Synthesis and biological evaluation of pyrrolopyridazine derivatives as novel HER-2 tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.09.038

A series of novel pyrrolopyridazine derivatives have been discovered to be HER-2 inhibitors. These compounds selectively inhibited HER-2 kinase activity at low nanomolar concentrations. Compound 7d was identified as a potent HER-2 inhibitor with an IC₅₀ of 4 nM.

Full text of DOI:10.1016/j.bmcl.2009.09.038

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6437–6440
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of pyrrolopyridazine derivatives
as novel HER-2 tyrosine kinase inhibitors
*
Tang Peng Cho , Feng Jun, Huang Li, Xu Zhe, Cheng Ling, Zhang Xu, Zhang Lei, Hu Bing
Shanghai Hengrui Pharmaceuticals Co., Ltd., 279 Wenjing Road, Shanghai 200245, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2009
Revised 26 August 2009
Accepted 11 September 2009
Available online 17 September 2009
A series of novel pyrrolopyridazine derivatives have been discovered to be HER-2 inhibitors. These com-
pounds selectively inhibited HER-2 kinase activity at low nanomolar concentrations. Compound 7d was
identified as a potent HER-2 inhibitor with an IC50 of 4 nM.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Pyrrolopyridazine derivatives
Selective HER-2 kinase inhibitors
Receptor tyrosine kinases (RTKs) play a crucial role in signal
transduction pathways that regulate cell differentiation, prolifera-
tion and angiogenesis. Inhibition of RTK activation has become a
compelling approach in the development of anticancer agents.1
The ErbB family of receptor tyrosine kinases comprises four closely
related members: the epidermal growth factor receptor (EGFR,
ErbB1 or HER-1), the human epidermal growth factor receptor 2
(ErbB2 or HER-2), ErbB3 (HER-3), and ErbB4 (HER-4).2,3 Over
expression of EGFR and HER-2 has been observed in many cancers
including non-small-cell lung cancer, and prostate, breast, colon
and stomach cancers. Small molecules that inhibit the kinase activ-
ity of EGFR and HER-2 are considered to be new therapeutic anti-
tumor agents.4,5
zin-4-one 3 was transformed into the 4-chloro-pyridazine deriva-
tive 4 upon treatment with POCl3. Acid catalysed displacement of
the chloride with the appropriate aniline was performed in isopro-
panol to give 5. Saponification of 5 followed by coupling with dif-
ferent amines afforded compounds 7a–e.11
Analogs with pyrrolo[3,4-d]pyridazine scaffold II and 7-substi-
tuted pyrrolo[2,3-d]pyridazine scaffold III were synthesized by
similar routes starting from different commercially available pyr-
roles as shown in Schemes 2 and 3. Pyrrole diester 812 and 1313
were transformed to aldehyde 9 and 14, respectively, under Vils-
meier–Haack reaction conditions, which were converted to 10
and 15 via intramolecular ring-closing condensations. Chlorination
of 10 and 15 followed by coupling with 3-chloro-4-(3-fluoro-ben-
zyloxy)-phenylamine in isopropanol afforded 12 and 17. Saponifi-
cation and amidation of 17 led to the desired compounds 18a–b as
shown in Scheme 3.
Inhibition of EGFR and HER-2 tyrosine kinase activity was eval-
uated in enzymatic and cellular assays using A431(overexpressing
EGFR) and SK-BR-3 (overexpressing HER-2).14,15 Table 1 summa-
rises the in vitro potency of representative compounds. No signif-
icant EGFR tyrosine kinase inhibitory activity was observed in
these compounds. Computational modeling studies provided a
possible explanation for this result. A docking model of compound
7a based on the crystal structure of EGFR complexed with
Lapatinib16 (PDB code 1XKK) was developed (Fig. 3). The docked
models were energy minimized in Sybyl7.3. This model showed
that compound 7a bound to EGFR in definitely different fashions
when compared with Lapatinib. The pyrrolopyridazine core of 7a
deviated from the quinazoline core of Lapatinib, and was oriented
in the ATP binding site such that there was a hydrogen bond be-
tween N-2 and the hinge region Met769 NH. The C-4 hydrophobic
Three small molecule drugs, Gefitnib (IressaÒ),6 Erlotinib
(TarcevaÒ),7 and Lapatinib (TykerbÒ),8 have been approved and mar-
keted for the treatment of cancer (Fig. 1). Several other ErbB family
inhibitors are currently being investigated in various phases. These
quinazoline-based small molecules have a common scaffold that
mimics the adenine moiety of ATP, and therefore are potent and
competitive inhibitors of ATP.9 During the course of our exploration
of non-quinazoline scaffolds, pyrrolopyridazine derivatives (I, II, III)
were found to be novel and potent HER-2 inhibitors (Fig. 2). Here, we
report our progress in the synthesis and biological evaluation of
these pyrrolopyridazine-based inhibitors.
As shown in Scheme 1, reaction of pyrrole diester 1 with ceric
ammonium nitrate (CAN) afforded pyrrole aldehyde 2,10 which
underwent a cyclization with hydrazine hydrate in acetic acid to
give pyrrolo[2,3-d]pyridazin-4-one 3. The pyrrolo[2,3-d]pyrida-
* Corresponding author. Tel.: +86 21 54752877; fax: +86 21 54759072.
E-mail address: tangpc@shhrp.com (P.C. Tang).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.038

6438
P. C. Tang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6437–6440
F
O
F
HN
HN
N
O
Cl
Cl
HN
N
H
O
O
SO3H
N
N
O
O
O
O
N
S
N
O
N
O
O
2
.
.HCI
N
.H2O
Gefitinib (Iressa®)
Erlotinib (Tarceva®)
Lapatinib (Tykerb®)
Figure 1. Examples of launched ErbB family inhibitors.
R1
R1
R1
group extended out into the aqueous phase instead of extending
into the deep hydrophobic pocket. This might explain why com-
pound 7a was almost devoid of EGFR activity.
Moderate growth inhibitions toward A431 cells in compounds
7b, 7c and 7e might be caused by the inhibition of other kinases.
It was noticeable that compounds 7a and 7d exhibited similar po-
R2
R3
R4
HN
HN
HN
N
N
N
N
N
N
R2
R4
N
R3
N
N
R4
R2
R3
III
I
II
tency as Lapatinib (0.003
lM) against HER-2 kinase, but was found
to be >100 times more selective than the closely related EGFR ki-
Figure 2. General structure of the pyrrolopyridazine-based compounds.
O
O
O
O
O
O
O
O
O
O
NH
N
O
O
H
a
b
N
H
N
N
H
H
3
2
1
R1
R1
Cl
HN
HN
O
O
N
N
O
c
e
d
N
N
N
H
N
O
N
N
O
N
HO
H
H
4
5
6
R1
HN
f
O
N
N
R5
N
H
N
H
7a-e
Scheme 1. Synthesis of type I derivatives. Reagents and conditions: (a) 4.0 equiv CAN, THF/HOAc/H2O, rt, 1.5 h, 65%; (b) 1.1 equiv NH2NH2ꢀH2O, HOAc, 100 °C, 2 h, 79%; (c)
2.0 equiv POCl3, CH3CN, 80 °C, 1.5 h, 95%; (d) 1.5 equiv R1NH2, 2.5 equiv Et3N, i-PrOH, aq HCl, 80 °C, 4 h, 50%; (e) 1 N NaOH, EtOH, reflux, then 1 N HCl, pH 1, 91%; (f) 1.5 equiv
R5NH2, Et3N, EDCI, HOBt, DMF, rt, 17–58%.
O
O
Cl
O
O
EtOOC
HN
EtOOC
HN
O
O
H
c
NH
N
a
b
N
N
O
O
N
N
H
O
H
O
10
11
9
8
O
F
Cl
HN
EtOOC
d
N
HN
N
12
Scheme 2. Synthesis of type II derivatives. Reagents and conditions: (a) 2.0 equiv POCl3, DMF, 100 °C, 2 h, 77%; (b) 1.5 equiv NH2NH2ꢀH2O, EtOH, 90 °C, 3 h, 72%; (c) 2.0 equiv
POCl3, CH3CN, 80 °C, 5 h; (d) 1.1 equiv 3-chloro-4-(3-fluoro-benzyloxy) -phenylamine, i-PrOH, 90 °C, 7 h, 51%.

P. C. Tang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6437–6440
6439
O
O
Cl
O
H
H
H
H
N
O
H
O
O
O
O
O
O
b
N
N
c
a
N
O
O
O
N
N
NH
N
O
15
13
14
16
O
O
F
F
d
e
f
Cl
HN
HN
Cl
H
N
H
O
O
N
N
N
N
N
R5
N
H
O
18a-b
17
Scheme 3. Synthesis of type III derivatives. Reagents and conditions: (a) 2.0 equiv POCl3, DMF, 100 °C, 3 h, 21%; (b) 1.5 equiv NH2NH2ꢀH2O, EtOH 90 °C, 1.5 h, 44%; (c)
2.0 equiv POCl3, CH3CN, 80 °C, 3 h; (d) 1.1 equiv 3-chloro-4-(3-fluoro-benzyloxy)-phenylamine, i-PrOH, 90 °C, 3 h, 34%; (e) 1 N NaOH, EtOH, reflux, then 1 N HCl, pH 1, 85%; (f)
1.5 equiv R5NH2, Et3N, EDCI, HOBt, DMF, rt, 62–85%.
Table 1
Enzyme and cellular inhibitory activity assay results
R1
R5
EGFR IC50
1.28
(lM)
A431 IC50
1.16
(lM)
HER-2 IC50
0.007
(lM)
SK-BR-3 IC50
0.96
(lM)
a
a
a
a
No.
O
7a
F
F
F
F
N
Cl
N
O
7b
7c
7d
7e
>10
>10
5–10
>10
0.98
ND
2.24
0.843
>5
N
Cl
N
O
O
O
0.257
12.65
0.867
ND
N
N
N
Cl
O
0.004
ND
Cl
O
>5
N
Cl
12
—
—
—
>10
>10
1.21
ND
ND
0.934
3.84
18a
77.22
N
N
18b
—
>10
15.14
ND
0.62
TarcevaÒb
TykerbÒb
—
—
—
—
0.015
0.019
0.42
0.14
1.52
1.89
0.003
0.124
ND: not determined.
a
Average values (at least two experiments).
b
TarcevaÒ and TykerbÒ were prepared according to the literatures.7,8

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P. C. Tang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6437–6440
References and notes
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4838.
8. Spector, N.; Raefsky, E.; Hurwitz, H., et al Proc. Am. Assoc. Clin. Oncol. 2003, 193.
9. Vema, A.; Panigrahi, S. K.; Rambabu, G.; Gopalakrishinan, B.; Sarma, J. A. R. P.;
Desiraju, G. R. Bioorg. Med. Chem. 2003, 11, 4643.
10. Thyrann, T.; Lightner, D. A. Tetrahedron Lett. 1995, 36, 4345.
11. All compounds were characterized by 1H NMR and MS etc. Preparation of
compound 7d is described herein. A stirred solution of 4-[3-chloro-4-(3-fluoro-
benzyloxy)-phenylamino]-3-methyl-1H-pyrrolo[2,3-d] pyridazine-2-carboxylic
acid 6 (1 mmol) in N,N-dimethylformamide (15 mL) was added N-ethyl-N0-
(dimethylamino propyl)-carbodiimide hydrochloride (1.5 mmol), 1-hydroxy-
benzotriazol (1.5 mmol) and triethylamine (2.5 mmol), the mixture was stirred
for 5 min at room temperature, then 2-morpholin-4-yl-ethylamine (1.5 mmol)
was added, the reaction mixture was stirred overnight at this temperature until
the start material disappeared as monitored by TLC. The mixture was poured into
ice water (80 mL) and extracted with ethyl acetate (4 ꢁ 30 mL). The combined
organic extracts were washed with saturated sodium chloride aqueous solution
(50 mL), dried over anhydrous magnesium sulfate, filtered and concentrated
underreduced pressure, the residueispurifiedbycolumn chromatographytogive
4-[3-chloro-4-(3-fluoro-benzyloxy)-phenyl amino]-3-methyl-1H-pyrrolo[2,3-d]-
pyridazine-2-(2-morpholin-4-yl-ethyl)-formamide 7d (176 mg, yellow solid,
yield 30.2%). 1H NMR (400 MHz, DMSO-d6): d 8.87 (s, 1H), 7.89 (s, 1H), 7.55 (d,
1H), 7.40 (m, 1H), 7.30 (m, 2H), 7.17 (m, 2H), 5.22 (s, 2H), 3.59 (t, 4H), 3.44 (t, 2H),
2.71 (s, 3H), 2.49 (t, 2H), 2.40 (m, 4H). MS m/z (ESI): 539 [M+1].
Figure 3. Predicted binding mode of compound 7a (yellow) modeled in the X-ray
structure of the Lapatinib/EGFR kinase complex. The Lapatinib molecule is shown in
magenta.
12. Takaya, Hikaru; Kojima, Sachiko; Murahashi, Shun-Ichi Org. Lett. 2001, 3, 421.
13. Barker, Peter; Gendler, Paul; Rapoport, Henry J. Org. Chem. 1978, 43, 4849.
14. In vitro kinase assays. An enzyme linked immunosorbent assay (ELISA) was
conducted to measure the kinase activity of EGFR and HER-2 in vitro. The assay
was performed in 96-well plates (PerkinElmer Life Sciences #AAAND-0005).
nase. The HER-2 selectivities of the compounds 7a and 7d cannot
be explained adequately due to lack of an HER-2 X-ray structure.
Further studies are planned to investigate these selectivities. Com-
pounds 7a, 7c, 12 and 18 were also found to be low-micromolar
inhibitors toward SK-BR-3 cells.
In conclusion, pyrrolopyridazine derivatives were prepared and
found to be low nanomolar HER-2 selective inhibitors over EGFR.
Further studies of these compounds are in progress.
EGFR HTRF assay. 30 ng EGFR was used to phosphorylate 1.5
Gastrin Precursor (Tyr 87) peptide in the presence of 20 M ATP, 5 mM MgCl2,
5 mM MnCl2, 3 M Na3VO4, 1.25 mM DTT, and 60 mM HEPES (Ph 7.5). A 30
portion of 50 mM EDTA was added to the reaction as a negative control. The
30 L kinase reaction with or without inhibitors in 5% DMSO was carried out at
room temperature for 30 min and then stopped by 30 L of 50 mM EDTA. A
100 L portion of Phospho-Tyrosine mAb (P-Tyr-100) (1:1000) was added to
each well and incubated at room temperature for 60 min. Then a 100
portion of dilute Europium labeled anti-mouse IgG was added to each well and
incubated at room temperature for 30 min. 100
portion of DELFIAÒ
lM Biotin–
l
l
lL
l
l
l
lL
Acknowledgments
A
lL
enhancement solution was added to the well. The plate was incubated at room
temperature for 5 min and read on the BMG Nova star in the time-resolved
fluorescence mode by exciting at 340 nm and reading the emission at 615 nm.
HER-2 HTRF assay was conducted in a similar way.
The authors thank Mr. Sun Piao Yang for his vision and sup-
port in new drug discovery in China, and Mr. Wu Qian and Xue
Zhen Dong of Analytical Chemistry Group of Shanghai Hengrui
Pharmaceuticals Co., Ltd. for 1H NMR and Mass Spectroscopy
services.
15. Deng, B. C.; Tang, P. C.; Feng, J.; Zhang, L. WO2007082470.
16. Wood, E. R.; Truesdale, A. T.; McDonald, O. B., et al Cancer Res. 2004, 64, 6652.