Synthesis and anti-angiogenic activity of 6-(1,2,4-thiadiazol-5-yl)-3-amino pyridazine derivatives

Article abstract of DOI:10.1016/S0960-894X(01)00805-8

General screening for inhibitors of microvessel growth in vitro in the rat aortic ring assay led to the discovery of a novel series of thiadiazole pyridazine compounds with potential anti-angiogenic activity. Chemical optimization produced orally active compounds with potent in vitro and in vivo anti-angiogenesis and anti-tumor activities.

Full text of DOI:10.1016/S0960-894X(01)00805-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 589–591
Synthesis and Anti-Angiogenic Activity of 6-(1,2,4-Thiadiazol-5-
yl)-3-amino Pyridazine Derivatives
Jean-Pierre Bongartz,a,* Raymond Stokbroekx,a Marcel Van der Aa,a Marcel Luyckx,a
Marc Willems,a Marc Ceusters,a Lieven Meerpoel,a Gerda Smets,a Tine Jansen,a
Walter Wouters,a Charlie Bowden,a Lisa Valletta,b Mark Herb,b Rose Tominovichb
and Robert Tumanb
aJanssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, Belgium
bRW Johnson Pharmaceutical Research Institute, Drug Discovery, Welsh and McKean Roads, PO Box 776,
Spring House, PA 19477-0776, USA
Received 10 October 2001; accepted 26 November 2001
Abstract—General screening for inhibitors of microvessel growth in vitro in the rat aortic ring assay led to the discovery of a novel
series of thiadiazole pyridazine compounds with potential anti-angiogenic activity. Chemical optimization produced orally active
compounds with potent in vitro and in vivo anti-angiogenesis and anti-tumor activities. # 2002 Elsevier Science Ltd. All rights
reserved.
The formation of new blood vessels, called angiogenesis,
is a tightly controlled process in healthy tissue. Normal
vascular proliferation occurs during embryonic devel-
opment, the female reproductive cycle and wound heal-
ing. Uncontrolled hyperproliferation of blood vessels
can be observed in diseases such as cancer, diabetic
retinopathy and arthritis.1 The hypothesis that tumor
growth could be arrested by anti-angiogenic treatment
hasreceived a lot of attention in cancer reesarch. Sev-
eral anti-angiogenic drugsare currently in clinical
trials.2
models. A synthetic program was initiated to optimize
the anti-angiogenic propertiesof thislead compound
and to remove the undesirable eye toxicity.4
Initial effortsto replace the thiadiazolyl moiety by iso-
steric replacement failed to maintain the inhibitory
potency (data not shown). Systematic modifications of
the other structural features led to new insights in the
structure–activity relationships as discussed in this
paper.
To identify novel small molecule angiogenesis inhibi-
tors, the Janssen strategic compound collection was
screened using the rat aortic ring assay, an in vitro
functional model of angiogenesis.3 We identified
R 90324 (1), a novel compound with nanomolar
potency for microvessel growth.
Chemistry
All compoundshsown in Table 1 were prepared fol-
lowing two routesasoutlined in Scheme 1 and exem-
plified for compounds 1 and 2.5
In the first approach, 6-(1,2,4-thiadiazol-5-yl)-3-chloro-
pyridazine isa key intermediate obtained by a one-pot
and two-step transformation of 3-chloro-6-methyl-pyr-
idazine. Thisintermediate must be purified immediately
and cannot be stored for more than a few weeks. The
subsequent amine adducts (1, 6–26) are chemically
stable. The 2,4-substituted pyrimidines (4, 5) are
obtained following the same route A. In the second
approach, route B, the amine iscoupled first, followed
Further in vivo profiling of thiscompound showed that
it caused severe eye toxicity in rodent models after
repeated oral dosing. This precluded further use of this
compound for testing in in vivo anti-tumor xenograft
*Corresponding author. Fax +32-14-60-53-44; e-mail: jbongart@
janbe.jnj.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00805-8

590
J.-P. Bongartz et al. / Bioorg. Med. Chem. Lett. 12 (2002) 589–591
Scheme 1. Synthesis of thiadiazole derivatives. (a) SOCl2, reflux; (b) optionally substituted amidines, 50% NaOH, CH2Cl2; (c) optionally substituted
sec. amines, Na2CO3, DMF, 60 ꢀC; (d) Lawesson’s reagent, toluene, reflux; (e) (CH3)2NC(CH3)(OCH3)2; (f) NH2OSO2OH, MeOH, EtOH, pyridine.
by a three-step conversion of the arylamide to a 1,2,4-
thiadiazol-5-yl structure, as described for compound 2.
Compound 3 is synthesized starting from 4-fluoro-
benzamide, which isconverted to the 1-(1,2,4-thiadia-
diazol-5-yl)-4-fluorophenyl intermediate, following steps
d, e and f and finally coupled to the appropriate aryl
piperazine according to condition c.
While we cannot exclude other possibilities, it seems
that the pyridazine moiety iscloes to optimal for pri-
mary anti-angiogenic activity (1 vs 2–5). On the other
hand, replacement of the pyridazine by other aromatic
cyclic structureswassufficient to get rid of the eye toxicity.
Replacement of the methyl substituent at the 3-position
of the thiadiazole ring by other groups, while keeping
the pyridazine moiety, removed the eye toxicity aswell
(1 vs 6–10). Thisreduced the rat aortic ring activity to
different extents( 1 vs 6–10) but could be improved
again by optimizing the portion of the molecule to the
right side of the thiadiazolyl pyridazine group (21–26).
Pharmacology
Anti-angiogenic activity ismeasured by quantitation of
microvessel growth in rat aortic rings cultured in a fibrin
clot for 8 daysand compared to vehicle-treated control
rings. Quantitation is performed using an image analy-
sis system, consisting of a light microscope, a CCD
camera and an automated, custom-designed image ana-
lysis program.3,6
The substituted phenyl ring is essential for activity.
Removal or replacement by other structures completely
abolished inhibitory activities (data not shown). As
exemplified by compounds 1 and 11–20 the optimal
phenyl substituents are electron-withdrawing groups,
preferably but not exclusively at the meta position. For
most of the lead optimization work we continued using
a 3- or 3,5-CF3-substituted phenyl group. None of the
tested phenyl variants abolish the eye toxicity if a 3-
methyl-substituted thiadiazolyl pyridazine moiety is
present in the molecule.
Ocular toxicity was assessed after oral dosing of the
compoundsat 40 mg/kg b.i.d. for 4 daysin male Wistar
rats. The nature of the ocular toxicity was studied in
detail and will be the subject of a future publication.
In vivo anti-tumor and anti-metastatic activities were
measured after oral dosing b.i.d. for 28 days to B6D2F1
mice bearing metastatic 3LL Lewis lung tumors. Anti-
tumor efficacy wasdetermined by the decreaes in pri-
mary tumor weight and the anti-metastatic activity was
measured by reduction of the number of lung mets and
decrease in dry lung weight.
Avoidance of the 3-methyl substitution of the thiadia-
zole ring with modificationsof the central piperazine
spacer enabled us to obtain potent non-toxic in vitro
and in vivo active angiogenesis inhibitors with anti-
metastatic activity (21–26). Compound R 123942 (22)
also showed dose-dependent (7.5–60 mg/kg b.i.d. Â 28
days) reductions in primary tumor weight. No overt
toxicity or loss of body weight was associated with
repeated oral dosing up to 60 mg/kg b.i.d. Â 28 days.
However, further evaluation of thiscompound versusa
panel of human tumor xenograftsdid not confirm these
promising results. Attempts to characterize the mode of
Results and Discussion
The systematic modification of every part of the lead
compound (1) enabled usto elucidate the important
chemical featuresrequired for activity (Table 1).

J.-P. Bongartz et al. / Bioorg. Med. Chem. Lett. 12 (2002) 589–591
591
Table 1. Rat aortic ring microvessel growth inhibition, eye toxicity and 3LL Lewis Lung tumor mets formation inhibition results for
No.
R1
R2
R3
X
Aortic ring IC50 (nM)
0.6
Eye toxicity
YesNT
% Inhibition (mpk/b.i.d.)
1
CH3
I
II
3-Cl3
2
3
4
5
CH3
CH3
CH3
CH3
II
II
II
II
3-CF3
3-CF3
3-CF3
3-CF3
5
10
No
No
No
NT
NT
NT
NT
NT
>100
>100
6
7
8
9
H
CH3CH2
NH2
NHCH3
N(CH3)2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-F
70
8
20
3
6
0.3
6
No
No
No
No
NT
NT
NT
NT
NT
10
11
12
13
14
15
16
17
18
19
20
21
No
YesNT
NT
NT
YesNT
NT
NT
YesNT
NT
YesNT
YesNT
No
NT
NT
3-CN
3
3
3-NO2
3-NH2
3-OCH3
2-CF3
4-CF3
3,5-CF3
3,5-Cl
3,5-CF3
>100
7
NT
NT
0.2
12
<0.1
<0.1
<1
NT
24% (15)
42% (15)
22
H
H
I
3-CF3
0.7
No
23
I
3-CF3
0.3
No
54% (15)
III
24
25
26
N(CH3)2
N(CH3)2
NH(CH3)2
I
I
I
III
II
II
3-CF3
3-CF3
<1
0.1
0.4
Very weak
No
No
25% (15)
13% (15)
39% (15)
NT, not tested.
4. Tuman, R.; Herb, M.; Thomas, S.; Tominovich, R.; Wal-
lace, N.; Valetta, L.; Bongartz, J. P.; Smets, G.; Stokbroekx,
R.; Van Ginckel, R.; Wouters, W. New Drugs in Cancer
Therapy, 11th NCI-EORTC-AACR Symposium, Nov 7–10,
2000.
5. (a) Stokbroekx, R. A.; Van Der Aa, M. J. M.; Willems, M.;
Meerpoel, L.; Luyckx, M. G. M.; Tuman, R. W. WO Patent
9726258, 1997. (b) Stokbroekx, R. A.; Ceusters, M. A.; Van
Der Aa, M. J. M.; Luyckx, M. G. M.; Willems, M.; Tuman,
R. W. WO Patent 9858919, 1998. (c) Stokbroekx, R. A.;
Ceusters, M. A.; Van Der Aa, M. J. M.; Luyckx, M. G. M.;
Willems, M.; Tuman, R. W. WO Patent 9858929, 1998.
6. Nissanov, J.; Tuman, R. W.; Gruver, L. M.; Fortunato,
J. M. Lab. Invest. 1995, 73, 734.
action of thisesriesof compoundsfailed. They are
inactive against a panel of tyrosine and serine/threonine
kinases including VEGF and EGF. They are also in-
active against matrix metalloproteinases 1, 2, 3, 7, 8 and
9 and other proteolytic enzymes such as urokinase, cal-
pain, tissue kallikrein and chymotrypsin. The mechan-
ism responsible for the anti-angiogenic activity remains
undefined.7
References and Notes
1. Auerbach, W.; Auerbach, R. Pharmac. Ther. 1994, 63, 265.
2. Deplanque, G.; Harris, A. L. Euro. J. Cancer 2000, 36,
1713.
7. Smets, G.; Tuman, R.; Voina, S.; Thomas, S.; Koolwijk, P.;
Peeters, E.; Van Hinsbergh, V.; Bongartz, J.-P.; Wouters, W.
New Drugs in Cancer Therapy, 11th NCI-EORTC-AACR
Symposium, Nov 7–10, 2000.
3. Nicosia, R. F.; Ottinetti, A. Lab. Invest. 1990, 63, 115.