Tyrosine kinase inhibitors. 19. 6-alkynamides of 4-anilinoquinazolines and 4-anilinopyrido[3,4-d]pyrimidines as irreversible inhibitors of the erbB family of tyrosine kinase receptors

Article abstract of DOI:10.1021/jm050936o

Structure-activity relationships for inhibition of erbB1, erbB2, and erbB4 were determined for a series of alkynamide analogues of quinazoline- and pyrido[3,4-ui]pyrimidine-based compounds. The compounds were prepared by coupling the appropriate 6-aminoquinazolines or 6-aminopyrido[3,4-d]pyrimidines with alkynoic acids, using EDCI·HCl in pyridine. The compounds showed pan-erbB enzyme inhibition but were on average about 10-fold more potent against erbB1 than against erbB2 and erbB4. For cellular inhibition, the nature of the alkylating side chains was an important determinant, with 5-dialkylamino-2- pentynamide type Michael acceptors providing the highest potency. This is suggested to be due to an improved ability of the amine to participate in an autocatalysis of the Michael reaction with enzyme cysteine residues. Pyrido[3,4-d]pyrimidine analogue 39 was selected for in vivo evaluation and achieved tumor regressions at 10 mg/kg in the A431 human epidermoid carcinoma and at 40 mg/kg for the SF767 human glioblastoma and the SKOV3 human ovarian carcinoma. Complete stasis was observed at 40 mg/kg in the BXPC3 human pancreatic carcinoma as well as in the H125 human non-small-cell lung carcinoma.

Full text of DOI:10.1021/jm050936o

J. Med. Chem. 2006, 49, 1475-1485
1475
Tyrosine Kinase Inhibitors. 19. 6-Alkynamides of 4-Anilinoquinazolines and
4-Anilinopyrido[3,4-d]pyrimidines as Irreversible Inhibitors of the erbB Family of Tyrosine
Kinase Receptors
Sylvester R. Klutchko,† Hairong Zhou,† R. Thomas Winters,† Tuan P. Tran,† Alexander J. Bridges,† Irene W. Althaus,†
Danielle M. Amato,† William L. Elliott,† Paul A. Ellis,† Mary Ann Meade,† Billy J. Roberts,† David W. Fry,†
Andrea J. Gonzales,† Patricia J. Harvey,† James M. Nelson,† Veronica Sherwood,† Hyo-Kyung Han,† Gerry Pace,†
Jeff B. Smaill,‡ William A. Denny,‡ and H. D. Hollis Showalter*,†,§
Pfizer Global Research and DeVelopment, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48106-1047, and Auckland
Cancer Society Research Centre, School of Medical Sciences, The UniVersity of Auckland, PriVate Bag 92019, Auckland 1020, New Zealand
ReceiVed September 20, 2005
Structure-activity relationships for inhibition of erbB1, erbB2, and erbB4 were determined for a series of
alkynamide analogues of quinazoline- and pyrido[3,4-d]pyrimidine-based compounds. The compounds were
prepared by coupling the appropriate 6-aminoquinazolines or 6-aminopyrido[3,4-d]pyrimidines with alkynoic
acids, using EDCI‚HCl in pyridine. The compounds showed pan-erbB enzyme inhibition but were on average
about 10-fold more potent against erbB1 than against erbB2 and erbB4. For cellular inhibition, the nature
of the alkylating side chains was an important determinant, with 5-dialkylamino-2-pentynamide type Michael
acceptors providing the highest potency. This is suggested to be due to an improved ability of the amine to
participate in an autocatalysis of the Michael reaction with enzyme cysteine residues. Pyrido[3,4-d]pyrimidine
analogue 39 was selected for in vivo evaluation and achieved tumor regressions at 10 mg/kg in the A431
human epidermoid carcinoma and at 40 mg/kg for the SF767 human glioblastoma and the SKOV3 human
ovarian carcinoma. Complete stasis was observed at 40 mg/kg in the BXPC3 human pancreatic carcinoma
as well as in the H125 human non-small-cell lung carcinoma.
Introduction
increasing their potency against erbB2. This has generally been
achieved by incorporating additional lipophilic bulk in the
4-position of the aniline ring, such as in Lapatinib (GW572016;
3),18 which is a potent dual inhibitor of erbB1 and erbB2
currently in phase III clinical trials against breast cancer.4
Lapatinib is reported to have a slow inhibitor off-rate consistent
with its prolonged effect on down-regulating receptor tyrosine
phosphorylation in tumor cells. This slow off-rate may in part
be explained by the crystal structure of Lapatinib bound to an
inactive-like conformation of erbB1.19 BMS-599626 (4) is
another example of a reversible inhibitor with dual activity
against erbB1and erbB2 reported to be in phase I clinical
trials.20,21 AEE788 (6), an analogue of the dual erbB1/erbB2
inhibitor PKI-166 (5),22 is also in phase I trials and represents
the next generation of multitargeted tyrosine kinase inhibitors
possessing potent activity against erbB1, erbB2, and the VEGF
receptors.23
Crystal structures of 2 binding to the ATP site of the erbB1
kinase domain24 and of the related 4-anilinoquinazoline (7)
bound to the related p38 serine-threonine kinase25 show that
the quinazoline chromophore binds to a narrow hydrophobic
pocket in the N-terminal domain normally occupied by the
adenine of ATP. It is stabilized by hydrogen bonds from the
enzyme to the quinazoline N1 and N3 atoms (the latter through
a bridging water molecule) with positions C-6 and C-7 of the
quinazoline pointing toward the mouth of the channel. These
observations are broadly supportive of the binding model
proposed earlier.7 This model, in conjunction with a homology
model built by the Wyeth group, has been used in the design
of the quinoline-3-carbonitriles (e.g., 8) by replacing the N3
atom with a cyano group that can displace the bound water
molecule.26
A significant proportion of human tumors overexpress growth
factor receptor tyrosine kinase enzymes of the erbB family, and
this overexpression is associated with poor prognosis of the
disease.1-3 Inhibitors of growth factor signaling through these
pathways, especially via erbB1 (EGFr, HER-1) and erbB2
(HER-2, neu), have thus been sought as potential anticancer
drugs.4 The most widely studied class of small molecule
inhibitors are the 4-anilinoquinazolines5 and related 4-anili-
nopyrido[d]pyrimidines.6 These have proved to be potent and
selective reversible inhibitors of erbB1 receptor tyrosine kinase
activity, through competitive binding to the ATP site of the
enzyme.7 The 4-anilinoquinazolines gefitinib (ZD-1839, Iressa;
1)8,9 and erlotinib (OSI-774, Tarceva; 2)10 have both been
approved for the treatment of non-small-cell lung cancer
(NSCLC). Responses to treatment with gefitinib11,12 and erlo-
tinib13 have particularly been observed in a subgroup of NSCLC
patients that were identified to have somatic mutations in the
tyrosine kinase domain of erbB1. It has, however, been reported
that the binding affinities of gefitinib and erlotinib to nine
clinically relevant NSCLC erbB1 mutants are comparable to
those of the wild-type receptor,14 supporting the possibility that
cells containing an activating mutation are intrinsically more
sensitive to erbB1 inhibition.15 A secondary mutation (T790M)
in the kinase domain of erbB1 has been reported in three of six
cases of acquired resistance to gefitinib and erlotinib treat-
ment.16,17(See Chart 1 for formulas.)
Further development of reversible inhibitors has focused on
improving the clinical efficacy of this class of molecules by
* Corresponding author. Telephone: 734-649-5478. Fax: 734-647-8430.
E-mail: showalh@umich.edu.
† Pfizer Global Research and Development.
‡ The University of Auckland.
Because these compounds are considered to work by a
blockade of the binding of ATP, which is at high concentrations
in cells, the use of irreversible inhibitors is attractive. The
§ Current address. College of Pharmacy, University of Michigan, Ann
Arbor, MI 48109.
10.1021/jm050936o CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/01/2006

1476 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Chart 1. Formulas
Klutchko et al.
observation that the erbB family of enzymes contains a unique
unpaired cysteine residue (Cys 773 in erbB1, Cys 784 in erbB2,
and Cys 778 in erbB4) close to the entrance of the ATP binding
site, in a position normally occupied by a glutamine or serine
in other kinases,27 prompted work on irreversible inhibitors
possessing Michael acceptors at the 6-position, positioned to
react with this cysteine.28 Development of this concept29,30
resulted in the drug canertinib dihydrochloride (CI-1033; 9),
which is currently in phase II clinical trials.31,32 In this compound
the Michael acceptor of choice is the acrylamide moiety. Other
Michael acceptors have also been evaluated, including vinyl-
sulfonamides, vinylsulfones, vinylsulfines,33 and butyna-
mides.34,35 All but the vinylsulfines had some utility for
providing irreversible inhibition of erbB1. Despite 9 featuring
a similar erbB1 selective reversible binding core to gefitinib, it
possesses more potent activity against all members of the erbB
family (see Tables 1 and 2). Clearly alkylation of the key
cysteine of erbB2 and erbB4 can result in improved potency
against these receptors. Potent inhibition of erbB3 (which does
not contain a cysteine in this region) by this compound is
consistent with erbB3 only being able to acquire signaling
potential through heterodimerization with another erbB recep-
tor.4
that amines pendent off the R- and â-carbons of the Michael
acceptor through a methylene spacer are well-tolerated and can
in some instances provide potent irreversible inhibition of
erbB1.34 The dimethylamino crotonamide Michael acceptor of
compound 10 is the most widely studied, with the dimethyl-
amino moiety proposed to serve as an intramolecular base
catalyst between the Cys 773 sulfhydryl group and the Michael
acceptor through a cyclic five-membered ring mechanism.34,36
Compound 10 and its dimethylamino butynamide analogue (11)
showed potent inhibition of erbB1 (isolated enzyme IC50’s 0.011
and 0.006 µM, respectively) and erbB2 (isolated enzyme IC50’s
0.301 and 0.014 µM, respectively).
Applying the dimethylamino crotonamide Michael acceptor
to the quinoline-3-carbonitrile class of compounds has led to
EKB-569 (12)36 which is in clinical trial.37 Interestingly this
compound is a potent irreversible inhibitor of erbB1 (isolated
enzyme IC50 ) 0.08 µM) but is less effective against erbB2
(isolated enzyme IC50 ) 1.23 µM) suggesting that this Michael
acceptor will provide erbB2 potency on a quinazoline core (such
as for 10) but not on an analogous quinoline-3-carbonitrile core.
Application of this Michael acceptor to the 1,7-naphthyridine
core as in compound 13 also provides potent and presumably
irreversible inhibition of erbB1; however no erbB2 data have
been reported.38 We have previously reported in the 6-acryla-
midoquinazoline series that introduction of a benzyloxy or
phenoxy substituent at the 4-position of the aniline ring, such
as in compounds 15 and 16, is well-tolerated and can provide
irreversible inhibitors with cellular potency against erbB1 and
erbB2.29 Optimization of the quinoline-3-carbonitriles to im-
prove their erbB2 potency has proceeded along similar lines
In our initial structure-activity studies around the Michael
acceptor we probed the acrylamide nitrogen, the R-carbon, and
the â-carbon with a methyl substituent33 and found that only
the acrylamide nitrogen would tolerate an alkyl substituent while
still providing fully irreversible inhibition of erbB1. Further
elaboration to introduce a soluble side chain at this position
was not tolerated. The Wyeth group has independently shown

IrreVersible Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1477
Table 1. Kinase Inhibitory Properties of 6-Substituted 4-Anilinoquinazolines and 4-Anilinopyrido[3,4-d]pyrimidines Bearing Alkynamide Michael
Acceptors
IC50 (nM)a
compd.
X
Y
Z
erbB1
erbB2
erbB4
autophos (EGF)b
autophos (HER)c
7.4
9
19d
20e
21
22
23
24
25
26
27
28d
29
30
31
32
33
34
35
36
37
38
39
40
3.0
1.6
0.2
1.4
3.1
4.2
40
20
1.9
17
2.1
46
2.8
464
300
2.6
2.3
2.4
1.1
3.8
5.4
597
>103
67
31
1.7
12
7.3
310
11
624
229
16
4.3
13
0.5
2.4
7.1
644
538
73
6.2
1.0
1.0
1.8
1.2
0.8
1.7
2.7
2.5
25
6.8
11
11
31
4.5
12
15
2.5
9.6
4.5
48
73
49
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
N
N
N
N
N
N
N
N
3-Br
3-Br
H
Me
CH2OH
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Br
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Br, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-Br, 4-F
43
7.5
65
32
173
23
CH2N(Et)2
CH2N(i-Pr)2
CH2N(n-Bu)2
CH2Nmorpholide
(CH2)2N(Et)2
(CH2)2Nmorpholide
H
86
1.8
1.7
0.6
0.3
1.7
15
15
44
17
3.1
1.3
1.5
0.9
0.5
1.1
7.6
Me
n-Pr
n-Bu
24
170
20
116
36
320
22
5
CH2N(Et)2
CH2N(i-Pr)2
CH2N(n-Bu)2
CH2Nmorpholide
(CH2)2Nmorpholide
(CH2)2Nmorpholide
(CH2)2Npiperidine
(CH2)2N(4-Me-pz)f
(CH2)2N(4-Me-pz)f
26
14
0.8
0.7
2.1
0.7
0.5
1.8
4.5
4.3
1.9
2.7
N
N
N
N
0.5
2
1
3.5
a Concentration for 50% inhibition of phosphorylation of a Glu/Tyr copolymer (see Experimental Section). Values are the average of at least two separate
determinations, with a CV of (22%. b Inhibition of erbB1 autophosphorylation in NIH3T3 cells transfected with the full-length human erbB1 (see Experimental
Section). c Inhibition of heregulin-stimulated erbB autophosphorylation in MDA-MB-453 human breast carcinoma cells (see Experimental Section). d Reference
55. e References 34 and 55. f 4-Methylpiperazine.
by introducing a 2-pyridinylmethoxy group in the 4-position
of the aniline ring resulting in HKI-272 (14), a potent irreversible
inhibitor of both erbB1 and erbB2, which is reported to be in
clinical trial.39 HKI-272 and analogues have been shown to be
effective in vitro against NSCLC cells that have acquired
resistance to gefitinib, encouragingly including those that have
the T790M mutation.40
Irreversible inhibitors of the erbB family of tyrosine kinases
continue to be of considerable interest clinically. In certain
instances the Michael acceptor can confer pan-erbB activity to
an otherwise erbB1 selective core structure. In this paper we
report on structure-activity relationships for alkynamide ana-
logues of both the quinazoline and pyrido[3,4-d]pyrimidine core
structures. We identify a novel Michael acceptor that provides
potent pan-erbB activity, and we report data for this against
the erbB1, erbB2, and erbB4 kinases.
The propargylic acids 18a-i were either commercially
available or synthesized by the Wyeth procedure.34 The
homologous pentynoic acids 18j-m were synthesized by
modification of a procedure described many years ago,43 with
details shown in Scheme 1. Thus, condensation of commercially
available 4-(tosyloxy)-1-butyne (41) with a range of secondary
amines provided 4-(substituted amino)-1-butynes (42) in non-
optimized yields (31-82%). Subsequent lithiation of 42 with
n-butyllithium in THF or ether and carboxylation provided target
pentynoic acids 18j-m in 59-77% yield. These either were
coupled to 17a-e as their isolated lithium salts or were
converted to their neutral form by passage over an ion-exchange
resin. When the lithium salt was utilized, an equivalent of
pyridinium hydrochloride was added to buffer the reaction
medium.
Results and Discussion
Chemistry
Table 1 lists the structures and kinase inhibitory properties
of a series of 6-alkynamide derivatives of both 4-anilino-
quinazolines and 4-anilinopyrido[3,4-d]pyrimidines. As in our
previous studies,33 the anilino ring substituent was kept constant
(generally 3-chloro-4-fluoro; the substitution pattern used in both
gefitinib and canertinib dihydrochloride) to facilitate intercom-
parisons of the different Michael acceptors. The known com-
pounds 9 (canertinib dihydrochloride), 19, 20, and 28 were also
included for wider comparison.
All of the compounds in Table 1 were investigated for
inhibition of erbB1, erbB2, and erbB4, using cytoplasmic fusion
proteins made by cloning the erbB1 sequence (Met-668 to Ala-
1211), erbB2 (Ile-675 to Val-1256), and erbB4 sequence (Gly-
259 to Gly-690) into baculovirus. The IC50 values shown were
determined by an enzyme-linked immunosorbent assay based
The compounds reported in this paper were made by the
generalized routes outlined in Scheme 1. 6-Aminoquinazolines
(17a41 and 17b29) and the corresponding pyrido[3,4-d]pyrim-
idines (17c,42 17d, 17e29) were coupled with a wide range of
alkynoic acids (18a-m) to provide target compounds 19-40
(Table 1). In general, the 6-amino moiety of heterocycles 17a-e
was found to be weakly reactive toward the range of activated
acids utilized, and this was reflected in the poor yields of purified
products (5-32% for quinazolines; 2-28% for pyrido[3,4-d]-
pyrimidines). The most generalized conditions for this amidation
were found to be the use of 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCI‚HCl) in pyridine at
room temperature. Optimization of coupling conditions was not
extensively explored.

1478 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Klutchko et al.
Scheme 1a
a (i) 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDCI‚HCl), pyridine, or DMF, 0-25 °C; (ii) HNR1R2, DMF, or CH3CN or THF,
25-85 °C; (iii) n-BuLi, THF, -78 °C, then CO2, ether, -78 to 25 °C.
Table 2. Comparison of the in Vitro and Cellular Kinase Activities of
Compound 39 with Other ErbB Family Inhibitors
(ELISA-based) receptor tyrosine kinase assay, with poly(Glu-
Tyr) as substrate, and are technically apparent IC50 values.29
IC50 (nM)
They are an average of at least two separate determinations.
Overall, for the compounds where there was a full set of data
there was a good correlation between the degree of inhibition
of the different isozymes, suggesting that the general class of
6-alkynamides is composed of pan-erbB inhibitors (eqs 1-3;
data for eq 2 from compounds 19-40 of Table 1; data for eqs
1 and 3 omit compound 33). These equations also show that
the compounds were on average about 10-fold more potent
against erbB1 than against erbB2 and erbB4 (coefficient of IC50
term in eqs 1 and 2).
activity
39
canertinib gefitinib erlotinib
EGF receptor (erbB1)a
0.5
0.7
3.0
20
3.1
343
0.6
511
ErbB2 receptora
PDGF receptor
FGF receptor
insulin receptor
EGF receptor
>50 000 >50 000
>50 000 >50 000
>50 000 >50 000
1.9
0.8
1
1.7
14.4
599
19.3
299
autophosphorylationb
ligand-regulated erbB2
autophosphorylationc
HER-regulated tyrosine
autophosphorylationd
16
5.1
log IC50 (erbB1) ) 0.54((0.11) log IC50 (erbB2) -
0.05((0.13) (1)
a See footnotes to Table 1. b In erbB1-transfected NIH3T3 cells. c In T24
NIH fibroblasts expressing a chimeric receptor with the extracellular binding
domain of erbB1 and the intracellular kinase domain of erbB2. d In MDA-
MB-453 human breast carcinoma cells.
n ) 21, r ) 0.76, s ) 0.46, F2,19 ) 25
log IC50 (erbB1) ) 0.54((0.11) log IC50 (erbB4) -
0.11((0.15) (2)
to that reported previously.34 Compounds 37, 39, and 40 have
been confirmed as irreversible inhibitors of erbB1 in a modified
A431-cellular autophosphorylation assay containing a drug
wash-out protocol28,29 (data not shown).
n ) 22, r ) 0.76, s ) 0.48, F2,20 ) 28
Table 1 also reveals that there is a greater potency in cellular
versus enzyme assays for a number of compounds (e.g., 24 and
33). This is viewed as not being due to mixed erbB inhibition
in cells but perhaps to differences in response to different
enzyme constructs (i.e., enzyme assays use GST-fusion proteins
where GST is fused to the intracellular domain of the erbBs,
whereas in the HER-stimulated autophosphorylation cell assay,
the wild-type, full length forms are present).
Of the compounds evaluated, the pyrido[3,4-d]pyrimidine 39
had the best overall potencies in both the enzymic and cellular
assays and was selected for further studies. Table 2 shows that
39 has broadly similar in vitro activity to the clinical irreversible
inhibitor canertinib dihydrochloride (9) and is significantly more
potent (particularly in the erbB2 cellular assay) than the
clinically approved reversible inhibitors gefitinib and erlotinib.
Furthermore, compound 39 is selective for the erbB family of
tyrosine kinases over the PDGF, FGF, and insulin receptor
tyrosine kinases. The pan-erbB nature of this compound is
assessed in three cellular assays: first, in the EGF-stimulated
erbB1 autophosphorylation assay (in erbB1-transfected NIH3T3
cells) (see Figure 1, part A), second, in the ligand-stimulated
erbB2 autophosphorylation assay (in T24 NIH fibroblasts
log IC50 (erbB2) ) 0.91((0.08) log IC50 (erbB4) -
0.06 ((0.11) (3)
n ) 21, r ) 0.93, s ) 0.37, F2,19 ) 117
Over the whole class, there was a less close correlation between
the erbB1/EGF autophosphorylation and erbB2/HER autophos-
phorylation data (r values of 0.61 and 0.64, respectively).
However, there was a clear advantage in pan-erbB autophos-
phorylation potency for compounds bearing the 5-dialkylamino-
2-pentynamide class of Michael acceptor (such as 26, 27, 36-
40). These derivatives were more potent inhibitors of both the
isolated enzymes and of autophosphorylation in cells with
average IC50 values of 1.25, 1.4, 3.9, 4.5, and 6.1 nM in erbB1,
erbB2, erbB4, and EGF- and HER-stimulated autophosphoryl-
ation, respectively, compared with average values of 15, 91,
181, 27, and 94 nM, respectively, for all the other compounds
in these assays. The latter average figures did not vary greatly
if the non-amine-bearing analogues were excluded, suggesting
that the important factor was the ability of the amine to
participate in an autocatalytic reaction with the key cysteine of
the erbB family via a six-membered-ring mechanism, analogous

IrreVersible Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1479
Table 3. In Vivo Antitumor Efficacy of 39 in Mice against a Spectrum
of Implanted Human Tumor Xenografts
toxicity and in general was very mild. The large decrease in
weight for SKOV3 implanted mice appears to be due to effects
on the mice by the tumor rather than the drug, since untreated
tumor-bearing mice had similar weight losses.
dosea
(mg/kg)
weight
tumor
schedule
change (%)b T-Cc GDId
A431 epidermoid
BXPC3 pancreatic
H125 NSC lung
SF767 glioblastoma
SKOV3 ovarian
10
40
40
40
40
(Q1D × 5) × 2; D15
(Q1D × 5) × 3; D12
D17-31
-1.0
-0.6
0
-0.4
-6.5
26.9 269
16.2 108
17.1 114
23.8 159
21.4 187
For 39 the inhibition of target receptor activation remained
significantly inhibited for at least 24 h in tumors after a single
dose over a relatively wide dose range. The most sensitive tumor
was the A431 human epidermoid carcinoma, where significant
tumor regression was clearly evident (Table 3). Tumor regres-
sion in response to 39 was also apparent in the SF767 human
glioblastoma and the SKOV3 human ovarian carcinoma. The
compound produced complete stasis in the BXPC3 human
pancreatic carcinoma as well as in the H125 human non-small-
cell lung carcinoma under continuous daily dosing. In general,
39 was highly active in all five tumor models and was as
efficacious as canertinib dihydrochloride (9) in A431 xe-
nografts.30
(Q1D × 5) × 3; D15
D17-31
a Dose is PO. b Maximum treatment related weight loss, expressed as a
percent of initial group weight. c T-C is the difference, in days, for the
median treated and control tumors to reach a fixed evaluation size, usually
750 mg. d GDI (growth delay index) ) [(T-C)/treatment days] × 100.
Detailed pharmacokinetic studies of 39 were undertaken in
rat, monkey, and dog, and the results are summarized in Table
4. In rat and monkey the plasma clearance was higher than
hepatic blood flow; this may be related to low metabolic stability
in these species (unpublished data) and possibly to irreversible
binding of 39 to the target. The total amount of the unchanged
parent drug excreted into bile and urine was less than 1% in
rats, so that the contribution of biliary and renal clearance to
the total plasma clearance was minimal. The volume of
distribution was higher than total body water in all species,
suggesting that 39 likely distributed to tissue. It exhibited a short
plasma half-life (<2 h) in all three species, although the duration
of action did not solely rely on this because of its irreversible
inhibition mechanism. The bioavailability of 39 (F) was 11.4%
in rat and 67.8% in dog following a single oral administration
of 15 mg/kg under a fasted condition. In monkeys, the
bioavailability was 6.75% at a 30 mg/kg dose (Table 4).
Figure 1. A: Inhibition of erbB1 tyrosine kinase activity in NIH 3T3
fibroblasts expressing human erbB1. B: Inhibition of erbB2 tyrosine
kinase activity in T24 NIH fibroblasts expressing a chimeric receptor
with the extracellular binding domain of erbB1 and the intracellular
kinase domain of erbB2.
Conclusions
The alkynamides in general are potent inhibitors of erbB1,
erbB2, and erbB4, being on average the most potent (10×)
against erbB1. Analogues bearing a 5-dialkylamino-2-pentyna-
mide class of Michael acceptor are irreversible inhibitors of
erbB1 (and presumably erbB2 and erbB4), and possess a potency
advantage (6- to 80-fold) in terms of pan-erbB activity in both
isolated enzyme and cellular autophosphorylation assays. This
is likely due to an improved ability of the amine to participate
in autocatalysis of the Michael reaction with cysteine.34 The
pyrido[3,4-d]pyrimidine 39 had the best overall potencies in
both the enzymatic and cellular assays. This compound was
evaluated in a panel of five human tumor xenografts in nude
mice, by use of a multiple dose schedule. It was highly active
in all the models as measured by the growth delay index.
expressing a chimeric receptor with the extracellular binding
domain of erbB1 and the intracellular kinase domain of erbB2)
(see Figure 1, part B), and third, in the heregulin-stimulated
tyrosine autophosphorylation assay (in erbB2, -3, and -4
expressing MDA-MB-453 cells). Low nanomolar potency across
these three assays, as shown for 9 and 39, is considered
indicative of pan-erbB inhibition.
Compound 39 was evaluated for antitumor effects in a variety
of implanted human tumor xenografts in immune-deficient mice.
These models were chosen due to their expression of erbB
receptor family members. A summary of the data is shown
(Table 3), depicting the activity obtained at either the maximum
tolerated dose (MTD) or the dose where the highest efficacy
was obtained. Loss of weight was taken as an indication of
Experimental Section
Analyses were performed by the Analytical Department, Pfizer
Global Research and Development, Ann Arbor Laboratories.
Table 4. Mean Pharmacokinetic Parameters of 39 in Rat, Dog, and Monkey, Following a Single Dose Administration
dose
Cmax
tmax
t1/2
CL
Vdss
AUC(0-tlqc)
F
species
mg/kg
route
N
ng/mL
(h)
(h)
(mL/min)/kg
(L/kg)
(ng‚h/mL)
(%)
rat
dog
monkey
rat
dog
5
5
5
15
15
30
iva
iva
iva
pob
pob
pob
3
3
3
3
3
2
0.83
1.86
0.37
118.8
11.5
85.8
3.1
0.93
1.7
703
7470
1190
240
15 200
485
56.3
4180
83.5
4.7
1.0
3.5
11.4
67.8
6.75
monkey
a iv dosing vehicle: 5% DMSO/95% 0.05N methanesulfonic acid in saline (rat); 5% DMA/95% 50 mM lactic acid (pH 4.0) (dog and monkey). b po
dosing vehicle: 5% PEG/95% methylcellulose (0.5%) (rat); methylcellulose (0.5%) (dog and monkey).

1480 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Klutchko et al.
Melting points were determined using a Gallenkamp digital melting
point apparatus and are as read. NMR spectra were measured on a
Varian Unity 400 MHz spectrometer and referenced to Me4Si. Mass
spectra were recorded on a Finnigan MAT 900Q spectrometer.
Column chromatography was carried out with flash silica gel. All
reactions were run under an inert atmosphere of nitrogen or argon.
All reaction solvents were anhydrous, analytical reagent quality.
Solvent drying during extractive workups was carried out with Mg2-
SO4 unless otherwise noted. 4-(Tosyloxy)-1-butyne (41) was
purchased from Lancaster Synthesis.
Hz, 1H), 4.28 (d, J ) 5.8 Hz, 2H). APCIMS m/z (relative intensity)
371 (M+, 100). Anal. (C18H12ClFN4O2‚0.9H2O) C, H, N.
N-[4-[(3-Chloro-4-fluorophenyl)amino]-6-quinazolinyl]-4-(di-
ethylamino)-2-butynamide (22). A similar reaction of 17b and
4-(diethylamino)-2-butynoic acid34 (18f) provided 22 as a light
yellow solid (9%); mp 108-110 °C. 1H NMR [(CD3)2SO] δ: 10.9
(s, 1H), 9.89 (s, 1H), 8.65 (s, 1H), 8.52 (s, 1H), 8.07 (dd, J ) 6.8,
2.5 Hz, 1H), 7.8-7.73 (m, 3H), 7.39 (t, J ) 9.2 Hz, 1H), 3.57 (s,
2H), 2.49 (q, J ) 7.1 Hz, 4H), 0.97 (t, J ) 7.1 Hz, 6H). APCIMS
m/z (relative intensity): 426.1(M+, 100). Anal. (C22H21ClFN5O‚
0.3C3H6O‚0.8H2O) C, H, N.
N-[4-[(3-Chloro-4-fluorophenyl)amino]-6-quinazolinyl]-4-(bis-
(1-methylethyl)amino)-2-butynamide (23). A similar reaction of
17b and 4-[bis(1-methylethyl)amino]-2-butynoic acid34 (18g) pro-
vided 23 as a light yellow solid (32%); mp 120-122 °C. 1H NMR
[(CD3)2SO] δ: 10.87 (s, 1H), 9.88 (s, 1H), 8.66 (s, 1H), 8.51 (s,
1H), 8.07 (dd, J ) 6.9, 2.7 Hz, 1H), 7.79-7.72 (m, 3H), 7.39 (t,
J ) 9.3 Hz, 1H), 3.55 (s, 2H), 3.3-3.07 (m, 2H), 1.01 (d, J ) 6.6
Hz, 12H). APCIMS m/z (relative intensity): 454 (M+, 100). Anal.
(C24H25ClFN5O‚0.5H2O) C, H, N.
N-[4-[(3-Bromophenyl)amino]-6-quinazolinyl]-2-propyna-
mide (19). A stirred solution of 4-[3-bromophenylamino]-6-
aminoquinazoline41 (17a) (158 mg, 0.5 mmol) and propiolic acid
(18a) (0.08 mL, 1.1 mmol) in DMF (1.5 mL) was treated with
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI‚
HCl) (200 mg, 1.04 mmol) under nitrogen at 0 °C. The solution
was stirred at 0 °C for 30 min and then quenched with water. The
resulting fine precipitate was collected, dissolved in MeOH, and
purified by preparative TLC on silica gel. It was eluted with MeOH/
CHC13 (9:1) to give 19 as a yellow solid (21 mg, 12%); mp > 310
1
°C. H NMR [(CD3)2SO] δ: 11.18 (br s, 1H), 9.94 (s, 1H), 8.75
N-[4-[(3-Chloro-4-fluorophenyl)amino]-6-quinazolinyl]-4-(4-
morpholinyl)-2-butynamide (25). A similar reaction of 17b and
4-(4-morpholinyl)-2-butynoic acid34 (18i) provided 25 as an off-
(s, 1H), 8.59 (s, 1H), 8.15 (s, 1H), 7.85-7.79 (m, 3H), 7.37-7.28
(m, 2H), 4.53 (s, 1H). APCIMS m/z (relative intensity): 366
(79BrM+, 13), 367 (79BrMH+, 50), 368 (81BrM+, 24), 369 (81BrMH+,
47). Anal. (C17H11N4BrO) C, H, N.
1
white powder (20%); mp 167-169 °C. H NMR [(CD3)2SO] δ:
11.02 (s, 1H), 9.93 (s, 1H), 8.69 (s, 1H), 8.55 (s, 1H), 8.10 (dd, J
) 6.8, 2.7 Hz, 1H), 7.84-7.77 (m, 3H, H-8), 7.43 (t, J ) 9.2 Hz,
1H), 3.61 (br, 4H), 3.55 (s, 2H), 2.53 (br, 4H). APCIMS m/z
(relative intensity): 440 (M+, 100). Anal. (C22H21ClFN5O‚0.3C3H6O‚
0.3H2O) C, H, N.
N-[4-[(3-Bromophenyl)amino]-6-quinazolinyl]-2-butynam-
ide34 (20). A stirred solution of 2-butynoic acid (18b) (196 mg,
2.3 mmol) and EDCI‚HCl (385 mg, 2.0 mmol) in DMF (5 mL)
was treated with 4-[3-bromophenylamino]-6-aminoquinazoline41
(17a) (316 mg, 1.0 mmol) over 20 min at 25 °C. The resulting
solution was stirred at 25 °C, and two further lots of EDCI‚HCl
(206 mg, 1.0 mmol) and 2-butynoic acid (82 mg, 1.0 mmol) were
added after 14 and 22 h, respectively. The mixture was stirred at
25 °C for an additional 12 h and then quenched with water. The
resulting precipitate was collected and dissolved in MeOH and
purified by preparative TLC on silica gel. It was eluted with EtOAc/
acetone (1:1) to give 20 as a solid (20 mg, 5%); mp 281-283 °C.
1H NMR [(CD3)2SO] δ: 10.97 (br s, 1H), 9.93 (s, 1H), 8.76 (s,
1H), 8.57 (s, 1H), 8.14 (s, 1H), 7.84-7.76 (m, 3H), 7.34 (t, J )
8.1 Hz, 1H), 7.29 (d, J ) 7.8 Hz, 1H), 2.09 (s, 3H). APCIMS m/z
(relative intensity): 381 (79BrMH+, 95), 382 (81BrM+, 23), 383
(81BrMH+, 100). Anal. (C18H13BrN4O‚0.5H2O) C, H, N.
N-[4-[(3-Chloro-4-fluorophenyl)amino]-6-quinazolinyl]-5-(4-
diethylamino)-2-pentynamide (26). A similar reaction of 17b and
5-(diethylamino)-2-pentynoic acid (18j) provided 26 as a tan powder
1
(11%); mp 138-140 °C. H NMR [(CD3)2SO] δ: 10.87 (s, 1H),
9.89 (s, 1H), 8.68 (s, 1H), 8.51(s, 1H), 8.05 (dd, J ) 6.9, 2.7 Hz,
1H), 7.74-7.71 (m, 3H), 7.41 (t, J ) 9.1 Hz, 1H), 2.69 (t, J ) 7.2
Hz, 2H), 0.93 (t, J ) 7.1 Hz, 6H). APCIMS m/z (relative
intensity): 440 (M+, 100). Anal. (C23H23ClFN5O‚0.3H2O) C, H,
N.
N-[4-[(3-Chloro-4-fluorophenyl)amino]-6-quinazolinyl]-5-(4-
morpholinyl)-2-pentynamide (27). A similar reaction of 17b and
5-(4-morpholinyl)-2- pentynoic acid (18k) provided 27 as a cream
powder (11%); mp 155-157 °C. 1H NMR [(CD3)2SO] δ: 10.9 (s,
1H), 9.89 (s, 1H), 8.67 (s, 1H), 8.51 (s, 1H), 8.05 (dd, J ) 6.9, 2.5
Hz, 1H), 7.4-7.71 (m, 3H), 7.39 (t, J ) 9.1 Hz, 1H), 3.53 (t, J )
4.4 Hz, 4H), 2.58-2.53 (m, 4H), 2.38-2.34 (m, 4H). APCIMS
m/z (relative intensity): 454 (M+, 100). Anal. (C23H21ClFN5O2‚
0.6H2O) C, H, N.
N-[4-[(3-Bromophenyl)amino]pyrido[3,4-d]pyrimidin-6-yl]-2-
propynamide (28). A stirred solution of 6-amino-4-[(3-bromophe-
nyl)amino]pyrido[3,4-d]pyrimidine42 (17c) (94 mg, 0.3 mmol) and
propiolic acid (18a) (66 µL, 1.05 mmol) in pyridine (1.2 mL) under
nitrogen at -20 °C was treated with EDCI‚HCl (294 mg, 1.5
mmol). After 2.25 h, additional propiolic acid (33 µL) and EDCI‚
HCl (147 mg) were added, and after a total reaction time of 7.5 h
the mixture was diluted with DMF and then poured into EtOAc/
water (1:1). The aqueous phase was extracted with EtOAc (2×),
and the combined organic phases were washed with brine (2×),
dried, and filtered through silica gel. The filtrate was concentrated
and purified by column chromatography, then eluted with EtOAc.
Product fractions were pooled and concentrated and the residue
was triturated in EtOAc/tert-butyl methyl ether (1:1) to provide 28
(16 mg, 14%); mp >150 °C (dec). 1H NMR [(CD3)2SO] δ: 11.69
(s, 1H), 10.31 (s, 1H), 9.05 (s, 1H), 8.83 (s, 1H), 8.68 (s, 1H), 8.15
(s, 1H), 7.87 (d, J ) 7.2 Hz, 1H), 7.40-7.33 (m, 2H), 4.54 (s,
1H). APCIMS m/z (relative intensity): 367 (79BrM+, 28), 368
(79BrMH+, 100), 369 (81BrM+, 50), 370 (81BrMH+, 14). Anal.
(C16H10BrN5O‚0.2H2O) C, H, N.
N-[4-[(3-Chloro-4-fluorophenyl)amino]-6-quinazolinyl]-4-(dibu-
tylamino)-2-butynamide (24). A mixture of 6-amino-4-(3-chloro-
4-fluoroanilino)quinazoline29 (17b) (93 mg, 0.32 mmol), pyridine
(0.03 mL, 0.37 mmol), and 4-(dibutylamino)-2-butynoic acid (18h)
(69 mg, 0.33 mmol) in DMF (1.5 mL) at 0° C was treated with
EDCI‚HCl (76 mg, 0.40 mmol). The reaction was slowly warmed
to room temperature and quenched with water after 7 h. The mixture
was extracted with EtOAc, and the combined organic phases were
washed with water and brine, dried, and concentrated to a residue
that was purified first by silica gel column chromatography (elution
with 2.5% MeOH/CH2Cl2) and then by preparative TLC (elution
with 10% MeOH/CH2Cl2). The purified material was triturated in
ether to afford 24 as a light yellow solid (30 mg, 19%); mp 78-79
1
°C. H NMR [(CD3)2SO] δ: 10.9 (s, 1H), 9.89 (s, 1H), 8.66 (s,
1H), 8.52 (s, 1H), 8.07(dd, J ) 6.6, 2.5 Hz, 1H), 7.8-7.7 (m, 3H),
7.39 (t, J ) 9.2 Hz, 1H), 3.55 (s, 2H), 1.4-1.2 (m, 8H), 0.84 (t, J
) 7.1 Hz, 6H). APCIMS m/z (relative intensity): 482 (M+, 100).
Anal. (C26H29ClFN5O‚0.7H2O) C, H, N.
4-(Dibutylamino)-2-butynoic acid (18h) was synthesized by the
Wyeth procedure.34 1H NMR (CD3OD) δ: 4.18 (s, 2H), 3.23 (m,
4H), 1.69 (m, 4H), 1.43 (dq, J ) 15.0, 7.4 Hz, 4H), 1.00 (t, J )
7.4 Hz, 6H). APCIMS m/z (relative intensity): 212 (M+, 100), 168
(75).
N-[4-[(3-Chloro-4-fluorophenyl)amino]-6-quinazolinyl]-4-hy-
droxy-2-butynamide (21). A similar reaction of 17b and 4-hy-
droxy-2-butynoic acid44 (18e) provided 21 as a light yellow solid
N-[4-[(3-Chloro-4-fluorophenyl)amino]pyrido[3,4-d]pyrimi-
din-6-yl]-2-butynamide (29). A -30 °C stirred solution of
6-amino-4-[(3-chloro-4-fluorophenyl)amino]pyrido[3,4-d]pyrimi-
dine29 (17d) (0.290 g, 1.0 mmol), 2-butynoic acid (18b) (0.092 g,
1
(23%); mp 231-233 °C. H NMR [(CD3)2SO] δ: 11.0 (s, 1H),
9.91 (s, 1H), 8.71 (s, 1H), 8.51 (s, 1H), 8.05 (dd, J ) 6.8, 2.7 Hz,
1H), 7.74-7.70 (m, 3H), 7.39 (t, J ) 9.3 Hz, 1H), 5.55 (t, J ) 6.1

IrreVersible Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1481
1.1 mmol), and pyridine (5 mL) was treated with EDCI‚HCl (0.242
g, 1.26 mmol). After it was stirred overnight, the dark solution was
poured into 40 mL of saturated aqueous NaHCO3. The formed
precipitate was collected, washed with water, and then dissolved
in CHCl3/MeOH (10:1). The solution was purified by column
chromatography and was eluted with CHCl3/MeOH (20:1) to obtain
nearly pure product, which was dissolved in MeOH/CH2Cl2 (4:1).
The solution was clarified with charcoal and concentrated to 5 mL
at which point crystallization occurred. The precipitate was col-
lected, washed with MeOH and then ether, and dried to provide
29 (0.04 g, 11%); mp 258-261 °C. 1H NMR [(CD3)2SO] δ: 11.38
(s, 1H), 10.28 (s, 1H), 8.97 (s, 1H), 8.74 (s, 1H), 8.59 (s, 1H), 8.07
(m, 1H), 7.74 (m, 1H), 7.41 (t, J ) 9 Hz, 1H), 2.02 (s, 3H).
APCIMS m/z (relative intensity): 356 (M+, 100). Anal. (C17H11-
ClFN5O) C, H, N.
(18i) (0.445 g, 2.541 mmol), EDCI‚HCl (0.633 g, 3.30 mmol), and
pyridine (5 mL) in CH2Cl2 (10 mL), followed by workup,
chromatography, and trituration from CH2Cl2/tert-butylmethyl ether
1
provided 35 (0.036 g, 5%); mp 198 °C (dec). H NMR [(CD3)2-
SO] δ: 11.58 (s, 1H), 10.28 (s, 1H), 8.97 (s, 1H), 8.75 (s, 1H),
8.58 (s, 1H), 8.06 (m, 1H), 7.74 (m, 1H), 7.41 (t, J ) 9 Hz, 1H),
3.56 (m, 4H), 3.49 (s, 2H), 2.45 (m, 4H). APCIMS m/z (relative
intensity): 441 (M+, 100). Anal. (C21H18ClFN6O2‚0.4CH2Cl2‚
0.3MTBE) C, H, N.
N-[4-[(3-Chloro-4-fluorophenyl)amino]pyrido[3,4-d]pyrimi-
din-6-yl]-5-(4-morpholinyl)-2-pentynamide (36). A similar reac-
tion of 17d (0.290 g, 1.0 mmol), 5-(4-morpholinyl)-2-pentynoic
acid (18k) (0.202 g, 1.1 mmol), EDCI‚HCl (0.242 g, 1.26 mmol),
and pyridine (5 mL), followed by workup, chromatography, and
crystallization from EtOAc, gave 36 (0.090 g, 20%); mp 205-209
1
N-[4-[(3-Chloro-4-fluorophenyl)amino]pyrido[3,4-d]pyrimi-
din-6-yl]-2-hexynamide (30). A similar reaction of 17d (0.290 g,
1.0 mmol), 2-hexynoic acid (18c) (0.123 g, 1.1 mmol), EDCI‚HCl
(0.242 g, 1.26 mmol), and pyridine (5 mL) followed by workup
and crystallization from EtOAc provided 30 (0.043 g, 11%); mp
233-235 °C. 1H NMR [(CD3)2SO] δ: 11.41 (s, 1H), 10.27 (s, 1H),
8.97 (s, 1H), 8.74 (s, 1H), 8.59 (s, 1H), 8.07 (m, 1H), 7.75 (m,
1H), 7.41 (t, J ) 9 Hz, 1H), 2.37 (t, J ) 7 Hz, 2H), 1.53 (q, J )
7 Hz, 2H), 0.96 (t, J ) 7 Hz, 3H). APCIMS m/z (relative
intensity): 384 (M+, 100). Anal. (C19H15ClFN5O) C, H, N.
N-[4-[(3-Chloro-4-fluorophenyl)amino]pyrido[3,4-d]pyrimi-
din-6-yl]-2-heptynamide (31). A similar reaction of 17d (0.29 g,
1.0 mmol), 2-heptynoic acid (18d) (0.139 g, 1.1 mmol), EDCI‚
HCl (0.242 g, 1.26 mmol), and pyridine (5 mL) followed by
workup, chromatography, and crystallization from EtOAc gave 31
°C (dec). H NMR [(CD3)2SO] δ: 11.45 (s, 1H), 10.32 (s, 1H),
9.01 (s, 1H), 8.78 (s, 1H), 8.63 (s, 1H), 8.11 (m, 1H), 7.80 (m,
1H), 7.45 (t, J ) 9 Hz, 1H), 3.55 (m, 4H), 2.48 (m, 4H), 2.40 (m,
4H). APCIMS m/z (relative intensity): 455 (M+, 100). Anal.
(C22H20ClFN6O2‚0.2H2O) C, H, N.
N-[4-[(3-Bromo-4-fluorophenyl)amino]pyrido[3,4-d]pyrimi-
din-6-yl]-5-(4-morpholinyl)-2-pentynamide (37). A similar reac-
tion of 6-amino-4-[(3-bromo-4-fluorophenyl)amino]pyrido[3,4-d]-
pyrimidine29 (17e) (0.334 g, 1.0 mmol), 5-(4-morpholinyl)-2-
pentynoic acid (18k) (0.202 g, 1.1 mmol), EDCI‚HCl (0.242 g,
1.26 mmol), and pyridine (5 mL), followed by workup and
chromatography, gave 37 (0.042 g, 8%); mp 190-192 °C (dec).
1H NMR [(CD3)2SO] δ: 11.40 (s, 1H), 10.26 (s, 1H), 8.97 (s, 1H),
8.74 (s, 1H), 8.59 (s, 1H), 8.15 (m, 1H), 7.81 (m, 1H), 7.38 (t, J )
9 Hz, 1H), 3.52 (m, 4H), 2.55 (m, 4H), 2.38 (m, 4H). APCIMS
m/z (relative intensity): 501 (81BrMH+, 100). Anal. (C22H20-
BrFN6O2‚0.3CHCl3) C, H, N.
1
(0.025 g, 6%); mp 220-225 °C. H NMR [(CD3)2SO] δ: 11.40
(s, 1H), 10.27 (s, 1H), 8.97 (s, 1H), 8.74 (s, 1H), 8.59 (s, 1H), 8.07
(m, 1H), 7.74 (m, 1H), 7.41 (t, J ) 9 Hz, 1H), 2.39 (t, J ) 7 Hz,
2H), 1.49 (m, 2H), 1.39 (m, 2H), 0.86 (t, J ) 7 Hz, 3H). Anal.
(C20H17ClFN5O) C, H, N.
N-[4-[(3-Chloro-4-fluorophenyl)amino]pyrido[3,4-d]pyrimi-
din-6-yl]-5-(1-piperidinyl)-2-pentynamide (38). A similar reaction
of 17d (4.70 g, 16.2 mmol), 5-(1-piperidinyl)-2-pentynoic acid,
lithium salt (18l) (6.03 g, 32.2 mmol), pyridine hydrochloride (4.62
g, 40 mmol), EDCI‚HCl (7.70 g, 40 mmol), and pyridine (80 mL),
followed by workup, chromatography, and crystallization from
N-[4-[(3-Chloro-4-fluorophenyl)amino]pyrido[3,4-d]pyrimi-
din-6-yl]-4-(diethylamino)-2-butynamide (32). A similar reaction
of 17d (0.602 g, 2.08 mmol), 4-(diethylamino)-2-butynoic acid34
(18f) (0.67 g, 4.16 mmol), EDCI‚HCl (1.04 g, 5.41 mmol), and
pyridine (5 mL) in CH2Cl2 (10 mL), followed by workup and
chromatography, gave 32 (0.047 g, 5%); mp 234-236 °C. 1H NMR
[(CD3)2SO] δ: 11.52 (s, 1H), 10.26 (s, 1H), 8.97 (s, 1H), 8.75 (s,
1H), 8.60 (s, 1H), 8.07 (m, 1 H), 7.75 (m, 1H), 7.41 (t, J ) 9 Hz,
1H), 3.55 (s, 2H), 2.48 (m, 4H), 0.96 (t, J ) 7 Hz, 6H). APCIMS
m/z (relative intensity): 427 (M+, 100). Anal. (C21H20ClFN6O‚
1.0H2O) C, H, N.
1
EtOAc, gave 38 (0.35 g, 5%); mp 290-300 °C (dec). H NMR
[(CD3)2SO] δ: 11.40 (s, 1H), 10.27 (s, 1H), 8.97 (s, 1H), 8.74 (s,
1H), 8.59 (s, 1H), 8.07 (m, 1H), 7.74 (m, 1H), 7.42 (t, J ) 9 Hz,
1H), 2.52 (m, 2H), 2.50 (m, 2H), 2.33 (m, 4H), 1.43 (m, 4H), 1.31
(m, 2H). APCIMS m/z (relative intensity): 453 (M+, 100). Anal.
(C23H22ClFN6O‚H2O) C, H, N.
N-[4-[(3-Chloro-4-fluorophenyl)amino]pyrido[3,4-d]pyrimi-
din-6-yl]-5-(4-methyl-1-piperazinyl)-2-pentynamide (39). A simi-
lar reaction of 17d (2.35 g, 8.1 mmol), 5-(4-methyl-1-piperazinyl)-
2-pentynoic acid, lithium salt (18m) (3.49 g, 16 mmol), pyridine
hydrochloride (2.31 g, 20 mmol), EDCI‚HCl (3.85 g, 20 mmol),
and pyridine (40 mL), followed by workup, chromatography, and
crystallization from EtOAc, gave 39 (0.78 g, 20%); mp 170-180
°C. 1H NMR (CD3OD) δ: 8.93 (s, 1H), 8.71 (s, 1H), 8.57 (s, 1H),
8.05 (m, 1H), 7.70 (m, 1H), 7.26 (t, J ) 9 Hz, 1H), 2.68 (m, 4H),
2.55 (br m, 8H), 2.28 (s, 3H). APCIMS m/z (relative intensity):
468 (M+, 100). Anal. (C23H23ClFN7O‚0.5H2O) C, H, N.
N-[4-[(3-Chloro-4-fluorophenyl)amino]pyrido[3,4-d]pyrimi-
din-6-yl]-4-(bis(1-methylethyl)amino)-2-butynamide (33). A simi-
lar reaction of 17d (0.580 g, 2.0 mmol), 4-[bis(1-methylethyl)-
amino]-2-butynoic acid34 (18g) (0.440 g, 2.2 mmol), EDCI‚HCl
(0.480 g, 2.5 mmol), and pyridine (10 mL), followed by workup,
chromatography, and crystallization from EtOAc, gave 33 (0.11 g,
1
12%); mp 200-230 °C (dec). H NMR [(CD3)2SO] δ: 11.46 (s,
1H), 10.26 (s, 1H), 8.98 (s, 1H), 8.75 (s, 1H), 8.60 (s, 1H), 8.07
(m, 1H), 7.74 (m, 1H), 7.42 (t, J ) 9 Hz, 1H), 3.54 (s, 2H), 3.10
(m, 2H), 1.00 (m, 12H). APCIMS m/z (relative intensity): 455 (M+,
100). Anal. (C23H24ClFN6O‚H2O) C, H, N.
N-[4-[(3-Bromo-4-fluorophenyl)amino]pyrido[3,4-d]pyrimi-
din-6-yl]-5-(4-methyl-1-piperazinyl)-2-pentynamide (40). A simi-
lar reaction of 17e (0.334 g, 1.0 mmol), 5-(4-methyl-1-piperazinyl)-
2-pentynoic acid (18m) (0.216 g, 1.1 mmol), EDCI‚HCl (0.242 g,
1.3 mmol), and pyridine (5 mL), followed by workup, chromatog-
raphy, and crystallization from EtOAc, gave 40 (0.027 g, 5%); mp
N-[4-[(3-Chloro-4-fluorophenyl)amino]pyrido[3,4-d]pyrimi-
din-6-yl]-4-(dibutylamino)-2-butynamide (34). A similar reaction
of 17d (0.145 g, 0.5 mmol), 4-(dibutylamino)-2-butynoic acid (18h)
(0.116 g, 0.55 mmol), EDCI‚HCl (0.120 g, 0.63 mmol), and
pyridine (2.5 mL), followed by workup, chromatography, and
crystallization from EtOAc, gave 34 (0.063 g, 28%); mp 172-174
1
190-195 °C. H NMR (CD3OD) δ: 8.96 (s, 1H), 8.73 (s, 1H),
1
°C (dec). H NMR [(CD3)2SO] δ: 11.56 (s, 1H), 10.30 (s, 1H),
8.59 (s, 1H), 8.10 (m, 1H), 7.78 (m, 1H), 7.26 (t, J ) 9 Hz, 1H),
2.68 (m, 4H), 2.55 (br m, 8H), 2.30 (s, 3H). APCIMS m/z (relative
intensity): 514 (81BrMH+, 100). Anal. (C23H23BrFN7O‚0.8H2O) C,
H, N. Alternatively, a similar reaction of 17e (0.538 g, 1.61 mmol),
5-(4-methyl-1-piperazinyl)-2-pentynoic acid, lithium salt (18m)
(0.697 g, 3.22 mmol), pyridine hydrochloride (0.462 g, 4.00 mmol),
EDCI‚HCl (0.770 g, 4.0 mmol), and pyridine (8 mL) provided pure
40 (35% yield). Anal. (C23H23BrFN7O‚H2O) C, H, N.
9.02 (s, 1H), 8.79 (s, 1H), 8.64 (s, 1H), 8.10 (m, 1H), 7.81 (m,
1H), 7.46 (t, J ) 9 Hz, 1H), 3.57 (s, 2H), 2.42 (m, 4H), 1.35 (m,
8H), 0.87 (m, 6H). APCIMS m/z (relative intensity): 483 (M+,
100). Anal. (C25H28ClFN6O‚0.5H2O) C, H, N.
N-[4-[(3-Chloro-4-fluorophenyl)amino]pyrido[3,4-d]pyrimi-
din-6-yl]-4-(4-morpholinyl)-2-butynamide (35). A similar reaction
of 17d (0.515 g, 1.78 mmol), 4-(4-morpholinyl)-2-butynoic acid34

1482 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Klutchko et al.
Synthesis of 5-(Aminosubstituted)-2-pentynoic Acids. 5-(1-
1-butyne47 (42; R1 ) R2 ) ethyl) (0.816 g, 6.5 mmol), and THF
(20 mL) at -78 °C, followed by carboxylation and workup, gave
18j as a light yellow solid (0.652 g, 59% yield), which was utilized
Piperidinyl)-2-pentynoic Acid (18l). 1-(3-Butynyl)piperidine (42;
R1, R2 ) piperidinyl) was synthesized as previously reported.45 1
H
1
NMR (CDCl3) δ: 1.38 (ddd, J ) 11.3, 6.0, 5.8 Hz, 2H), 1.54 (dt,
J ) 11.3, 5.8 Hz, 4H), 1.92 (t, J ) 2.7, Hz, 1H), 2.34 (m, 6H),
2.53 (m, 2H).
directly without further processing. H NMR [(CD3)2SO] δ: 3.79
(br, 1H), 2.85 (t. J ) 7.3 Hz, 2H), 2.74 (q, J ) 7.0, 5.0 Hz, 4H),
2.52-2.4 (m, 2H), 1.03 (t. J ) 7.2 Hz, 6H). APCIMS m/z (relative
intensity): 170 (MH+, 100).
A stirred -78 °C solution of 42 (R1, R2 ) piperidinyl) (2.56 g,
18.7 mmol) in ether (65 mL) was treated with n-BuLi (2.3 mL of
a 10 M solution in hexanes). After 3 h at -78 °C, powdered CO2
was added to bring the solution volume to ca. 250 mL while
maintaining the temperature at -78 °C. The cooling bath was
removed, and the mixture was stirred until all excess CO2 had
evaporated (ca. 16 h). The resultant lithium salt was collected by
filtration and then dissolved in 30 mL of water. The solution was
adjusted to pH 5 with 5% aq HCl and then loaded onto a column
of 29 g of Dowex 50X8-200 ion-exchange resin (H+ form,
prewashed with 800 mL of deionized water). The column was eluted
with ca. 2 column volumes of water and then with 500 mL of 0.2
N aq NH4OH, while 8 mL fractions were collected. The fractions
containing product were pooled and concentrated under reduced
pressure to leave a residue that was coevaporated twice with MeOH
to leave 18l (2.42 g, 72%) as a light orange solid, which was utilized
without further processing. 1H NMR [(CD3)2SO] δ: 1.28 (m, 1H),
1.58 (m, 1H), 1.65 (m, 4H), 2.82 (m, 2H), 2.91 (t, J ) 7.5 Hz,
2H), 3.17 (t, J ) 7.5 Hz, 2H), 3.33 (d, J ) 12.2 Hz, 2H).
5-(4-Methyl-1-piperazinyl)-2-pentynoic Acid (18m). Utilizing
a method similar to that of Turner et al.,46 we stirred a mixture of
4-(tosyloxy)-1-butyne (41) (8.80 g, 40 mmol), N-methylpiperazine
(8.02 g, 80 mmol), dry, powdered K2CO3 (25 g), and DMF (25
mL) overnight at room temperature. Ether (100 mL) was added,
the mixture was filtered, and the filter cake was washed with ether.
The filtrate was concentrated at 20 mm and 40 °C to remove ether
and most of the DMF, and residual DMF was removed by silica
gel chromatography and eluted sequentially with CHCl3/hexanes
(1:1), then CHCl3 (to remove DMF), and finally CHCl3/MeOH (10:
1) to strip off the product. Concentration of product fractions
provided 42 (R1, R2 ) 4-methyl-1-piperazinyl) (3.78 g, 62%) as
5-(4-Morpholinyl)-2-pentynoic Acid (18k). A similar reaction
of n-BuLi (15 mL of a 2.18 M solution in THF), 4-(3-butynyl)-
morpholine30 (42; R1, R2 ) morpholinyl) (5.06 g, 36 mmol), and
THF (100 mL), followed by carboxylation and workup, gave 18k
as a dull yellow solid (5.08 g, 77%), which was used directly
1
without further processing. H NMR [(CD3)2SO] δ: 7.2-7.4 (br,
1H), 3.54 (m, 4H), 2.49-2.36 (m, 8H). APCIMS m/z (relative
intensity): 184 (MH+, 100).
ELISA-Based erbB Kinase Assay. The erbB1, erbB2, and
erbB4 cytoplasmic fusion proteins were made by cloning the erbB1
sequence (Met-668 to Ala-1211), erbB2 (Ile-675 to Val-1256), and
erbB4 sequence (Gly-259 to Gly-690) into the baculoviral vector
pFastBac using PCR. Proteins were expressed in baculovirus-
infected Sf9 insect cells as GST fusion proteins. The proteins were
purified by affinity chromatography using glutathione sepharose
beads. Inhibition of erbB tyrosine kinase activity was assessed using
an ELISA-based receptor tyrosine kinase assay. Kinase reactions
(50 mM HEPES, pH 7.4, 125 mM NaCl, 10 mM MgCl2, 100 µM
sodium orthovanadate, 2 mM dithiothreitol, 20 µM ATP, test
compound or vehicle control, and 1-5 nM GST-erbB per 50 µL
of reaction mixture) were run in 96-well plates coated with 0.25
mg/mL poly-Glu-Tyr (Sigma). The reactions were incubated for 6
min at room temperature while being shaken. Kinase reactions were
stopped by removal of the reaction mixture, then the wells were
washed with wash buffer (0.1% Tween 20 in PBS). Phosphorylated
tyrosine residues were detected by adding 0.2 µg/mL anti-
phosphotyrosine antibody (Oncogene Ab-4; 50 µL/well) coupled
to horseradish peroxidase (HRP) diluted in PBS containing 3% BSA
and 0.05% Tween 20 for 25 min while being shaken at room
temperature. The antibody was removed, and plates were washed
in wash buffer. HRP substrate (SureBlue3,3′,5,5′-tetramethyl ben-
zidine or TMB, Kirkegaard & Perry Labs) was added (50 µL per
well) and incubated for 10-20 min while it was shaken at room
temperature. The TMB reaction was stopped with the addition of
50 µL of stop solution (0.09 N H2SO4). The signal was quantified
by measuring absorbance at 450 nm. IC50 values were determined
for test compounds using the median effect method.48
Inhibition of erbB1 and erbB2 Autophosphorylation in Cells
Using BioVeris Technology. Inhibition of erbB1 tyrosine kinase
activity was evaluated in NIH3T3 cells transfected with the full-
length human erbB1 receptor, and inhibition of erbB2 tyrosine
kinase activity was evaluated in T24 NIH 3T3 cells transfected
with a chimeric receptor with the extracellular binding domain of
erbB1 and the intracellular kinase domain of erbB2. The derivation
of these lines has been previously described by Cohen et al.49 Cells
were plated in a 96-well plate at a density of 20 000 cells per well
in DMEM/F12 media (Gibco BRL) containing 10% fetal bovine
serum (Gibco, BRL) and 10 ng/mL Gentamicin (Gibco, BRL). Cells
were cultured at 37 °C in 5% CO2. Prior to drug treatment, the
cells were cultured in serum-free medium for 12-24 h. The cells
were then treated with the test compound dissolved in DMSO for
2 h (final DMSO concentration of <0.5%) at 37 °C and 5% CO2.
After the 2 h drug treatment, the cells were stimulated with 100
ng/mL EGF (Sigma) for 10 min at 37 °C and 5% CO2. The medium
was removed, and the cells were immediately lysed in buffer (50
mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM EDTA,
1% Triton, 10 mM â-glycerophosphate, 0.1 mM NaVa, 1 mM NaF,
0.25% deoxycholic acid, 10 µg/mL aprotinin, 10 µg/mL leupeptin)
for 15 min at room temperature, mixing gently.
1
an oil, which was utilized without further processing. H NMR
(D2O) δ: 2.38 (t, J ) 7 Hz, 2H), 2.19 (t, J ) 7 Hz, 2H), 2.06 (s,
1H), 2.01 (s, 3H), 1.9-2.7 (br m, 8H). APCIMS m/z (relative
intensity): 153 (MH+, 100).
A stirred -78 °C solution of 42 (R1, R2 ) 4-methyl-1-
piperazinyl) (1.06 g, 7 mmol) in dry THF (15 mL) was treated
with n-BuLi (5.0 mL of 1.6 M solution in hexane). The mixture
was stirred for 1 h at -78 °C, and then dry CO2 gas was bubbled
in at a moderate rate at -78 °C for 1 h and then while allowing
the mixture to warm to room temperature. After further stirring
for 1 h at room temperature, the solution was ice-cooled and treated
with 1 N aq HCl (8 mL). The mixture was concentrated, and the
residue was coevaporated with absolute EtOH (2 × 25 mL). The
resulting sticky solid was dissolved in hot absolute EtOH (15 mL),
and ether (4 mL) was added to precipitate a solid. The solid, free
of LiCl, was collected and then recrystallized by dissolution in warm
water (1 mL) followed by addition of MeCN (3 mL). The product
was collected, washed with MeCN (1 mL) and ether, and dried to
1
give 18m (0.97 g, 67%); mp 207-209 °C (dec). H NMR (CD3-
OD) δ: 3.2 (br m, 4H), 2.8-3.0 (br m, 4H), 2.80 (s, 3H), 2.76 (t,
J ) 7 Hz, 2H), 2.52 (t, J ) 7 Hz, 2H). APCIMS m/z (relative
intensity): 197 (MH+, 100). Anal. (C10H16N2O2‚0.6H2O) C, H, N.
5-(4-Methyl-1-piperazinyl)-2-pentynoic Acid, Lithium Salt
(18m). A stirred -65 °C solution of 42 (R1, R2 ) 4-methyl-1-
piperazinyl) (50 g, 0.33 mol) and dry THF (155 mL) was treated
with n-BuLi (135 g of a 1.6 M solution in hexanes) followed by
dry CO2 (15 g, 395 mmol). The mixture was stirred at -65 °C for
ca. 3 h, warmed to 25 °C, and then diluted with 110 mL of isopropyl
alcohol and 10 mL of water. After warming to reflux, the solution
was cooled to allow crystallization, and then the product was
collected by filtration and vacuum-dried at ca. 65 °C to give 18m,
lithium salt (50 g, 75%) hydrated with e2% water.
Inhibition of erbB1 autophosphorylation was detected using
BioVeris technology. Anti-erbB1 antibody tagged with biotin was
added to the lysates to capture erbB1 (NeoMarkers Clone 528; 100
ng/mL per well). An antiphospho-erbB1 antibody (Cell Signaling
p-Y1068; 100 ng/mL per well) labeled with ruthenium (BioVeris)
5-(Diethylamino)-2-pentynoic Acid (18j). A similar reaction
of n-BuLi (2.7 mL of a 2.18 M solution in THF), 4-(diethylamino)-

IrreVersible Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1483
was simultaneously added to detect phosphorylated erbB1. Plates
were mixed gently for 60 min at room temperature. Magnetic beads
coated with streptavidin (Dynabeads M 289 streptavidin, Dynal
Biotech Inc.) were added at a final concentration of 50 ng/mL and
mixed gently at room temperature for 30 min. The antibodies and
beads were diluted in the following buffer: 150 mM NaCl, 50 mM
Tris-HCl pH 7.4, 1% BSA, 0.02% Na azide. After the incubation,
samples were analyzed using a BioVeris M8 analyzer. IC50
determinations were determined using the median effect method.
Inhibition of erbB2 autophosphorylation was also detected using
BioVeris technology. Anti-erbB1 tagged with biotin was added to
the lysate to capture erbB1/erbB2 chimeric protein (NeoMarkers
Clone 528; 200 ng/mL per well). An antiphospho-erbB2 antibody
(Cell Signaling p-Y1248, 200 ng/mL per well) labeled with
ruthenium (BioVeris) was simultaneously added to detect phos-
phorylated erbB2. The remaining methods followed procedures
outlined for inhibition of erbB1 autophosphorylation.
Efficacy was also assessed by calculating the growth delay index
(GDI), which allows for a better comparison of the magnitude of
tumor growth suppression between studies since it factors in
differences in dosing schedules. GDI is calculated by dividing the
T-C by the number of treatment days and multiplying by 100%. A
GDI of 0 indicates no effect; a GDI below 100 indicates a slowing
of tumor growth, but growth is still occurring during treatment. A
GDI of 100 would mean complete tumor stasis, and a GDI above
100 means that drug treatment delayed tumor growth for longer
than the duration of treatment. Compound 39 was considered to
have activity if it could produce a GDI of greater than 50.
Acknowledgment. This work was partially supported by the
Auckland Division of the Cancer Society of New Zealand. The
T24 NIH 3T3 lines were a gift from Dr. Bruce Cohen at the
National Cancer Institute, Bethesda, MD.
Inhibition of EGF and Heregulin-Stimulated erbB Auto-
phosphorylation in Cells Using Western Blotting. Inhibition of
EGF-stimulated or heregulin-stimulated erbB receptor autophos-
phorylation was assessed in either NIH 3T3 fibroblasts expressing
full length human erbB1, the T24 NIH 3T3 fibroblasts expressing
a chimeric receptor with the extracellular binding domain of erbB1
and the intracellular kinase domain of erbB2, or the human breast
carcinoma cell line MDA-MB-453 by antiphosphotyrosine Western
blots. Western blotting procedures have been previously described.50
To detect phospho-erbB1, an antiphospho-erbB1 antibody was used
(p-Y1068, Cell Signaling). To detect phospho-erbB2, an antiphos-
pho-erbB2 antibody was used (p-Y1248, Cell Signaling). To detect
phospho-erbB signals in MDA-MB-453 cells, an antiphospho-Tyr
antibody was used as described.50 Band intensities from the anti-
phosphotyrosine Western blots were determined using a Molecular
Dynamics laser densitometer and expressed as percent control. IC50
determinations were determined using the median effect method.
Pharmacokinetics. All animals were fasted overnight prior to
dosing the next morning, and food was withheld until 4 h following
dosing. Water was allowed ad libitum throughout the study. For
each study, blood samples were collected from a jugular cannula
into EDTA/ascorbic acid tubes. Sampling occurred prior to dosing
and at 7-9 different time points up to 24 h after the iv and the po
dose. Plasma drug concentration was determined by a LC/MS/MS
assay. Briefly, 39 was isolated from plasma using protein precipita-
tion with acetonitrile. Supernatant was injected directly into an
amide column with mobile phase consisting of acetonitrile/10 mM
ammonium formate (pH ) 4.0) (35:65, v/v), with mass spectro-
metric detection. Drug concentration below the limit of quantitation
(<6.5 ng/mL) was treated as zero for pharmacokinetic and statistical
calculations.
In Vivo Antitumor Effects of 39. Immune-deficient CB.17
SCID mice and nude mice (NCr nu/nu) were purchased from
Charles River. Mice were housed in microisolator cages within a
barrier facility on a 12 h light/dark cycle and received food and
water ad libitum. Animal housing was in accord with AAALAC
guidelines. All experimental protocols involving animals were
approved by the institutional animal care and use committee. The
A431 epidermoid carcinoma, the H125 non-small-cell lung carci-
noma line, and the SKOV3 ovarian carcinoma line were maintained
by serial passage in nude mice (NCr nu/nu). The SF-767 glioblas-
toma and the BxPC-3 pancreatic tumor lines were maintained by
serial passage in SCID mice.
Tumors were implanted subcutaneously in immune-deficient
mice and allowed to grow to a size of 100-150 mg before dosing
was initiated. The drug was dissolved in 0.05 N Na lactate buffer,
pH 4.0, and the route of drug administration for all studies was
oral. Animals were dosed either daily for 14 days or daily 5 days
a week for 2-3 weeks. Tumor measurements were made 2-3 times
per week until tumors reached 750 mg. Efficacy was assessed by
tumor growth delay (T-C) and tumor growth delay index (GDI).
Tumor growth delay is the difference, in days, for the treated (T)
and control (C) tumors to reach 750 mg. Calculation of tumor
growth delay (T-C) was performed as described previously.51-54
Supporting Information Available: Combustion analytical
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
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