In vivo effective dibenzo[b,d]furan-1-yl-thiazoles as novel PDE-4 inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.09.011

Herein we report the synthesis, PDE-4B and TNF-α inhibitory activities of a few dibenzo[b,d]furan-1-yl-thiazole derivatives. The hydroxycyclohexanol amide derivatives 14, 18, 24, 29, 31 and 33 exhibited promising in vitro PDE-4B and TNF-α inhibitory activities. Compound 24 showed good systemic availability in preclinical animal models and was also found to be non-toxic (exploratory mutagenicity test). Further it exhibited promising results in in vivo asthma/COPD and Uveitis models.

Full text of DOI:10.1016/j.bmc.2016.09.011

Accepted Manuscript
In-vivo effective Dibenzo[b,d]furan-1-yl-thiazoles as Novel PDE-4 Inhibitors
Gopalan Balasubramanian, Sukunath Narayanan, Lavanya Andiappan,
Thirunavukkarasu Sappanimuthu, Saravanan Thirunavukkarasu,
Shamundeeswari Sundaram, Saravanakumar Natarajan, Naresh Sivaraman,
Sridharan Rajagopal, Fakrudeen Ali Ahamed Nazumudeen, Sanjeev Saxena,
Santosh L. Vishwakarma, Shridhar Narayanan, Ganapavarapu V.R. Sharma,
ChidambaramV. Srinivasan, Narasimhan Kilambi
PII:
S0968-0896(16)30698-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.011
BMC 13264
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
22 August 2016
7 September 2016
Please cite this article as: Balasubramanian, G., Narayanan, S., Andiappan, L., Sappanimuthu, T., Thirunavukkarasu,
S., Sundaram, S., Natarajan, S., Sivaraman, N., Rajagopal, S., Nazumudeen, F.A.A., Saxena, S., Vishwakarma, S.L.,
Narayanan, S., Sharma, G.V.R., Srinivasan, ChidambaramV., Kilambi, N., In-vivo effective Dibenzo[b,d]furan-1-
yl-thiazoles as Novel PDE-4 Inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/
j.bmc.2016.09.011
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In-vivo effective Dibenzo[b,d]furan-1-yl-thiazoles as
Novel PDE-4 Inhibitors
Gopalan Balasubramaniana, Sukunath Narayanana, Lavanya Andiappana,
Thirunavukkarasu Sappanimuthua, Saravanan Thirunavukkarasua, Shamundeeswari
Sundarama, Saravanakumar Natarajana, Naresh Sivaramana, Sridharan Rajagopala,
Fakrudeen Ali Ahamed Nazumudeenb, Sanjeev Saxenab, Santosh L. Vishwakarmab,
Shridhar Narayananb, Ganapavarapu V. R. Sharmaa, ChidambaramV. Srinivasana,
Narasimhan Kilambia,*
a
b
Department of Medicinal Chemistry, Department of Biology, Drug Discovery
Research, Orchid Pharma Ltd, Chennai-600119, India.
* Corresponding author. Tel.: +91 (44) 24503137 x 609; fax: +91 (44) 24501650.
E-mail Address: narasimhank@orchidpharma.com/ k_narasimhan99@yahoo.com (or)
ipm@orchidpharma.com
A B S T R A C T
Herein we report the synthesis, PDE-4B and TNF- inhibitory activities of a few
Dibenzo[b,d]furan-1-yl-thiazole derivatives. The hydroxycyclohexanol amide
derivatives 14, 18, 24, 29, 31 and 33 exhibited promising invitro PDE-4B and TNF-
inhibitory activities. Compound 24 showed good systemic availability in preclinical
animal models and was also found to be non-toxic (exploratory mutagenicity test).
Further it exhibited promising results in invivo Asthma / COPD and Uveitis models.
Key Words: PDE-4 inhibitors; TNF-; Dibenzofuran.
1. Introduction
The cyclic nucleotide phosphodiesterases (PDEs) are a family of enzymes that
selectively catalyze the hydrolysis of 3’ cyclicphosphate bonds of the cyclic
adenosine monophosphate (cAMP) and/ or cyclic guanosine monophosphate (cGMP)
which in turn act as key regulators of many important physiological processes. There
are more than 200 splice isoforms for 21 human PDE genes that encode for the 12
PDE isoenzymes by variation in their amino acid sequence. The sequence identity
between different families is only 30-40%, but these isozymes differ enough in their
kinetic and physical characteristics, substrate selectivity, tissue distribution and sub-

cellular localization [1-4]. Many cell types express more than one PDE and
distribution of isoenzymes between the cells varies markedly.
Phosphodiesterase type 4 (PDE-4) is a cAMP-specific and Ca2+ independent
enzyme and hydrolyses cAMP in mast cells, basophils, eosinophils, monocytes and
lymphocytes. The human recombinant PDE-4 exists in four isoforms A, B, C and D.
Isoforms A, B, and D are expressed in many inflammatory cells such as eosinophils,
basophils, neutrophils, monocytes, mast cells, macrophages, T cells, CD4+, CD8+, B
cells, various smooth muscle cells, epithelial cells, endothelial cells and fibroblasts.
PDE-4C enzyme is mainly found in testis, skeletal muscle, the central nervous system
(CNS) and human fetal lung [5, 6].
Tumor necrosis factor (TNF-α), a cell signaling protein involved in systemic
inflammation, is one of the cytokines that makes the acute phase reaction. It is
produced chiefly by activated macrophages, although it can be produced by many
other cell types such as CD4+ lymphocytes, NK cells, neutrophils, mast cells,
eosinophils and neurons. It has been well demonstrated that TNF-α production in pro-
inflammatory cells becomes attenuated by an elevation of intracellular cyclic
adenosine 3’,5’-monophosphate (cAMP). This second messenger is regulated by the
PDE family of enzymes [7, 8]. Excessive or unregulated TNF-α production has been
implicated in mediating or exacerbating a number of undesirable physiological
conditions in diseases including osteoarthritis and other arthritic conditions septic
shock, endotoxic shock and respiratory distress syndrome. Since TNF-α also
participates in the onset and progress of autoimmune diseases, PDE-4 inhibitors may
find utility as therapeutic agents for rheumatoid arthritis, multiple sclerosis and
Crohn’s disease [9, 10].
Inhibition of the PDE-4 in these cells effectively elevates the intracellular
cAMP levels, thereby activating specific protein phosphorylation cascades and
regulating cellular functions. This in turn inhibits the release of inflammatory
mediators such as TNF-, interleukin-2 (IL-2), interleukin-12 (IL-12). The
association between cAMP elevation in inflammatory cells with airway smooth
muscle relaxation and inhibition of mediator release has led to widespread interest in
the design of PDE-4 inhibitors [11, 12]. Unfortunately, clinical utility of several
PDE-4 inhibitors is limited due to their undesirable side effect profile which includes
2

nausea, emesis and gastric acid secretion due to action on PDE-4 in parietal cells in
the gut [13, 14].
At present the most advanced PDE-4 inhibitor compounds are Roflumilast
(Nycomed/Forest/Tanabe) [15-17] and Cilomilast (GlaxoSmithKline) [18, 19], which
have been studied quite extensively. Roflumilast got EU and FDA approval for COPD
after completion of four Phase III trials world wide. Another PDE-4 inhibitor of
interest which has reached (Phase III) clinical trials is Tetomilast (Otsuka) [20] and is
reported to be in Phase III for the treatment of Crohn’s disease and ulcerative colitis
[21]. Apremilast (Celgene) [22-25] is a selective PDE-4 inhibitor with additional
activities inhibiting cytokines. Initially it was developed for asthma and COPD and is
now approved for psoriasis and other chronic inflammatory diseases. Oglemilast
(Glenmark/Forest/Teijin) [25, 26] a potent PDE-4 inhibitor which entered Phase IIb
clinical trials for asthma and COPD is now discontinued.
Other compounds of interest are Revamilast (Glenmark), MEM 1414
(Memory pharmaceuticals) which have entered Phase II clinical trials in the field of
inflammation. ELB353 (Biotie Therapies) exhibits good efficacy profile inhibiting
LPS-induced pulmonary neutrophilia in rats similar to that of Roflumilast. This
compound completed Phase I clinical trials with better adverse effect profile
compared to other competitors. Furthermore GPD 1116 (ASKA Pharma) and its
metabolite 1133 exhibited interesting preclinical results [25, 27-29].
3

Rationale of design: In the literature thiazole bearing compounds are found to
inhibit both PDE-IV and TNF- enzymes [30-32]. Tetomilast, a PDE-IV inhibitor
from Otsuka contains thiazole moiety in it [20]. As part of our program for
development of PDE-4 inhibitors in the field of inflammation, we came up with a new
series of dibenzofuran (Oglemilast/ Revamilast fragment) fused thiazole (Tetomilast
fragment) compounds carrying cyclohexyl (Cilomilast fragment) derivatives.
2. Results and discussion
2.1 Chemistry
To begin with the key synthon thioamides, were synthesised by following either
of two routes. Route-I, where direct installation of desired thioamide group on
dibenzofuran was carried out using potassium thiocyanate in acidic medium (yield,
70-80%). Route-II was followed for the compounds bearing a substitution at R3
position of second phenyl ring of dibenzofuran moiety. The desired starting
carboxylic acids for this route were prepared using literature procedure [33] and
converted to corresponding thioamides in three steps. Amidation of carboxylic acid
4

was performed using aqueous ammonia followed by treatment with TFAA to get
corresponding nitrile compounds. Reaction of phosphorous pentasulphide with these
nitrile compounds gave the key thioamides.
Thiazole moiety on dibenzofuran ring was constructed by reacting -
bromoketones
such
as
2-bromo-1-(4-methoxyphenyl)ethanone,
4-(2-
bromoacetyl)benzonitrile and ethyl 6-(2-bromoacetyl)picolinate with the thioamides
in presence of sodium bicarbonate in ethanol at room temperature (RT) (compounds
1-3), yield 30-46%. Esters of compounds 6, 25 and 28 were synthesised using
corresponding -bromoketones (ethyl 3-bromo-2-oxopropanoate/ ethyl 5-bromo-4-
oxopentanoate/ ethyl 4-bromo-3-oxobutanoate) and thioamides in dimethoxy ethane
and water at 80 °C (yield 50-73%), whereas, esters of 17 and 32 were synthesised
using ethyl 3-bromo-2-oxobutanoate with corresponding thioamide in presence of
potassium carbonate in ethanol and acetone at 80 °C (yield 10-15%). Ester bearing
compounds were hydrolysed to corresponding acids using potassium hydroxide in
ethanol - water mixture at RT to get compounds 4, 6, 25, 28 and 32 (yield 45-75%).
Some of these acids were made into amides 7-24, 26, 27, 29-31, 33 & 34 using
various aliphatic, alicyclic and aromatic amines in presence of a coupling agent [34].
5

6

7

2.2Biology
2.2.1 In-vitro experiments:
The synthesised Dibenzo[b,d]furan-1-yl thiazole derivatives were evaluated
for their ability to inhibit the PDE-4B enzyme (purified human enzyme) and
inhibition of production of TNF- in human Peripheral Blood Mononuclear cells
(PBMC). Interesting compounds were tested for their IC50 values and are summarized
in Table-I & II. Apremilast and Roflumilast were used as positive controls. GRC 3886
which belongs to dibenzofuran class of compounds inhibited the PDE-4 enzyme at
1.4nM and TNF at 190nM [25].
Compounds 1-4 (compound 4 is with tetomilast side chain), where R2 is an
aryl group connected directly to thiazole ring fused to dibenzofuran moiety exhibited
low to moderate PDE-4B inhibition (5 - 19.4%) at 100 nM concentration. Compound
5 where R2 is a simple ethyl ester retained moderate PDE-4B inhibition (17.6%) and
interestingly its acid 6 demonstrated a two fold increased activity (36.0%) at 100 nM
and three fold activity (80.4%) at 1000 nM concentration.
The carboxylic group of compound 6 was coupled with various aromatic, alicyclic
and aliphatic amines to prepare corresponding amides. Among all the aromatic
amides, compound 10 where R2 is N-(3,5-dichloropyridin-4-yl)acetamide, a side
chain of Oglemilast/ Roflumilast exhibited PDE-4B enzyme inhibitory activity of
70.3% at 100 nM concentration (IC50 48.4 nM) which is almost two folds more active
than the parent acid 6. A significant decrease in the activity was observed with
lengthening of carbon chain of these amides 15 (8.9%) and 16 (32.7%) at 100 nM
concentration. Replacement of aromatic amides with alicyclic amides increased the
enzyme inhibitory activities substantially. Particularly 4-hydroxycyclohexanol amides
14 and 18 exhibited PDE-4B enzyme inhibitory activity of 71.8% (IC50 49.5 nM) and
62.6% (IC50 43.8 nM). No significant change in inhibitory activity was observed for
substituting R1 = H or R1 = CH3 on the thiazole ring.
All the interesting compounds (includes all the cyclohexanolamides and few
selective compounds based on its side chain) were tested for inhibition of production
of TNF- (IC50) in human Peripheral Blood Mononuclear Cells (PBMC). Except
compound 26, all the cyclohexanol amides 14 (37.7 nM), 18 (27.7 nM), 24 (31 nM),
29 (39.6 nM), 31 (18.4 nM) and 33 (54.4 nM) showed the inhibitory activity (IC50) at
nano molar concentrations (nM) whereas all other tested compounds 9 (0.386M), 10
8

(1.75M), 16 (2.78M) and 21 (2.21M) exhibited the TNF- inhibitory activity at
micro molar concentrations (M).
Table-I: PDE-IVB and TNF-inhibitory data of compounds 1-20
PDE-4 inhibition nM
(human PDE-4B)
TNF- inhibition nM
(human PBMC)
S.NO
R1
R2
1000
41.5
100
IC50
ND
1000
ND
100
ND
IC50
ND
1.
2.
H
H
19.4
15.8
5.2
ND
ND
ND
ND
15.6
26.4
5.8
ND
ND
ND
ND
ND
ND
ND
ND
3.
4.
H
H
18.2
24.4
80.4
7.4
17.6
36.0
2.4
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
5.
6.
7.
H
H
H
5.2
1.2
ND
15.81
2.98
ND
8.
H
43.8
92
15.4
70.3
ND
69.8
34.2
35.5 385.7
9.
H
H
48.4
ND
ND
1753
ND
10.
59.3
25.8
ND
ND
11.
H
9

52.9
ND
21.9
39.6
ND
ND
ND
22
ND
ND
ND
ND
12.
13.
H
H
ND
71.8
49.5
ND
63.2
37.7
14.
H
38.5
87.8
8.9
ND
ND
ND
21
ND
8
ND
15.
16.
H
H
32.7
2780
ND
ND
24.6
62.6
ND
23.8
9.9
ND
17.
18.
-CH3
-CH3
43.8
103.1
82.7
27.7
ND
ND
1.6
ND
ND
39.1
9.9
21.9
0.9
ND
ND
19.
20.
-CH3
-CH3
11.1
*ND: Not done
Table-II: PDE-IVB and TNF- inhibitory data of compounds 21 - 34
PDE-4 inhibition nM
(human PDE-4B)
TNF- inhibition nM
(human PBMC)
S.NO
R1
R2
R3
10

1000
88.6
100
IC50
1000
100
IC50
42.3 127.1 28.3
14.5 2208
21.
H
H
ND
ND
ND
ND
ND
31.9
ND
26.7
8.5
ND
ND
22.
23.
H
H
H
H
35.7
ND
65.3
63.5
93.8
77.7
31
24.
H
H
ND
ND
59.3
51.5
ND
ND
ND
ND
-4.2
ND
ND
25.
26.
H
H
CF3
CF3
17.2
ND
27.4
ND
ND
14
ND
27.
H
CF3
ND
ND
56.5
69
ND
ND
15.5
83.4
ND
28.
29.
H
H
NHCOMe
NHCOMe
40.4
91.9
39.6
ND
ND
43.4
67
ND
ND
25.1
78.6
ND
30.
31.
H
H
NHSO2Me
NHSO2Me
31.4
97.8
18.4
ND
ND
62.6
67.6
ND
ND
ND
ND
32.
33.
CH3
CH3
NHSO2Me
NHSO2Me
24.7
92.8
61.8
54.4
ND
81.2
ND
ND
35.4
ND
34.
CH3
NHSO2Me
11

ND
ND
ND
ND
74
ND
ND
ND
ND
77
Apremilast
Roflumilast
0.2
4.3
1.4
190
GRC 3886 (lit. value)
*ND: Not done
In Table-III & Figure-I interesting compounds have been grouped with
varying substitution at R2 position and their activities for easy reference. This table
includes the compounds bring the side chains selected from some of the standard
PDE-4 inhibitors viz. Tetomilast (pyridyl carboxylic acid), Rouflumilst/ Oglemilast
(dichloropyridyl amide) and Cilomilast (cyclohexane).
Table-III: PDE-IVB and TNF- inhibitory data of Interesting compounds
PDE-4 inhibition nM
(human PDE-4B)
TNF- inhibition nM
S.NO
(As
allotted
in Table-
(human PBMC)
R
R1
R2
R3
I and II)
100
IC50
100
ND
IC50
ND
Tetomilast side chain bearing (at R2) compound:
18.2 ND
-OCH3
-H
-H
4.
Roflumilast / Oglemilast side chain bearing (at R2) compounds:
70.3
11.1
42.3
43.4
48.4
ND
ND
0.9
1753
ND
-OCH3
-OCH3
-H
-CH3
-H
-H
-H
10.
20.
21.
30.
127.1
ND
14.5
25.1
2208
ND
-OCHF2
-OCHF2
-H
-NHSO2Me
-H
Cyclohexanol side chain bearing (at R2) compounds:
71.8
62.6
65.3
49.5
43.8
63.5
63.2
82.7
77.7
37.7
27.7
31
-OCH3
-OCH3
-H
-CH3
-H
-H
-H
-H
14.
18.
24.
-OCHF2
12

51.5
69
ND
40.4
31.4
24.7
17.2
83.4
78.6
61.8
ND
39.6
18.4
54.4
-OCHF2
-OCHF2
-OCHF2
-H
-H
-H
-CF3
26.
29.
31.
33.
-NHCOMe
-NHSO2Me
-NHSO2Me
66.9
67.6
-OCHF2 -CH3
PDE-4 and TNF- activities of interesting cyclohexanol amides (IC50 nm)
100
90
80
70
60
50
40
30
20
10
0
PDE-4 activity
TNFa activity
ND
t
t
s
a
l
i
s
14
18
24
26
29
31
33
a
l
i
m
m
u
l
e
f
r
Compound
o
p
A
R
Figure-I: Invitro enzyme inhibitory activities of interesting compounds
Mutagenicity Test – Exploratory
Compound 24 was subjected to Salmonella typhimurium reverse mutation assay and
found it is non-mutagenic and non-cytotoxic up to 5mg/plate (Table-IV).
Table-IV: Mutagenicity test results of compound 24
TA98 and TA100
Strains used
0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5 & 5
Rat liver S9 fraction
Dose Concentrations (mg/plate)
Metabolic activation
Result
Non-mutagenic and non-cytotoxic up to 5 mg/plate
Ames (TA 98 & TA 100) - Negative
2.2.2 In-vivo experiments:
In the process of lead compound identification a few invitro active molecules
were subjected to following preliminary invivo experiments:
Exp 1. Asthma and COPD - Rat model
13

Exp 2. Estimation of TNF-- Mice model
Further compound 24 was explored with the following other four in-vivo experiments.
Exp 3. Pharmoco Kinetics (PK) - Rat model
Exp 4. LPS induced total cell count - Rat model
Exp 5. LPS induced Uveitis - Rat model and
Exp 1: LPS induced Neutrophil influx in BALF of SD rats for asthma and COPD
LPS induced neutrophilia for asthma and COPD was performed in male
Sprague Dawley rats with selected in-vitro active compounds, the details such as oral
dosage, LPS induced neutrophilia (% of inhibition) and number of animals grouped
for the experiments are tabulated in Table-V.
Table-V: LPS induced Neutrophil influx in BALF of SD rats after 6 h (n = 8)
Compound No.
24
Dose
(mg/kg)
LPS induced neutrophilia
(% inhibition)
10
5
58
35.7
51.2
19.3
73.2
29
33
10
10
10
Roflumilast
Exp 2: LPS induced sepsis model in mice for the measurement of TNF-
The LPS induced sepsis model was performed in female Swiss Albino mice for
the measurement of TNF-. The fasted mice were dosed orally with the test
compound at 10 mg/kg body weight; the percentage of inhibition was tabulated in
Table-VI. Out of the test compounds 10 (37.8%); 14 (46.7%) and 23 (40.7%)
exhibited similar activity though they showed different invitro potency, whereas
compound 24 efficiently inhibited 82.2% of the enzyme at much lower concentration
(3mg/kg body weight).
Table-VI: LPS induced TNF-α in Swiss Mice (n = 6)
Compound No. Dose (mg/kg, p.o.)
TNF- Inhibition (%)
10
10
37.8
46.7
10
14
14

10
0.3
1
40.7
16.4
34.2
82.2
59.2
53.9
72.5
23
24
3
3
Roflumilast
GRC 3886
1
10
Exp 3: Pharmacokinetic parameters of compound 24 in male wistar rats
Following a single dose oral and intravenous administration, the plasma
concentration of compound 24 declined in a time dependent manner, with the
pharmacokinetic parameters adequately described by non-compartmental analysis
against plasma concentration versus time data. Compound 24 is absorbed slowly and
peak concentrations were reached 4.8 h post-dose, when studied at pharmacological
dose in rats (Table-VII). Volume of distribution after intravenous administration was
about 0.94 L/kg and it was about 1.5 times of the total body water in rats, indicating
that it may be distributed in extra cellular as well as intracellular fluid. Clearance after
intravenous administration of compound 24 in rat was 0.35 L/h/kg (Figure-II). The
Clearance was low in rat which was about 10% of the rat liver blood flow. Compound
24 has a terminal half-life of about 1.9 h in rats. The oral bioavailability of compound
24 was studied in rat at 10 mg/kg and the absolute oral bioavailability is about 42%.
Table-VII : Pharmacokinetic parameters of compound 24 in male wistar rats
Estimates
Parameters
Unit
Oral
I.V
mg/kg
hr
10
4.8
1.4
-
5
-
Dose
Tmax
Cmax
Co
mg/L
mg/L
hr*mg/L
L/kg
L/hr/kg
Hr
-
4
11.5
-
13.7
1
AUClast
Vd
-
0.4
1.9
CL
5.0
t1/2
%
42
F
15

Figure-II. Mean plasma concentration profile of Compound 24 (oral and intravenous
administration) in male wistar rats
Exp 4: LPS induced Total cell count in BALF of SD rats
A single dose oral administration of compound 24 at 10 mg/kg concentration
produced inhibition of 75.2% on par with the internal standard Roflumilast which
showed the inhibition of 73.2% of LPS induced total cells BALF of male SD rats after
6 h. The explorative dose response studies of the compound produced inhibition of
52.8, 34.6, 60.7, 70.9, 75.2 and 78.7% at concentrations 0.1, 0.3, 1, 3, 10 and 30
mg/kg respectively (Figure-III). Flattening of inhibition was observed from 3mg/kg
body weight onwards and maximum inhibition (78.7%) was observed at 30mg/kg
weight.
Normal
LPS (100 g/mL)
Compound 24 (0.1 mg/kg,p.o.)
Compound 24 (0.3 mg/kg,p.o.)
Compound 24 (1 mg/kg,p.o.)
15.0
##
Compound 24 (3 mg/kg,p.o.)
12.5
Compound 24 (10 mg/kg,p.o.)
Compound 24 (30 mg/kg,p.o.)
Roflumilast (10 mg/kg,p.o.)
10.0
7.5
5.0
2.5
0.0
@
**
**
**
**
**
Treatment
Figure-III. Compound 24 - LPS induced Total cell count in BALF of SD rats
Exp 5: LPS induced Uveitis in Female Wistar Rats
Endotoxin-induced uveitis (EIU) was induced in male Wistar rats by a footpad
injection of lipopolysaccharide (LPS). LPS 250 µg/animal was injected to both left
16

and right hind paw (each hind paw receives 125 µg). After 24 hr of LPS injection, the
animal was injected orally with the Compound 24 @ 0.1, 0.5, 1 and 3 mg/kg and
Dexamethsone @ 10 µg/10 µL/eye. One hr after drug administration, aqueous humor
was collected from both eyes and total number of infiltrating cells was counted using
a hemacytometer and the percentage of inhibition was calculated. Compound 24
produced 43.6, 54.4, 90.3, and 96.9 % inhibition at 0.1, 0.5, 1 and 3 mg/kg
respectively whereas Dexamethasone @ 10 µg/10µL/eye showed 79.1 % inhibition
(Figure- IV). The number of infiltrating cells in the anterior segment of the eye was
reduced in rats treated with Compound 24 (**p<0.01) when compared with that with
LPS and vehicle treatment (nꢀ=ꢀ10). The results showed the total cell number in
aqueous humor was significantly reduced at the oral dose of 1 mg/kg (p<0.01) itself
and there was only slight increase in the response at 3 mg/kg. Preliminary result of
this study shows that oral administration of Compound 24, at the dose of 1 mg/kg,
produces anti-inflammatory effects against LPS-induced ocular inflammation.
120
Normal
##
100
80
60
40
20
0
LPS 250ug/animal
Compound 24 @ 0.1mg/kg p.o.
Compound 24 @0.5mg/kg p.o.
Compound 24 @ 1mg/kg p.o.
Compound 24 @ 3mg/kg p.o.
Dexamethasone 10ug/10ul/eye
*
**
**
**
**
Treatment
Figure-IV. Compound 24 - LPS induced Uveitis in female wistar rats
3. Conclusion
Thirty four new Dibenzo[b,d]furan-1-yl-thiazole derivatives (1-34) were synthesised
and tested their PDE-4B and TNF- inhibitory activities. The promising invitro
potent compounds were screened for their metabolic stability in mouse liver
microsomes (MLM), human liver microsomes (HLM) and they exhibited moderate to
good stability in both microsomes (data not shared). The invivo PK experiments (both
oral and intravenous) on compound 24 shows good systemic availability. Hence this
compound was screened for Asthma/ COPD and TNF- inhibition in mice and the
results are encouraging. Moreover, the results from LPS induced Uveitis on this
compound 24 were found to be interesting and the exploratory mutagenicity test
17

reveals that the compound is non toxic. Further developmental studies on this
compound and the backup (31) are underway.
Experimental
4.1. Chemistry
Starting materials were procured from Sigma Aldrich and Johnson Matthey,
Alfa aesar (lab grade) and are used as such without further purification. Penultimate
compounds were purified by silica gel Column chromatography (silica gel 60-100
mesh, E. Merck). All the solvents were from commercial sources and are dried
according to standard procedures. Reaction progress was monitored by thin layer
chromatography (TLC) silica on aluminium sheet obtained from Merck. Melting
points were recorded on a Buchi apparatus and are uncorrected. IR spectra (KBr
pellets) were recorded on a JASCO spectrometer and frequencies were expressed in
cm-1. Mass spectra (GC/MS) were recorded on Agilent MSD VL mass spectrometer
1
and M+1 of the compounds were expressed in %. H NMR spectra were recorded on
a Bruker Avance 400 MHz spectrometer in CDCl3 or DMSO-d6 solution using TMS
as an internal standard and the chemical shifts are reported in ppm (δ). Coupling
constants (J) are expressed in Hz.
4.2 Preparation of key synthon thioamides:
Two routes were followed to synthesise the thioamides, Route-I where R3 is
only hydrogen and Route-II for compounds substituted with either of -CF3 or -NO2
group. The nitro group was further reduced to amine as depicted in the schemes
described.
Route – I: Synthesis of 4-methoxy dibenzo[b,d]furan-1-carbothioamide:
To a mixture of 4-methoxy dibenzo[b,d]furan (1 g, 5.05 mmol) in methane
sulphonic acid (11 mL), was added potassium thiocyanate (1 g, 10.29 mmol) at 0-5
°C and the reaction mass was stirred at RT. The reaction mixture was poured into
crushed ice and the solid obtained was filtered. It was triturated with n-hexane and
dried to get a pale brown coloured solid (0.9 g). Yield - 70%; TLC - Rf 0.25 (EtOAc:
1
n-Hexane, 3:7); HPLC purity - 91.4%; H-NMR (400MHz, CDCl3): δ 4.08 (s, 3H, -
OCH3), 6.97 (d, J = 8.2 Hz, 1H, Ar-H), 7.28 (m, 1H, Ar-H), 7.35 (m, 1H, Ar-H), 7.65
(d, J = 8.2 Hz, 1H, Ar-H), 7.69 (d, J = 7.8Hz, 1H, Ar-H), 7.94 (d, J = 8.0Hz, 1H, Ar-
H), 8.31 (d, J = 8.2 Hz, 1H, Ar-H); Mass m/z: 258.1 (M+1).
18

Route – II: General procedure for the thioamides with a substitution of –CF3 and –
NO2 at R3.
Step1: Synthesis of 4-(difluoromethoxy)dibenzo[b,d]furan-1-carboxylic acid:
Followed the procedure from reference 33.
Step 2: Synthesis of 4-(difluoromethoxy)dibenzo[b,d]furan-1-carboxamide
To a solution of 4-(difluoromethoxy)dibenzo[b,d]furan-1-carboxylic acid (8.7
mmol) in THF (5 mL), was added triethylamine (21.5 mmol) followed by
ethylchloroformate (41.9 mmol) at 0-5 °C and the reaction mass was stirred at the
same temperature for 2 h. Aqueous ammonia (5 mL) was added and the mass was
kept stirring overnight at RT. The reaction mixture was poured into crushed ice and
the solid settled was filtered. It was triturated with n-hexane and dried to furnish the
amide. Yield: 55 - 70%; TLC - Rf 0.45 to 0.55 (Chloroform: methanol, 9:1); HPLC
purity: 90 - 95%.
Step 3: Synthesis of 4-(difluoromethoxy)dibenzo[b,d]furan-1-carbonitrile:
To a solution of 4-(difluoromethoxy)dibenzo[b,d]furan-1-carboxamide (0.34
mmol) in DCM (5 mL), was added triethylamine (1.1 mmol) followed by
trifluoroacetic anhydride (1.1 mmol) at 0-5 °C and the reaction mass was stirred at RT
for 2 h. The reaction mixture was poured into cold water and extracted with MDC
(2x25 mL). Organic layer was further washed with brine and dried over anhydrous
sodium sulphate. The organic layer was concentrated under reduced pressure to give
the desired nitrile compound. Yield: 70- 85%; TLC - Rf 0.31 (EtOAc: n-Hexane, 3:7);
HPLC purity: 70 - 80%.
Step 4: Synthesis of 4-(difluoromethoxy)dibenzo[b,d]furan-1-carbothioamide:
Phosphorous pentasulfide (49 mmol) in ethanol (10 mL) was refluxed for 30
minutes and 4-(difluoromethoxy)dibenzo[b,d]furan-1-carbonitrile (R’ = -CF3 or -NO2,
9.8 mmol) was added and refluxing was continued for 6 h. The reaction mixture was
concentrated under reduced pressure, cold water (50 mL) was added and extracted
with EtOAc (2x 50 mL). Organic layer was washed with brine and dried over sodium
sulphate and concentrated under reduced pressure to get the corresponding thioamide.
Yield: 50-70%; TLC - Rf 0.25 (EtOAc: n-Hexane, 3:7); HPLC purity: 85 - 90%.
4.3 Synthesis of 2-(4-methoxydibenzo[b,d]furan-1-yl)-4-(4-methoxyphenyl)
thiazole (1):
19

General procedure-A: To a stirred solution of 4-methoxydibenzo[b,d]furan-1-
carbothioamide (50 mg, 0.194 mmol) in ethanol (5 mL) was added sodium
bicarbonate
(15
mg,
0.179
mmol)
followed
by
2-bromo-1-(4-
methoxyphenyl)ethanone (45 mg, 0.196 mmol) and the mixture was stirred at RT for
1 h. Subsequently the reaction mixture was poured into cold water (25 mL) and the
obtained precipitate was filtered to get a peach coloured solid (30mg), Yield - 40%;
1
TLC - Rf 0.62 (EtOAc : Hexane 3:7); HPLC purity - 98.6%; M.P.153-156 °C; H-
NMR (CDCl3): δ 3.88 (s, 3H, -OCH3), 4.13 (s, 3H, -OCH3), 7.00-7.06 (m, 3H, Ar-H),
7.28-7.32 (m, 3H, Ar-H), 7.46-7.52 (m, 2H, Ar-H), 7.63-7.67 (m, 2H, Ar-H), 8.02 (d,
J = 8.72 Hz, 2H, Ar-H), 8.83 (d, J = 7.96 Hz, 1H, Ar-H);Mass m/z: 388.1 (M+1).
4.4 Synthesis of 4-(2-(4-methoxydibenzo[b,d]furan-1-yl)thiazol-4-yl) benzonitrile (2):
Prepared by General procedure-A using 4-(2-bromoacetyl)benzonitrile in the
reaction. Pale pink coloured solid, Yield - 50%; TLC - Rf 0.32 (EtOAc : n-Hexane,
3:7); HPLC purity - 94.8%; M.P. 212-215 °C; 1H-NMR (400 MHz, CDCl3): δ 4.15 (s,
3H, -OCH3), 7.07 (d, J = 8.44 Hz, 1H, Ar-H), 7.30 (d, J = 7.84 Hz, 1H, Ar-H), 7.51-
7.55 (m, 1H, Ar-H), 7.64-7.70 (m, 2H, Ar-H), 7.76-7.81 (m, 3H, Ar-H), 8.19 (d, J =
8.28 Hz, 2H, Ar-H), 8.72 (d, J = 8 Hz, 1H, Ar-H);Mass m/z: 383.1 (M+1).
4.5 Synthesis of ethyl 6-(2-(4-methoxydibenzo[b,d]furan-1-yl)thiazol-4-yl)
picolinate (3):
To a stirred solution of 4-methoxydibenzo[b,d]furan-1-carbothioamide (140
mg, 0.544 mmol) in ethanol (5 mL) was added sodium bicarbonate (60 mg, 0.714
mmol) followed by ethyl 6-(2-bromoacetyl)picolinate (150 mg, 0.551 mmol) and the
mixture was stirred at RT for 20 h. Subsequently the reaction mixture was poured into
cold water (25 mL) and extracted with ethyl acetate (2x25 mL). Organic layer was
dried over anhydrous sodium sulphate, filtered and evaporated at reduced pressure,
which was further purified by column chromatography using a gradient of EtOAc in
hexane (0-6%) to get a pale pink colour solid (0.065 g); Yield - 30%; TLC - Rf 0.5
1
(EtOAc: n-Hexane, 3:7); HPLC purity - 98.1%; M.P - N/R; H-NMR (400 MHz,
CDCl3): δ 1.48-1.51 (m, 3H, -CH3), 4.14 (s, 3H, -OCH3), 4.50-4.55 (m, 2H, -OCH2),
7.07 (d, J = 8.32 Hz, 1H, Ar-H), 7.49-7.53 (m, 1H, Ar-H), 7.65-7.69 (m, 2H, Ar-H),
7.94-7.98 (m, 1H, Ar-H), 8.09 (d, J = 7.6 Hz, 1H, Ar-H), 8.42 (s, 1H, Ar-H), 8.47 (d,
J = 7.76 Hz, 1H, Ar-H), 8.73 (d, J = 7.76 Hz, 1H, Ar-H); Mass m/z: 431.1 (M+1).
4.6 Synthesis of ethyl 2-(4-methoxydibenzo[b,d]furan-1-yl)thiazole-4-carboxylate
(5):
20

General procedure-B: To a stirred solution of 4-methoxydibenzo[b,d]furan-1-
carbothioamide (0.584 mmol) in dimethoxyethane and water (2:1) was added ethyl 3-
bromo-2-oxopropanoate (0.717 mmol) at RT. After 10 minutes, the reaction mixture
was refluxed at °C for 2 h, subsequently it was poured into cold water (25 mL) and
the precipitate obtained was filtered to get white solid, Yield - 73%; TLC - Rf 0.53
(EtOAc : n-Hexane, 3:7); HPLC purity - 97.4%; M.P. 151-154 °C; 1H-NMR (CDCl3):
δ 1.50-1.53 (m, 3H, -OCH2CH3), 4.13 (s, 3H, -OCH3), 4.48-4.53 (m, 2H, -CH2), 7.04
(d, J = 8.4 Hz, 1H, Ar-H), 7.35-7.39 (m, 1H, Ar-H), 7.50-7.55 (m, 1H, Ar-H), 7.65-
7.69 (m, 2H, Ar-H), 8.24 (s, 1H, Ar-H), 9.20 (d, J = 8 Hz, 1H, Ar-H); Mass m/z:
354.1 (M+1).
4.7 Synthesis of ethyl 3-(2-(4-methoxydibenzo[b,d]furan-1-yl)thiazol-4-yl)
propanoate (5a):
Prepared by General procedure-B using ethyl 5-bromo-4-oxopentanoate in
place of ethyl 3-bromo-2-oxopropanoate, white solid, Yield - 68%. Mass m/z: 382.1
(M+1).
4.8 Synthesis of ethyl 2-(2-(4-methoxydibenzo[b,d]furan-1-yl)thiazol-4-yl)acetate
(5b):
Prepared by General procedure-B using ethyl 4-bromo-2-oxobutanoate in
place of ethyl 3-bromo-2-oxopropanoate. White solid, Yield - 53%. Mass m/z: 368.1
(M+1).
4.9 Synthesis of ethyl 2-(4-methoxydibenzo[b,d]furan-1-yl)-5-methylthiazole-4-
carboxylate (5c):
General procedure-C: To a stirred solution of 4-methoxydibenzo[b,d]furan-1-
carbothioamide (1.0 mmol) in ethanol (5 mL) was added potassium carbonate (1.5
mmol) followed by 3-bromo-2-oxobutanoate (1.2 mmol) at RT. The reaction mixture
was refluxed at °C for 2 h, subsequently it was poured into cold water (25 mL) and
the precipitate obtained was filtered to get a peach coloured solid, Yield -10%; Mass
m/z: 340.0 (M+1).
4.10 Synthesis of ethyl 2-(4-difluoromethoxydibenzo[b,d]furan-1-yl)-5-
methylthiazole-4-carboxylate:
21

Prepared by General procedure-C using 4-difluoromethoxydibenzo[b,d]furan-
1-carbothioamide in place of 4-methoxydibenzo[b,d]furan-1-carbothioamide to get
peach coloured solid. Yield - 15%. Mass m/z: 404.09 (M+1).
4.11 General procedure for the hydrolysis of esters to acids (4, 6 Carboxylic acids
of 15-24, 25, 28 and 32)
4.11.1 Synthesis of 6-(2-(4-methoxydibenzo[b,d]furan-1-yl)thiazol-4-yl)picolinic
acid (4)
General procedure-D: To a slurry of ethyl 6-(2-(4-methoxy dibenzo[b,d]furan-1-
yl)thiazol-4-yl) picolinate (50 mg, 0.116 mmol) in ethanol (5 mL) was added
potassium hydroxide (30 mg, 0.535 mmol) followed by water (0.5 mL) and the mass
was stirred at RT for 2 h. Subsequently the reaction mixture was poured into cold
water, acidified with 1N HCl to a pH of 3-4; and the precipitated solid was filtered
and triturated with ether followed by n-hexane to give a off-white solid; Yield - 42%;
1
TLC - Rf 0.23 (CHCl3: MeOH, 8.5:1.5); HPLC purity- 96%; M.P. N/R; H-NMR
(400MHz, CDCl3): δ 4.15 (s, 3H, -OCH3), 7.09 (d, J = 8.4 Hz, 1H, Ar-H), 7.26-7.32
(m, 1H, Ar-H), 7.51-7.55 (m, 1H, Ar-H), 7.67-7.71 (m, 2H, Ar-H), 8.08-8.12 (m, 1H,
Ar-H), 8.24 (d, J = 6.56 Hz, 2H, Ar-H), 8.56 (d, J = 7.84 Hz, 1H, Ar-H), 8.75 (d, J =
7.92 Hz, 1H, Ar-H); Mass m/z: 403.1(M+1).
4.11.2 2-(4-Methoxydibenzo[b,d]furan-1-yl)-1,3-thiazole-4-carboxylic acid (6)
Off-white solid, Yield - 70%; TLC - Rf 0.38 (CHCl3: MeOH, 9:1); HPLC
purity - 98.5%; M.P. 228-230 °C ; 1H-NMR (DMSO-d6): δ 4.08 (s, 3H, -OCH3), 7.33-
7.39 (m, 2H, Ar-H), 7.58-7.62 (m, 1H, Ar-H), 7.78 (d, J = 8.28 Hz, 1H, Ar-H), 7.85
(d, J = 8.44 Hz, 1H, Ar-H), 8.58 (s, 1H, Ar-H), 9.27 (d, J = 7.96 Hz, 1H, Ar-H);
Mass m/z: 326.0 (M+1).
Followed the same procedure in synthesising the other acids 6a and 6b.
4.11.3
2-[4-(Difluoromethoxy)-8-(trifluoromethyl)dibenzo[b,d]furan-1-yl]-1,3-
thiazole-4-carboxylic acid (25)
Pale brown coloured solid, Yield - 76%; TLC - Rf 0.41 (CH2Cl2 : MeOH, 9:1);
1
HPLC purity - 94.1%; M.P. 246-248 °C; H-NMR (DMSO-d6): δ 7.42-7.59 (m, 2H,
Ar-H), 8.02-8.09 (m, 3H, Ar-H), 8.69 (s, 1H, Ar-H), 10.02 (s, 1H, Ar-H), 13.38 (s,
1H, -COOH);Mass m/z: 430.0 (M+1)..
4.11.4 2-[8-(Acetylamino)-4-(difluoromethoxy)dibenzo[b,d]furan-1-yl]-1,3-thiazole-
4-carboxylic acid (28)
22

Off-white solid, Yield - 77%; TLC - Rf 0.29 (CH2Cl2 : MeOH, 8:2); HPLC
1
purity – 96.8%; M.P. 301.5-303.5 °C; H-NMR (DMSO-d6): δ 2.03 (s, 3H, -
NHCOCH3), 7.37-7.56 (m, 2H, Ar-H), 7.74-7.78 (m, 3H, Ar-H), 8.62 (s, 1H, Ar-H),
8.78 (s, 1H, Ar-H), 9.90 (s, 1H); Mass m/z: 417.0 (M-1).
4.11.5
2-(4-(Difluoromethoxy)-8-(methylsulfonamido)dibenzo[b,d]furan-1-yl)-5-
methyl-1,3-thiazole-4-carboxylic acid (32)
Off-white solid, Yield - 45%; TLC - Rf 0.49 (CH2Cl2 : MeOH, 9:1). HPLC
1
purity - 94.7%; M.P. 278-283 °C; H -NMR (DMSO-d6): δ 2.84 (s, 3H, -CH3), 2.97
(s, 3H, -SO2CH3), 7.37-7.56 (m, 3H, Ar-H), 7.74 (d, J = 8.52 Hz, 1H, Ar-H), 7.83 (d,
J = 8.84 Hz, 1H, Ar-H), 8.74 (d, J = 2.16 Hz, 1H, Ar-H), 9.61 (s, 1H, Ar-H), 13.14
(s, 1H, -COOH); Mass m.z: 469.0 (M+1).
4.12 General procedure for the Synthesis of amides (7-24, 26, 27, 29-31 & 32).
Acid compound (6, 6a, 6b, 6c or 25 or 28 or 32; 1.0 mmol), EDCI (1.0 mmol),
HOBt (1.0 mmol), DMAP (0.5 mmol) and DIPEA (5 mmol) were taken in DMF (4
mL for each 100 mg of acid), stirred for 20 minutes and cooled to 0 °C.
Corresponding amine (1.2 mmol) was added and the temperature was brought to RT.
The reaction mixture was stirred till completion of the reaction, cold water was added
and the precipitate settled was filtered. If the precipitate could not be filtered, it was
treated with MDC (3x 25 mL) and water (30 mL). The organic layer was separated,
washed with brine solution, dried over anhydrous sodium sulphate and concentrated
under reduced pressure. The resulting crude product was purified by column
chromatography over silica gel using MDC/MeOH solvent combinations (up to 10%
MeOH in MDC) to get the corresponding title compounds.
4.12.1 2-(4-Methoxydibenzo[b,d]furan-1-yl)-N-phenyl-1,3-thiazole-4-carboxamide (7)
Pale yellow coloured solid, Yield - 31%; TLC - Rf 0.53 (EtOAc : n-Hexane,
°
1
3:7); HPLC purity - 99.2%; M.P. 206-208 C; H-NMR (CDCl3): δ 4.16 (s, 3H, -
OCH3), 7.09 (d, J = 8.36 Hz, 1H, Ar-H), 7.14-7.18 (m, 1H, Ar-H), 7.31-7.41 (m, 3H,
Ar-H), 7.53-7.57 (m, 1H, Ar-H), 7.62 (d, J = 8.32 Hz, 1H, Ar-H), 7.71-7.74 (m, 3H,
Ar-H), 8.32 (s, 1H, Ar-H), 8.52 (d, J = 8 MHz, 1H, Ar-H), 9.39 (s, 1H, Ar-H); Mass
m/z: 401.1 (M+1).
4.12.2
carboxamide (8)
Off-white solid, Yield - 20%; TLC - Rf 0.6 (EtOAc : n-Hexane, 3:7); HPLC
N-(4-Chlorophenyl)-2-(4-methoxydibenzo[b,d]furan-1-yl)-1,3-thiazole-4-
purity - 98.4%; M.P. 225-228 °C; 1H-NMR (CDCl3): δ 4.16 (s, 3H, -OCH3), 7.09 (d, J
23

= 8.4 Hz, 1H, Ar-H), 7.26-7.35 (m, 3H, Ar-H), 7.54 (d, J = 7.24 Hz, 1H, Ar-H), 7.57
(m, 1H, Ar-H), 7.61 (d, J = 8.36 Hz, 2H, Ar-H), 7.66-7.73 (m, 1H, Ar-H), 8.32 (s, 1H,
Ar-H), 8.47 (d, J = 8.4 Hz, 1H, Ar-H), 9.39 (s, 1H, Ar-H);Mass m/z: 435.0 (M+1)
4.12.3 2-(4-Methoxydibenzo[b,d]furan-1-yl)-1,3-thiazole-4-carboxamide (9)
Off-white solid, Yield - 35%; TLC - Rf 0.53 (EtOAc : n-Hexane, 7:3); HPLC
purity – 92.1%; M.P. 213-215 °C; 1H-NMR (DMSO-d6): δ 4.08 (s, 3H, -OCH3), 7.32-
7.37 (m, 2H, Ar-H), 7.59 (s, 1H, Ar-H), 7.73-7.80 (m, 4H, Ar-H), 8.43 (s, 1H, Ar-H),
8.61 (s, 1H, Ar-H); Mass m/z: 325.0 (M+1)
4.12.4
thiazole-4-carboxamide (10)
White solid, Yield - 46%; TLC - Rf 0.61 (EtOAc : n-Hexane, 1:1); HPLC
N-(3,5-Dichloropyridin-4-yl)-2-(4-methoxydibenzo[b,d]furan-1-yl)-1,3-
purity – 96.5%; M.P. 233-238 °C; 1H-NMR (CDCl3): δ 4.14 (s, 3H, -OCH3), 7.09 (d,
J = 8.44 Hz, 1H), 7.27-7.34 (m, 1H, Ar-H), 7.49-7.55 (m, 1H, Ar-H), 7.61 (d, J =
8.32 Hz, 1H, Ar-H), 7.64-7.70 (m, 1H, Ar-H), 8.41 (s, 1H, Ar-H), 8.45-8.51 (m, 1H,
Ar-H), 8.61 (d, J = 7.28 Hz, 2H, Ar-H), 9.17 (s, 1H, -NH);Mass m/z: 470.0 (M+).
4.12.5
(2-(4-Methoxydibenzo[b,d]furan-1-yl)-1,3-thiazol-4-yl)(4-(pyrimidin-2-
yl)piperazin-1-yl)methanone (11)
Off-white solid, Yield - 25%; TLC - Rf 0.61 (EtOAc : n-Hexane, 7:3); HPLC
1
purity – 99.7%; M.P. 177-180 °C; H-NMR (DMSO-d6): δ 3.82-3.86 (m, 8H, -
piperdine CH2), 4.08 (s, 3H), 6.67-6.71 (m, 1H, Ar-H), 7.34-7.38 (m, 2H, Ar-H),
7.58-7.61 (m, 1H, Ar-H), 7.78 (d, J = 8.16 Hz, 1H, Ar-H), 7.83 (d, J = 8.52 Hz, 1H,
Ar-H), 8.30 (s, 1H, Ar-H), 8.37 (d, J = 4.64 Hz, 2H, Ar-H), 8.87 (d, J = 8.04 Hz, 1H,
Ar-H); Mass m/z: 472.1 (M+1).
4.12.6
(2-(4-Methoxydibenzo[b,d]furan-1-yl)-1,3-thiazol-4-yl)(4-(pyridin-2-
yl)piperazin-1-yl)methanone (12)
Off-white solid, Yield - 25%; TLC - Rf 0.61 (EtOAc : n-Hexane, 7:3); HPLC
1
purity - 96.5%; M.P. N/R; H-NMR (DMSO-d6) δ 3.54-3.86 (m, 8H, - piperdine
CH2), 4.08 (s, 3H, -OCH3), 6.65-6.68 (m, 1H, Ar-H), 6.84 (d, J = 8.56 Hz, 1H, Ar-H),
7.34-7.39 (m, 2H, Ar-H), 7.52-7.60 (m, 2H, Ar-H), 7.77-7.84 (m, 2H, Ar-H), 8.11 (d,
J = 3.28 Hz, 1H, Ar-H), 8.30 (s, 1H, Ar-H), 8.88 (d, J = 7.88 Hz, 1H, Ar-H); Mass
m/z: 472.1 (M+1).
4.12.7 2-(4-Methoxydibenzo[b,d]furan-1-yl)-N-morpholino-1,3-thiazole-4-carboxamide
(13)
24

Green coloured solid, Yield - 28%; TLC - Rf 0.23 (EtOAc : n-Hexane, 7:3);
1
HPLC purity - 99.9%; M.P. 205-209 °C; H-NMR (CDCl3): δ 2.98 (s, 4H, -
NCH2CH2), 3.90 (s, 4H, -OCH2CH2), 4.14 (s, 3H, -OCH3), 7.07 (d, J = 8.36 Hz, 1H,
Ar-H), 7.29 (d, J = 7.52 Hz, 1H, Ar-H), 7.52-7.60 (m, 2H, Ar-H), 7.70 (d, J = 7.96
Hz, 1H), 8.23 (s, 1H, Ar-H), 8.28 (s, 1H, Ar-H), 8.46 (d, J = 7.88 Hz, 1H, Ar-H);
Mass m/z: 410.0 (M+1).
4.12.8 N-(4-Hydroxycyclohexyl)-2-(4-methoxydibenzo[b,d]furan-1-yl)-1,3-thiazole-
4-carboxamide (14)
Off-white solid, Yield -28%; TLC - Rf 0.43 (CH2Cl2 : MeOH, 9.4:0.6); HPLC
1
purity - 98.3%; M.P. 211-213 °C; H-NMR (CDCl3): δ 1.33-1.48 (m, 6H, -CH2),
2.02-2.04 (m, 2H, -CH2), 2.14-2.17 (m, 2H, -CH2), 3.41-3.47 (m, 1H, -CH), 3.82-3.85
(m, 1H, -CH), 4.14 (s, 3H, -OCH3), 7.07 (d, J = 8.36 Hz, 1H, Ar-H), 7.35 (d, J = 8.68
Hz, 1H, Ar-H), 7.51-7.55 (m, 1H, Ar-H), 7.59 (d, J = 8.32 Hz, 1H, Ar-H), 7.69 (d, J =
8.28 Hz, 1H, Ar-H), 8.19 (s, 1H, Ar-H), 8.46 (d, J = 8 Hz, 1H, Ar-H); Mass m/z:
423.1 (M+1).
4.12.9
thiazol-4-yl)acetamide (15)
Pale brown coloured solid, Yield - 25%; TLC - Rf 0.69 (EtOAc : n-Hexane,
N-(3,5-Dichloropyridin-4-yl)-2-(2-(4-methoxydibenzo[b,d]furan-1-yl)-1,3-
1:1); HPLC purity - 94.5%; M.P. 236-240 °C; 1H-NMR (CDCl3): δ 4.13-4.18 (m, 5H,
CH2,-OCH3), 7.06 (d, J = 8.4 Hz, 1H, Ar-H), 7.19-7.23 (m, 1H, Ar-H), 7.32 (s, 1H,
Ar-H), 7.45-7.49 (m, 1H, Ar-H), 7.60 (d, J = 8.36 Hz, 1H, Ar-H), 7.65 (d, J = 8.32
Hz, 1H, Ar-H), 8.38 (s, 2H, Ar-H), 8.51 (d, J = 8 Hz, 1H, Ar-H), 9.50 (s, 1H, -
NHCO); Mass m/z: 484.0 (M+).
4.12.10 N-(3,5-Dichloropyridin-4-yl)-3-(2-(4-methoxydibenzo[b,d]furan-1-yl)-1,3-
thiazol-4-yl)propanamide (16)
Pale pink coloured solid, Yield - 41%; TLC - Rf 0.48 (EtOAc : n-Hexane, 1:1);
HPLC purity - 91.2%; M.P. N/R; 1H-NMR (CDCl3): δ 2.99-3.02 (m, 2H, -CH2), 3.38-
3.41 (m, 2H, -CH2CO), 4.13 (s, 3H, Ar-H), 7.04-7.15 (m, 3H, Ar-H), 7.56 (d, J = 8.32
Hz, 2H, Ar-H), 8.28 (s, 2H, Ar-H), 8.33 (d, J = 7.92 Hz, 1H, Ar-H), 9.36 (s, 1H, -
NH); Mass m/z: 498.0 (M+).
4.12.11
N-Ethyl-2-(4-methoxydibenzo[b,]furan-1-yl]-5-methyl-1,3-thiazole-4-
carboxamide (17)
Off-white solid, Yield - 21%; TLC - Rf 0.45 (EtOAc : n-Hexane, 3:7); HPLC
1
purity - 97.5%; M.P. 146-150 °C; H-NMR (CDCl3): δ 1.17-1.21 (m, 3H, -
25

NCH2CH3), 2.88 (s, 3H. -CH3), 3.39-3.46 (m, 2H, -CH3), 4.06 (s, 3H, -OCH3), 6.97
(d, J = 8.36 Hz, 1H, Ar-H), 7.19-7.23 (m, 1H, Ar-H), 7.42-7.49 (m, 3H, Ar-H), 7.61
(d, J = 8.28 Hz, 1H, Ar-H), 8.39 (d, J = 7.96 Hz, 1H, Ar-H);Mass m/z: 367.1 (M+1).
4.12.12 N-(4-Hydroxycyclohexyl)-2-(4-methoxydibenzo[b,d]furan-1-yl)-5-methyl-
1,3-thiazole-4-carboxamide (18)
Off-white solid, Yield - 26%; TLC - Rf 0.53 (CH2Cl2 : MeOH, 9.5:0.5); HPLC
1
purity - 96.1%; M.P. 216-220 °C; H-NMR (DMSO-d6): δ 1.34-1.42 (m, 4H,
aliphatic CH2), 1.83-1.90 (m, 4H, aliphatic CH2), 2.83 (s, 3H, -CH3), 3.33-3.38 (m,
1H, -NHCH), 3.71-3.89 (m, 1H, -OHCH), 4.06 (s, 3H, -OCH3), 4.57 (d, J = 4.22 Hz
1H), 7.30-7.35 (m, 2H, Ar-H), 7.58-7.62 (m, 1H, Ar-H), 7.67 (d, J = 8.4 Hz, 1H, Ar-
H), 7.78-7.85 (m, 2H, Ar-H), 8.62 (d, J = 8 Hz, 1H, Ar-H);Mass m/z: 437.1 (M+1).
4.12.13 N-Cyclopentyl-2-(4-methoxydibenzo[b,d]furan-1-yl)-5-methyl-1,3-thiazole-
4-carboxamide (19)
Off-white solid, Yield - 35%; TLC - Rf 0.54 (EtOAc : n-Hexane, 3:7); HPLC
1
purity - 98.8%; M.P. 147-150 °C; H-NMR (CDCl3): δ 1.51-1.54 (m, 2H, aliphatic
CH2), 1.62-1.73 (m, 4H, aliphatic CH2), 2.06-2.10 (m, 2H, aliphatic CH2), 2.95 (s, 3H,
CH3), 4.13 (s, 3H, -OCH3), 4.41-4.45 (m, 1H, -CH), 7.04 (d, J = 8.4 Hz, 1H, Ar-H),
7.52-7.57 (m, 3H, Ar-H), 7.68 (d, J = 8.24 Hz, 1H, Ar-H), 8.54 (d, J = 7.04 Hz, 1H,
Ar-H); Mass m/z: 407.1 (M+1).
4.12.14
N-(3,5-Dichloropyridin-4-yl)-2-(4-methoxydibenzo[b,d]furan-1-yl)-5-
methyl-1,3-thiazole-4-carboxamide (20)
Off-white solid, Yield - 24%; TLC - Rf 0.51 (EtOAc : n-Hexane, 4:6); HPLC
1
purity - 92.2%; M.P. 229-231 °C; H-NMR (DMSO-d6): δ 2.87 (s, 3H, -CH3), 4.08
(s, 3H, -OCH3), 7.30-7.36 (m, 2H, Ar-H), 7.57-7.61 (m, 1H, Ar-H), 7.70 (d, J = 8.4
Hz, 1H, Ar-H), 7.78 (d, J = 8.5 Hz, 1H, Ar-H), 8.63 (d, J = 8 Hz, 1H, Ar-H), 8.76 (s,
2H, Ar-H), 10.44 (s, 1H, -CONH);Mass m/z: 484 (M+).
4.12.15
N-(3,5-dichloropyridin-4-yl)-2-(4-(difluoromethoxy)dibenzo[b,d]furan-1-
yl)-1,3-thiazole-4-carboxamide (21)
Pale pink coloured solid, Yield - 26%; TLC - Rf 0.65 (EtOAc : n-Hexane,
1:1); HPLC purity - 95.6%; M.P. 179-182 °C; 1H-NMR (DMSO-d6): δ 7.26-7.34 (m,
1H, Ar-H), 7.41 (d, J = 8 Hz, 1H, Ar-H), 7.55-7.61 (m, 2H, Ar-H), 7.66-7.71 (m, 1H,
Ar-H), 8.44 (d, J = 7.8 Hz, 1H, Ar-H), 8.48 (s, 1H, Ar-H), 8.60 (s, 2H, Ar-H), 9.14 (s,
1H, Ar-H);Mass m/z: 506 (M+1).
26

4.12.16 2-((4-Difluoromethoxy)dibenzo[b,d]furan-1-yl)-N-(2,2,2-trifluoroethyl)-1,3-
thiazole-4-carboxamide (22)
Off-white solid, Yield - 32%; TLC - Rf 0.41 (EtOAc : n-Hexane, 3:7); HPLC
1
purity - 98.6%; M.P. 234-236 °C; H-NMR (CDCl3): δ 4.14-4.17 (m, 2H, -
CH2CH2CF3), 6.74-7.10 (m, 1H, Ar-H), 7.31-7.35 (m, 1H, Ar-H), 7.39 (d, J = 8.4 Hz,
1H, Ar-H), 7.56-7.61 (m, 2H, Ar-H), 7.68-7.73 (m, 2H, Ar-H), 8.36 (d, J = 10.16 Hz
2H, Ar-H);Mass m/z: 443 (M+1).
4.12.17 2-((4-Difluoromethoxy)dibenzo[b,d]furan-1-yl)-N-methoxy-1,3-thiazole-4-
carboxamide (23)
Off-white solid, Yield - 21%; TLC - Rf 0.39 (EtOAc : n-Hexane, 1:1); HPLC
1
purity - 98.2%; M.P. 162-164 °C; H-NMR (CDCl3): δ 3.95 (s, 3H, -OCH3 ), 6.74-
7.10 (m, 1H, Ar-H), 7.33-7.39 (m, 2H, Ar-H), 7.55-7.60 (m, 2H, Ar-H), 7.69 (d, J =
8.32 Hz, 1H, Ar-H), 8.28 (d, J = 7.92 Hz, 1H, Ar-H), 8.37 (s, 1H, Ar-H), 9.77 (s, 1H,
Ar-H); Mass m/z: 391 (M+1).
4.12.18 2-((4-Difluoromethoxy)dibenzo[b,d]furan-1-yl)-N-(4-hydroxycyclohexyl)-
1,3-thiazole-4-carboxamide (24)
Off-white solid, Yield - 41%; TLC - Rf 0.49 (CH2Cl2 : MeOH, 9.4:0.6); HPLC
1
purity - 96.5%; M.P. 160-163 °C; H-NMR (CDCl3): δ 1.26-1.52 (m, 4H, Aliphatic
CH2), 2.04 (d, J = 9.12 Hz, 2H, Aliphatic CH2), 2.16 (d, J = 9.52 Hz, 2H, Aliphatic
CH2), 3.55-3.71 (m, 1H, -NHCH), 3.97-4.09 (m, 1H, -OHCH), 6.73-7.09 (m, 1H, Ar-
H), 7.30 (d, J = 7.22 Hz, 1H, Ar-H), 7.38 (d, J = 8.36 Hz, 1H, Ar-H), 7.56-7.60 (m,
2H, Ar-H), 7.70 (d, J = 8.24 Hz, 1H, Ar-H), 8.26 (s, 1H, Ar-H), 8.40 (d, J = 8 Hz, 1H,
Ar-H); Mass m/z: 459 (M+1).
4.12.19
2-(4-(Difluoromethoxy)-8-(trifluoromethyl)dibenzo[b,d]furan-1-yl)-N-(4-
hydroxycyclohexyl)-1,3-thiazole-4-carboxamide (26)
Pale brown coloured solid, Yield - 77%; TLC - Rf 0.46 (CH2Cl2 : MeOH, 9:1);
1
HPLC purity - 95.4%; M.P. 217-219 °C; H-NMR (DMSO-d6): δ 1.25-1.31 (m, 2H,
aliphatic CH2), 1.36-1.42 (m, 2H, aliphatic CH2), 1.84 (d, J = 10.24 Hz, 4H, aliphatic
CH2), 3.33-3.38 (m, 1H, -NHCH), 3.72-3.82 (m, 1H, -OHCH), 4.57 (d, J = 4.32 Hz
1H, -OH), 7.42-7.67 (m, 1H, Ar-H), 7.92 (d, J = 8.52 Hz, 1H, Ar-H), 8.02 (d, J = 8.84
Hz, 1H, Ar-H), 8.07 (d, J = 7.96 Hz, 1H, Ar-H), 8.12 (d, J = 8.64 Hz, 1H, Ar-H), 8.52
(s, 1H, Ar-H), 9.06 (s, 1H, Ar-H);Mass m/z: 527 (M+1).
4.12.20
(2-(4-(Difluoromethoxy)-8-(trifluoromethyl)dibenzo[b,d]furan-1-yl)-1,3-
thiazol-4-yl)(4-hydroxypiperidin-1-yl)methanone (27)
27

Pale brown coloured solid, Yield - 73%; TLC - Rf 0.47 (CH2Cl2 : MeOH, 9:1);
1
HPLC purity - 95.9%; M.P. 212-214 °C; H-NMR (CDCl3): δ 1.21-1.23 (m, 1H),
1.24-1.30 (m, 1H), 1.42-1.48 (m, 2H), 1.60-1.65 (m, 1H, aliphatic CH2), 1.80-1.85 (m,
1H), 3.74-3.83 (m, 2H), 4.01-4.05 (m, 1H), 4.78 (d, J = 3.52 Hz, 1H, -OH), 7.42-7.67
(m, 2H, Ar-H), 8.03 (d, J = 8.44 Hz, 2H, Ar-H), 8.11 (d, J = 8.6 Hz, 1H, Ar-H), 8.31
(s, 1H, Ar-H), 9.49 (s, 1H, Ar-H);Mass m/z: 513 (M+1).
4.12.21 2-(8-Acetamido-4-(difluoromethoxy)dibenzo[b,d]furan-1-yl)-N-(4-hydroxy
cyclohexyl)-1,3-thiazole-4-carboxamide (29)
Off-white solid, Yield - 32%; TLC - Rf 0.57 (CH2Cl2 : MeOH, 8:2); HPLC
1
purity - 99.1%; M.P. 225-227 °C; H-NMR (DMSO-d6): δ 1.23-1.31 (m, 2H), 1.40-
1.51 (m, 2H), 1.78-1.88 (m, 4H), 2.05 (s, 3H, -COCH3), 3.82-3.85 (m, 2H), 4.57 (d, J
= 4.32 Hz, 1H, -OH), 7.52-7.55 (m, 1H, Ar-H), 7.73-7.80 (m, 4H, Ar-H), 8.50 (s, 1H,
Ar-H), 8.69 (s, 1H, Ar-H), 10.12 (s, 1H, -CONH); Mass m/z: 516 (M+1).
4.12.22
N-(3,5-Dichloropyridin-4-yl)-2-(4-(difluoromethoxy)-8-(methyl
sulfonamido) dibenzo[b,d]furan-1-yl)-1,3-thiazole-4-carboxamide (30)
Off-white solid, Yield - 22%; TLC - Rf 0.44 (EtOAc : n-Hexane, 6:4); HPLC
1
purity - 98.9%; M.P. 237-240 °C; H-NMR (DMSO-d6): δ 2.87 (s, 3H, -SO2CH3),
7.53-7.68 (m, 3H, Ar-H), 7.81-7.85 (m, 2H, Ar-H), 8.16 (s, 1H, Ar-H), 8.74 (s, 2H,
Ar-H), 8.81 (s, 1H, Ar-H), 9.63 (s, 1H, -NH), 10.42 (s, 1H, -NH); Mass m/z: 599
(M+).
4.12.23 2-(4-(Difluoromethoxy)-(methylsulfonamido)dibenzo[b,d]furan-1-yl)-N-(4-
hydroxycyclohexyl)-1,3thiazole-4-carboxamide (31)
Yellow coloured solid, Yield - 84%; TLC - Rf 0.37 (CHCl3 : MeOH, 9:1);
1
HPLC purity - 97%; M.P. N/R; H-NMR (CDCl3): δ 1.24-1.27 (m, 2H), 1.45-1.48
(m, 2H), 1.70-1.91 (m, 4H), 2.91 (s, 3H, -CH3), 3.32-3.36 (m, 1H), 3.72-3.92 (m, 1H),
4.54 (d, J = 4.16 Hz, 1H, -OH), 7.49-7.57 (m, 3H, Ar-H), 7.77 (d, J = 8.44 Hz, 1H,
Ar-H), 7.81-7.86 (m, 2H, Ar-H), 8.22 (s, 1H, Ar-H), 8.51 (s, 1H, Ar-H), 9.74 (s, 1H, -
CONH); Mass m/z: 552 (M+1).
4.12.24 2-(4-(Difluoromethoxy)-8-(methylsulfonamido)dibenzo[b,d]furan-1-yl)-N-
(4-hydroxycyclohexyl)-5-methyl-1,3thiazole-4-carboxamide (33)
Off-white solid, Yield - 66%; TLC - Rf 0.59 (CH2Cl2 : MeOH, 9:1); HPLC
purity - 97%; M.P. 244-246 °C; 1H-NMR (DMSO-d6): δ 1.33-1.53 (m, 4H, aliphatic
CH2), 1.80-1.88 (m, 4H, aliphatic CH2), 2.58 (s, 3H, -CH3), 2.88 (s, 3H, -NHCOCH3),
28

3.33-3.40 (m, 1H, -CH), 3.72-3.85 (m, 1H,-CH), 4.53 (d, J = 3.6 Hz, 1H, -OH), 7.48-
7.55 (m, 2H, Ar-H), 7.67-7.72 (m, 2H, Ar-H), 7.84 (d, J = 8.88 Hz, 1H, Ar-H), 8.21
(s, 1H, Ar-H), 9.76 (s, 1H, -NHCO); Mass m/z: 564 (M-1).
4.12.25 2-(4-(Difluoromethoxy)-8-(methylsulfonamido)dibenzo[b,d]furan-1-yl)-N-
methoxy-5-methyl-1,3-thiazole-4-carboxamide (34)
Off-white solid, Yield - 22%; TLC - Rf 0.56 (EtOAc : n-Hexane, 8:2); HPLC
purity - 95.5%; M.P. N/R; 1H-NMR (DMSO-d6): δ 2.87 (s, 3H, -CH3), 2.94 (s, 3H, -
SO2CH3), 3.70 (s, 3H, -NHOCH3), 7.37-7.55 (m, 4H, Ar-H), 7.68-7.84 (m, 1H, Ar-
H), 8.23 (s, 1H, Ar-H), 9.76 (s, 1H, -NH), 11.48 (s, 1H, -NH); Mass m/z: 498 (M+1)
4.2 Biology
4.2.1 Phosphodiesterase- 4 screening assay: PDE-4 (Phosphodiesterase type IV)
enzymes convert cyclic AMP (cAMP) into AMP. The assay is performed to
determine the effect of test compounds on the inhibition of purified human PDE-4B
enzyme. It involves the detection of the tritiated AMP (product) using SPA beads
known as yittrium silicate. The linear AMP binds preferentially to SPA yittrium
silicate beads compared to cyclic nucleotides in the presence of zinc sulphate. The
binding of the radiolabelled product to the bead brings the isotope into close
proximity to allow radiation from the tritium to excite the scintillant within the bead
to emit light. The unbound radiolabel is not close enough to allow this energy transfer
and the light emitted due to the binding of tritiated AMP is detected as cpm.
PDE Activity (SPA Based Assay Protocol)
PDE-4B2 activity was inhibited by the compounds according to the invention in a
modified SPA (scintillation proximity assay) test, supplied by GE Healthcare Life
Sciences (see procedural instructions "Phosphodiesterase [3H]-cAMP SPA enzyme
assay, code TRKQ 7090"), carried out in 96-well microtitre plates. The test volume is
100 μL and contains 50 mM Tris buffer (pH 7.4), 8.3 mM Mg2+ in the presence of
inhibitor or test compound, and containing PDE-4B2 enzyme (sufficient to ensure that
10-20% of the cAMP is converted under the said experimental conditions). The final
concentration of DMSO in the assays (1% v/v) does not substantially affect the
activity of the PDEs investigated. After a pre-incubation of 5 minutes at 37 °C the
reaction is started by adding the substrate (cAMP; 0.5 μM cAMP, including about
50,000 cpm of [3H]-cAMP) and the assays are incubated for a further 10 minutes;
after that they are stopped by adding SPA beads containing 18 mM ZnSO4 (50 μL).
After the beads have been sedimented (>30 minutes) the microtitre plates are analyzed
29