Synthesis and biological activity of aminophthalazines and aminopyridazines as novel inhibitors of PGE2 production in cells

Article abstract of DOI:10.1016/j.bmcl.2012.11.030

This Letter reports the synthesis and biological evaluation of a collection of aminophthalazines as a novel class of compounds capable of reducing production of PGE₂ in HCA-7 human adenocarcinoma cells. A total of 28 analogs were synthesized, a

Full text of DOI:10.1016/j.bmcl.2012.11.030

Bioorganic & Medicinal Chemistry Letters 23 (2013) 528–531
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of aminophthalazines and aminopyridazines
as novel inhibitors of PGE2 production in cells
Federico Medda a,c, Earlphia Sells b, Hui-Hua Chang b, Justin Dietrich a,c, Shashi Chappeta c,
Breland Smith a,d, Vijay Gokhale a, Emmanuelle J. Meuillet b, Christopher Hulme a,c,d,
⇑
a BIO5 Oro Valley, The University of Arizona, 1580 E. Hanley Blvd., Oro Valley, AZ 85737, United States
b Departments of Nutritional Sciences and Molecular and Cellular Biology, The University of Arizona, 1515 N. Campbell Ave., Tucson, AZ 85724, United States
c Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721, United States
d Department of Chemistry & Biochemistry, The University of Arizona, 1306 E. University Blvd., Tucson, AZ 85721, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
This Letter reports the synthesis and biological evaluation of a collection of aminophthalazines as a novel
class of compounds capable of reducing production of PGE2 in HCA-7 human adenocarcinoma cells. A
total of 28 analogs were synthesized, assayed for PGE2 reduction, and selected active compounds were
evaluated for inhibitory activity against COX-2 in a cell free assay. Compound 2xxiv (R1 = H, R2 = p-
Received 9 October 2012
Revised 1 November 2012
Accepted 7 November 2012
Available online 21 November 2012
CH3O) exhibited the most potent activity in cells (EC50 = 0.02
ity (3% at 5
models exhibiting good anti-cancer activity.
l
M) and minimal inhibition of COX-2 activ-
l
M). Furthermore, the anti-tumor activity of analog 2vii was analyzed in xenograft mouse
Keywords:
Aminophthalazines
PGE2
Ó 2012 Elsevier Ltd. All rights reserved.
COX-2
Cancer
Prostaglandin E2 (PGE2) is well known to play a pivotal role in
processes associated with inflammation, pain and pyresis and is
over expressed in various tumors where chronic inflammation
has been linked to the growth of various cancerous tissues. Indeed,
PGE2 has been identified as the major prostaglandin associated
with the progression of various tumor malignancies including that
of the colon, lung, and breast.1–5
Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly
used as analgesics that function by inhibiting the activity of cyclo-
oxygenase (COX) enzymes which are involved in the biosynthesis
of a variety of prostaglandins, including PGE2. At high doses NSA-
IDs have been shown to reverse the growth of colorectal tumors
and patients using NSAIDs over long periods of time for chronic
disorders (i.e rheumatoid arthritis) exhibit a lower risk of develop-
ing colon cancer.1–4 It is postulated that the anti-cancer properties
elicited by NSAIDs are primarily due to a reduction in PGE2 levels
supported by the observation that exogenous treatment with
PGE2 is shown to impede NSAID induced tumor regression. Histor-
ically, side effects with non-selective COX-1/2 inhibitors (Aspirin,
Tylenol, Ibuprofen) driven by COX-1 mediated gastrointestinal
intolerance, promoted efforts to develop specific COX-2 inhibitors
(CelebrexÒ, BextraÒ and VioxxÒ). However, the latter two drugs
were subsequently withdrawn from the market due to a drug in-
duced imbalance of prostacyclin (PGI2) and thromboxane A2
(TXA2) leading to cases of myocardial infarction and thrombo-
sis.1–4 Thus the search for colon cancer modifying therapeutics is
now geared towards the development of PGE2 reducing drugs that
do not alter COX activity.6–11
In search of novel anti-tumor small molecules with the capacity
to reduce cellular levels of PGE2 without affecting in vitro COX-2
activity, our group has recently reported a series of 2-aminothiaz-
oles with interesting biological profiles (Fig. 1).11 In particular, 1a
exhibited the most potent cellular PGE2 reducing activity of the en-
tire series (EC50 90 nM), with only a nominal COX-2 inhibition [IC50
>5 lM]. Furthermore, analog 1b exhibited promising anti-cancer
activity in mouse xenograft models.11 In continuation of our stud-
ies, we herein report the biological evaluation of a novel series of
aminophthalazines in Table 1 analogs 2 with PGE2 reducing char-
acter in HCA-7 colon cancer cells, Figure 1.
The aminophthalazines Table 1 (2i–xxiv), were assembled using
methodology depicted in Scheme 1.12,13 Briefly, 1,4-dichloroph-
thalazine 3 was treated at reflux with an equivalent amount of ani-
line 4 affording intermediates 5 in good yield. Compounds 5
smoothly underwent Suzuki-coupling with select boronic acids
enabling formation of final aminophthalazine analogs 2i–xxiv
in moderate yields. Analogs 6i–ii were obtained via standard
N-methylation methodology from 2vi and 2vii respectively.
Pyridazines 9i–ii were prepared from commercially available 1,4-
dichloropyridazine 7 and p-toluidine 4 in a similar fashion to the
⇑
Corresponding author.
E-mail address: hulme@pharmacy.arizona.edu (C. Hulme).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.030

F. Medda et al. / Bioorg. Med. Chem. Lett. 23 (2013) 528–531
529
R1
R3
H
N
H
N
N N 4
1
N
N
N
Cl
H3C
H3C
S
S
O
O
R2
OH
1a
1b
2
Figure 1. Structures of 2-aminothiazoles 1a–b and general structure of aminophthalazines 2.
R2
CH3
NH2
X
X
N
N
N N
N N
N
N
R2
N
NH
Cl
Cl
Cl
NH
iii
i
ii
+
R1
R1
R1
R1
3
4
2i-xxiv
5
6i-ii
Scheme 1. Synthesis of aminophthalazines 2i–xxiv and 6i–ii (R1, R2 and X vary, see Table 1).13 Reagents and conditions: (i) (a) EtOH, reflux, 0.5 h, (b) NaOH (aq); (ii) Boronic
acid (2 equiv), K2CO3 (2 equiv), Bis(triphenylphosphine)palladium(II) dichloride (0.05 equiv), dioxane–H2O (4:1), microwave irradiation, 100 °C, 1 h; (iii) NaH (1.5 equiv), MeI
(1.5 equiv), DMF, 0 °C to rt.
Cl
NH2
N N
N N
i
ii
R1
NH
N
N
+
Cl
NH
Cl
7
CH3
4
8
9i-ii
CH3
CH3
Scheme 2. Synthesis of aminopyridazines 9i–ii. Reagents and conditions: (i) (a) EtOH, reflux, 0.5 h, (b) NaOH (aq); (ii) Boronic acid (2 equiv), K2CO3 (2 equiv),
Bis(triphenylphosphine)palladium(II) dichloride (0.05 equiv), dioxane–H2O (4:1), microwave irradiation, 100 °C, 1 h.
aminophthalazines in Scheme 2. Final compounds exhibited puri-
ties >95%, as judged by LC/MS (254 and 214 nm) and evaporative
light scattering (ELS).
of PGE2 levels (2xi, 94%; EC50 0.42 lM), albeit 10-fold less than
2vii. Enlargement of the para-methoxy (R2) to ethoxy (2xii) or iso-
propoxy (2xiii) resulted in partial loss of activity (75 and 98%; EC50
All compounds were screened for their ability to reduce PGE2
0.57 and 0.70 lM, respectively). A consistent reduction in activity
production in HCA-7 colon cancer cells at 1
l
M concentration
was also observed when a bulkier 3,4-methylenedioxy substituent
and activities are summarized as percentage reduction of PGE2 lev-
was added to the phenyl ring R2 (2xiv, 59%).
els Table 1.14 Compounds that exhibited reduction of PGE2 levels
Introduction of a bulky hydrophobic substituent at the meta po-
sition of the C-1 phenyl ring (R2), including chloro (2xv, 75%; EC50
higher than 70% were tested for COX-2 inhibition at 5 lM in an
in vitro cell free assay, with Celecoxib incorporated as a positive
control in both PGE2 and COX-2 assays.15 IC50 values for COX-2
inhibition were not determined, as no inhibitory activity against
COX-2 >50% was observed. Compounds exhibiting >70% reduction
of PGE2 levels, and <50% COX-2 inhibition were pushed forward for
EC50 determinations of PGE2 reducing level ability.
Aminophthalazines 2i–v, characterized by various polar substit-
uents at both the C-1 phenyl (R2) and C-4 aniline (R1) rings, gener-
ally exhibited poor reduction of cellular PGE2 levels (0–39%),
possibly due to a reduced ability to permeate the cellular mem-
brane. Replacement with R1 = p-CH3 (2vi–xvii) resulted in good
to high levels of inhibition of cellular PGE2 levels (59–99%), the
only exception being 2x (R2 = H, X = N, 15%), characterized by a po-
lar pyrimidine ring instead of the phenyl ring. In this group of ana-
logs, the activity was influenced by the nature and position of the
substituent R2 on the C-1 phenyl ring. In detail, when this ring was
unsubstituted (2vi), a strong reduction of PGE2 levels was observed
0.86
activity, whilst movement of a chlorine atom from the meta to
the ortho position (2xvi) improved activity (97%; EC50 0.38 M).
This result may be explained by restricted rotation of the phenyl
ring and associated lowering of entropic barriers to binding,
resulting from bulky substituent in the ortho position. Elongation
of the para methyl group on the C-4 of the phenyl ring (R1) to ethyl
lM) or phenyl (2xvii, 85%; EC50 0.54 lM) led to moderate
l
(2xviii, 85%; EC50 0.93
l
M) resulted in slightly reduced levels of
activity when compared to 2vii (R2 p-CH3O; EC50 0.032
lM).
Movement of the para methyl group on the C-4 aniline ring to
the meta position led to a decrease in the observed activity (R1,
2xix and 2xx, 74 and 87%; EC50 0.76 and 0.64
compared with the activity previously observed for 2vi–vii (99
and 98%; EC50 0.031 and 0.032 M). Replacement of the meta
methyl R1 with a bioisosteric chlorine (2xxi, 79%; EC50 0.5
M)
lM, respectively),
l
l
resulted in a similar level of activity to 2xix. Introduction of a para
phenoxy substituent on the C-1 phenyl ring (R2, 2xxii–xxiii)
maintained strong reduction of PGE2 cellular levels, surprisingly
only when the C-4 anilino ring (R1) was substituted with a para
(99%; EC50 0.031
(R2) led to the same level of inhibitory activity as 2vi (2vii, 98%;
EC50 0.032
M). Movement of the methoxy group R2 to the meta
and ortho positions resulted in a reduction of activity (2viii and
2ix, 62 and 79%, respectively; 2ix EC50 0.59 M). Similar to what
lM). Surprisingly, a para-methoxy substituent
l
phenoxy group (92%; EC50 = 0.09 lM). Interestingly, removal of
the methoxy group from the aniline ring (R1 = H, R2 = p-CH3O)
resulted in slight increase of cellular activity (2xxiv, 97%;
l
was observed for analog 2vii, a dimethylamine substituent at the
EC50 = 0.02
pound observed in cells.
lM), delivering the most functionally potent com-
para position of the C-1 phenyl ring (R2) led to potent reduction

530
F. Medda et al. / Bioorg. Med. Chem. Lett. 23 (2013) 528–531
Table 1
Biological activity of aminophthalazines analogs 2i–xxiv, 6i–ii and pyridazines 9i–iiabcdef
R2
N
X
X
N
CH3
N4
N
N
N 4
1
1
4
1
R2
N
R2
NH
NH
R1
6i-ii
R1
R1
2i-xxiv
9i-ii
Compd
R1
R2
X
PGE2 (%)d
39.2 13
COX-2 (%)e
PGE2 EC50/l
Mf
2i
2ii
2iii
2iv
2v
2vi
2vii
2viii
2ix
p-CH3O
p-OH
p-C(O)NH(CH2)2OH
H
H
H
C
C
C
C
C
C
C
C
C
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
—
—
—
—
—
—
—
—
—
—
—
—
—
26.7
NR
5
p-SO2NH2
p-COOEt
p-OCH2C(O)NH2
p-CH3
p-CH3
p-CH3
p-CH3
p-CH3
p-CH3
p-CH3
p-CH3
p-CH3
p-CH3
p-CH3
p-CH3
p-Et
m-CH3
m-CH3
m-Cl
p-PhO
p-PhO
H
CH3
CH3O
CH3
CH3
18.4
NR
3
p-CH3O
H
p-CH3O
m-CH3O
o-CH3O
H
p-N(CH3)2
p-OEt
p-OCH(CH3)2
3,4-OCH2O-
m-Cl
—
98.9
98.3
62.0
77.2
15.1
94.4
75.3
76.7
59.0
75.2
97.2
84.2
1
3
8
0
4
1
9
3
8
8
23.0
NR
—
NR
—
14.2
NR
NR
—
1
1
0.031 0.07
0.032 0.02
—
0.59 0.04
—
0.42 0.002
0.57 0.06
0.70 0.25
—
0.86 0.10
0.38 0.01
0.54 0.05
0.93 0.01
0.76 0.30
0.64 0.34
0.50 0.4
—
2x
2xi
2xii
2xiii
2xiv
2xv
2xvi
2xvii
2xviii
2xix
2xx
2xxi
2xxii
2xxiii
2xxiv
6i
NR
NR
NR
NR
NR
NR
10.1
NR
NR
o-Cl
1
m-Ph
p-CH3O
H
p-CH3O
H
1
85.0 11
74.1
87.5
79.4
6
5
9
7
H
71.2 15
p-CH3O
p-CH3O
CH3O
H
H
CH3O
92.6
97.3
87.0
92.4
62.5
49.0
5
1
2
1
1
4
0.09 0.05
0.02 0.01
0.05 0.02
0.220 0.006
—
—
3.0 10
NR
21.4 27
—
—
6ii
9i
9ii
a
b
c
d
e
f
Previously described inhibitor of cellular PGE2 levels, 1b, was used as a positive control (PGE2 10.9 4.5 and EC50 = 0.33 0.09 l
M).11
NR, no observed reduction.
— Not determined.
% of inhibition of PGE2 levels in HCA-7 cells at 1
lM concentration SD (n = 3).
% of inhibition of COX-2 levels in vitro at 5 M concentration SD (n = 3).
l
EC50 for PGE2 level reduction in HCA-7 cells SD (n = 3).
N-Methylation of 2vi–vii to afford 6i and 6ii respectively, inter-
anti-cancer activity with tumor versus control value (T/C) of 34%
for HCA-7 human colonic adenocarcinoma cell lines, at a dosing
schedule of 100 mg/kg ip over 10 days. Work is now on going to
elucidate the mode of action of these molecules with tagged
probes. Future synthetic efforts will focus on further development
of new scaffolds with improved physicochemical properties to
further develop established SAR and promote discovery of new
candidates for in vivo evaluation in mouse xenograft models.
estingly only slightly impaired the capacity to reduce cellular PGE2
levels (87 and 92%; EC50 0.05 and 0.220 M) when compared to
l
structurally related analogs 2vi–vii. Finally, replacement of the
phthalazine scaffold with a pyridazine ring resulted in reduced le-
vel of activity (62 and 49%, 9i and 9ii, respectively) and no further
efforts were pursued with this heterocycle.
Remarkably for this class of compounds, the most functionally
active compounds in cells exhibited only negligible inhibition of
COX-2 activity in vitro at 5 lM, with % inhibition comprised be-
tween 0 and 30%. As such, no dose response curves for COX-2 were
Acknowledgments
determined.
The Authors would like to thank financial support provided by
NIH training Grant research fellowship (BCP fellowship grant,
#5T32GM008804-09 and BMCB grant #T32GM08659), the Univer-
sity of Arizona College of Pharmacy for start up funds to CH and the
NCI CA138702 grant to EJM.
In conclusion, we have prepared and evaluated twenty-six
aminophthalazine and two pyridazine analogs for their capacity
to reduce cellular levels of PGE2 in HCA-7 cells. The in vitro inhib-
itory activity against COX-2 was also determined, leading to the
identification of potent inhibitors of PGE2 levels with negligible
activity against COX-2. Compounds 2vi–vii, xvi and xxiv exhibited
the highest reduction of PGE2 levels, with percentage of PGE2
reduction between 97.2 and 98.9% and EC50 values comprised
References and notes
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between 0.038 and 0.02 lM respectively. Furthermore, 2vii was
tested for its effect on tumor growth in mouse xenograft
models expressing HCA-7 cells and was confirmed to have good

F. Medda et al. / Bioorg. Med. Chem. Lett. 23 (2013) 528–531
531
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reflux for 0.5 h. After cooling down to room temperature, aqueous NaOH (aq)
was added at once and the product 5 precipitated as a white microcrystalline
solid which was isolated by suction filtration and used in the next reaction
without further purification (1.72 g, 6.05 mmol, 80%). 5 (200 mg, 0.70 mmol,
1 equiv) was dissolved in dioxane-H2O (4:1, 5 mL) and phenylboronic acid
(171 mg, 1.40 mmol, 2 equiv), Bis(triphenylphosphine)palladium(II) dichloride
(25 mg, 0.03 mmol, 0.05 equiv) and K2CO3 (193 mg, 1.39 mmol, 2 equiv) were
added. The reaction was subjected to microwave irradiation for 1 h at 100 °C.
After filtering through celite, H2O (50 mL) was added and the aqueous layer
extracted with DCM (3 Â 50 mL). The organic layers were collected, washed
with brine (50 mL), dried (MgSO4) and the solvent removed under reduced
pressure. The final product 2xxiv was purified by silica-gel column
chromatography (hexane/EtOAc, 0–50%) using an ISCOTM system and
obtained as a yellow microcrystalline powder (50 mg, 0.33 mmol, 48%). Mp:
210–213 °C; 1H NMR (400 MHz, DMSO-d6) d 9.11 (br s, 1H), 8.61 (d, J = 8.3 Hz,
1H), 7.97 (ddd, J = 8.3, 6.8, 1.5 Hz, 1H), 7.89 (ddd, J = 8.0, 6.8, 1.1 Hz, 1H), 7.86–
7.77 (m, 3H), 7.66–7.60 (m, 2H), 7.58–7.47 (m, 3H), 6.98–6.92 (m, 2H), 3.75 (s,
3H). 13C NMR (101 MHz, DMSO-d6) d 155.4, 153.3, 152.4, 137.4, 134.0, 132.4,
131.9, 130.1, 128.9, 128.8, 126.1, 126.1, 123.6, 123.0, 118.7, 114.1, 55.7. LC–MS
[MH]+ 328.00. Purity (LC–MS): >99% (ELSD-LT); >99% (254 nm); 90% (214 nm).
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overnight in DMEM/10% FBS. They were serum starved for the next 18 h. Cells
were then treated with 10 ng/ml IL-1b and increasing concentration of
compounds (dissolved in DMSO) in 1 mL serum-free medium. After 72 h of
incubation, the supernatants were collected for PGE2 level detection using the
PGE2 EIA kit (R&D Systems).
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13. General procedure for the preparation of analog 2xxiv: 1,4-Dichlorophthalazine
(3, 1.50 g, 7.57 mmol, 1 equiv) and para-anisidine (4, 940 mg, 7.57 mmol,
1 equiv) were mixed together in ethanol (10 mL) and the reaction stirred at
15. COX-2 cell-free assay: COX-2 activity was measured by a COX Fluorescent
inhibitor screen assay kit following the manufacturer’s instructions (Cayman
Chemical, http://www.caymanchem.com).