New C-5 substituted pyrrolotriazine dual inhibitors of EGFR and HER2 protein tyrosine kinases

Article abstract of DOI:10.1016/j.bmcl.2007.01.002

Novel C-5 substituted pyrrolotriazines were optimized for dual EGFR and HER2 protein tyrosine kinase inhibition. The lead compound exhibited promising oral efficacy in both EGFR and HER2 driven human tumor xenograft models. It is hypothesized that its C-5

Full text of DOI:10.1016/j.bmcl.2007.01.002

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2036–2042
New C-5 substituted pyrrolotriazine dual inhibitors of
EGFR and HER2 protein tyrosine kinases
Harold Mastalerz,a,* Ming Chang,b Ping Chen,b Pierre Dextraze,c Brian E. Fink,b
Ashvinikumar Gavai,b Bindu Goyal,b Wen-Ching Han,b Walter Johnson,a David Langley,a
Francis Y. Lee,b Punit Marathe,b Arvind Mathur,b Simone Oppenheimer,b
Edward Ruediger,c James Tarrant,a John S. Tokarski,b Gregory D. Vite,b
Dolatrai M. Vyas,a Henry Wong,a Tai W. Wong,b Hongjian Zhangb and Guifen Zhanga
aDepartments of Oncology Chemistry, Discovery Biology, Pharmaceutical Candidate Optimization, Computer Aided Drug Design,
and Chemical Synthesis, 5 Research Parkway, Wallingford, CT 06492-1951, USA
bBristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA
c100 Boul. de l’Industrie, Candiac, Que., Canada J5R 1J1
Received 29 November 2006; revised 19 December 2006; accepted 5 January 2007
Available online 12 January 2007
Abstract—Novel C-5 substituted pyrrolotriazines were optimized for dual EGFR and HER2 protein tyrosine kinase inhibition. The
lead compound exhibited promising oral efficacy in both EGFR and HER2 driven human tumor xenograft models. It is hypothe-
sized that its C-5 morpholine side chain binds in the ribose phosphate portion of the ATP binding pocket.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The epidermal growth factor receptor (EGFR, ErbB1 or
HER1) and the human epidermal growth factor receptor
2 (HER2, ErbB2) are members of the ErbB family of
receptor tyrosine kinases and have been clinically validat-
ed as targets for cancer therapy.1 Their frequent co-ex-
pression in a variety of tumor types and their capacity
to form heterodimers with other members of the ErbB
family provide a strong rationale for simultaneously tar-
geting both of these receptors.2 There are currently sever-
al small molecule, ATP-competitive, reversible, dual
EGFR, and HER2 kinase inhibitors in clinical develop-
ment. These include: lapatinib (GW572016),3 AEE-
788,4 and BMS-599626 (1).5 The latter utilizes the bicyclic
pyrrolo[2,1-f][1,2,4]triazine ring system as a scaffold for
the construction of an ATP mimic.6 Its lipophilic C-4 sub-
stituent provides potent and selective kinase inhibition,
N
F
N
Ar
RO
HN
N
HN
Me
6
O
4
5
NH
O
3
N
N
HN
N
N
2
O
N
1
7
2, R = solublizing group
1
Figure 1. Pyrrolotriazine dual EGFR and HER2 kinase inhibitors.
Keywords: EGFR; HER2; Receptor tyrosine kinase inhibitor; Pyrrolotriazine.
*
Corresponding author. Tel.: +1 203 677 7773; e-mail: harold.mastalerz@bms.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.002

H. Mastalerz et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2036–2042
2037
while its C-6 solubilizing side chain imparts good phar-
macokinetics and further potency. We explored the
SAR of analogs of 1 (Fig. 1) where the solubilizing group
is tethered to C-5 via a methylene ether linkage, that is 2,
and describe our results in this report.
sulfide, 30 (Scheme 2). Bromination of the 5-methyl group
followed by reaction with (S)-tert-butyl 2-(hydroxymeth-
yl)morpholine-4-carboxylate8 gave ether intermediate 31.
Oxidation of the sulfide group followed by reaction with
different anilines and then deprotection gave the C-4
analogs.
5-Methyl-pyrrolotriazines with different C-4 substituents
(compounds 4–10 in Table 1) were prepared by reaction
of 4-chloro-5-methyl-pyrrolotriazine 36 with anilines in
the presence of a base (Scheme 1). To prepare C-5 meth-
ylene ether analogs (compounds 12–27 in Table 2), the
5-methyl group of 3 was first brominated to give 11.7 This
was treated with an excess of the alcohol of interest fol-
lowed by 1-(3-fluorobenzyl)-1H-indazol-5-amine to give
the ether analogs. A more efficient procedure was used
with alcohols of limited availability. For this, 11 was con-
verted into alcohol 28 by solvolysis in aqueous acetoni-
trile followed by reaction with 1-(3-fluorobenzyl)-1H-
indazol-5-amine. Treatment of 28 with thionyl chloride
gave a relatively unstable 5-chloromethyl intermediate,
29, that was reacted with a slight excess of the alcohol
of interest to give the corresponding ether analog.
Analogs 35–38 (Table 3) have chiral phenylethyl groups
attached to N-1 of the indazole residue. To prepare the
aminoindazole that was used to make 35, nitroindazole
39 was coupled with (S)-1-phenylethanol using a Mits-
unobu reaction (Scheme 3).9 This gave a 2:1 mixture
of the desired N-1 alkylation product, 40, and its N-2
regioisomer. The isomers were separated by chromatog-
raphy and their regiochemistry was established by 2D
NMR (COSY). Reduction of the nitro group gave the
aminoindazole, 41, that was used to make 35 as outlined
above. Analog 36 was similarly prepared from (R)-1-
phenylethanol. Chiral HPLC analysis of 35 and 36 indi-
cated that they were homochiral and that the Mitsunobu
reaction had proceeded with complete inversion of con-
figuration (ee > 99%). Their m-fluoro analogs, 37 and
38, were similarly prepared as a racemic mixture from
1-(3-fluorophenyl)ethanol. They were separated by pre-
parative chiral HPLC and their configuration was
The C-4 analogs of 24 (compounds 32–38 in Table 3) were
prepared by first converting
3 to the 4-methyl-
Table 1. Structure–activity relationship for the C-4 position
Ar
HN
Me
N
N
N
Compound
Ar
HER2a,b IC50 (lM)
EGFRb IC50 (lM)
4
>1
0.10
F
Cl
H
N
5
6
>1
0.21
0.25
N
O
F
0.43
Cl
N
7
8
0.11
0.31
0.25
0.088
0.086
0.20
1.8
N
N
N
N
N
N
N
N
N
9
10
0.081
F
a Recombinant HER2 cytoplasmic sequence is expressed in Sf9 insect cells as an untagged protein and purified by ion-exchange chromatography.
HER2 kinase activity is measured under the same conditions as for EGFR. See Refs. 5 and 6 for assay conditions.
b IC50 values are reported as means of at least three determinations. Variability around the mean value was <15%.

2038
H. Mastalerz et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2036–2042
Ar
N
noma) cell proliferation assays. These are cell lines that
HN
Cl
N
Me
Me
do not depend on EGFR or HER2 signaling and pro-
vide a check for off-target antiproliferative effects. The
5-hydroxymethyl derivative, 28, and its ether analogs,
12 and 13, were less potent kinase inhibitors than the
5-methyl compound, 10 (Table 2). Acid 14 showed good
kinase inhibition and modeling suggested that the acid
group could either interact with the conserved Lys745
normally involved in binding the phosphate group of
ATP or Cys773 of the solvent-exposed hinge region
(see Fig. 2A for the position of Cys773). It did not show
antiproliferative activity, presumably because of poor
cell permeability. The neutral ester and amide analogs,
15 and 16, were less potent kinase inhibitors. Analogs
with acyclic basic amino groups (compounds 17–19)
showed better potency in both the kinase and cell assays.
The best of these was 18 which had a propylene spacer
between the amino and ether groups. It was more potent
than its N,N-dimethyl analog, 20, and also showed good
stability when incubated with mouse liver microsomes.
The latter observation was found to be general; pyrrol-
otriazine analogs with basic primary or secondary amine
substituents showed better microsomal stability than
their tertiary or N-acylated counterparts. This has been
seen with other chemotypes and it has been suggested
that the greater polarity and basicity of the primary or
secondary amines reduces their rate of metabolism by
the CYP3A4 isozyme.13 The piperidine ether, 21,
showed similar kinase and antiproliferative activity,
while homolog 22 and its 3-piperidyl isomer, 23, were
less potent. Morpholine analog 24 showed good kinase
inhibition and significantly enhanced antiproliferative
activity. The latter appears to be due to EGFR and
HER2 kinase inhibition since 24 did not show signifi-
cant activity against the A2780 cell line. Its improved
cellular potency relative to analogs like 18 may be the
result of better cell permeability with the less basic mor-
pholine side chain.14 Its R-enantiomer, 25, showed a
similar profile and modeling suggested that the morpho-
line group of both enantiomers can make similar hydro-
gen bond contacts in the ribose-phosphate portion of
the ATP binding pocket (see below). Transposed analog
26 and racemic oxazepine 27 were less potent.
a
N
N
N
N
4-10
3
N
F
N
R
HN
N
b
N
N
12 - 27
Cl
Br
f
N
29, R = Cl
N
d
N
e
11
28, R = OH
Scheme 1. Reagents and conditions: (a) ArNH2 (1.0 equiv), NaHCO3
(2 equiv), CH3CN, rt; (b) NBS (1.05 equiv), benzoyl peroxide
(catalytic), CCl4, N2, reflux, 1 h (74%); (c) ROH (10 equiv), CH3CN,
NaHCO3 (2 equiv), rt and then 1-(3-fluorobenzyl)-1H-indazol-5-amine
(0.9 equiv), NaHCO3 (2 equiv), CH3CN, rt and then deprotection
(33% TFA, CH2Cl2, rt) if required; (d) NaHCO3 (2.5 equiv), CH3CN/
H2O = 10:1, rt, 3 days, then 1-(3-fluorobenzyl)-1H-indazol-5-amine
(0.9 equiv), NaHCO3 (0.8 equiv), rt, 18 h (65%); (e) SOCl2 (1.1 equiv),
CH2Cl2, rt; (f) ROH (1.5 equiv), DIPEA (1.1 equiv),CH2Cl2, rt, 3 days
and then deprotection (33% TFA, CH2Cl2, rt) if required.
assigned according to their biochemical potency relative
to that of the des-fluoro analogs, 35 and 36.
The 6-methoxy analog of 24 (compound 45, Table 3)
was prepared from 5-methyl ester 4210 as outlined in
Scheme 4. The ester was reduced to alcohol 43 which
was then converted to acetate 44. Heating 44 with an
excess of (S)-tert-butyl 2-(hydroxymethyl)morpholine-
4-carboxylate followed by deprotection gave 45.
The SAR of 5-methyl-pyrrolotriazines with different C-4
substituents (Table 1) was found to parallel that report-
ed for 5,6-disubstituted pyrrolotriazines.11 Analogs with
small C-4 anilines or bicyclic heterocycles (compounds 4
and 5) were selective EGFR inhibitors. Appending a
lipophilic benzyl group to the C-4 aniline increased
HER2 kinase inhibition without reducing EGFR inhibi-
tion (compounds 6 and 7). The 2-picolyl analog, 8,
showed a slight drop in overall potency, while its 3-pic-
olyl isomer, 9, was a HER2 selective inhibitor. The m-
fluorobenzylindazolylamino side chain of 10 was found
to provide the best dual EGFR and HER2 kinase inhi-
bition and was therefore kept as the C-4 substituent for
the SAR study of different C-5 methylene ether solubiliz-
ing groups.
The SAR of the C-4 substituent of 24 was then exam-
ined to see if further improvements were possible (Table
3). Benzyl ether 32 and picolyl analogs, 33 and 34, were
found to be less potent. The R-(1)-phenylethyl analog,
35, was more potent than its enantiomer, 36, suggesting
that its C-4 substituent experiences a better fit in the
hydrophobic specificity pocket. The corresponding fluo-
ro derivatives, 37 and 38, were more potent with the
former showing a profile similar to that of 24. Finally,
methoxy analog 45 was prepared to gauge the effect of
substitution at C-6. The observed drop in potency indi-
cated that this was not tolerated.
Potential binding modes for compound 24 in the ATP
binding site were modeled after the published X-ray
structure of the complex between lapatinib and the
EGFR.15 Two binding modes were considered possible
and these are shown in Figure 2. In both cases, the pyr-
rolotriazine core is oriented in the ATP binding site such
Promising C-5 analogs were screened for their antipro-
liferative activity against the N87 cell line. This is a hu-
man gastric carcinoma that overexpresses both EGFR
and HER2.5,12 Analogs were also screened in HT29 (hu-
man colon carcinoma) or A2780 (human ovarian carci-

H. Mastalerz et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2036–2042
Table 2. Structure–activity relationship for the C-5 position
2039
F
N
N
HN
R
N
N
N
a
Compound
R
IC50 (lM)
Metabolic stabilitye
HER2
EGFR
N87b
HT29c or A2780d
10
28
12
H
0.088
0.11
0.42
0.081
0.12
0.34
0.38
7.4
nd
>10c
>10c
nd
0.14
nd
HO
MeO
0.24
O
13
14
15
0.42
0.018
0.28
0.49
0.030
0.46
nd
nd
nd
nd
nd
MeO
HO2C
O
>10
1.7
>10d
nd
MeO2C
O
O
16
17
18
19
20
21
0.11
0.09
0.06
0.13
0.56
0.07
0.14
0.09
0.064
0.16
0.62
0.073
1.7
1.7
0.64
nd
>10d
8.3d
7.4d
nd
nd
O
O
H2N
nd
H2N
H2N
O
0.01
nd
O
H2N
Me2N
O
nd
nd
0.12
0.02
O
0.33
6.0d
HN
HN
22
23
24
0.12
0.17
0.055
0.13
0.19
0.061
0.72
0.98
0.083
4.9d
7.4d
8.7d
nd
O
nd
HN
HN
HN
O
O
O
O
O
0.07
25
26
0.060
0.59
0.083
0.64
0.13
3.0
>10d
>10d
nd
nd
NH
O
O
O
27
0.14
0.21
2.0
>10d
nd
O
HN
a IC50 values are reported as means of at least three determinations. Variability around the mean value was <15%.
b Cell line N87 is a human gastric carcinoma that overexpresses both EGFR and HER2.5
c,dCell lines HT29 (human colon carcinoma) and A2780 (human ovarian carcinoma) do not depend on EGFR or HER2 signaling.5
e Rate of metabolism (nmol minꢀ1 mg proteinꢀ1) by mouse liver microsomes after a 10-min incubation at 3 lM in the presence of NADPH.

2040
H. Mastalerz et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2036–2042
Table 3. Structure–activity relationship for the C-4 position of 24
a
Compound
Ar
IC50 (lM)
HER2
0.56
EGFR
N87
0.61
O
F
32
33
34
0.57
Cl
N
N
0.38
0.41
0.50
0.81
0.24
0.55
N
N
N
N
Me
N
35
36
37
0.13
0.85
0.041
0.12
0.69
0.068
0.66
N
N
N
Me
N
2.2
Me
N
0.13
F
Me
N
38
0.51
0.25
0.40
0.43
nd
N
N
F
F
N
45b
3.4
a IC50 values are reported as means of at least three determinations. Variability around the mean value was <15%.
b Compound 45 is the 6-methoxy analog of 24.
O
R2
SMe
O
Me
b
a
N
N
R1
N
3
N
N
N
N
31, R1 = Boc, R2 = SMe
30
c
32 - 38, R1 = H, R2 = NHAr
Scheme 2. Reagents and conditions: (a) NaSMe (1.5 equiv), THF, rt; (b) NBS (1.05 equiv), benzoyl peroxide (catalytic), CCl4, N2, reflux, 10 min,
then (S)-tert-butyl 2-(hydroxymethyl)morpholine-4-carboxylate (1.5 equiv), toluene, reflux, 55%; (c) m-CPBA (2.1 equiv), CH2Cl2, 0 ꢁC to rt, 30 min,
then ArNH2 (1.0 equiv), rt, then 15% TFA/CH2Cl2.

H. Mastalerz et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2036–2042
2041
the OPLS-AA force field17 and the GBSA continuum
model,18 an implicit solvation model. The resultant ener-
gy calculations (not shown) favor the latter model. The
C-5 side chains of quinazoline EGFR and HER2 kinase
inhibitors have recently been hypothesized to similarly
extend into the ribose portion of the ATP binding
pocket.19
Me
H
N
a
N
N
N
b
O2N
R
39
40, R = NO2
41, R = NH2
The preliminary data for 24 showed promise and there-
fore it underwent further evaluation. It exhibited good
selectivity in a small panel of kinases (Table 4) and a
dose-dependent inhibition of HER2 protein phosphory-
lation in N87 cells (Fig. 3) that was consistent with the
cell proliferation assay data.
Scheme 3. Reagents and condition: (a) (S)-(+)-1-phenylethanol (2.0
equiv), 1,10-azobis(N,N-dimethyl formamide) (2.0 equiv) PBu3 (2.0
equiv), toluene, 80 ꢁC, 64%; (b) H2 (1 atm), catalytic Pd(OH)2/C,
MeOH, 81%.
42, R = CO2Me
Ar
N
a
b
c
HN
N
R
Table 4. Kinase selectivity profile for 24
43, R = CH2OH
44, R = CH2OAc
a
Kinase
IC50 (lM)
MeO
N
EGFR
HER2
Met
0.061
0.055
6.5
CH2O
45, R =
LCK
>5
VEGFR2
CDK2
p38
>10
>50
>50
>50
O
NH
Scheme 4. Reagents and conditions: (a) LiAlH4 (5 equiv), THF, 0 ꢁC
to rt, 68%; (b) AcCl (1.2 equiv), DIPEA (1.5 equiv) DCM, 0 ꢁC, 75%;
(c) (S)-tert-butyl 2-(hydroxymethyl)morpholine-4-carboxylate (20
equiv), DCE, 85 ꢁC, 18 h then 50% TFA, DCE, 24%.
PKA
a IC50 values are reported as means of at least three determinations.
Variability around the mean value was <15%.
that there is a hydrogen bond between N-1 and the hinge
region Met769 NH and the C-4 benzyl indazole group
extends back into a deep hydrophobic pocket formed
partially by the C-helix. The C-5 substituent can either
extend out toward the solvent (Fig. 2A) or into the ri-
bose phosphate pocket (Fig. 2B). In the former model
the protonated morpholine NH can hydrogen bond with
the side chains of Cys773 and Asp776. In the latter, the
protonated morpholine NH can hydrogen bond with
the side chains of Asp831 and Asn818, and also with
the backbone carbonyl oxygen of Arg817. Also in this
model there exists an intramolecular hydrogen bond be-
tween the C4 aniline NH and the C5 ether oxygen. The
docked poses were energy-minimized in Maestro16 using
Anti-pTyr
Anti-HER2
Figure 3. Compound 24 inhibits receptor phosphorylation in N87
Cells. These were treated with compound 24 for 1 h at the concentra-
tion indicated and cell lysates were analyzed by Western blotting with
anti-phosphotyrosine and anti-HER2 antibodies.
Figure 2. Predicted binding modes of compound 24 modeled in the X-ray structure of the lapatinib/EGFR kinase complex15. In part A of the figure
the C-5 side chain extends out toward the solvent where the protonated morpholine NH hydrogen bonds with Cys773 and Asp776. In part B of the
figure the C-5 side chain extends into the ribose phosphate binding region where the protonated morpholine NH hydrogen bonds with the Asp831,
Asn818, and Arg817. Image created with Pymol from DeLano Scientific LLC, San Carlos, CA, USA. http://www.pymol.org.

2042
H. Mastalerz et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2036–2042
Table 5. In vivo antitumor activity of 24 against GEO and N87
xenografts implanted subcutaneously in athymic mice
3. (a) Rusnak, D. W.; Lackey, K.; Affleck, K.; Wood, E. R.;
Alligood, K. J.; Rhodes, N.; Kieth, B. R.; Murray, K. G.;
Knight, W. B.; Mullin, R. J.; Gilmer, T. M. Mol. Cancer
Ther. 2001, 1, 85; (b) Burris, H. A. The Oncologist 2004,
9(Suppl. 3), 10.
4. Traxler, P.; Allegrini, P. R.; Brandt, R.; Brueggen, J.;
Cozens, R.; Fabbro, D.; Grosios, K.; Lane, H. A.;
McSheehy, P. L.; Mestan, J.; Meyer, T.; Tang, C.;
Wartmann, M.; Wood, J.; Caravatti, G. Cancer Res.
2004, 64, 4931.
5. Wong, T. W.; Lee, F. Y.; Yu, C.; Luo, F. R.; Oppenhei-
mer, S.; Zhang, H.; Smykla, R. A.; Mastalerz, H.; Fink, B.
E.; Hunt, J. T.; Gavai, A. V.; Vite, G. D. Clin. Cancer Res.
2006, 12, 6186.
6. Hunt, J. T.; Mitt, T.; Borzilleri, R.; Gullo-Brown, J.;
Fargnoli, J.; Fink, B.; Han, W-C.; Mortillo, S.; Vite, G.;
Wautlet, B.; Wong, T.; Yu, C.; Zheng, X.; Bhide, R. J.
Med. Chem. 2004, 47, 4054.
Tumor model Dosea (mg kg-1) Schedule % TGIb Pc
N87
30
60
QD · 21
QD · 14
28
51
0.3329
0.0037
0.0001
0.0001
120
240
70
111
GEO
30
60
21
55
71
91
0.3329
0.0528
0.0037
0.0037
120
240
a Vehicle: propylene glycol/water (50:50).
b Percent tumor growth inhibition during treatment. Tumor cells were
implanted subcutaneously in athymic mice and staged to approxi-
mately 100 mg prior to the initiation of drug therapy. Activity is
defined as %TGI P 50%.
c Probability for median tumor weight at the end of drug treatment.
7. For experimental procedures, see: Mastalerz, H.; Zhang,
G.; Tarrant, J.; Vite, G. D. US 6,908,916.
8. Brenner, E.; Baldwin, R. M.; Tamagnan, G. Org. Lett.
2005, 7, 937.
9. (a) Fernandez, P. A.; Bellamy, T.; King, M.; Madge, D. J.;
Selwood, D. L. Heterocycles 2001, 55, 1813; (b) Tsunoda,
T.; Yamamiya, Y.; Ito, S. Tetrahedron Lett. 1993, 34,
1639.
10. Mastalerz, H.; Gavai, A. V.; Fink, B.; Struzynski, C.;
Tarrant, J.; Vite, G. D.; Wong, T. W.; Zhang, G.; Vyas, D.
M. Can. J. Chem. 2006, 84, 528.
11. Fink, B. E.; Vite, G. D.; Mastalerz, H.; Kadow, J. F.;
Kim, S.-H.; Leavitt, K. J.; Du, K.; Crews, D.; Mitt, T.;
Wong, T. W.; Hunt, J. T.; Vyas, D. M.; Tokarski, J. S.
Bioorg. Med. Chem. Lett. 2005, 15, 4774.
12. Additional cell proliferation data (not shown) using the
HER2 overexpressing BT-474 human breast tumor cell line
and a constitutively activated Sal2 murine salivary gland
tumor that expresses a CD8HER2 fusion protein wer found
to track with these observed for the N87 cell line.
13. (a) Smith, A. S.; van de Waterbeemd, H. In Pharmaco-
kinetics and Metabolism in Drug Design; Wiley-VCH:
Weinheim, 2001; p 84; (b) Smith, D. A.; Jones, C. J.;
Walker, D. K. Med. Res. Rev. 1996, 16, 243.
Compound 24 showed acceptable permeability in the
Caco-2 bilayer model (Pc = 64 nm/s) suggesting that
good oral absorption may be achieved in vivo. The free
fraction (fu) of the compound in mouse serum was 2.6%
as determined by equilibrium dialysis at 10 lM. It was
administered orally (50 mg kgꢀ1 dose) to male Balb/C
mice and showed a 4 h AUC of 29 lM h and a 4 h plas-
ma concentration of 5.8 lM. This was about 70-fold
higher than its antiproliferative IC50 versus the N87 cell
line in vitro and therefore it was evaluated in vivo in the
N87 human gastric carcinoma (HER2 and EGFR driv-
en) and the GEO human colon carcinoma (EGFR driv-
en) xenograft models. Orally administered 24 was found
to be well tolerated in both studies; treated animals
exhibited no clinically observable sign of toxicity or
weight loss at the highest test dose of 240 mg/kg. It elic-
ited robust and dose-proportional tumor responses over
the dose range of 60–240 mpk in both tumor models
(Table 5).20
14. Based on pKa’s (H2O) of 8.3 for morpholine versus 10.2
for 1-amino-3-hydroxypropane from: Perrin, D.D. Disso-
ciation Constants of Organic Bases in Aqueous Solution.
Supplement 1972; Butterworths: London, UK, 1972.
15. Wood, E. R.; Truesdale, A. T.; McDonald, O. B.; Yuan,
D.; Hassell, A.; Dickerson, S. H.; Ellis, B.; Pennisi, C.;
Horne, E.; Lackey, K.; Alligood, K. J.; Rusnak, D. W.;
Gilmer, T. M.; Shewchuk, L. Cancer Res. 2004, 64, 6652.
16. Schrodinger, L.L.C. 2003. 32nd Floor, Tower 45, 120
West Forty-Fifth Street, New York, 10036.
17. Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.;
Jorgensen, W. L. J. Phys. Chem. B 2001, 105, 6474.
18. Yu, Z.; Jacobson, M. P.; Friesner, R. A. J. Comput. Chem.
2006, 27, 72.
19. (a) Ballard, P.; Bradbury, R. H.; Harris, C. S.; Hennequin,
L. F. A.; Hickinson, M.; Johnson, P. D.; Kettle, J. G.;
Kinowska, T.; Leach, A. G.; Morgentin, R.; Pass, M.;
Ogilvie, D. J.; Olivier, A.; Warin, N.; Williams, E. J.
Bioorg. Med. Chem. Lett. 2006, 16, 1633; (b) Ballard, P.;
Bradbury, C. S.; Hennequin, L. F. A.; Hickinson, M.;
Johnson, P. D.; Kettle, J. G.; Kinowska, T.; Morgentin,
R.; Ogilvie, D. J.; Olivier, A. Bioorg. Med. Chem. Lett.
2005, 15, 4226.
In summary, it was found that pyrrolotriazines with a
basic solubilizing group that is tethered to C-5 via a
methylene ether linkage can show potent, selective inhi-
bition of both the EGFR and the HER2 kinases. Mod-
eling studies suggest that the C-5 solubilizing group may
extend into the ribose-phosphate binding region of the
ATP binding pocket where it can participate in multiple
hydrogen bonding interactions. The lead compound, 24,
exhibited good kinase selectivity, antiproliferative
potency, oral exposure, and efficacy in tumor xenograft
models. This effort was followed by a broader examina-
tion of C-5 solubilizing groups and the results will be de-
scribed in the future.
References and notes
1. (a) Baselga, J.; Arteaga, C. L. J. Clin. Oncol. 2005, 23,
2445; (b) Kamath, S.; Buolamwini, J. K. Med. Res. Rev.
2006, 26, 569, and references therein.
2. Baselga, J. Ann. Oncol. 2002, 13, 8.
20. For experimental details, see Ref. 5.