Synthesis and stereochemical effects of pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines as EGFR and ErbB-2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.11.023

A novel class of pyrrolidinyl-acetyleneic thieno[3,2-d]pyrimidines has been identified which potently inhibit the EGFR and ErbB-2 receptor tyrosine kinases. Synthetic modifications of the pyrrolidine carbamate moiety result in a range of effects on enzyme and cellular potency. In addition, the impact of the absolute stereochemical configuration on cellular potency and oral mouse pharmacokinetics is described.

Full text of DOI:10.1016/j.bmcl.2008.11.023

Bioorganic & Medicinal Chemistry Letters 19 (2009) 21–26
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and stereochemical effects of pyrrolidinyl-acetylenic
thieno[3,2-d]pyrimidines as EGFR and ErbB-2 inhibitors
b
a
a,
Kirk L. Stevens a, , Krystal J. Alligood , Jennifer G. Badiang Alberti , Thomas R. Caferro
,
*
Stanley D. Chamberlain a,à, Scott H. Dickerson c, Hamilton D. Dickson a, Holly K. Emerson a, Robert J. Griffin d,
Robert D. Hubbard a,§, Barry R. Keith b, Robert J. Mullin b,–, Kimberly G. Petrov a, Roseanne M. Gerding a,
Michael J. Reno a, Tara R. Rheault a, David W. Rusnak b, Douglas M. Sammond a,||, Stephon C. Smith a,
David E. Uehling a, Alex G. Waterson a, Edgar R. Wood e
a Department of Oncology Medicinal Chemistry, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
b Department of Oncology Biology, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
c Department of Computational and Structural Chemistry, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
d Department of Drug Metabolism and Pharmacokinetics, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
e Department of Biochemical and Cellular Targets, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of pyrrolidinyl-acetyleneic thieno[3,2-d]pyrimidines has been identified which potently
inhibit the EGFR and ErbB-2 receptor tyrosine kinases. Synthetic modifications of the pyrrolidine carba-
mate moiety result in a range of effects on enzyme and cellular potency. In addition, the impact of the
absolute stereochemical configuration on cellular potency and oral mouse pharmacokinetics is described.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 9 October 2008
Accepted 10 November 2008
Available online 13 November 2008
Keywords:
Epidermal growth factor receptor
EGFR
ErbB-2
Inhibitor
Thieno[3,2-d]pyrimidines
Proline
Overexpression of the epidermal growth factor receptor tyrosine
kinases1 EGFR (ErbB-1) and/or HER-2 (ErbB-2) has been implicated
in poor prognosis for a variety of cancers.2 Efforts to disrupt the
ErbB-family signaling pathway with either monoclonal antibodies3
or small molecule ATP-competitive kinase inhibitors have yielded no-
vel anticancer agents.4 The 4-anilinoquinazoline chemical class has
provided reversible and irreversible ErbB-family inhibitors (Fig. 1). Ir-
essaTM (gefinitib5), TarcevaTM (erlotinib6), and TykerbTM (lapatinib7)
are reversible ErbB-family kinase inhibitors approved in the U.S. by
the FDA. Additionally, a numberof irreversiblequinazolineErbB-fam-
ily inhibitors shown in Figure 1 are in clinical trials (CI-1033,8 HKI-
272,9 and BIBW-299210). These inhibitors contain a Michael acceptor
that forms a covalent bond with a cysteine residue near the front of
the ATP-binding pocket (Cys797 in EGFR and Cys805 in ErbB-2). A po-
tential advantage of an irreversible agent is prolonged inhibition of
the enzyme, effectively competing with the high cellular concentra-
tions of ATP to impair the ErbB-signaling pathway.11
Previously, we described our efforts to find effective dual inhib-
itors of EGFR and ErbB-2 in the pyrrolidinyl-acetylenic thienopyr-
imidine chemical class.12,13 These compounds (1) are potent and
selective EGFR/ErbB-2 enzyme inhibitors, with the ability to react
covalently with Cys797 of EGFR at carbon ‘a’ of the acetylene. Prior
SAR exploration indicated that the most desirable analogs con-
tained a basic pyrrolidine nitrogen with a C-4 carbamate.14 This
paper describes further characterization of a variety of chiral car-
bamates around the 2,4-disubstituted pyrrolidine in this class of
ErbB-family tyrosine kinase inhibitors.
O
* Corresponding author. Tel.: +1 919 483 7707; fax: +1 919 483 6053.
E-mail addresses: Kirk.L.Stevens@GSK.com, stevens@chemist.com (K.L. Stevens).
Present address: Pfizer Ltd, Sandwich, UK.
F
O
R1
HN
N
Cl
N
R2
O
5
4
3
à
Present address: Inhibitex Inc., Alpharetta, GA, USA.
Present address: Abbott Labs, Abbott Park, IL, USA.
Present address: Piedmont Research Center, Morrisville, NC, USA.
a
b
S
§
N
–
2
1
N
||
H
Present address: Array BioPharma, Inc., Boulder, CO, USA.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.023

22
K. L. Stevens et al. / Bioorg. Med. Chem. Lett. 19 (2009) 21–26
Reversible
F
O
F
O
S
O
H
HN
N
N
O
HN
N
Cl
HN
N
Cl
O
O
N
O
O
O
O
N
O
N
N
IressaTM (AstraZeneca)
Market
EGFR/ ErbB-2 (nM): 33/ 2700
HN5/ BT474 (nM): 10/ 300
TarcevaTM (OSI/Genentech/Roche)
Market
EGFR/ ErbB-2 (nM): 20/ 3500
HN5/ BT474 (nM): Unknown
TykerbTM (GlaxoSmithKline)
Market
EGFR/ ErbB-2 (nM): 5/ 5
HN5/ BT474 (nM): 15/ 20
Cl
O
Irreversible
F
F
N
O
O
O
HN
N
N
HN
N
Cl
HN
N
Cl
N
HN
O
HN
O
HN
O
N
N
N
O
N
O
CI-1033 (Pfizer)
HKI-272 (Wyeth)
Phase II
BIBW-2992 (Boehringer Ingelheim)
Phase II
Phase II
EGFR/ ErbB-2 (nM): 5/ 80
HN5/ BT474 (nM): 50/ 10
EGFR/ ErbB-2 (nM): 1/ 1
HN5/ BT474 (nM): Unknown
EGFR/ ErbB-2 (nM): 6/ 2
HN5/ BT474 (nM): Unknown
Figure 1. Chemical structures for reversible and irreversible EGFR/ErbB-2 kinase inhibitors. Enzyme inhibition data shown (EGFR/ ErbB-2) are IC50 values; cellular
proliferation inhibition data shown (HN5/ BT474 cell lines) are EC50 values. Values are the mean of two or more experiments.7
Synthesis of the thienopyrimidine analogs was initiated from
the known acetylenic pyrrolidine (2)15 which is derived from the
natural (S)-enantiomer of 4-hydroxy proline (Scheme 1). The C-4
alcohol in compound 2 was transformed to a variety of carbamates
(3a–j) via reaction with commercially available carbamoyl chlo-
rides or via reaction with diphosgene followed by amine addition
(Scheme 1). These products were coupled with the known thieno-
pyrimidinyl bromide 516 via standard palladium-catalyzed Sono-
gashira conditions. Subsequent deprotection with TFA in CH2Cl2
yielded the desired acetylenic thieno[3,2-d]pyrimidines 6a–j.
Analogs were initially assayed for their intrinsic potency against
EGFR and ErbB-2 kinases. Since these carbamate analogs possess
the ability to covalently modify either EGFR or ErbB-2, the IC50 val-
ues are time-dependent and may vary significantly as a function of
incubation time.17 The data presented are the apparent IC50 values
obtained with a 40-min incubation. These carbamate analogs were
also tested for inhibition of cellular proliferation against HN5 (head
and neck) and BT474 (breast) transformed cell lines whose prolif-
eration is driven by the overexpression of EGFR and ErbB-2, respec-
tively.18 EGFR covalent modification was determined at 3 h of
incubation with methods described in Ref. 12. The compounds
were also evaluated in mouse oral PK. The details are summarized
in Table 1.
As expected, all compounds showed good enzyme potency,
since the carbamate was identified as one of the most potent sub-
stituents on the pyrrolidine described in prior work from this ser-
ies.13 Smaller alkyl groups, both mono- and disubstituted,
appeared to have the greatest EGFR and ErbB-2 enzyme potency,
although the differences among the analogs were minor. For cellu-
lar potency in HN5 and BT474, the small alkyl substituents were
again the best analogs (Table 1, 6b–d). Interestingly, the morpho-
line analog (6h) showed comparable cell potency on the BT474 cell
O
Cl
F
O
R1
Cl
HN
HO
(R)
N
O
(R)
R2
S
(S)
(S)
S
N
a or b
N
c
Br
Br
+
N
N
N
N
t-BOC
t-BOC
2
3a-j
5
4
d, e
O
F
O
R1
HN
Cl
N
O
R2
S
(R)
(S)
N
N
N
H
6a-j
Scheme 1. Reagents and conditions: (a) NaH, 0 °C to rt, DMF, then R1R2NCOCl (50–80%); (b) ClC(O)OCCl3, CH2Cl2, TEA, then R1R2NH2, 0 °C to rt, CH2Cl2 (50–80%); (c) 3-chloro-
4-(3-fluorobenzyloxy)aniline, 60 °C, i-PrOH (80%); (d) (Ph3P)2PdCl2, CuI, TEA, 60 °C, THF (50–80%); (e) TFA, 0 °C to rt, CH2Cl2 (90%).

K. L. Stevens et al. / Bioorg. Med. Chem. Lett. 19 (2009) 21–26
23
Table 1
EGFR and ErbB-2 kinase inhibition, cell proliferation, covalent modification of EGFR, and mouse oral PK
O
F
HN
N
Cl
R
(R)
O
H
S
N
O
(S)
N
H
Compound
R-
Enzyme IC50 (nM)a,b
Cellular EC50 (nM)a
BT474
EGFR Modificationc
% @ 3 h
Mouse Oral DNAUCe
(ng h/mL/mg/kg)
EGFR
ErbB-2
HN5
100
HFF
6a
6b
6c
6d
6e
6f
H2N
40
16
25
5500
—
12f
18
61
93
25
—
50
32
28
20
43
68
60
95
30
5000
4200
2800
4300
3000
3500
4900
5100
8200
17d
24
11
8
N
H
30
N
H
N
100
250
240
120
160
400
30
30
*
*
63
50
80
N
50
32
90
—
N
N
*
*
6g
6h
6i
99
32
60
8
63
91
—
65
63
40
84
20
32
30
5d
9d
20d
O
N
100
290
HN
O
N
S
6j
O
0
N
H
a
Average values, n P 2.
b
Homogeneous time resolved fluorescence assay. As these analogues may covalently modify either EGFR or ErbB-2, the apparent IC50 results are time-dependent and may
vary significantly as a function of the 40 min incubation time (* denotes scintillation proximity assay). See Ref. 7.
c
Value represents % covalent modification after 3 h at 0.002 mM compound concentration. For details see Ref. 12.
Experiment run at 0.006 mM compound concentration.
10 mg/kg dosed as a solution in methanesulfonic acid:hydroxypropyl-b-cyclodextrin unless noted.
Dosed as a solution in PolyEthylene Glycol:Tween 20.
d
e
f
line to the alkyl substituents, although the potency dropped off on
the HN5 cell line. Most of the analogs displayed good selectivity
over the HFF (Human Foreskin Fibroblast) control cell line (ꢀ30–
150Â). All carbamate analogs that were tested had measurable
activity in the EGFR covalent modification assay. No obvious trend
in the SAR could be determined, but all compounds modified EGFR
at 3 h from 5% at the low end (6h) to 24% at the high end (6c).
Compounds that had promising enzyme and cell potency were
advanced to mouse oral PK studies. In general, compounds in this
series had poor to moderate oral exposure in mouse. Several com-

24
K. L. Stevens et al. / Bioorg. Med. Chem. Lett. 19 (2009) 21–26
HO
(S)
HO
(S)
(S)
e, f, g, h
(S)
CO2Me
N
N
t-BOC
t-BOC
(S, S) analog (8)
7
HO
(R)
HO
(R)
(S)
e, f, g, h
(S)
CO2Me
N
N
t-BOC
t-BOC
9
(S, R) analog (2)
HO
(R)
HO
(R)
HO
(R)
(R)
(R)
(S)
a, b, c, d
e, i, j, g, h
CO2H
CO2Me
N
N
N
t-BOC
t-BOC
t-BOC
10
11
(R,R) analog (12)
k
HO
(S)
(R)
N
t-BOC
(R,S) analog (13)
Scheme 2. Reagents and conditions: (a) Ac2O, AcOH, 90 °C; (b) 2 N HCl, 100 °C; (c) SOCl2, MeOH; (d) Boc2O, TEA, DMAP (50%, four steps); (e) TBDPSCl, imidazole, DMAP (90%);
(f) DIBAL-H, À78 °C then MeOH; (g) Bestmann–Ohira reagent, K2CO3, rt (75–88%, two steps); (h) TBAF, THF (90%); (i) LiBH4, THF, 0 °C; (j) Dess–Martin Periodinane, CH2Cl2, rt
(70%, two steps); (k) PPh3, DIAD, pNO2-PhCOOH (80%).
pounds had little or no exposure in these studies (6a, 6b, 6e, and
6j). The ethyl, dimethyl, and morpholine derivatives (6c, 6d, and
6h) had the best combination of enzyme/cell potency, selectivity
over HFF cells, and oral exposure in mouse PK studies. Compounds
from this (2S, 4R)-enantiomer failed to demonstrate better mouse
oral exposure than Tykerb (DNAUC = 150 ng h/mL/mg/kg).
Therefore, we elected to explore the other stereoisomers of the
2,4-disubstituted pyrrolidine utilizing the three most promising
carbamate substituents (ethyl, dimethyl, and morpholine). The
synthesis of the enantiomers is shown in Scheme 2.
Amino acid derivatives of the natural 2-(S)-enantiomer of proline
are readily available. The 4-(R)- and 4-(S)-diastereomers of commer-
cially available N-Boc protected 4-hydroxy proline methyl ester (7
and 9, Scheme 2) were converted to acetylenes (8 and 2) in a conve-
nient four step method. The alcohol is first protected as its TBDPS
ether, followed by low temperature, selective DIBAL-H reduction
of the ester to the corresponding aldehyde which is then converted
in-situ to the acetylene using the Bestmann–Ohira modification of
the Gilbert–Seyferth reagent.19 The silyl group is removed with TBAF
to provide the free hydroxyl acetylene derivatives 8 and 2. For the
compoundsderived from the natural enantiomer of proline, a conve-
nient one-pot transformation has since been developed which
avoids the need for alcohol protection.20
Amino acid derivatives of the unnatural 2-(R)-enantiomer of
proline are not readily available or are extremely expensive.21
For preparation of more than milligram quantities, we required a
workable synthesis of unnatural 2-(R)-proline analogs. Commer-
cially available 4-(R)-N-Boc-protected hydroxy 2-(S)-proline (10)
was epimerized to a 2-(R)-proline analog by treatment in refluxing
acetic anhydride followed by treatment in refluxing HCl.22 The
product crystallizes as the HCl salt which was free of the trans
compound. Simple ester formation via SOCl2 and MeOH followed
by reaction with BOC anhydride gave the desired unnatural hydro-
xy proline derivative (11) in 50% yield over four steps. Acetylene
analog (12) in the 2-(R)-proline series was prepared similarly to
the previously described conditions, with one change being a two
step conversion of the ester to aldehyde via LiBH4 reduction and
subsequent Dess–Martin periodinane oxidation. To our knowledge,
the low temperature DIBAL-H reduction to the aldehyde should
work as well as the one-pot protocol that was developed at a later
date. The last of the four enantiomers (13) can be readily prepared
by Mitsunobu reaction on alcohol (12). The enantiomeric set of
nine compounds 14–22 was prepared from acetylenes 8, 12, and
13 utilizing conditions shown in Scheme 1 for carbamate analogs
6a–j. Data for this set of compounds is shown in Table 2 with com-
parison to the original (2S,4R)- compounds (6c, 6d, and 6h).
All compounds in this set showed a similar level of enzyme
inhibition (<100 nM in the EGFR and ErbB-2 assays). There were,
however, interesting differences in the cell potencies that seem
to correlate with the configuration around the pyrrolidine. In gen-
eral, the trans isomers (2S,4R)- and (2R,4S)- were more potent on
the BT474 cell line than their cis isomer counterparts (2S,4S)-
and (2R,4R)-. The ethyl (20) and dimethyl (21) carbamates were
the exceptions to this trend, which were much more potent than
the morpholine carbamate in the (2R,4R) isomer. In order to quan-
tify the inhibition of intracellular autophosphorylation, a DELFIA
assay was developed for ErbB-2.23 The results from the DELFIA as-
say clearly indicate the (2S,4R)-configuration is the most potent
stereoisomer (Table 2). None of the tested enantiomeric com-
pounds (14–22) had improved oral mouse PK compared with the
original 6a–j carbamates in the (2S,4R)-configuration. Overall, the
original (2S,4R)-stereochemistry appeared to be the most preferred
configuration across the three carbamates shown.
In conclusion, we have described a novel class of substituted
pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines which are potent
inhibitors of both the EGFR and ErbB-2 receptor tyrosine kinases
and possess good anti-proliferative activity against EGFR and
ErbB-2 overexpressing tumor lines. Synthetic modifications of the
pyrrolidine carbamate moiety are found to result in a range of ef-
fects on enzyme and cellular potency. In addition, absolute stereo-
chemical configuration around the pyrrolidine was found to impact
the cellular potency and oral mouse pharmacokinetics. The natural,

K. L. Stevens et al. / Bioorg. Med. Chem. Lett. 19 (2009) 21–26
25
Table 2
EGFR and ErbB-2 kinase inhibition, cell proliferation, cellular autophosphorylation, and mouse oral PK
O
F
O
HN
N
Cl
R
O
S
N
N
H
Compound
Diastereomer
(2S, 4R)
Enzyme IC50 (nM)a,b
EGFR
Cellular IC50 (nM)a
BT474
ErbB-2 Delfiac
Mouse oral DNAUCd (ng h/mL/mg/kg)
R=
ErbB-2
43
6c
6d
6h
32
28
65
30
30
30
10
10
50
61
93
91
N
H
N
(2S, 4R)
68
84
(2S, 4R)
O
N
14
15
16
17
18
19
20
21
(2R, 4S)
(2R, 4S)
(2R, 4S)
(2S, 4S)
(2S, 4S)
(2S, 4S)
(2R, 4R)
(2R, 4R)
(2R, 4R)
50
40
32
40
32
63
63
40
20
63
63
60
30
50
30
1
—
37
—
3
N
H
N
N
N
40
50
50
O
O
O
N
N
N
125
79
130
230
140
20
130
220
130
10
N
H
79
—
32
1
N
H
79
10
20
6
22
50
120
120
—
a
Average values, n P 2.
b
HTRF assay. As these analogues may covalently modify either EGFR or ErbB-2, the apparent IC50 results are time-dependent and may vary significantly as a function of the
40 min incubation time. See Refs. 7 and 12.
c
See Ref. 23.
d
10 mg/kg dosed in methanesulfonic acid:hydroxypropyl-b-cyclodextrin.

26
K. L. Stevens et al. / Bioorg. Med. Chem. Lett. 19 (2009) 21–26
Rusnak, D. W.; Spehar, G. M.; Wood, E. R.; Griffin, R. J. Bioorg. Med. Chem. Lett.,
submitted for publication.
14. Hubbard, R. D.; Dickerson, S. H.; Emerson, H. K.; Griffin, R. J.; Reno, M. J.;
Hornberger, K. R.; Rusnak, D. W.; Wood, E. R.; Uehling, D. E.; Waterson, A. G
Bioorg. Med. Chem. Lett. 2008, 18, 5738.
2-(S)-proline configuration was found to have the best overall
properties in the series.
References and notes
15. Trybulski, E. J.; Kramss, R. H.; Mangano, R. M.; Rusinko, A. J. Med. Chem. 1990,
33, 3190.
16. Compound
4 (CAS#: 225385-03-5) has recently become commercially
1. Seymour, L. K. Curr. Drug Targets 2001, 2, 117.
2. Yarden, Y. Eur. J. Cancer 2001, 37, S3.
available through Medinoah Inc., Wallingford, CT. Synthetic details can be
found in PCT Int. Appl. WO 99/24440. Compound 5 is prepared by displacement
3. Rowinsky, E. K. Annu. Rev. Med. 2004, 55, 433.
on the chloro of
4 with 3-chloro-4-(3-fluorobenzyloxy)aniline which is
4. Dumas, J. Curr. Opin. Drug Discov. Dev. 2001, 4, 378.
available through Affinitis Pharma LLC, New Haven, CT. Synthetic details can
be found in PCT Int. Appl. WO 98/02437.
17. The modification data was only acquired for EGFR using methods described in
Ref. 12. These analogs have not been assayed versus ErbB-2 for potential
reactivity.
18. Thienopyrimidine analogs (6a–j) were found to be selective for the
transformed cell lines (HN5 and BT474) versus a control human foreskin
fibroblast cell line (HFF). Experimental protocols are described in Ref. 7.
19. Ohira, S. Synth. Commun. 1989, 19, 561.
20. Dickson, H. D.; Smith, S. C.; Hinkle, K. W. Tetrahedron Lett. 2004, 45, 5597.
21. (4R)-4-Hydroxy-(2R)-proline 5g, $153; (4S)-4-Hydroxy-(2R)-proline 5g, $291,
both from AstaTech, Bristol, PA. Advanced derivatives of the 2-(S) enantiomer,
such as 10, are occasionally available, 5g, $169 from AstraTech.
22. Baker, G. L.; Fritschel, S. J.; Stille, J. R.; Stille, J. K. J. Org. Chem. 1981, 46,
2954.
5. Baker, A. J.; Gibson, K. H.; Grudy, W.; Godfrey, A. A.; Barlow, J. J.; Healy, M. P.;
Woodburn, J. R.; Ashton, S. E.; Curry, B. J.; Scarlett, L.; Henthorn, L.; Richards, L.
Bioorg. Med. Chem. Lett. 2001, 11, 1191.
6. Bonomi, P. Expert Rev. Anticancer Ther. 2003, 3, 367.
7. Rusnak, D. W.; Lackey, K. E.; Wood, E. R.; Alligood, K. J.; Rhodes, N.; Knight, W.
B.; Gilmer, T. M. Mol. Cancer Ther. 2001, 1, 85.
8. Allen, L. F.; Eiseman, I. A.; Fry, D. W.; Lenehan, P. F. Semin. Oncol. 2003, 30, 65.
9. Rabindran, S. K.; Discafani, C. M.; Rosfjord, E. C.; Baxter, M.; Floyd, M. B.; Golas,
J.; Hallett, W. A.; Johnson, B. D.; Nilakantan, R.; Overbeek, E.; Reich, M. F.; Shen,
R.; Shi, X.; Tsou, H.-R.; Wang, Y.-F.; Wissner, A. Cancer Res. 2004, 64, 3958.
10. Reid, A.; Vidal, L.; Shaw, H.; de Bono, J. Eur. J. Cancer 2007, 433, 481.
11. Fry, D. W. Anti-Cancer Drug Des. 2000, 15, 3.
12. Wood, E. R.; Shewchuk, L. M.; Ellis, B.; Brignola, P.; Brashear, R. L.; Caferro, T. R.;
Dickerson, S. D.; Dickson, H. D.; Donaldson, K. H.; Gaul, M.; Griffin, R. J.; Hassell,
A. M.; Keith, B.; Mullin, R.; Petrov, K. G.; Reno, M. J.; Rusnak, D. W.; Tadepalli, S.
M.; Ulrich, J. C.; Wagner, C. D.; Vanderwall, D. E.; Waterson, A. G.; Williams, J.
D.; White, W. L.; Uehling, D. E. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2773.
13. Waterson, A. G.; Petrov, K. G.; Hornberger, K. R.; Hubbard, R. D.; Sammond, D.
M.; Smith, S. C.; Hinkle, K. W.; Stevens, K. L.; Uehling, D. E.; Dickerson, S. H.;
23. Dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), see:
Braunwalder, A. F.; Yarwood, D. R.; Sills, M. A.; Lipson, K. E. Anal. Biochem.
1996, 238, 159. BT474 cells and their culture conditions are described in
Ref. 7.