tris-Cl, 50 mM NaCl, 2 mM EDTA, 0.1 mM
PMSF, 1mH DTT, protease inhibitors 1 µg/µl. The homogenate was
centrifuged at 500 X g for 5 minute at 4ºC. Then supernatant was
Neutrophil isolation
Neutrophils were isolated from peripheral blood of healthy
individuals as described earlier45
. Briefly, venous blood was
collected from healthy individuals in heparin solution (20 U/ml) and
erythrocytes were removed by sedimentation against 6% dextran
solution. Plasma, rich in white blood cells, was layered over Ficoll-
Hypaque solution, followed by centrifugation (300 g for 20 min,
20 °C). The top saline layer and the Ficoll-Hypaque layer were
aspirated leaving neutrophils/RBC pellet. The residual red blood
cells were removed by hypotonic lysis. Isolated cells were washed
with PBS and re-suspended in PBS containing 5 mM glucose, 1 mM
CaCl2, and 1 mM MgCl2 at a final concentration of 6 × 105 cells/ml.
Cell Adhesion assay
further centrifuged at 40,000
X g for 30 minute (ultra
centrifugation). After centrifugation, supernatant was collected for
phosphodiesterase assay. Protein concentration was estimated and
for each assay, 2.5 µg protein (PDE enzyme preparation) was used.
All compounds tested for PDE 4 activity were dissolved in DMSO.
Phosphodiesterase 4 assay was conducted with [3H] cAMP SPA
enzyme assay (GE Amersham, UK) using 2.5 µg enzyme preparation
for each well. Into each well 60 µl water, 10 µl assay buffer supplied
with the kit, 10 µl compound (1-13), 10 µl PDE enzyme or
This was performed as described earlier45. Briefly, the endothelial
homogenizing buffer (blank) and 10 µl radiolabel cAMP ([3H]cAMP, cells plated in 96-well culture plates were incubated with or without
GE Amersham, UK) were added. To check the activity of the compound 11 at desired concentrations for 2 h, followed by
induction with TNF-α (10 ng/ml) for 6 h. Endothelial monolayers
were washed with PBS and neutrophils (6 × 104/well) were added
over it and were allowed to adhere for 1 h at 37 °C. The non-
adherent neutrophils were washed with PBS and neutrophils bound
to endothelial cells were assayed by adding a substrate solution
consisting of o-phenylenediamine dihydrochloride (40 mg/100 ml in
citrate phosphate buffer, pH 4.5), 0.1% cetrimethyl ammonium
bromide, and 3-amino-1,2,4 triazole (1 mM). The absorbance was
read at 490 nm using an automated microplate reader (Model 680,
Bio-Rad, USA).
enzyme without inhibitor 10 µl DMSO was added in place of
inhibitor to a separate well. The reaction mixtures were incubated
for 30 min at 30ºC. The reaction was stopped by adding 50 µl SPA
bead supplied with the kit. It was then mixed well and allowed to
stand at room temperature for 20 min. Breakdown of cAMP in each
tube was monitored by β counter. The reading in each tube was
obtained in counts per minute (CPM). The reading of the blank tube
(without enzyme) was subtracted from each tube to normalize the
counts. The enzyme activity was calculated by considering the CPM
reading without any inhibitor (compound) as 100.
Pharmacokinetics
High throughput phosphodiesterase profiling
The compound 11 was administered orally (20 mg/kg) or through
intravenous route (10 mg/kg i.v) as a fine suspension in 1% Gum
acacia. Blood samples were drawn from retro-orbital plexus at
designated times in pre-labeled heparinised tubes and centrifuged
(3500 rpm x 10 minutes) to obtain the plasma. Blood samples were
collected at 0, 5, 15 and 30 min, and 1,2,4,6, 8, 16 and 24 hrs post-
administration after oral/ iv route.
To determine the inhibitory activity and selectivity of triazine-aryl-
bis-indole derivative on the PDE superfamily high throughput
profiling platform containing a panel of 13 isozymes was utilized
(CEREP, France). Inhibitory activity of compound 11 was examined
in 5 different concentrations (0.1, 1, 2, 5 and 10 µg/ml). Inhibitory
activity was expressed in terms of IC50 value obtained from the plot
of 5 different concentrations in duplicates.
Measurement of Cellular level of cAMP
43,44
Aliquots of plasma were mixed with acetonitrile (1:2), swirled for
two minutes (2500 rpm) and centrifuged at 5000 rpm for 10 min.
The organic layer was collected and evaporated to dryness using
solvent evaporator (Model: SPD 111V, Thermo Electron
Human Lung epithelial cell line (L-132) cells
were grown to
70% confluence. After overnight serum starvation, the cells were
pre-treated with the compound (10 µg/ml) for 5 min followed by
addition of forskolin, (40µM, Sigma aldrich) and the reactions were
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Corporation, MA). The dried samples were reconstituted in mobile
phase for HPLC analysis. In a similar manner, samples were also
prepared from aliquots of plasma collected from untreated animals
which were spiked with compound 11 (0.25 µg – 10 µg/ ml). These
were used to draw calibration curves which were found to be linear
(r2 = 0.9998). Reproducibility of the method was defined by both
intra-, and inter-day variance. The retention time of compound 11
was 10.688 min, at which no other interfering peak was observed.
The lower limit of detection (LOD) was 11 ng /ml; limit of
quantitation (LOQ) was 32 ng/ml; recovery was 90 ± 2%. HPLC
determinations were done on Shimadzu chromatograph (Model:
LC-10 Atvp equipped with a diode array detector). Compound 7 was
determined at 276 nm using RP-18 column (5 µm x 25 cm). Mobile
phase consisted of acetonitrile: water (55:45) with a flow rate of 1.0
2. M. D. Houslay, P Schafer, K. Y. J. Zhang.,Drug Discovery
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3. V. Dal Piaz, M. P. Giovannoni.,Eur. J. Med. Chem.2000,35,
463-480.
4. N. A. Molfino.Respiration.,2005, 72, 105-112.
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6. M. M.Teixeira, R. W Gristwood, N. Cooper, P. G. Hellewell.
Trends Pharmacol. Sci., 1997,18, 164-170.
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269-97.
9. W. M. Brown.,International Journal of COPD. 2007,2, 517-
533.
ml/min.
A concentration-time curve for compound 11 was
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established and pharmacokinetic parameters AUC, Cmax, and tmax
were determined by a non-compartmental analysis using TOPFIT
software package. Absolute bioavailability was determined using
the following formula: AUCoral x dose iv / AUC iv x Dose oral
.
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Cytotoxicity Assay: Basal cytotoxicity of compound 11 was
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Pauls, G. Liang, K.Sides, D. Cramer, J. Cairns, Y. Luo,
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performed to measure IC50. Balb/c 3T3 Neutral Red Dye Uptake
(NRU) cytotoxicity assay procedure was adopted46
. In brief, 3T3
cells were grown in 96 well microtitre plates and exposed to
compound 11, vehicle control or untreated vehicle control. After
48-hours incubation, the test material was removed, cells rinsed
twice with 250 µL of pre-warmed Dulbecco’s Phosphate Buffered
Saline (D-PBS) and thereafter incubated with Neutral red (NR)
medium at 37°C, 90% humidity and 5% CO2/Air for ~3h. Wells
showing NR crystalizations were rejected. After incubation, the NR
medium was removed, and cells rinsed with 250 µL pre-warmed D-
PBS and added 100 µL NR Desorb (ETOH/acetic acid) solution to all
wells, including blanks. NR was extracted from the cells by placing
the microtiter plate on a microtiter plate shaker for 30 minutes and
17.
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Choi, Molecules.,2013,.18, 6620-6662.
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R.D.Jones, C. Lane,R.A.Lewthwaite, S. Mantell, C. Perros-
Huguet, D.A.Price, R.Webster, Bioorg Med Chem
Lett., 2007,17, 6188-6191.
absorption measured at 540nm in
a microtiter plate reader
(spectrophotometer), using the blanks as a reference. A calculation
of cell viability expressed as NRU was made for each concentration
of compound 11 by using the mean NRU of 4 to 6 replicate values
per test concentration. The cell viability value was compared with
the mean NRU of all vehicle control values and relative cell viability
was then expressed as percent of untreated vehicle control.
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Cancer Ther. 2006,5, 3285-3293.
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269,326-338.
Statistical Analysis
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M. Sugiura, Y. Inoue, C.Fukaya, Y. Naito, T. Imagawa,
N. Nakamura,J Med Chem. 1998.,41, 2985-2993.
The data are presented as the mean ± S.E.M. All the experiments
were repeated independently at least 3 times. Data between two
groups were evaluated by Students t-test (independent) using
Microcal Origin 6.0 (Microcal Software Inc, MA, USA). A level of
p<0.05 was set as the threshold for statistical significance between
the control and various experimental groups.
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Conclusion
We demonstrate synthesis of novel triazine-aryl-bis-indole
derivatives which inhibit phosphodiesterase and stabilized cAMP in
in vivo conditions in association with inhibition of adhesion
molecules. These compounds might have therapeutic potential for
the treatment of various inflammatory diseases.
Acknowledgements
This work is supported by grants (NWP 033 and BSC 0116) from
Council of Scientific and Industrial Research, New Delhi, India.
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ARTICLE
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DOI: 10.1039/C5RA11495K
Journal Name
A
100
*
IICT/TAꢀ67
Rolipram
*
*
*
80
60
40
20
*
*
*
*
*
0
10
g/ml
20
µ
B
Figure 1
*
*
Figure 1. Inhibition of phosphodiesterase activity by triazine-aryl-
bis-indole’ compound in vitro. Enzyme preparations were
incubated in the absence or presence of different concentrations
(1-20 µg/mL) of compound 11 or rolipram and breakdown of cAMP
was monitored by β counter. Enzyme activity was expressed as
percentage of inhibition considering CPM value of enzyme without
inhibitor as 100. Data represents mean ± SEM of 3 experiments
*
*
(N=3).
*
indicates significantly different vs control without
compound.
Figure 3. Compound 11 inhibits TNF-α induced expression ICAM-1
and VCAM-1 on HUVECs. HUVECs were seeded in 96-well plates at
a cell density of 2x104 cells/well. Cells were pretreated with various
concentrations of compound 11 for 2 hours followed by induction
with TNF-α for 16 hours for ICAM-1 (A) and VCAM-1 (B). The cell
surface expression of cell adhesion molecules was measured by
ELISA. Experiment was repeated 3 times. Asterick mark indicate
significantly different compared to control experiment in the
absence of compound 11. P<0.05
120
100
80
60
40
20
0
TA67FSK
FSK
*
*
*
*
0
5
10
15
20
Time (min)
Figure 2. Effect of compound 11 on cellular cAMP level
Cellular level of cAMP was measured in L-132 cells pre-treated with
the compound (14.8 µM) for 5 min followed by addition of forskolin
(40µM). The reactions were terminated by the addition of ice-cold
95% ethanol at different time periods. The cells were harvested and
centrifuged to remove cell debris. Measurement of cAMP was
conducted using enzyme immunoassay kit (GE healthcare) as
described by the manufacturer. The experiment was repeated 3
times with different batch of cells in duplicate wells for each time
point. * denotes significantly different at P<0.01 vs corresponding
time points in the absence of the compound.
10 | J. Name., 2012, 00, 1-3
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Journal Name
ARTICLE
tubes and centrifuged (3500 rpm x 10 minutes) to obtain
the plasma. Concentration-time curves for compound 11
were established and pharmacokinetic parameters were
calculated as described in the experimental biology
section. Values are mean ± SEM (n = 6).
*
*
*
*
120
100
80
60
40
20
0
*
*
*
0
50
100
150
200
Comp 11 (mg/ml)
Figure 4. Compound 11 inhibits the adhesion of Neutrophils on
TNF-alpha induced HUVECs. HUVECs were seeded in 96-well plates
at a cell density of 2x104 cells/well. Cells were pretreated with
various concentrations of compound 11 for 2 hours followed by
induction with TNF-α for 6 hours. Human blood Neutrophils were
added onto the endothelial cells monolayer and allowed to adhere
for one hour at 37 oC. The number of cells that remained adhered
to the monolayer was estimated by measuring the peroxidase
activity. . *Significantly (P<0.05) different compared to control
experiment in the absence of compound 11.
Figure 6. Determination of IC50 of compound 11 on 3T3cells
using the NRU cytotoxicity assay. Cytotoxicity is expressed as a
concentration dependent reduction of the uptake of NR after
compound 11 exposure, thus providing a sensitive, integrated signal
of both cell integrity and growth inhibition.
A
120
96
72
48
24
Inflammation
ICAM/VCAM
Bronchodilation
AC
PDE
c AMP
AMP
ATP
0
24
0
2
4
6
8
16
Time (hrs)
HN
O
N
400
300
200
100
0
H
N
B
COOH
O
HN
COOH
N
N
NH
Comp 11
Figure 7. Schematic representation of probable mechanism of
action of Compound 11. Scheme depicts conversion of ATP to
cAMP by adenylate cyclase (AC) which is normally hydrolyzed to
0 30 60
240
120
adenosine
monophosphate
(AMP)
by
endogenous
phosphodiesterase (PDE). Compound 11 inhibits (┬) PDE and
helps maintaining increased (↑) level of cAMP which is known to
cause bronchodilation (23, 24, 27-30). Compound 11 also inhibits
(┬) cell adhesion molecules, ICAM and VACM which likely (broken
arrow) to help bronchodilation by preventing (down dashed
arrow) ongoing inflammatory process in respiratory diseases.
Time (min)
Figure 5. Pharmacokinetic profile of compound 11
Pharmacokinetics profiles of compound 11 either by oral
(A) administration (20 mg/kg) or through intravenous (B)
route (10 mg/ kg i.v). Blood samples were drawn from
retro-orbital plexus at indicated times in heparinised
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