Discovery of thiazolyl-phthalazinone acetamides as potent glucose uptake activators via high-throughput screening

Article abstract of DOI:10.1016/j.bmcl.2013.07.067

With the aim to discover orally active small molecules that stimulate glucose uptake, high throughput screening of a library of 5000 drug-like compounds was conducted in differentiated skeletal muscle cells in presence of insulin. N-Substituted phthalazinone acetamide was identified as a potential glucose uptake modulator. Several novel derivatives were synthesized to establish structure activity relationships. Identified lead thiazolyl- phthalazinone acetamide (7114863) increased glucose uptake (EC₅₀ of 0.07 ± 0.02 μM) in differentiated skeletal muscle cells in presence of insulin. Furthermore, 7114863 was superior to rosiglitazone under similar experimental conditions without inducing PPAR-γ agonist activity thus making it a very interesting scaffold.

Full text of DOI:10.1016/j.bmcl.2013.07.067

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5740–5743
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of thiazolyl-phthalazinone acetamides as potent glucose
uptake activators via high-throughput screening
a
a
a
a
Madhavi Agrawal a, , Prashant Kharkar , Sonali Moghe , Tushar Mahajan , Vaishali Deka ,
Chandni Thakkar b, Amrutha Nair c, Chirag Mehta c, Julie Bose c, Asha Kulkarni-Almeida b, Dilip Bhedi a,
Ram A. Vishwakarma a
⇑
a Department of Medicinal Chemistry, Piramal Enterprises Limited, 1, Nirlon Complex, Goregaon (E), Mumbai, India
b Department of High-Throughput Screening, Piramal Enterprises Limited, 1, Nirlon Complex, Goregaon (E), Mumbai, India
c Department of Pharmacology, Piramal Enterprises Limited, 1, Nirlon Complex, Goregaon (E), Mumbai, India
a r t i c l e i n f o
a b s t r a c t
Article history:
With the aim to discover orally active small molecules that stimulate glucose uptake, high throughput
screening of a library of 5000 drug-like compounds was conducted in differentiated skeletal muscle cells
in presence of insulin. N-Substituted phthalazinone acetamide was identified as a potential glucose
uptake modulator. Several novel derivatives were synthesized to establish structure activity relation-
ships. Identified lead thiazolyl-phthalazinone acetamide (7114863) increased glucose uptake (EC50 of
Received 14 March 2013
Revised 24 July 2013
Accepted 30 July 2013
Available online 7 August 2013
0.07 0.02 lM) in differentiated skeletal muscle cells in presence of insulin. Furthermore, 7114863
Keywords:
was superior to rosiglitazone under similar experimental conditions without inducing PPAR-
c agonist
Thiazolyl-phthalazinone acetamides
Glucose uptake activators
Type 2 diabetes
activity thus making it a very interesting scaffold.
Ó 2013 Elsevier Ltd. All rights reserved.
Obesity
PPAR-
c
Type 2 diabetes is a chronic metabolic disorder that refers to a
condition created by non-responsiveness to or deficiency of insu-
lin. Causes for this disease are many and quite complex. It has been
observed that about 80% of the type 2 diabetes patients are obese.
Obesity indicates an increase in the number or size of adipocytes
leading to an overproduction of certain hormones like leptin and
cytokines, some of which appear to cause cellular resistance to
insulin.1 While adipocytes contribute significantly towards the
development of insulin resistance, skeletal muscles are also known
to play an important role in glucose homestatsis.2–4 It has been
observed that people with type 2 diabetes have reduced insulin
dependent glucose transport in skeletal muscles.5 At present, var-
ious treatments for type 2 diabetes mellitus aim at reducing these
hyperglycemic conditions. The therapeutic regimens include
to reverse the effect of insulin resistance and yet not produce any
undesirable side effect.
In an attempt to discover orally active small molecules that
stimulate glucose uptake, we devised a high throughput screening
(HTS) assay to identify compounds which could improve the ability
of skeletal muscle to take up glucose under in vitro conditions.4,5
A
HTS assay measuring glucose uptake in differentiated skeletal
muscle cells pulsed with radioactive glucose in presence of insulin
was established. A library comprising of 5000 drug-like com-
pounds was screened and the compound 7114863 was found to
be most potent compound in inducing glucose uptake in differen-
tiated skeletal muscle cells (EC50 = 0.070 0.002
pound increased glucose uptake without activating PPAR-
l
M). This com-
. We
c
evaluated its potential by measuring the effect on glucose uptake
in human skeletal muscle cells (Fig. 1, Table 1). The activity of
7114863 (1a) was superior to that of rosiglitazone under similar
among other classes, sulfonylureas, metformin, PPAR-
(thiazolidinediones), -glucosidase inhibitors and insulin itself.
PPAR- full agonists enhance insulin action and are the mainstay
c agonists
a
c
experimental conditions without inducing PPAR-c agonist activity
drugs for the treatment of type 2 diabetes. However in recent times
thiazolidinedione treatment has been linked to increased inci-
dences of weight gain and congestive heart failure.6 It is therefore
essential to develop a therapeutic that follows alternate pathways
thus making it a very interesting scaffold. Based on these interest-
ing results, we explored structure–activity relationship around
7114863 (1a) molecule. Chemistry and biological results are dis-
cussed in this Letter.
The active molecule obtained from high-throughput screening
7114863 (EC50 of 0.070 0.002 lM) was synthesized to validate
its biological activity. Retrosynthetic analysis of 7114863 revealed
that a key precursor acid 6a was required. The resynthesized
⇑
Corresponding author. Tel.: +91 22 3081 8310; fax: +91 22 3081 8334.
E-mail address: madhavi.agrawal@piramal.com (M. Agrawal).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.07.067

M. Agrawal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5740–5743
5741
O
O
N
5
4
3
2
A
7114863, EC50=0.07 0.02μM
Rosiglitazone, EC50=4.49 0.41 µM
O
Ph3P
O
O
COOEt
b
O
O
a
NH
O
O
O
4
O
N
2
3
S
N
H
c
d
N
N
7114863
O
O
O
OH
OH
O
O
1
N
NH
N
N
N
N
d
0.001
0.01
0.1
1
10
100
Concentration (µM)
O
O
O
6b
6a
5
B
% hPPAR- γ activation
Compound
(n=3)
Scheme 1. Reagents and conditions: (a) CHCl3, reflux, 5 h, 51%; (b) hydrazine
hydrate, EtOH, addition at rt and then reflux, 90 °C, 2 h, 71%; (c) MeI, KOtBu, DMF,
30 min, 80%; (d) LiOH, THF:MeOH:water (1:1:1), rt, 8–10 h, 81%.
7114863
0.3
100
Rosiglitazone, 1 μM
Figure 1. Effect of 7114863 and rosiglitazone on (A). Glucose uptake by L6
myotubes and (B). PPAR- activity in human PPAR- receptor transfected CV1 cells.
R2
O
c
c
O
N
R3
OH
R
S
N
a
R4
N
N
compound (1a) showed a reproducible effect on glucose uptake
with an EC50 of 0.090 0.006 M. The key precursor 6a along with
N
N
l
R1
O
6a-b
its non-methylated analog 6b were synthesized from commercially
available phthalic anhydride in 3–4 steps as depicted in Scheme 1.
Phthalic anhydride on treatment with Witting reagent PPh3@CH–
COOEt in chloroform as solvent under reflux conditions produced
the desired ester 3 in 51% yield. Ester 3 was then treated with
hydrazine hydrate to yield the phthalazine product 4 (71% yield).
The phthalazine ester on further treatment with methyl iodide, fol-
lowed by ester hydrolysis using LiOH produced the desired phthal-
azine acetic acid 6a. Similarly its non-methylated analog 6b was
synthesized.
O
1a: R1 = CH3, R2 = Ph, R3 = R4 = H
1b: R1 = CH3, R2 = Ph(3-F), R3 = R4 = H
1c: R1 = R3 = CH3, R2 = Ph(4-Br), R4 = H
1d: R1 = CH3, R2 = Ph, R3 = COOEt, R4 = H
1e: R1 = R3 = CH3, R2 = Ph, R4 = H
1f: R1 = CH2CH2OH, R2 = Ph, R3 = R4 = H
1g: R1 = CH3, R2 = Ph(2-OH), R3 = R4 = H
1h: R1 = CH3, R2 = Ph, R3 = COOH, R4 = H
1i: R1 = CH3, R2 = R4 = H, R3 = Ph
1j: R1 = CH3, R2 = Ph(4-F), R3 =R4 = H
1k R1 = CH3, R2 = Ph(4-Br), R3 = R4 = H
1l: R1 = CH3, R2 = CH3, R3 = COOEt, R4 = H
1m:R1 = CH3, R2 = Ph(4-CF3), R3 = R4 = H
1n: R1 = CH3, R2 =P h(4-OCH3), R3 = CH3, R4 = H
1o: R1 = CH3, R2 = R3 = R4 = H
After synthesis of key precursors, acid 6a was treated with cor-
responding thiazol-2-yl amine to get desired 7114863 (1a) in 46%
yield. Similarly by varying acids and different thiazole-amines, a
series of analogs 1b–1s were synthesized as depicted in Scheme 2.
Phthalizine ester 4 on treatment with phenacyl bromide in dry
DMF in presence of potassium tert-butoxide produced N-alkylated
product 7 in 70% yield, which further on LiOH hydrolysis produced
corresponding acid 8 (Scheme 3).
1p: R1 = CH3, R2 = Ph(4-Cl), R3 = R4 = H
1q R1 = R3=R4 = H, R2 = Ph
1r: R1 = CH3, R2 = Ph, R3 = H, R4 = CH2COOEt
1s R1 = R4 = CH3, R2 = Ph, R3 = H
Scheme 2. Reagents and conditions: (a) TBTU, DIPEA, heterocyclic amine, CH2Cl2,
rt, 48 h, 46%.
hydrazide 9 in 70% yield. Hydrazide 9 on treatment with 4-phenyl
substituted acyl chloride (prepared by treating corresponding acid
with oxalyl chloride) produced product 10 in 25% yield (Scheme 4).
Phthalazine acetic acid 6b on treatment with hydrazine hydrate
under reflux conditions for 30 min produced the corresponding
Table 1
Results of preliminary screening at 10 l
Ma
Entry
Fold increase over control
Remarks
Entry
Fold increase over control
1.8
Remarks
Active
7114863
1a
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
1.8
1.8
3.4
1.3
3.4
0.8
1.3
1.3
1.6
2.7
3.6
0.9
3.2
2.9
1.7
3.4
1.8
1.9
Active
Active
Active
Not active
Active
Not active
Not active
Not active
Not active
Active
Active
Not active
Active
1s
7
8
10
11a
11b
11c
11d
11e
11f
11g
11h
11i
14a
14b
Rosiglitazone
1.1
0.9
0.9
1.2
1.9
1.3
2.0
1.0
0.7
4.9
1.3
0.8
0.9
1.2
1.8
Not active
Not active
Not active
Not active
Active
Not active
Active
Not active
Not active
Active
Not active
Not active
Not active
Not active
Active
Active
Active
Active
Active
Active
a
Fold increase of insulin (200 nM) over control was found to be 1.3. Values comparable to the effect of rosiglitazone at the same concentration were considered as active.

5742
M. Agrawal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5740–5743
Table 2
O
O
N
O
Dose response studies of the actives
O
O
OH
Entry
EC50 SE (
l
M)
Entry
EC50 SE (lM)
b
a
N
N
O
N
N
O
a
7114863
1aa
1c
1e
1k
0.070 0.002
0.090 0.006
0.003 0.0004
0.010 0.002
0.1 0.02
1q
1r
1s
0.040 0.009
0.28 0.17
5.96 2.7
NH
O
7
O
4
O
8
11b
11d
11g
Rosiglitazone
7.47 1.7
2.56 0.85
0.17 0.02
4.49 0.41
Scheme 3. Reagents and conditions: (a) phenacyl bromide, pot. tert-butoxide, DMF,
1l
0.04 0.03
rt, 30 min, 70%; (b) LiOH, THF:MeOH:water (1:1:1), rt, 8–10 h, 80%.
1n
1o
0.009 0.002
0.12 0.01
a
7114863 and 1a are same compounds, 7114863 was purchased from Chem-
Bridge library and 1a was synthesized by us.
O
N
O
O
N
O
S
NH2
OH
NH NH
NH
N
a
b
N
NH
NH
NH
Substituted thiazoles or
O
or
substituted pyrimidone
isoquinoline prefered, replacement
with other heterocycles looses
activity
O
O
R4 = Amidic substitution
decreases activity
O
6b
10
9
N
Scheme 4. Reagents and conditions: (a) hydrazine hydrate, reflux, 30 min, 70%; (b)
Et3N, rt, heterocyclic amine, 48 h, 25%.
R4
N
N
R2 = Phenyl ring
R1
R2
substituted at
4-posision with
halogen or OMe
prefered
O
O
O
N
R3
R
R1 = Me is prefered,
replacement with H retains the
activity (with some decrease),
other substitutions leads to
loss of activity.
OH
N
S
H
a
N
N
N
N
R3 = H, CH3 tolerated.
CH3 is prefered over H
O
O
6b
11a-i
Figure 2. Structure–activity relationship for thiazolyl-phthalazinone acetamide
series as potent glucose uptake activators.
O
N
F
O
N
Ph
11c:
HN
HN
N
R =
11b:
11a:
11d:
11g:
R =
R =
R =
R =
O
N
5
A
1n
CF3
Cl
Rosiglitazone
4
11e:
R =
11f:
11i:
R =
R =
OMe
OH
O
H
3
O
N
N
O
O
N
N
11h:
R=
S
N
H
N
N
1n
2
O
O
1
Scheme 5. Reagents and conditions: (a) TBTU, DIPEA, heterocyclic amine, CH2Cl2,
rt, 48 h, 40–70%.
0.000001
0.0001
0.01
1
100
Concentration (µM)
O
B
% hPPAR- γ activation
OH
Compound
(n=3)
a
S
1n
1
S
N
Rosiglitazone, 1 μM
100
N
NH2
N
12
O
H
13
Figure 3. Effect of 1n and rosiglitazone on (A). Glucose uptake by L6 myotubes and
(B). PPAR- activity in human PPAR- receptor transfected CV1 cells.
c
c
b
O
OR
Phthalazine acetic acid 6b on treatment with different hetero-
cyclic amines in presence of TBTU and DIPEA produced corre-
sponding amides 11a–i as depicted in Scheme 5.
S
N
N
O
We also synthesized adipic acid linked 4-phenyl thiazole-2-yl
compounds 14a and 14b as depicted in Scheme 6. All synthesized
compounds were characterized by NMR and MS data.7
In order to establish the structure–activity relationship around
identified novel lead compound, all synthesized analogs were
14a: R = H
14b: R = Me
Scheme 6. Reagents and conditions: (a) polymeric adipic anhydride, pyridine, rt,
overnight, 46%; (b) MeI, DMF, pot-tert-butoxide, rt, 30 min, two products 14a and
14b.

M. Agrawal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5740–5743
5743
screened for glucose uptake in L6 rat skeletal muscle cells8,9 and
human skeletal muscle myotubes10,11 and results are summarized
in Table 1. From the biological results, we found that compounds
from Schemes 3, 4 and 6 were inactive. The most promising com-
pounds were found from Schemes 2 and 5.
Based on preliminary screening results, active compounds were
selected for dose–response studies and EC50 values were deter-
mined as depicted in Table 2.
Based on the obtained biological results, key structural features
essential for glucose uptake activity have been identified. The N-
substituted phthalazimyl acetamides are tolerated. As depicted in
Figure 2, the presence of substituted thiazoles 1, substituted pyr-
imidone 11b and isoquinoline 11d yielded most active compounds.
Among all active analogs, most of them consist of the thiazole moi-
ety linked to phthalazine-acetamide skeleton. Further there was a
significant effect of substituent variation on the thiazole-linked
phthalazine acetamide skeleton. Methyl substitution on N1 of
phthalazine is preferred over H or other alkyl chains. Substitution
on amidic NH resulted in decreased activity. A halogen or OMe
substituted phenyl ring at 4th position of thiazole ring is preferred.
Methyl substitution at 5th position of thiazole is preferred over
unsubstituted position.
2. Krützfeldt, J.; Kausch, C.; Volk, A.; Klein, H. H.; Rett, K.; Häring, H. U.; Stumvoll,
M. Diabetes 2000, 49, 992.
3. Korsheninnikova, E.; Seppälä-Lindroos, A.; Vehkavaara, S.; Goto, T.; Virkamäki,
A. Diabetes Metab. Res. Rev. 2002, 18, 209.
4. Kulkarni-Almeida, A. A.; Brahma, M. K.; Padmanabhan, P.; Mishra, P. D.; Parab,
R. R.; Gaikwad, N. V.; Thakkar, C. S.; Tokdar, P.; Ranadive, P. V.; Nair, A. S.;
Damre, A. A.; Bahirat, U. A.; Deshmukh, N. J.; Doshi, L. S.; Dixit, A. V.; George, S.
D.; Vishwakarma, R. A.; Nemmani, K. V. S.; Mahajan, G. B. AMB Express 2011, 1,
42.
5. Castaneda, F.; Layne, J. E.; Castaneda, C. Int. J. Med. Sci. 2006, 3, 84.
6. Doshi, L. S.; Brahma, M. K.; Bahirat, U. A.; Dixit, A. V.; Nemmani, K. V. Expert
Opin. Invest. Drugs 2010, 19, 489.
7. Spectral
dihydrophthalazin-1-yl)-N-(4-phenylthiazol-2-yl)acetamide
(petroleum ether:EtOAc, 1:1); yield: 1.4 g (46%); purity (HPLC): 96.64%;
mp 306–308 °C; 1H NMR (DMSO-d6, 300 MHz):
12.68 (s, 1H), 8.32 (d,
data
for
selected
compounds.
2-(3-Methyl-4-oxo-3,4-
(1a): Rf = 0.4
d
J = 7.8 Hz, 1H), 7.85–7.97 (m, 5H), 7.64 (s, 1H), 7.44 (t, J = 7.5 Hz, 2H), 7.31–
7.36 (m, 1H), 4.26 (s, 2H), 3.73 (s, 3H); ESI-MS: m/z 377.1 [M+H]+, 399
[M+Na]+; HRMS: m/z observed 377.1074 [M+H]+ for C20H16N4O2S + H+
(377.1067); analysis (C20H16N4O2S). N-(4-(4-Bromophenyl)-5-methylthiazol-
2-yl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)acetamide (1c): yield:
51%; purity (HPLC): 98.16%; 1H NMR (DMSO, 300 MHz):
d 12.54 (br s,
1H), 8.32 (d, J = 7.5 Hz, 1H), 7.94–7.84 (m, 3H), 7.66 (d, J = 8.7 Hz, 2H) 7.61
(d, J = 8.7 Hz, 2H), 4.22 (s, 2H), 3.72 (s, 3H), 2.45 (s, 3H); ESI-MS: m/z 471.1
[M+H]+. N-(4-(4-Methoxyphenyl)-5-methylthiazol-2-yl)-2-(3-methyl-4-oxo-
3,4-dihydrophthalazin-1-yl)acetamide (1o): yield: 14 mg; purity (HPLC):
96.64%; 1H NMR (DMSO-d6, 300 MHz): d 12.50 (s, 1H), 8.31 (d, J = 7.8 Hz,
1H), 7.85–7.95 (m, 3H), 7.50 (d, J = 3.6 Hz, 1H), 7.22 (d, J = 3.6 Hz, 1H), 4.23
(s, 2H), 3.72 (s, 3H); ESI-MS: m/z 301.1 [M+H]+, 323.1 [M+Na]+. N-(4-(4-
Chlorophenyl)thiazol-2-yl)-2-(3-methyl-4-oxo-3,4-dihydroph-thalazin-1-
yl)acetamide (1q): yield: 15 mg; purity (HPLC): 96.84%; 1H NMR (DMSO-d6,
300 MHz): d 12.66 (s, 1H), 8.29 (d, J = 7.8 Hz, 1H), 7.82–8.0 (m, 5H), 7.64 (s,
1H), 7.42–7.47 (m, 2H), 7.31–7.36 (m, 1H), 4.22 (s, 2H); ESI-MS: m/z 363
[M+H]+, 385 [M+Na]+.
In conclusion, we have discovered new scaffolds 1 and 11 for in-
creased glucose uptake in skeletal muscle cells with potential for
anti-diabetic activity. Of the above mentioned scaffolds our lead
molecule 1n showed eightfold improvement in glucose uptake
8. Sweeney, G.; Somwar, R.; Ramlal, T.; Volchuk, A.; Ueyama, A.; Klip, A. J. Biol.
Chem. 1999, 274, 10071.
activity as compared to 7114863, without inducing PPAR-c agonist
activity (Fig. 3). Additionally, a 23-fold increase in glucose uptake
activity for compound 1c over 7114863 was observed. No further
studies were performed on the latter due to the presence of unde-
sirable bromine atom on the molecule.
9. Glucose uptake assay in L6 myotubes. L6 rat skeletal muscle cells (ATCC, USA)
were cultured in 96 well plates (Nunc) in MEM alpha medium (AMIMED)
containing 10% serum and 1% penicillin–streptomycin solution. Differentiation
was induced in confluent myotube cultures by culturing the cells in 2% serum.
The differentiated fused myotubes were further starved in serum free media,
treated with the samples and incubated overnight. After 24 h the cells were
pulsed with C14 tagged deoxy glucose (GE Healthcare, UK) for 10 min in
presence of insulin (Sigma, St. Louis, MO, USA). Glucose uptake measured by
lysing the cells with Microscint PS (Perkin Elmer, USA). The plates were read
using a Top Count Reader (Perkin Elmer, USA).
Acknowledgements
The authors wish to thank Dr. Somesh Sharma, Dr. Rajiv Shar-
ma, Dr. Veena Agarwal and Dr. Arun Balakrishnan of Piramal Enter-
prises Limited, India for supporting this study and providing the
infrastructure needed for carrying out the research work reported
in this Letter. We also thank Mr. T.T. Matan and Mr. S. Senthil Ku-
mar for their excellent technical support.
10. Jackson, S.; Bagstaff, S. M.; Lynn, S.; Yeaman, S. J.; Turnbull, D. M.; Walker, M.
Diabetes 2000, 49, 1169.
11. PPAR-c luciferase assay: CV-1 cells were seeded in a 96 well plate (25,000 cells/
well) and incubated overnight. Cells were then transiently co-transfected with
a luciferase gene under the control of Gal4 DNA binding elements and a
plasmid containing the ligand binding domain for PPAR-c fused to the GAL4
DNA binding domain. All transfections were performed using Lipofectamine
2000 (Invitrogen) according to the manufacturer’s instructions. Six hours after
transfection, cells were treated with test compounds and incubated for an
additional 24 h and the luciferase assay (Promega) was performed according to
the manufacture’s protocol. Luminescence was measured using POLARStar
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.07.
067.
Optima (BMG-Labtech). Transactivation of human PPAR-
c
by
1 lM
rosiglitazone was considered as 100% activation and hence data for test
compounds were calculated in comparison to rosiglitazone which was used as
a positive control in the assay. The entire assay was performed in triplicates
and repeated twice.
References and notes
1. Front Diabetes. Diabetes and Cancer. Epidemiological Evidence and Molecular
Links; Masur, K, Thévenod, F., Zänker, KS, Eds.; Karger publishers: Basel, 2008.