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H. Mastalerz et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2828–2833
Table 2. Pharmacokinetic parametersa for 1p in mice
Cmax (nM) po
Tmax (h) po
AUC
48913
(nM h) po
t1/2 (h) iv
MRT (h) iv
4.0
Cl (mL/min/kg) iv
39
Vss (L/kg) iv
Fpo (%)
77
0–8 h
7951
3
3.7
9.5
a pK parameters were obtained from composite plasma concentration–time profiles with an average of three male Balb/C mice at each time point.
Mice were dosed at 90 (po) and 10 (iv) mg kgÀ1 using Tween 80/PEG400/water (10:40:50, v/v) as the vehicle.
in the ATP binding site such 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
alpha-C-helix. The C-5 substituent extends out into the
ribose phosphate pocket where the protonated homo-
piperazine NH can hydrogen bond with the side chains
of Asp831, Asn818 and/or the backbone carbonyl oxy-
gen of Arg817. In this model, there is also an intramo-
lecular hydrogen bond between the C-4 aniline NH
and the hompiperazine tertiary nitrogen atom. This
binding model is similar to that previously suggested3a
for morpholine analog 1b and has been hypothesized17
for quinazoline EGFR and HER2 kinase inhibitors with
solubilizing side chains at C-5. It also offers an explana-
tion for why (R)-3-hydroxy-homopiperazine 1t is more
potent than its S-enantiomer, 1u. The hydroxy group
of the former homopiperazine is better situated to
hydrogen bond with Asp831 and/or the conserved
Lys745 that are normally involved in binding the phos-
phate group of ATP.
Compound 1p was evaluated in EGFR/HER2 driven
N87 human gastric and EGFR driven GEO human co-
lon carcinoma xenograft models in athymic mice. In
both studies it was administered orally, twice a day
(BID), for 14 consecutive days. It was robustly active
in the N87 model yielding tumor growth inhibition of
97 to 121% over the dose-range of 90–180 mg/kg (Table
3). A clear maximum-tolerated dose (MTD) was reached
at the 180 mg/kg dose which was associated with
approximately 12% drug related mortality. In the
GEO model, 1p was tested at 90 and 135 mg/kg and
was active at both dose levels. The 4-hydroxy-homopip-
erazine, 1t, was also evaluated in vivo but it did not
demonstrate an advantage over 1p.
In summary, pyrrolotriazines with diamino solubilizing
groups that are tethered to C-5 showed potent inhibition
of both EGFR and HER2 kinases. Modeling studies
suggested that the solubilizing group can extend into
the ribose–phosphate binding region of the ATP binding
pocket where it can participate in multiple hydrogen
bonding interactions. The homopiperazine analog, 1p,
emerged as a key lead and it exhibited potent kinase
inhibition, antiproliferative activity, and oral efficacy
in tumor xenograft models. This encouraged a broader
examination of other C-5 amino and diamino solubiliz-
ing groups and the results will be described in future
publications.
The PK parameters for 1p in mice are summarized in
Table 2. Absorption of the compound after oral admin-
istration was excellent (77% bioavailable) and this was
consistent with its high permeability in Caco-2 cells.
After intravenous administration, 1p showed a moderate
plasma clearance of 39 mg/min/kg, with a half-life (t1/2
)
and mean residency time (MRT) of 3.7 and 4 h, respec-
tively. A relatively high steady-state volume of distribu-
tion (Vss) was observed in mice. The compound was
98.4% bound to mouse serum proteins, yielding a free
fraction of 1.6% in that species. The aqueous solubility
of 1p was pH-dependent, ranging from 9.7 mg/mL with
a solution pH of 3.9 for the HCl salt to 0.012 mg/mL in
a pH 7.5 phosphate buffer. The relatively low aqueous
solubility at neutral pH is similar to that of other ATP
competitive EGFR kinase inhibitors.18
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.
3. (a) Mastalerz, H.; Chang, M.; Chen, P.; Dextraze, P.;
Fink, B. E.; Gavai, A.; Goyal, B.; Han, W.-C.; Johnson,
W.; Langley, D.; Lee, F. Y.; Oppenheimer, S.; Ruediger,
E.; Tarrant, J.; Tokarski, J. S.; Vite, G. D.; Vyas, D. M.;
Wong, T. W.; Zhang, H.; Zhang, G. Bioorg. Med. Chem.
Lett. in press; Quinazolines with a C-4 benzylindazolyl-
amino side chain similarly show potent dual EGFR and
HER2 kinase inhibition, see: (b) Brignola, P. S.; Lackey,
K.; Kadwell, S. H.; Hoffman, C.; Horne, E.; Carter, H. L.;
Stuart, J. D.; Blackburn, K.; Moyer, M. B.; Alligood, K.
J.; Knight, W. B.; Wood, E. R. J. Biol. Chem. 2002, 277,
1576, and references therein.
4. For experimental details, see: Mastalerz, H; Zhang, G.;
Tarrant, J.; Vite, G. D.; US 6,908,916.
5. Sarri, W. S.; Raab, A. W.; King, S. W. J. Org. Chem.
1971, 36, 1711.
6. Griffith, W. P.; Ley, S. V. Aldrichimica Acta 1990, 23, 13.
7. Bergeron, R. J.; Ludin, C.; Muller, R.; Smith, R. E.;
Phanstiel, O. J. Org. Chem. 1997, 62, 3285.
8. Kan, T.; Kobayashi, H.; Fukuyama Synlett 2002, 697, The
diazapine 8 most likely forms via the 7-endo ring closure
Table 3. In vivo antitumor activity of orally administered 1p against
established N87 and GEO xenografts implanted subcutaneously in
athymic micea
Tumor model Dosea, mg kgÀ1 Schedule % TGIb Pc
N87
90
135
180
BID · 14
BID · 14
97
111
121
0.0037
0.0001
0.0018
GEO
90
135
60
78
0.0001
0.0018
a Vehicle: Tween 80/PEG400/water, 10:40:50.
b Percent tumor growth inhibition during treatment. Activity is defined
as %TGI P50%.
c Probability for median tumor weight at the end of drug treatment,
compared with controls.
Mastalerz, Harold
