Novel phthalazinone and benzoxazinone containing thiazolidinediones as antidiabetic and hypolipidemic agents

Article abstract of DOI:10.1016/S0223-5234(01)01257-0

We report here the synthesis of a series of 5-[4-[2-[substituted phthalazinones-2(or 4)yl]ethoxy]phenylmethyl]thiazolidine-2,4-diones and 5-[4-[2-[2,3-benzoxazine-4-one-2-yl]ethoxy]phenylmethyl]thiazolidine-2, 4-diones and their plasma glucose and plasma triglyceride lowering activity in db/db mice. In vitro PPARγ transactivation assay was performed in HEK 293T cells. In vitro and in vivo pharmacological studies showed that the phthalazinone analogue has better activity. PHT46 (compound 5a), the best compound in this series, showed better in vitro PPARγ transactivation potential than troglitazone and pioglitazone. In insulin resistant db/db mice, PHT46 showed better plasma glucose and triglyceride lowering activity than the standard drugs. Pharmacokinetic study in Wistar rats showed good systemic exposure of PHT46. Subchronic toxicity study in Wistar rats did not show any treatment-related adverse effect.

Full text of DOI:10.1016/S0223-5234(01)01257-0

Eur. J. Med. Chem. 36 (2001) 627–637
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01257-0/FLA
Original article
Novel phthalazinone and benzoxazinone containing thiazolidinediones as
antidiabetic and hypolipidemic agents
Gurram R. Madhavana, Ranjan Chakrabartib, Sunil K.B. Kumarb, Parimal Misrab,
Rao N.V.S. Mamidib, V. Balrajua, Katneni Kasiramb, Ravi K. Babub, Juluri Sureshb,
Braj B. Lohrayb,1, Vidya B. Lohrayb,1, Javed Iqbala,*, Ramanujam Rajagopalanb,*
aDiscovery Chemistry, Dr. Reddy’s Research Foundation, Bollaram Road, Miyapur, Hyderabad 500 050, India
bDiscovery Biology, Dr. Reddy’s Research Foundation, Bollaram Road, Miyapur, Hyderabad 500 050, India
Received 5 March 2001; revised 18 June 2001; accepted 19 June 2001
Abstract – We report here the synthesis of a series of 5-[4-[2-[substituted phthalazinones-2(or 4)yl]ethoxy]phenylmethyl]thiazolidine-
2,4-diones and 5-[4-[2-[2,3-benzoxazine-4-one-2-yl]ethoxy]phenylmethyl]thiazolidine-2,4-diones and their plasma glucose and plasma
triglyceride lowering activity in db/db mice. In vitro PPARg transactivation assay was performed in HEK 293T cells. In vitro and
in vivo pharmacological studies showed that the phthalazinone analogue has better activity. PHT46 (compound 5a), the best
compound in this series, showed better in vitro PPARg transactivation potential than troglitazone and pioglitazone. In insulin
resistant db/db mice, PHT46 showed better plasma glucose and triglyceride lowering activity than the standard drugs. Pharmacoki-
netic study in Wistar rats showed good systemic exposure of PHT46. Subchronic toxicity study in Wistar rats did not show any
´
treatment-related adverse effect. © 2001 Editions scientifiques et me´dicales Elsevier SAS
phthalazinone / benzoxazinone / thiazolidinedione / PPARg / plasma glucose / triglyceride lowering / diabetes mellitus / insulin
resistance
1. Introduction
cose levels has revolutionised the treatment of
NIDDM [6]. Their mechanism of action is quite dif-
ferent from that of established antidiabetics such as
sulfonylureas [7] and biguanides [8]. These com-
Non-insulin dependent diabetes mellitus (NIDDM)
is characterised by hyperglycemia, hyperinsulinemia
and impaired insulin action [1]. Insulin resistance is
pounds are synthetic, high affinity ligands of Perox-
considered to be the underlying mechanism in the
isome Proliferator Activated Receptor gamma
pathogenesis of type 2 diabetes, which also leads to
(PPARg), a member of the nuclear receptor super-
dyslipidemia, hypertension and obesity, termed to-
gether as metabolic syndrome [2, 3]. The combination
family which controls the expression of genes in-
volved in lipid and carbohydrate metabolism in target
tissues [9, 10]. It is believed that this class of com-
pounds ameliorates insulin resistance through PPARg
activation. Many thiazolidinediones are in various
stages of clinical development. Reports of hepatotoxi-
city and idiosyncratic deaths associated with troglita-
zone treatment have validated the need for safer
insulin sensitisers to manage type 2 diabetes [11, 12].
We have previously reported [13] DRF-2189, which
was designed based on the constrained conformation
concept, conceptually obtained by cyclising the
of these disorders leads to the development of late
diabetic complications like coronary artery disease,
nephropathy, etc. [4, 5].
The advent of thiazolidinediones, which ameliorate
insulin resistance and normalise elevated blood glu-
* Correspondence and reprints
E-mail addresses: javediqbal@mailcity.com (J. Iqbal), drfra-
jagopalan@hotmail.com (R. Rajagopalan).
1 Present address: Zydus Research Centre, Ahmedabad, In-
dia.

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G.R. Madhavan et al. / European Journal of Medicinal Chemistry 36 (2001) 627–637
[15, 16]. The phthalazinones 2 were treated with 4-(2-
bromoethoxy)benzaldehyde (1) [17] in the presence of
K2CO3 in dimethyl formamide at 70–80 °C for 2–6 h
to furnish the aldehyde 3, which was reacted with
thiazolidine-2,4-dione in the presence of piperidine
benzoate to afford benzylidene thiazolidinedione ana-
logues 4 in a good range of yields. The olefinic bond
in 4 was hydrogenated in 1,4-dioxane using 10% Pd/C
as catalyst to give 5-benzyl-2,4-thiazolidinediones (5),
which were converted into their salts 6 by using metal
(Na, K) methoxide in methanol (figure 2). Similarly,
2,3-benzoxazininone compounds 7–11 [18] (figure 3)
were prepared following the protocol described for
phthalazinones. Accordingly, 4-hydroxymethyl-2-
methyl-1(2H)-phthalazinone (12) [19] (figure 4) was
coupled with 4-fluorobenzaldehyde (NaH–DMF) at
room temperature to give the aldehyde 13, which was
condensed with 2,4-thiazolidinedione followed by hy-
drogenation to afford the product 15.
Figure 1. Conversion of rosiglitazone to phthalazinones.
methyl group on nitrogen of rosiglitazone with and
without incorporation of a carbon or heteroatom. We
envisioned that cyclisation with a hetero atom would
lead to a lactam while preserving the link with the
nitrogen atom of the lactam ring (figure 1), thus one
can generate phthalazinone/benzoxazinone series of
compounds, with potential insulin sensitising activity.
It is known that a heterocycle containing a car-
bonyl group is more efficacious than a simple hetero-
cycle [14], so we anticipated that this type of
modification will yield potent thiazolidinediones. In
the present study, we are reporting some structure–
activity relation studies (SAR) of thiazolidinediones
having phthalazinones and benzoxazinones.
3. Pharmacology
Diabetic db/db mice, a commonly used animal
model of NIDDM [20], were used to assess the
plasma glucose and triglycerides lowering activity of
these compounds. Troglitazone, pioglitazone and
rosiglitazone were used as references in our studies.
2. Chemistry
A general strategy for the synthesis of thiazolidine-
2,4-diones is shown in figure 2. The starting materials
phthalazinones were prepared by reported methods
Figure 2. Synthesis of phthalazinone–thiazolidinediones 3–6. Reagents: (a) DMF, K2CO3, 4-(2-bromoethoxy)benzaldehyde (1),
70 °C; (b) thiazolidine-2,4-dione (TZD), toluene, piperidine-benzoate, reflux; (c) 10% Pd/C–dioxane, r.t.; (d) MeOH, MOMe
(M=metals like Na or K), r.t.

G.R. Madhavan et al. / European Journal of Medicinal Chemistry 36 (2001) 627–637
629
Figure 3. Synthesis of 5-[4-[2-[4-oxo-3,4-dihydro-1H-2,3-benzoxazine-3-yl]ethoxy]phenylmethyl]thiazolidine-2,4-dione (10).
Reagents: (a) DMF, K2CO3, 4-(2-bromoethoxy)benzaldehyde (1), 70 °C; (b) TZD, toluene, piperidine-benzoate, reflux; (c) 10%
Pd/C–dioxane, r.t.; (d) MeOH, MOMe(M=metals like Na or K), r.t.
Figure 4. Synthesis of 5-[4-[[2-methyl-1-oxo-1,2-dihydrophthalazin-4-yl]methoxy]phenylmethyl]thiazolidine-2,4-dione (15). Reagents:
(b) TZD, toluene, piperidine-benzoate, reflux; (c) 10% Pd/C–dioxane, r.t.; (e) NaH, DMF, 4-fluorobenzaldehyde, r.t.

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G.R. Madhavan et al. / European Journal of Medicinal Chemistry 36 (2001) 627–637
Table I. SAR studies of phthalazinone and benzoxazinone–thiazolidinediones 5–15.
Male db/db mice were treated with the compounds for 6 days and plasma glucose and triglyceride levels were measured. Percentage
reduction was calculated according to the formula: 1−[(TT/OT)/(TC/OC)]×100; TT: test day treated, OT: zero day treated, TC: test
day control, OC: zero day control; HET: heterocycle; DB: double bond; NE: no effect; PG: plasma glucose; TG: plasma triglyceride.
Male Wistar rats were used for pharmacokinetic stud-
ies to check the systemic exposure of the interesting
compound. Wistar rats of both sexes were used
for subchronic oral toxicity studies. Since thiazo-
lidinediones are known to activate nuclear receptor
PPARg, we have used a cell-based reporter assay
to check the activity of these compounds on
PPARg.

G.R. Madhavan et al. / European Journal of Medicinal Chemistry 36 (2001) 627–637
631
Table II. Effect of PHT46, troglitazone and pioglitazone in PPAR transactivation assay.
Concentration (mM)
PHT46 (fold activation)
Troglitazone (fold activation)
Pioglitazone (fold activation)
0.250
0.500
1.0
1.7
3.9
5.4
1.8
2.3
3.7
5.5
1.6
2.4
3.8
6.0
5.0
11.0
HEK 293T cells transfected with GAL4-PPARg1 and Pgl2-gal4×5-Luc plasmids (Section 6.2). Transfected cells were treated with
PHT46, troglitazone and pioglitazone at concentrations mentioned. Each data point represents mean of two experiments.
tion by phenyl (5d) or ethyl (5c) groups leads to the
loss of both glucose and triglycerides lowering activ-
ity. Interestingly, the substitution by hydrogen (5b)
retains the glucose lowering activity but not the
triglycerides lowering potential. On the other hand,
interchanging the methyl group and linker positions
as seen in compound 15 leads to the loss of biological
activity. We believe that this could be due to the size
[13, 22] of the substitution group, as methyl is smaller
and more planar compared to ethyl, phenyl and a
4-position substituted compound. The unsaturated
analogue 6b showed less euglycemic activity, as com-
pared to the saturated analogue 5. In view of its
potent in vitro and in vivo activity, PHT46 (5a) was
selected for further evaluation.
A comparative dose–response study was performed
to evaluate the PPARg transactivation potential of
PHT46 with troglitazone and pioglitazone as stan-
dard compounds. Interestingly, PHT46 showed much
better PPARg activation than both the standard com-
pounds in HEK 293T cells (table II). In an in vivo
dose–response study in db/db mice, PHT46 showed
dose-dependent reduction in plasma glucose and
triglycerides levels after 10 days of treatment (table
4. Results and discussion
The synthesis of phthalazinone derived thiazo-
lidinedione analogues 5 and 15 and benzoxazinone
derivatives 10 and 11 was achieved by the synthetic
protocols described earlier. Compounds 5 and 10
were prepared via a common route, but for com-
pound 15 slight modification was done at an interme-
diate step to prepare 13 which was obtained from
4-fluorobenzaldehyde. Compound 10 could not be
tested because of its gummy oil nature but its sodium
salt 11 was tested for biological activity. These com-
pounds were evaluated in db/db mice at 10 mg kg−1
dose (p.o.) for 6 days and plasma glucose and triglyc-
erides levels were measured. The results after 6 days
of treatment (table I) suggest that phthalazinone thia-
zolidinediones 5c, 5a, 15, 5d, 6b showed poor plasma
glucose (PG) reductions. However, phthalazinone 5b
(R=H) showed interesting glucose reduction (54%),
but no triglycerides reduction at 10 mg kg−1 dose
whereas 5a (R=CH3) showed both impressive
plasma glucose (61%) and triglyceride (40%) lowering
activity. The benzoxazinone derivative of thiazo-
lidinedione 10 was prepared as its sodium salt 11 and
it showed a moderate plasma glucose reduction
(37%), but impressive triglycerides reduction (60%).
In vitro PPARg transactivation assay of 5a showed
more potent activity than 11 (11-fold vs. 6-fold at 1
mM). In view of our interest in potent PPARg agonist,
thiazolidinediones having both sugar and triglyceride
lowering activity, we further explored the phthalazi-
none series. In view of the propensity of the racemisa-
tion of the C-5 chiral centre of thiazolidine-2,4-dione
at physiological pH [21], no attempts were made to
synthesise these compounds in optically pure forms.
It appears that the methyl derivative of phthalazi-
none with a linker from one of the nitrogen atom
adjacent to carbonyl 5a (named as PHT46) showed a
better profile compared to other derivatives. Substitu-
Table III. Effect of PHT46 on plasma glucose and triglyceride
levels in db/db mice.
Group
Plasma Glucose
(mg/dl)
Plasma triglyceride
(mg/dl)
Control
419.06919.46
347.13998.13
221.20958.60*
118.7095.15*
135.85918.48
75.4295.45
66.6596.40*
56.0191.61*
1 mg kg−1
3 mg kg−1
10 mg kg−1
Male db/db mice were treated with PHT46 at the doses
mentioned for 10 days. Values are mean9S.E. (n=5). *PB
0.05 vs. control (ANOVA).

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G.R. Madhavan et al. / European Journal of Medicinal Chemistry 36 (2001) 627–637
Table IV. Dose–response studies of selected thiazolidinediones in db/db mice.
Compound
PHT46
Dose
Percent reduction in plasma glucose
Percent reduction in plasma triglyceride
(mg kg−1 day−1
)
1
3
10
100
200
400
10
24.293.6
45.191.5
72.692.6
37.298.5
45.593.5
59.893.6
19.493.5
38.592.4
46.292.9
33.596.5
57.393.1
58.592.9
38.499.2
57.292.6
62.792.8
14.791.9
36.191.7
46.991.6
Troglitazone
Pioglitazone
30
100
Male db/db mice were treated with the compounds orally at the doses mentioned for 10 days. Values are mean9S.E. (n=5).
Percentage reduction was calculated as mentioned in table I.
5. Conclusion
III). This study indicates that the treatment with
PHT46 brings down the plasma glucose to the normal
level. PHT46 was compared with troglitazone and
pioglitazone in a 10-day dose–response study in db/
db mice. PHT46 at 3 mg kg−1 dose showed similar
efficacy to pioglitazone at 100 mg kg−1 and troglita-
zone at 200 mg kg−1 doses (table IV). PHT46 was
also compared with rosiglitazone at maximal effective
dose in a 10-day treatment schedule in db/db mice. In
this study, PHT46 at 10 mg kg−1 dose showed 72%
reduction of plasma glucose and 58% reduction of
triglyceride, whereas rosiglitazone at 30 mg kg−1
showed 67% reduction in plasma glucose and 40%
reduction in triglyceride. These studies indicate that
PHT46 is more potent and efficacious than the stan-
dard compounds.
Subchronic (14 days) oral administration of PHT46
to Wistar rats did not show any significant change in
body weight (control male rats: 192 7; female rats:
144 5; PHT46 treated male rats: 191 8; female rats:
150 5 g) and food consumption throughout the ex-
periment. The haematology examination after 14 days
showed non-significant reduction in haemoglobin
(6%) and packed cell volume (5%). There was a non-
significant increase (6%) of heart and liver weight.
The changes observed in haematology and organ
weights were within the acceptable limit. The bio-
chemical and histological findings are not suggestive
of any treatment related pathology.
Insulin resistance is considered as the primary de-
fect in NIDDM patients. Insulin resistance not only
leads to hyperglycemia, but also to hyperlipidemia.
Thiazolidinediones are known to ameliorate insulin
resistance through their action on PPARg. In this
communication, we have shown that our phathalazi-
none derivatives of thiazolidinediones have interesting
insulin sensitising property. PHT46, the best com-
pound in this series is a potent PPARg activator and
shows both glucose and triglycerides lowering activ-
ity. The compound shows better efficacy than the
reference thiazolidinediones (i.e. troglitazone, piogli-
tazone and rosiglitazone) in both in vitro and in vivo
animal studies. Subchronic toxicity study in Wistar
rats did not show any treatment related adverse ef-
fect. It is important to note that an efficient control of
triglyceride levels in type 2 diabetic patients is consid-
ered to be beneficial in diabetes related complications,
especially atherosclerosis and cardiovascular disease,
as such a drug that ameliorates insulin resistance and
also lowers triglyceride is preferable for treatment of
Table V. Pharmacokinetic parameters of PHT46 in Wistar
rats.
AUC(0–ꢀ) (mg h mL−1
)
13.17
4.15
3.00
0.42
1.67
Cmax (mg mL−1
Tmax (h)
)
Kel (h−1
t1/2 (h)
)
To check the systemic exposure of PHT46, a single
dose oral pharmacokinetic study was performed in
male Wistar rats, which indicated that the compound
has good oral exposure and pharmacokinetic parame-
ters (table V, figure 5).
Male Wistar rats were administered with a single dose of
PHT46 at 10 mg kg−1, p.o. Each value is mean from n=3
animals.

G.R. Madhavan et al. / European Journal of Medicinal Chemistry 36 (2001) 627–637
633
6.1.1. General method of preparation of 4-(2-heterocycle-
yl ethoxy)benzaldehyde
To a stirred suspension of K2CO3 (82.5 mmol) in
anhydrous dimethylformamide (50 mL) at 25–30 °C
was added a solution of the respective phthalazinone
(41.25 mmol) in dry DMF (100 mL). The reaction
mixture was stirred for 30 min at 25 °C and 4-(2-bro-
moethoxy)benzaldehyde (1) (41.25 mmol) was added.
The reaction mixture was immersed in a pre-heated oil
bath at 70 °C and stirred at 65–70 °C for 24 h. The
reaction mixture was cooled to room temperature (r.t.)
and water (50 mL) was added. The reaction mixture was
extracted with ethyl acetate (3×50 mL) and the com-
bined organic layers were washed with water and brine.
Drying the organic extract over anhydrous Na2SO4 fol-
lowed by concentration afforded the crude solids, which
were recrystallised from methanol to afford the pure
aldehydes in good yields.
6.1.2. 4-[2-[4-Methyl-1-oxo-1,2-dihydrophthalazin-2-
yl]ethoxy]benzaldehyde (3a)
Figure 5. Plasma concentration profile of PHT46. Male Wis-
tar rats were treated with 10 mg kg−1 of PHT46 and plasma
samples were collected at different time points. Values are
expressed as mean S.D. (n=3).
This compound was prepared by the general proce-
dure using 4-methyl-1(2H)phthalazinone (6.6 g, 41.25
mmol), aldehyde 1 (9.4 g, 41.25 mmol) and K2CO3 (11.3
1
g, 82.5 mmol) in 77% yield (9.7 g), m.p.: 90 °C. H-
NMR (CDCl3): l=2.50 (s, 3H), 4.50 (t, J=5.66 Hz,
2H), 4.64 (t, J=5.66 Hz, 2H), 7.03 (d, J=8.50 Hz, 2H),
7.20–7.90 (m, 5H), 8.46 (d, J=8.10 Hz, 1H), 9.85 (s,
1H).
type 2 diabetes. Therefore, PHT46 by virtue of its
potent antidiabetic and triglyceride lowering property
is a potential drug for the treatment of metabolic
syndrome. Further developmental work is in progress.
6.1.3. 4-[2-[1-Oxo-1,2-dihydrophthalazin-2-
yl]ethoxy]benzaldehyde (3b)
This compound was prepared by the general proce-
dure using 1(2H)-phthalazinone (0.8 g, 5.48 mmol),
aldehyde 1 (1.25 g, 5.48 mmol) and K2CO3 (1.5 g, 10.96
6. Experimental protocols
1
mmol) in 44% yield (0.7 g). H-NMR (CDCl3): l=4.53
6.1. Synthesis
(t, J=5.70 Hz, 2H), 4.69 (t, J=5.70 Hz, 2H), 7.04 (d,
J=8.72 Hz, 2H), 7.69–7.87 (m, 5H), 8.19 (s, 1H), 8.46
(d, J=8.10 Hz, 1H), 9.87 (s, 1H).
Thin layer chromatography was performed on pre-
coated silica gel plates (F254, Merck). Chromatography
was performed on silica gel (SRL 100–200 mesh). Melt-
ing points were recorded in a Veego Melting Point
6.1.4. 4-[2-[4-Ethyl-1-oxo-1,2-dihydrophthalazin-2-
yl]ethoxy]benzaldehyde (3c)
1
apparatus and are uncorrected. H-NMR spectra were
obtained with a Varian Gemini 200 MHz spectrometer
and are reported as parts per million (ppm) downfield
to TMS. The infrared spectra were recorded in a
Perkin–Elmer FT-IR 1600 spectrometer. 4-(2-Bro-
moethoxy)benzaldehyde (1) and phthalazinones [15, 16]
were prepared according to reported literature methods
[17], while 2(1H)-phthalazinone was obtained from
Merck.
This compound was prepared by the general proce-
dure using 4-ethyl-1(2H)-phthalazinone (0.45 g, 2.6
mmol), aldehyde 1 (0.6 g, 2.6 mmol) and K2CO3 (0.72 g,
5.2 mmol) in 54% yield (0.45 g), m.p.: 110 °C. 1H-NMR
(CDCl3): l=1.36 (t, J=7.05 Hz, 3H), 2.98 (q, J=7.05
Hz, 2H), 4.50 (t, J=5.70 Hz, 2H), 4.68 (t, J=5.70 Hz,
2H), 7.02 (d, J=8.72 Hz, 2H), 7.70–7.92 (m, 5H), 8.51
(d, J=8.10 Hz, 1H), 9.21 (s, 1H).

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G.R. Madhavan et al. / European Journal of Medicinal Chemistry 36 (2001) 627–637
6.1.5. 4-[2-[4-Phenyl-1-oxo-1,2-dihydrophthalazin-2-
yl]ethoxy]benzaldehyde (3d)
6.1.9. 5-[4-[2-[4-Methyl-1-oxo-1,2-dihydrophthalazin-2-
yl]ethoxy]phenylmethylene]thiazolidine-2,4-dione (4a)
This compound (3.3 g, 91%) was prepared according
to the general procedure using 3a (2.75 g, 8.93 mmol)
and thiazolidine-2,4-dione (1.05 g, 8.93 mmol), m.p.:
This compound was prepared by the general
procedure using 4-phenyl-1(2H)-phthalazinone (1.30 g,
5.85 mmol), aldehyde 1 (1.34 g, 5.85 mmol) and K2CO3
(1.60 g, 11.7 mmol) in 85% yield (2.0 g), m.p.: 136 °C.
1H-NMR (CDCl3): l=4.56 (t, J=5.70 Hz, 2H), 4.75 (t,
J=5.70 Hz, 2H), 7.03 (d, J=8.72 Hz, 2H), 7.55 (bs,
5H), 7.65–7.85 (m, 5H), 8.55 (d, J=8.10 Hz, 1H), 9.86
(s, 1H).
1
202 °C. H-NMR (DMSO-d6): l=2.60 (s, 3H), 4.48 (t,
J=5.30 Hz, 2H) 4.55 (t, J=5.30 Hz, 3H), 6.95 (d,
J=8.30 Hz, 2H), 7.40 (d, J=8.30 Hz, 2H), 7.72 (s, 1H),
7.75–7.90 (m, 3H), 8.49 (d, J=8.10 Hz, 1H).
6.1.10. 5-[4-[2-[1-Oxo-1,2-dihydrophthalazin-2-
yl]ethoxy]phenylmethylene]thiazolidin-2,4-dione (4b)
This compound (533 mg, 80%) was prepared accord-
ing to the general procedure using 3b (500 mg, 1.7
mmol) and thiazolidine-2,4-dione (200 mg, 1.7 mmol),
6.1.6. 4-[2-[4-Oxo-3,4-dihydro-(1H)-2,3-benzoxazin-3-
yl]ethoxy]benzaldehyde (8)
This compound was prepared by the general
procedure using 4-oxo-3,4-dihydro-(1H)-2,3-benzoxazin
(0.28 g, 1.87 mmol), aldehyde 1 (0.43 g, 1.87 mmol) and
K2CO3 (0.52 g, 3.74 mmol), m.p.: 102 °C. 1H-NMR
(CDCl3): l=4.23 (t, J=5.0 Hz, 2H), 4.35 (t, J=5.0
Hz, 2H), 5.11 (s, 2H), 7.04 (d, J=8.70 Hz, 2H), 7.10 (d,
J=7.00 Hz, 1H), 7.40–7.60 (m, 2H), 7.80 (d, J=8.70
Hz, 2H), 8.10 (d, J=7.20 Hz, 1H), 9.89 (s, 1H).
1
m.p.: 242 °C. H-NMR (DMSO+CDCl3): l=4.50 (t,
J=5.25 Hz, 2H), 4.58 (t, J=5.25 Hz, 2H), 7.05 (d,
J=8.72 Hz, 2H), 7.46 (d, J=8.72 Hz, 2H), 7.88–7.98
(m, 3H), 8.06 (s, 1H), 8.33 (d, J=7.20 Hz, 1H), 8.35 (s,
1H). 12.3 (bs, 1H, D2O exchangeable).
6.1.11. 5-[4-[2-[4-Ethyl-1,2-dihydrophthalazine-2-
yl]ethoxy]phenylmethylene]thiazolidine-2,4-dione (4c)
This compound (432 mg, 82%) was prepared accord-
ing to the general procedure using 3c (400 mg, 1.24
mmol) and thiazolidine-2,4-dione (145 mg, 1.24 mmol),
6.1.7. 4-[[2-Methyl-1-oxo-1,2-dihydrophthalazin-4-
yl]methoxy]benzaldehyde (13)
To
a
solution of 4-hydroxymethyl-2-methyl-
1
1(2H)phthalazinone (0.35 g, 1.84 mmol) in dry DMF
(30 mL) was added NaH (0.088 g, 3.68 mmol) in
portions over 30 min at 25–30 °C. This was followed by
dropwise addition of 4-fluorobenzaldehyde (0.228 g,
1.84 mmol) at 0–15 °C and the resulting reaction
mixture was stirred at r.t. for 6 h. Ice (200 g) was added
to the reaction mixture which was extracted with ethyl
acetate. The ethyl acetate layer was washed with water,
dried over anhydrous Na2SO4 and concentrated to
afford the title compound 13 (0.3 g, 50%), m.p.:
m.p.: 230 °C. H-NMR (DMSO+CDCl3): l=1.31 (t,
J=7.38 Hz, 3H), 2.98 (q, J=7.38 Hz, 2H), 4.38–4.65
(m, 4H), 7.05 (d, J=8.72 Hz, 2H), 7.45 (d, J=8.72 Hz,
2H), 7.80–7.98 (m, 3H), 7.80–7.98 (m, 3H), 8.09 (s,
1H), 8.35 (d, J=7.15 Hz, 1H), 12.34 (bs, 1H, D2O
exchangeable).
6.1.12. 5-[4-[2-[4-Phenyl-1-oxo-1,2-dihydrophthalazin-
2-yl]ethoxy]phenylmethylene]thiazolidine-2,4-dione (4d)
This compound (1.10 g, 87%) was prepared according
to the general procedure using 3d (1.0 g, 2.7 mmol) and
thiazolidine-2,4-dione (316 mg, 2.7 mmol), m.p.:
224 °C. 1H-NMR (DMSO+CDCl3): l=4.55 (t, J=
5.25 Hz, 2H), 4.65 (t, J=5.25 Hz, 2H), 7.04 (d, J=8.72
Hz, 2H), 7.44 (d, J=8.72 Hz, 2H), 7.40–7.60 (m, 5H),
7.60–7.89 (m, 3H), 7.96 (s, 1H), 8.45 (t, J=7.30 Hz,
1H), 12.20 (bs, 1H, D2O exchangeable).
1
168–170 °C. H-NMR (CDCl3): l=3.88 (s, 3H), 5.39
(s, 2H), 7.18 (d, J=8.72 Hz, 2H), 7.72–8.00 (m, 5H),
8.48 (d, J=8.10 Hz, 1H), 9.92 (s, 1H).
6.1.8. General method to prepare 5-[4-[2-(heterocyclyl)-
ethoxy]phenylmethylene]thiazolidine-2,4-dione (4)
A mixture of 4-[2-(heterocyclyl)ethoxy]benzaldehyde 3
(10 mmol), thiazolidine-2,4-dione (10 mmol), benzoic
acid (1.3 mmol) and piperidine (1.5 mmol) in 25 mL of
toluene was refluxed for 1 h with continuous removal of
water using a Dean Stark water separator. The reaction
mixture was cooled to r.t. and the resultant crystalline
compound filtered, washed with water and dried to
afford the pure compound 4 (70–99%).
6.1.13. 5-[4-[2-[4-Oxo-3,4-dihydro-1H-2,3-benzoxazin-
3yl]ethoxy]phenylmethylene]thiazolidine-2,4-dione (9)
This compound (310 mg, 67%) was prepared accord-
ing to the general procedure using 8 (350 mg, 1.18
mmol) and thiazolidine-2,4-dione (138 mg, 1.18 mmol),
1
m.p.: 182 °C. H-NMR (CDCl3): l=4.18 (t, J=5.40

G.R. Madhavan et al. / European Journal of Medicinal Chemistry 36 (2001) 627–637
635
Hz, 2H), 4.35 (t, J=5.40 Hz, 2H), 5.14 (s, 2H), 7.04 (d,
6.1.18. 5-[4-[2-[4-Ethyl-1-oxo-1,2-dihydrophthalazin-
J=8.70 Hz, 2H), 7.20 (d, J=7.40 Hz, 1H), 7.44 (d,
J=8.70 Hz, 2H), 7.50–7.60 (m, 2H), 7.71 (s, 1H), 8.02
(d, J=7.40 Hz, 1H), 12.15 (bs, 1H, D2O exchangeable).
2-yl]ethoxy]phenylmethyl]thiazolidine-2,4-dione (5c)
This compound was prepared according to the gen-
eral procedure using 4c (430 mg, 1.02 mmol) and 10%
Pd/C (860 mg) in 46% yield (200 mg), m.p.: 174 °C.
1H-NMR (CDCl3): l=1.33 (t, J=7.50 Hz, 3H), 2.96
(q, J=7.50 Hz, 2H), 3.04 (d, J=14.11, 9.59 Hz, 1H),
3.40 (dd, J=14.11, 3.92 Hz, 1H), 4.32 (t, J=5.90 Hz,
2H), 4.42 (dd, J=9.59, 3.92 Hz, 1H), 4.59 (t, J=5.90
Hz, 2H), 6.85 (d, J=8.72 Hz, 2H), 7.08 (d, J=8.72 Hz,
2H), 7.60–7.80 (m, 3H), 8.02 (bs, 1H, D2O exchange-
able), 8.46 (d, J=8.41 Hz, 1H).
6.1.14. 5-[4-[[2-Methyl-1-oxo-1,2-dihydrophthalazin-4-
yl]methoxy]phenylmethylene]thiazolidine-2,4-dione (14)
This compound (yield: 250 mg, 98%) was prepared
according to the general procedure using 13 (190 mg,
0.65 mmol) and thiazolidine-2,4-dione (75 mg, 0.65
mmol), m.p.: 262 °C. 1H-NMR (CDCl3+DMSO-d6):
l=3.79 (s, 3H), 5.47 (s, 2H), 7.28 (d, J=8.72 Hz, 2H),
7.62 (d, J=8.72 Hz, 2H), 7.70 (s, 1H), 7.90–8.15 (m,
3H), 8.35 (d, J=7.25 Hz, 1H).
6.1.19. 5-[4-[2-[4-Phenyl-1-oxo-1,2-dihydrophthalazin-
2-yl]ethoxy]phenylmethyl]thiazolidine-2,4-dione (5d)
This compound was prepared according to the gen-
eral procedure using 4d (1.0 g, 2.1 mmol) and 10% Pd/C
6.1.15. General procedure for preparation of 5-[4-[2-
[heterocyclyl]ethoxy]phenylmethyl]thiazolidine-2,4-diones
(5)
1
(2.0 g) in 60% yield (600 mg), m.p.: 198 °C. H-NMR
A solution of thiazolidine-2,4-dione 4 (1.0 g) in
1,4-dioxane (35 mL) was hydrogenated in the presence
of 10% Pd/C (2.0 g) at 60 PSI for 36–60 h at r.t. The
mixture was filtered through a bed of celite and the
filtrate was evaporated to dryness under reduced
pressure. The residue was chromatographed over silica
gel using a mixture of ethyl acetate and petroleum ether
as an eluent to yield pure compound 5.
(CDCl3): l=3.07 (1H, dd, J=14.12, 9.6 Hz), 3.44 (dd,
J=14.12, 3.74 Hz, 1H), 4.42 (dd, J=9.60, 3.74 Hz,
1H), 4.46 (t, J=5.90 Hz, 2H), 4.72 (t, J=5.90 Hz, 2H),
6.89 (d, J=8.62 Hz, 2H), 7.11 (d, J=8.62 Hz, 2H),
7.45–7.70 (m, 5H), 7.70–7.90 (m, 3H), 8.00 (bs, 1H,
D2O exchangeable), 8.55 (d, J=8.30 Hz, 1H).
6.1.20. 5-[4-[[2-Methyl-1-oxo-1,2-dihydrophthalazin-4-
yl]methoxy]phenylmethyl]thiazolidine-2,4-dione (15)
This compound was prepared according to the gen-
eral procedure using 14 (1.0 g, 2.54 mmol) and 10%
Pd/C (2.0 g) in 50% yield (500 mg), m.p.: 234 °C.
1H-NMR (CDCl3): l=3.08 (dd, J=14.11, 9.44 Hz,
1H), 3.46 (dd, J=14.11, 3.74 Hz, 1H), 3.86 (s, 3H), 4.46
(dd, J=9.44, 3.74 Hz, 1H), 5.28 (s, 2H), 6.99 (d,
J=8.12 Hz, 2H), 7.19 (d, J=8.12 Hz, 2H), 7.88–7.92
(m, 2H), 7.99 (d, J=7.30 Hz, 1H), 8.42 (d, J=7.30 Hz,
1H), 11.70 (bs, 1H, D2O exchangeable).
6.1.16. 5-[4-[2-[4-Methyl-1-oxo-1,2-dihydrophthalazin-
2-yl]ethoxy]phenylmethyl]thiazolidine-2,4-dione (5a)
This compound was prepared according to the gen-
eral procedure using 4a (9.0 g, 22 mmol) and 10% Pd/C
(18 g) in 80% yield (7.2 g), m.p.: 170 °C. 1H-NMR
(CDCl3): l=2.60 (s, 3H), 3.07 (dd, J=14.12, 9.50 Hz,
1H), 3.42 (dd, J=14.12, 3.70 Hz, 1H), 4.40 (t, J=6.10
Hz, 2H), 4.46 (dd, J=9.50, 3.70 Hz, 1H), 4.61 (t,
J=6.10 Hz, 2H), 6.88 (d, J=8.50 Hz, 2H), 7.11 (d,
J=8.50 Hz, 2H), 7.68–7.91 (m, 3H), 8.51 (d, J=8.10
Hz, 1H).
6.1.21. 5-[4-[2-[4-Oxo-3,4-dihydro-1H-2,3-benzoxazin-
3-yl]ethoxy]phenylmethyl]thiazolidine-2,4-dione (10)
This compound (310 mg, 67%) was prepared as a
gummy oil according to the general procedure using 9
(250 mg, 0.63 mmol) and 10% Pd/C (500 mg) in 40%
6.1.17. 5-[4-[2-[1-Oxo-1,2-dihydrophthalazin-2-yl]-
ethoxy]phenylmethyl]thiazolidine-2,4-dione (5b)
This compound was prepared by the general proce-
dure using 4b (385 mg, 0.98 mmol) and 10% Pd/C (770
mg) in 52% yield (200 mg), m.p.: 165 °C. 1H-NMR
(CDCl3): l=3.08 (dd, J=14.12, 9.32 Hz, 1H), 3.40 (dd,
J=14.12, 3.97 Hz, 1H), 4.42 (t, J=5.81 Hz, 2H), 4.46
(dd, J=9.32, 3.97 Hz, 1H), 4.65 (t, J=5.81 Hz, 2H),
6.88 (d, J=8.72 Hz, 2H), 7.11 (d, J=8.72 Hz, 2H),
7.60–7.90 (m, 3H), 8.07 (bs, 1H, D2O exchangeable),
8.20 (s, 1H), 8.45 (d, J=8.62 Hz, 1H).
1
yield (100 mg), m.p.: 140 °C. H-NMR (CDCl3): l=
3.12 (dd, J=14.11, 4.89 Hz, 1H), 3.41 (dd, J=14.11,
3.73 Hz, 1H), 4.18 (t, J=4.50 Hz, 2H), 4.24 (t, J=4.50
Hz, 2H), 4.50 (dd, J=9.22, 3.83 Hz, 1H), 5.09 (s, 2H),
6.88 (d, J=8.60 Hz, 2H), 7.21 (d, J=8.60 Hz, 2H),
7.40–7.60 (m, 3H), 8.00 (bs, 1H, D2O exchangeable),
8.08 (d, J=7.20 Hz, 1H).

636
G.R. Madhavan et al. / European Journal of Medicinal Chemistry 36 (2001) 627–637
6.1.22. Sodium salt of 5-[4-[2-[4-methyl-1-oxo-1,2-di-
hydrophthalazin-2-yl]ethoxy]phenylmethylene]-
thiazolidine-2,4-dione (6b)
All experimental protocols were approved by the DRF
animal ethics committee.
In preliminary studies db/db mice (8–9 weeks) were
treated with drugs (10 mg kg−1) for 6 days. Animals in
control groups received vehicle only (0.25% CMC, 10
mL kg−1). During the dose–response study, db/db mice
were treated with PHT46 at 1, 3 and 10 mg kg−1 doses
for 10 days. Blood samples were collected from animals
(in fed state) under mild ether anaesthesia from retro-or-
bital sinus 1 h after drug administration. Plasma sam-
ples were separated for glucose, triglycerides, free fatty
acids and insulin measurement.
For the subacute toxicity study, 6–8 week old Wistar
rats (obtained from National Institute of Nutrition,
Hyderabad) of either sex (110–160 g) were divided
randomly into two groups, each consisting of four males
and four females. PHT46 was administered orally daily
at 100 mg kg−1 dose, while the control group received
the vehicle only. On the day of termination (14th day),
blood was collected from retro-orbital sinus under light
ether anaesthesia for haematological parameters fol-
lowed by autopsy and macroscopic examination.
To 5-[4-[2-[4-methyl-1-oxo-1,2-dihydrophthalazin-2-
yl]ethoxy]phenylmethylene]thiazolidine-2,4-dione
(4)
(200 mg, 0.49 mmol) in dry methanol (5 mL) was added
NaOMe (freshly prepared from 45 mg of Na metal in 1
mL of dry methanol) and the mixture stirred at r.t. for
1 h. The reaction mixture was diluted with 40 mL of dry
diethylether and the white precipitate thus formed was
filtered and washed with methanol–ether (1:3) and dried
under vacuum, to give a white compound 6b (180 mg,
1
86%), m.p.: 280 °C. H-NMR (DMSO-d6): l=2.60 (s,
3H), 4.42 (t, J=5.20 Hz, 2H), 4.50 (t, J=5.20 Hz, 2H),
6.94 (d, J=8.72 Hz, 2H), 7.38 (d, J=8.72 Hz, 2H),
7.70–7.90 (m, 3H), 7.88 (s, 1H), 8.34 (d, J=7.13 Hz,
1H).
6.1.23. Sodium salt of 5-[4-[2-[4-oxo-3,4-dihydro-1H-
2,3-benzoxazin-3-yl]ethoxy]phenylmethyl]-
thiazolidine-2,4-dione (11)
This compound (50 mg, 48%) was prepared according
to the previous method using 10 (100 mg, 0.344 mmol)
and NaOMe (0.688 mmol), m.p.: 255 °C. 1H-NMR
(DMSO-d6): l=2.65 (dd, J=10.47, 3.23 Hz, 1H), 2.55
(dd, J=10.47, 4.27 Hz, 1H), 4.00–4.19 (m, 3H), 4.21 (t,
J=5.07 Hz, 2H), 5.15 (s, 2H), 6.85 (d, J=8.38 Hz, 2H),
7.10 (d, J=8.38 Hz, 2H), 7.34 (d, J=7.47 Hz, 1H),
7.45–7.70 (m, 2H), 7.91 (d, J=7.47 Hz, 1H).
6.2.3. PPAR transactivation assay
The response element (UASGAL4×5) is present up-
stream of pFR-Luc reporter (Promega, WI, USA) that
contains Simian virus early promoter for luciferase as-
say. GAL4 fusions were made by fusing the human
PPARg1 ligand binding domain (amino acids: 174–475)
to the C-terminal end of the yeast GAL4 DNA binding
domain (amino acids: 1–147) of the pM1 vector. The
pAdVantage vector was used to enhance luciferase
expression.
HEK 293T cells were transfected with relevant plas-
mids by superfect as per the instruction manual [23].
Forty-two hours after transfection, cells were treated for
18 h with the test compounds. DMSO (1:1000) was used
as blank. Luciferase activity was determined as fold
activation relative to untreated cells by using a Luclite
kit (Packard Instrument Co, Meriden, CT, USA) in a
Packard Top Count (Packard Instrument Co.).
6.2. Pharmacological methods
6.2.1. Materials
The standard compounds, troglitazone and pioglita-
zone, were synthesised in house by the published proce-
dure and were found to be 99% pure. Plasma glucose
and triglyceride were measured spectrophotometrically
using commercially available kits (Point Scientific,
USA). Carboxymethylcellulose (CMC) was obtained
from LOBA Chemicals Pvt. Ltd, Mumbai, India.
6.2.2. Animals and treatment
Male C57 BL/Ks J-db/db mice were from the breed-
ing stock of the DRF animal house generated from an
original stock of Jackson Laboratories, Maine, USA.
All animals were maintained under 12 h light and 12 h
dark cycles at 25 1 °C. All animals were given stan-
dard chow (National Institute of Nutrition, India) and
water ad libitum. All animal experiments were carried
out in accordance with internationally valid guidelines.
6.2.4. Pharmacokinetic studies
All studies were carried out in male Wistar rats
obtained from the National Institute of Nutrition
(Hyderabad, India). The animals (200–225 gm) were
fasted 12 h before starting the experiment and they had
free access to water throughout the experiment period.
Animals were fed 3 h after drug administration.

G.R. Madhavan et al. / European Journal of Medicinal Chemistry 36 (2001) 627–637
637
6.2.4.1. Single dose pharmacokinetics
Novo Nordisk, Bagsvaerd, Denmark, for providing the
PPAR constructs.
Animals were administered the drug at 10 mg kg−1
per orally as 0.5% CMC suspension and about 0.30 mL
of blood sample was collected into heparinised mi-
crofuge tubes at different time points from retro-orbital
sinus. To 0.1 mL of plasma, internal standard (another
thiazolidinedione) was added and drugs were extracted
with a suitable solvent mixture. The solvent was evapo-
rated and the residue was reconstituted with a mobile
phase and injected into the HPLC system. The samples
were analysed by reverse-phase HPLC to generate
plasma concentration time profiles.
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Millennium software and a Suplecosil C18 (ODS)
column (5 mm, 4.6 mm×250 mm). Analysis of PHT46
was carried out using 0.05 M NaH2PO4 buffer (pH
4.0)/methanol (30:70) as the mobile phase at a flow rate
of 1 mL min−1. The eluate from the column was moni-
tored by a UV detector (Waters LC Module) set at 280
nm. Under these conditions retention times for PHT46
and IS were, respectively, 13.7 and 9.9 min. Absolute
recovery was >95%, the limit of quantification was 0.1
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Acknowledgements
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41 (1999) 1927–1940.
The authors are thankful to the management of Dr
[23] Superfect Transfection Reagent Handbook, Qiagen, Max
Reddy’s group for encouragement. We are thankful to
Volmer Strabe 4, 40724 Hilden, Germany.