Design and synthesis of triazolopyridazines substituted with methylisoquinolinone as selective c-Met kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.09.066

A series of triazolopyridazines substituted with methylisoquinolinone were designed and synthesized. Some of the triazolopyridazines strongly inhibited c-Met kinase and showed good anti-proliferative activity against a panel of c-Met-amplified gastric cancer cell lines (MKN-45, SNU-5 and Hs746T).

Full text of DOI:10.1016/j.bmcl.2011.09.066

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7185–7188
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of triazolopyridazines substituted
with methylisoquinolinone as selective c-Met kinase inhibitors
Jae Wook Ryu a, Sun-Young Han a,b, Jeong In Yun a,c, Sang-Un Choi a, Heejung Jung a, Jae Du Ha a,
Sung Yun Cho a, Chong Ock Lee a, Nam Sook Kang a,d, Jong Sung Koh a, Hyoung Rae Kim a, Jongkook Lee a,
⇑
a Bio-Organic Science Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600, Republic of Korea
b College of Pharmacy and Research Institute of Life Sciences, Gyeongsang National University, Jinju, Gyeongnam 660-751, Republic of Korea
c College of Pharmacy, Chungnam National University, Yuseong, Daejeon 305-764, Republic of Korea
d Graduate School of New Drug Discovery & Development, Chungnam National University, Yuseong, Daejeon 305-764, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of triazolopyridazines substituted with methylisoquinolinone were designed and synthesized.
Some of the triazolopyridazines strongly inhibited c-Met kinase and showed good anti-proliferative
activity against a panel of c-Met-amplified gastric cancer cell lines (MKN-45, SNU-5 and Hs746T).
Ó 2011 Elsevier Ltd. All rights reserved.
Received 15 August 2011
Revised 16 September 2011
Accepted 17 September 2011
Available online 22 September 2011
Keywords:
c-Met
Cancer
Kinase inhibitor
Triazolopyridazine
Methylisoquinolinone
Receptor tyrosine kinases (RTKs) have been extensively studied
as targets of anti-cancer drugs. Several small molecule inhibitors
and a few antibodies against RTKs have been used successfully in
the clinic for cancer treatment.1,2 c-Met tyrosine kinase, a receptor
for hepatocyte growth factor/scatter factor (HGF/SF), has recently
attracted considerable interest as a therapeutic target for several
types of cancers because it regulates cell survival, proliferation,
migration and angiogenesis in both normal mammalian cell devel-
opment and cancer metastasis.3–5 Activating mutations or amplifi-
cation of c-Met have frequently been found in various cancers
including lung, gastric, renal, ovarian, prostate and liver cancers.3,4
At the molecular level, binding of HGF to the extracellular domain
of c-Met results in receptor dimerization and phosphorylation
of multiple tyrosine residues. The activated c-Met binds and
phosphorylates adaptor proteins including growth factor recep-
tor-bound protein 2 (Grb2), Grb2-associated-binding protein 1
(Gab-1), Shc and c-Cbl, and subsequently induces the activation
of a number of downstream signal transducers such as extracellu-
lar signal-regulated kinases 1 and 2 (ERK1/2), signal transducers
and activators of transcription (STAT), phosphoinositide-3-kinase
(PI3K), Akt and focal adhesion kinase (FAK).5
A number of c-Met kinase inhibitors have already been reported
and some of them have reached the clinical trial phase.4 Among the
known c-Met inhibitors, bicyclic triazole-based inhibitors showed
MeO
HO
N
N
O
S
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
1
SGX-523 (3)
2
N
N
F
F
HO
N
N
N
N
N
N
N
N
N
N
N
N
N
JNJ-38877605 (4)
PF-4217903 (5)
⇑
Corresponding author. Tel.: +82 42 860 7178; fax: +82 42 860 7160.
E-mail address: jongkook@krict.re.kr (J. Lee).
Figure 1. Structures of known bicyclic triazole-based inhibitors of c-Met kinase.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.066

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J. W. Ryu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7185–7188
high c-Met inhibitory potency and excellent selectivity against
other kinases (Fig. 1).6 Some of the bicyclic triazole-based inhibi-
tors have also been examined in clinical trials in which they exhib-
ited good efficacy. However, further development of these was
stopped or delayed due to toxicity7 or unknown reasons. Thus,
there is a need to discover new classes of bicyclic triazole-based
inhibitors against c-Met for the treatment of cancer patients.
Previously, we have reported a series of aminopyridine based-
inhibitors of c-Met kinase.8 In an ongoing effort to discover novel
c-Met inhibitors, we have designed and synthesized a new series
of triazolopyridazine-based inhibitors against c-Met kinase.
The synthesis of triazolopyridazines substituted with methyl-
isoquinolinone commenced with the incorporation of an acrylate
moiety into commercially available bromophenylacetic acid 6 by
the Heck reaction as shown in Scheme 1.9 Coupling of phenylacetic
acid 7 with hydrazine 8 and subsequent treatment of the resulting
hydrazide 9 with TsOH afforded triazolopyridazine 10.6a–c,e The
t-butyl ester group in triazolopyridazine 10 was hydrolyzed by
treatment with trifluoroacetic acid and the resulting carboxylic
acid 11 was transformed into acylazide 12. Subjection of 12 to
the Curtius rearrangement and concomitant cyclization yielded
isoquinolinone 13,10 which was converted to disubstituted triazo-
lopyridazines 14–18 and 23–33 by Suzuki coupling.6a,b,11 Substitu-
tion of the chloride in the triazolopyridazine ring of 13 with
pyrrolidine and piperidine furnished triazolopyridazines 21 and
22, respectively.
The syntheses of 19, 20, 34 and 35 are depicted in Scheme 2.
The respective removal of the THP group in 17 and the Boc group
in 18 was effected to produce alcohol 19 and piperidine 20, by
treatment with hydrogen chloride solution and trifluoroacetic acid,
respectively. N-Alkylation of isoquinolinone 29 with the corre-
sponding alkyl iodides gave triazolopyridazines 34 and 35.
To find a new class of triazole-based inhibitors that might target
the ATP-binding site of c-Met, triazolopyridazines 14 and 1512
substituted with methylisoquinolinone were initially designed
and synthesized (Table 1). Gratifyingly, both of these compounds
showed excellent potency against c-Met. These results imply that
the isoquinolinone ring works well as a hinge binding group, sim-
ilar to the hydroxyphenyl and quinoline groups in the known tria-
zole-based c-Met inhibitors 1–5. Moreover, 14 and 15 revealed
good selectivity in the kinase profiling assay against a panel of ki-
nases consisting of structurally relevant kinases such as Ron, Flt3,
O
Br
t-BuO
i
ii
O
O
HO
TrkA and Aur A (IC50s >10 lM). The potency and selectivity of 14
6
HO
and 15 were similar to those of 1–5. We sought to investigate
the structure–activity relationships (SAR) in triazolopyridazines
14 and 15, and the pyrazole moiety of 14 was modified first.
Replacement of the methyl group in the pyrazole ring of 14 with
a 2-hydroxyethyl group did not change the c-Met inhibitory
7
O
O
O
t-BuO
t-BuO
iii
H
N
N
N
H
Cl
N
N
N
H
N
H
N
N
O
O
N
Cl
THPO
HO
10
9
i
N
N
N
N
H
N
O
O
N
N
O
N
N
R
N
N
N
iv
N
vi
17
19
Cl
N
Cl
N
N
N
N
H
N
H
N
O
N
N
N
BocN
HN
11 : R = OH
12 : R = N3
xHCl
13
viii
ii
v
N
N
N
N
N
N
vii
N
N
N
N
H
N
H
N
N
N
O
O
18
20
R
N
H
N
O
O
N
R
N
N
N
N
n
F
N
N
N
F
iii
N
14 18, 23 33
N
-
-
21
: n = 1
N
N
N
N
N
22 : n = 2
N
N
Scheme 1. Reagents and conditions: (i) t-butyl acrylate, (o-tolyl)3P, n-Bu3N,
1,4-dioxane, Pd(OAc)2, reflux, 12 h, 94%; (ii) 3-chloro-6-hydrazinylpyridazine (8),
EDCI, CH2Cl2, 0 °C, 2 h, 79%; (iii) p-TsOH, THF, 60 °C, 12 h, 81%; (iv) CF3CO2H, CH2Cl2,
rt, 2 h, 95%; (v) Et3N, ClCO2Et, acetone, 0 °C 10 min then NaN3, H2O, 0 °C, 1 h, 83%;
(vi) n-Bu3N, Ph2O, 140–210 °C, 50 min, 47%; (vii) RB(OH)2 or RB(OR1)2, Cs2CO3 or
Na2CO3, Pd(dppf)Cl2 or Pd(Ph3P)2Cl2, DME or 1,4-dioxane, H2O, 80–110 °C, 2–12 h,
16–76%; (viii) pyrrolidine or piperidine, reflux, 2 h, 38–52%.
29
34
: R = Me
: R = Et
35
Scheme 2. Reagents and conditions: (i) 1 M aqueous HCl, CH2Cl2, rt, 2 h, 70%; (ii)
CF3CO2H then 4 M HCl in dioxane, MeOH, rt, 4 h, 80%; (iii) NaH, DMF, MeI or EtI,
0 °C, 1 h, 25–30%.

J. W. Ryu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7185–7188
7187
Table 1
Table 2
Triazolopyridazines substituted with methylisoquinolinone
SAR of triazolopyridazine derivatives
R2
N
H
O
N
O
R
N
R
N
N
N
N
N
N
N
a
Compound
R
c-Met IC50
0.002
(lM)
a
Compound
R1
R2
H
c-Met IC50
0.072
(lM)
N
N
14
15
16
26
0.003
Cl
N
NH
N
27
28
29
30
31
32
33
34
35
H
0.004
0.007
0.003
0.004
0.003
0.003
0.005
0.073
0.072
0.005
Cl
N
H
19
20
0.002
0.027
OH
F
N
N
H
NH
NO2
CN
OH
OMe
F
H
21
22
23
0.027
0.029
0.004
N
N
H
S
N
H
24
25
0.005
0.014
H
S
a
For assay conditions, see Ref. 8.
Me
Et
F
potency. However, attachment of a piperidinyl group to the pyra-
zole ring decreased c-Met inhibitory activity. The pyrazole group
in compound 14 was also replaced with other heteroaryl or hetero-
cycloalkyl groups. Of these, pyrrole 21 and piperidine 22 were not
as active as pyrazole 14, while thiophene 23 and pyridine 24 did
show good potency against c-Met.
a
For assay conditions, see Ref. 8.
We then turned our attention to the modification of the phenyl
ring of triazolopyridazine 15, and synthesized a series of deriva-
tives in which the phenyl group of 15 was replaced with a variety
of substituted phenyl groups. Most of the compounds exhibited
good c-Met inhibitory activity (Table 2). The potency in N-alkyl-
ated isoquinolinones 34 and 35 against c-Met had decreased,
which suggests the free NH on the isoquinolinone ring might inter-
act with a hinge residue of c-Met.
Modeling experiments suggested that triazolopyridazine 15
would dock strongly into the ATP-binding site of c-Met, as shown
in Figure 2. This analysis utilized a crystal structure of c-Met com-
plexed with 1 that was obtained from the Protein Data Bank (pdb
entry: 3CCN).6b Calculations for this docking analysis were carried
out using LibDock13 interfaced with Discovery Studio 3.0 (Accel-
rys). The crystal structure of 3CCN was regenerated very well with
a root mean square displacement (RMSD) of 0.7 Å. We set the H
atom in the peptide backbone of M1160 as an interaction site with
default parameters in LibDock. Triazolopyridazine 15 was bound
very tightly by c-Met when the same docking parameters were
used (DockScore, 137.0 for 15 vs 125.7 for 1). These calculated
values are in good agreement with the experimental results (c-
Met IC50 3 nM for 15 vs 6 nM for 1). The strong interaction of 15
with c-Met is attributed both to H-bonding of the isoquinolinone
moiety with hinge residues M1160 and P1158, and to
p-stacking
interactions between the triazolopyridazine moiety and Y1230.
Hydrophobic interactions of the triazolopyridazine moiety with
M1211 could also contribute to the tight binding. It is likely that
the low potencies of 26 and 27 compared with those of 15 and
28–33 arise from disfavorable interactions between the ortho-sub-
stituents on the phenyl ring and backbone carbonyl groups of
N1209 and R1208.
The anti-proliferative activity of the triazolopyridazines was
evaluated by performing a cytotoxicity assay on a panel of
c-Met-amplified gastric cancer cell lines (MKN-45, SNU-5 and
Hs746T).14,15 Most of the triazolopyridazines substituted with
methylisoquinolinones were potent growth inhibitors for all the
c-Met-amplified gastric cancer cell lines (Table 3). Several com-
pounds showed double digit nanomolar anti-proliferative activity,
but the proliferation of c-Met-independent N87 cells was hindered
only slightly by treatment with the triazolopyridazines, even at

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J. W. Ryu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7185–7188
Acknowledgments
This work was supported by the Korea Research Institute of
Chemical Technology (KRICT), Mid-career Researcher Program
through NRF Grant funded by the government of Korea (MEST)
(R01-2008-000-20205-0) and Basic Science Research Program
from NRF (2011-0010374, S.-Y.H.). We thank the Drug Discovery
Technology Platform team at KRICT for providing us with technical
assistance on the pharmacokinetic experiments.
References and notes
1. Knight, Z. A.; Lin, H.; Shokat, K. M. Nat. Rev. Cancer 2010, 10, 130.
2. Jänne, P. A.; Gray, N.; Settleman, J. Nat. Rev. Drug Disc. 2009, 8, 709.
3. Christensen, J. G.; Burrows, J.; Salgia, R. Cancer Lett. 2005, 225, 1.
4. (a) Comoglio, P. M.; Giordano, S.; Trusolino, L. Nat. Rev. Drug Disc. 2008, 7, 504;
(b) Sattler, M.; Hasina, R.; Reddy, M. M.; Gangadhar, T.; Salgia, R. Ther. Adv. Med.
Oncol. 2011, 3, 171; (c) Underiner, T. L.; Herbertz, T.; Miknyoczki, S. J. Anti-
Cancer Agents Med. Chem. 2010, 10, 7.
Figure 2. A proposed structure for the c-Met complex with triazolopyridazine 15
(yellow), 26 (green) and 28 (magenta). H-bonding interactions between the 15 and
c-Met are shown in blue dotted lines.
5. Jiang, W. G.; Martin, T. A.; Parr, C.; Davies, G.; Matsumoto, K.; Nakamura, T. Crit.
Rev. Oncol. Hematol. 2005, 53, 35.
6. (a) Zhang, F.-J.; Vojkovsky, T.; Huang, P.; Liang, C.; Do, S. H.; Koenig, M.; Cui, J.
WO2005010005, 2005.; (b) Albrecht, B. K.; Harmange, J.-C.; Bauer, D.; Berry, L.;
Bode, C.; Boezio, A. A.; Chen, A.; Choquette, D.; Dussault, I.; Fridrich, C.; Hirai, S.;
Hoffman, D.; Larrow, J. F.; Kaplan-Lefko, P.; Lin, J.; Lohman, J.; Long, A. M.;
Moriguchi, J.; O’Connor, A.; Potashman, M. H.; Reese, M.; Rex, K.; Siegmund, A.;
Shah, K.; Shimanovich, R.; Springer, S. K.; Teffera, Y.; Yang, Y.; Zhang, Y.; Bellon,
S. F. J. Med. Chem. 2008, 51, 2879; (c) Boezio, A. A.; Berry, L.; Albrecht, B. K.;
Bauer, D.; Bellon, S. F.; Bode, C.; Chen, A.; Choquette, D.; Dussault, I.; Hirai, S.;
Kaplan-Lefko, P.; Larrow, J. F.; Lin, M.-H. J.; Lohman, J.; Potashman, M. H.; Rex,
K.; Santostefano, M.; Shah, K.; Shimanovich, R.; Springer, S. K.; Teffera, Y.; Yang,
Y.; Zhang, Y.; Harmange, J.-C. Bioorg. Med. Chem. Lett. 2009, 19, 6307; (d)
Buchanan, S. G.; Hendle, J.; Lee, P. S.; Smith, C. R.; Bounaud, P.-Y.; Jessen, K. A.;
Tang, C. M.; Huser, N. H.; Felce, J. D.; Froning, K. J.; Peterman, M. C.; Aubol, B. E.;
Gessert, S. F.; Sauder, J. M.; Schwinn, K. D.; Russell, M.; Rooney, I. A.; Adams, J.;
Leon, B. C.; Do, T. H.; Blaney, J. M.; Sprengeler, P. A.; Thompson, D. A.; Smyth, L.;
Pelletier, L. A.; Atwell, S.; Holme, K.; Wasserman, S. R.; Emtage, S.; Burley, S. K.;
Reich, S. H. Mol. Cancer Ther. 2009, 8, 3181; (e) Lu, T.; Alexander, R.; Connors, R.
W.; Cummings, M. D.; Galemmo, R.; Hufnagel, H. R.; Johnson, D. L.; Khalil, E.;
Leonard, K. A.; Markotan, T. P.; Maroney, A. C.; Sechler, J. L.; Travins, J. M.;
Tuman, R. W. WO2007075567, 2007.; (f) Timofeevski, S. L.; McTigue, M. A.;
Ryan, K.; Cui, J.; Zou, H. Y.; Zhu, J. X.; Chau, F.; Alton, G.; Karlicek, S.;
Christensen, J. G.; Murray, B. W. Biochemistry 2009, 48, 5339.
Table 3
Anti-proliferative activity of triazolopyridazine derivatives against a panel of gastric
cancer cell lines
Compound
GI50
SNU-5
(l
M)a
MKN-45
Hs746T
N87
14
15
23
24
28
29
31
32
33
0.13
0.69
0.059
0.056
0.45
0.019
0.052
0.12
1.1
0.064
0.071
0.17
0.40
0.053
0.13
0.059
0.60
0.10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.038
0.032
0.42
0.011
0.042
0.036
0.47
0.095
0.13
a
For assay conditions, see Ref. 15.
7. Diamond, S.; Boer, J.; Maduskuie, T. P., Jr.; Falahatpisheh, N.; Li, Y.; Yeleswaram,
S. Drug Metab. Dispos. 2010, 38, 1277.
high concentration (10 lM). This difference represents a roughly
100-fold or more differential cytotoxicity toward c-Met-addicted
gastric cancer cell lines.
8. (a) Cho, S. Y.; Han, S.-Y.; Ha, J. D.; Ryu, J. W.; Lee, C. O.; Jung, H.; Kang, N. S.; Kim,
H. R.; Koh, J. S.; Lee, J. Bioorg. Med. Chem. Lett. 2010, 20, 4223; Han, S.-Y.; Lee, C.
O.; Ahn, S.-H.; Lee, M.-O.; Kang, S.-Y.; Cha, H.-J.; Cho, S. Y.; Ha, J. D.; Ryu, J. W.;
Jung, H.; Kim, H. R.; Koh, J. S.; Lee, J. Invest. New Drugs in press. doi:10.1007/
s10637-010-9584-2.
A preliminary pharmacokinetic study of triazolopyridazine 15
9. Kawata, S.; Ashizawa, S.; Hirama, M. J. Am. Chem. Soc. 1997, 119, 12012.
10. Briet, N.; Brookes, M. H.; Davenport, R. J.; Galvin, F. C. A.; Gilbert, P. J.; Mack, S.
R.; Sabin, V. Tetrahedron 2002, 58, 5761.
11. Thomson, A. E.; Hughes, G.; Batsanov, A. S.; Bryce, M. R.; Parry, P. R.; Tarbit, B. J.
Org. Chem. 2005, 70, 388.
showed a suitable elimination half life (t1/2), slow absorption (Tmax
and moderate bioavailability (F) (Table 4).
)
In summary, we have designed and synthesized a series of tria-
zolopyridazines substituted with methylisoquinolinone, a moiety
that strongly interacts with the hinge residues of c-Met, as selec-
tive c-Met kinase inhibitors. Some of the triazolopyridazines
showed good anti-proliferative activity against a panel of c-Met-
amplified gastric cancer cell lines, but the proliferation of c-Met
independent cells was little affected by treatment with any of
the triazolopyridazines. Further in vitro and in vivo pharmacolog-
ical evaluation of these compounds is planned and the results will
be reported in a due course.
12. Characterization data for triazolopyridazine 15: 1H NMR (500 MHz, DMSO-d6) d
11.31 (br s, 1H), 8.41 (d, J = 9.8 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 8.08–8.06 (m,
2H), 7.93 (d, J = 9.8 Hz, 1H), 7.76 (d, J = 6.8 Hz, 1H), 7.59–7.57 (m, 3H), 7.45 (t,
J = 7.7 Hz, 1H), 7.22 (t, J = 6.2 Hz, 1H), 6.91 (d, J = 7.4 Hz, 1H), 4.86 (s, 2H) d 13C
NMR (75 MHz, DMSO-d6) d 161.9, 152.8, 148.1, 143.3, 136.8, 133.97, 133.93,
131.1, 131.0, 129.18, 129.15, 127.3, 126.7, 126.1, 125.9, 125.1, 119.7, 101.3,
27.4; HRMS (EI) m/z calcd for C21H15N5O (M+) 353.1277, found 353.1282.
13. Diller, D. J.; Merz, M. L., III Proteins: Structure, Function, and Genetics 2001, 43,
113.
14. (a) Kuniyasu, H.; Yasui, W.; Kitadai, Y.; Yokozaki, H.; Ito, H.; Tahara, E. Biochem.
Biophys. Res. Commun. 1992, 189, 227; (b) Smolen, G. A.; Sordella, R.; Muir, B.;
Mohapatra, G.; Barmettler, A.; Archibald, H.; Kim, W. J.; Okimoto, R. A.; Bell, D.
W.; Sgroi, D. C.; Christensen, J. G.; Settleman, J. Proc. Natl. Acad. Sci. U.S.A. 2006,
103, 2316; (c) Asaoka, Y.; Tada, M.; Ikenoue, T.; Seto, M.; Imai, M.; Miyabayashi,
K.; Yamamoto, K.; Yamamoto, S.; Kudo, Y.; Mohri, D.; Isomura, Y.; Ijichi, H.;
Tateishi, K.; Kanai, F.; Ogawa, S.; Omata, M.; Koike, K. Biochem. Biophys. Res.
Commun. 2010, 394, 1042; (d) Rege-Cambrin, G.; Scaravaglio, P.; Carozzi, F.;
Ponzetto, C.; Comoglio, P. M.; Saglio, G. Cancer Genet. Cytogenet. 1992, 64, 170.
15. Cytotoxicity/MTS assay: MKN-45, SNU-5, Hs746T and N87 cells were
purchased from American Type Culture Collection (ATCC) or Korean Cell Line
Bank (KCLB). The cells were cultured in RPMI1640 or DMEM supplemented
Table 4
Preliminary pharmacokinetic profile for 15 in rats
Parameter
iva
poa
Cmax
Tmax
t1/2
(
lg/mL)
0.3
12.7
5.3
6.4
AUC (
l
g h/mL)
11.2
0.8
6.4
3.9
with 10% fetal bovine serum and gentamycin (50 lg/mL) at 37 °C and 5% CO2.
MKN-45, SNU-5, Hs746T and N87 cells were plated into 96-well plates (5000
or 10,000 cells/well) and treated with various concentrations of
triazolopyridazines or DMSO for 72 h in triplicate. The anti-proliferative
activity of the compounds was measured using CellTiter 96ÒAQueous
NonRadioactive Cell Proliferation Assay (Promega).
CL (L/kg h)
Vss (L/kg)
F (%)
35
a
Dose, 10 mg/kg (three rats were used).