Rational design, synthesis, and SAR of a novel thiazolopyrimidinone series of selective PI3K-beta inhibitors

Article abstract of DOI:10.1021/ml300045b

A novel thiazolopyrimidinone series of PI3K-beta selective inhibitors has been identified. This chemotype has provided an excellent tool compound, 18, that showed potent growth inhibition in the PTEN-deficient breast cancer cell line MDA-MB-468 under anchorage-independent conditions, and it also demonstrated pharmacodynamic effects and efficacy in a PTEN-deficient prostate cancer PC-3 xenograft mouse model.

Full text of DOI:10.1021/ml300045b

Letter
pubs.acs.org/acsmedchemlett
Rational Design, Synthesis, and SAR of a Novel Thiazolopyrimidinone
Series of Selective PI3K-beta Inhibitors
Hong Lin,*,† Mark J. Schulz,† Ren Xie,† Jin Zeng,† Juan I. Luengo,† Michael D. Squire,†
Rosanna Tedesco,† Junya Qu,† Karl Erhard,† James F. Mack,† Kaushik Raha,∥ Ramona Plant,‡
Cynthia M. Rominger,‡ Jennifer L. Ariazi,‡ Christian S. Sherk,‡ Michael D. Schaber,⊥
Jeanelle McSurdy-Freed,§ Michael D. Spengler,§ Charles B. Davis,§ Mary Ann Hardwicke,‡
and Ralph A. Rivero†
‡
§
∥
†Cancer Metabolism Chemistry; Cancer Metabolism Biology; Cancer Metabolism DMPK; Platform Technology
⊥
SciencesComputational Chemistry; and Platform Technology SciencesScreening & Compound Profiling; GlaxoSmithKline,
1250 South Collegeville Road, Collegeville, Pennsylvania 19426-0989, United States
S
* Supporting Information
ABSTRACT: A novel thiazolopyrimidinone series of PI3K-beta selective inhibitors has been
identified. This chemotype has provided an excellent tool compound, 18, that showed potent
growth inhibition in the PTEN-deficient breast cancer cell line MDA-MB-468 under anchorage-
independent conditions, and it also demonstrated pharmacodynamic effects and efficacy in a PTEN-
deficient prostate cancer PC-3 xenograft mouse model.
KEYWORDS: PI3K-beta inhibitor, PTEN-deficient, phosphatidylinositol 3-kinase, homology model, structure−activity relationship
ince 2006, numerous small molecule phosphatidylinositol
3-kinase (PI3K) inhibitors have entered a wide range of
S
clinical trials, primarily as targeted anticancer agents.1,2 These
molecules are either class I pan-PI3K inhibitors (with or
without mTOR activity) or PI3K-alpha selective or PI3K-delta
selective inhibitors. Despite many years of effort, it remains
unclear what PI3K target selectivity profile is required of an
inhibitor to provide a safe and effective agent for a specific
patient population. Recent preclinical studies have shown that,
in a PTEN-loss context, tissue-specific deletion of the PI3K-
beta isoform in the prostate specifically reduces PI3K signaling
and blocks the formation of aggressive prostate tumors.3 Driven
by these intriguing data, a couple of academic groups have
published their efforts on identifying novel beta-isoform selective
small molecule tools or using an existing tool molecule to
understand the biology and the pathway in depth.4 In previous
communications,5 we have reported on the discovery of two
novel series of PI3K-beta inhibitors, imidazo[1,2-a]-pyrimidin-
5(1H)-ones and 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones,
which are exemplified by compounds 1 and 2 (Figure 1).
Although these are excellent tool molecules for understanding
PI3K-beta biology at a cellular level, they are limited from in vivo
target validation due to the poor rodent pharmacokinetic profile.6
The modeling and SAR results from our two previous series
have suggested that a potent and selective PI3K-beta inhibitor
could be designed from a bicyclic core structure bearing sub-
stituents designed to make three key binding interactions: (a) a
carbonyl group to interact with the back-pocket Tyr-839; (b) a
morpholine to act as a hinge binder; and (c) a lipophilic group
Figure 1. Thiazolopyrimidinone, a novel series of PI3K-beta inhibitors
by design.
that can induce a selectivity-pocket formed by Met-779 and
Trp-787 (Figure 2A). We soon discovered that thiazolopyr-
imidinones with a substituted benzyl group at the N1-position
met the above requirements (Figure 1).
Received: February 20, 2012
Accepted: May 29, 2012
Published: May 29, 2012
© 2012 American Chemical Society
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Letter
Figure 2. (A) Compound 9 in the PI3K-beta homology model; (B) overlay of compounds 9 and 1.
Scheme 1. Synthesis of Compounds 16, 18, and 20
Reagents and conditions: (a) HCONH2, 150 °C, 80%; (b) LiHMDS (2 equiv), 1-(bromomethyl)-2-methyl-3-(trifluoromethyl)benzene, THF, 58%;
(c) NaOH, MeOH, 75 °C; (d) propionyl chloride, then NaOH, 30%; (e) AcCl, DMAP, Py, 80 °C, 82%; (f) K2CO3, 1-(bromomethyl)-2-methyl-3-
(trifluoromethyl)benzene, DMF, 90 °C, 81%; (g) K2CO3, sodium perborate hydrate, MeOH/THF/H2O 1:1:1, 55 °C, 32−50%.
Compound 9 was docked in our PI3K-beta homology model
based on public and in house unpublished structural inform-
ation.7 Figure 2A depicts the preferred pose of compound 9 in
the binding site as viewed from below the plane of the thiazolo-
pyrimidinone core. The morpholine-oxygen could accept an
H-bond from the hinge Val-854 while the carbonyl group of the
thiazolopyrimidinone core could interact with the back-pocket
Tyr-839, via a network of H-bonds involving a bridge water
molecule. Furthermore, the N1-o-toluene group of compound 9
may induce a conformational switch in the P-loop at the top of
the ATP binding site, creating a lipophilic pocket lined by Met-
779 and Trp-787, which we believe to be responsible for beta
isoform selectivity.
In addition, the 2-position of the thiazolopyrimidinone is
in the proximity of Met-779, Lys-805, Asp-923, and Asp-937,
which are reasonably flexible residues that could provide other
lipophilic or H-bond interactions. We predicted that a variety of
substituents at the 2-position could not only be tolerated but
also enhance potency. This predicted improvement had been
witnessed in the imidazopyrimidinone series.8
From the overlay, it appears that the benzene ring of com-
pound 9 occupies the space where the 2-methyl and 3-CF3
groups on the benzyl tail of compound 1 are. The ortho-methyl
group of compound 9 points toward the benzene ring of com-
pound 1, and a small lipophilic group adjacent to the methyl
group might be able to increase potency. This overlay indicates
that even though the two ortho-methyl groups in the two
molecules align in different directions, we could still predict
that, in the thiazolopyrimidinone series, 2,3-disubstituted benzyl
groups should provide potent and selective PI3K-beta inhibitors.
The synthesis of thiazolopyrimidinones is exemplified by the
preparation of compounds 16, 18, and 20 (Scheme 1). Methyl
4-amino-1,3-thiazole-5-carboxylate, prepared as described in the
literature,9 was heated in formamide at 150 °C overnight to
provide [1,3]thiazolo[4,5-d]pyrimidin-7(4H)-one (3).10 De-
protonation of 3 with 2 equiv of lithium bis(trimethylsilyl)-
amide (LiHMDS) in anhydrous tetrahydrofuran (THF) at
0 °C, followed by addition of 1-(bromomethyl)-2-methyl-3-
(trifluoromethyl)benzene, afforded the desired product 16 as
the major regioisomer, which was separable from the minor
regioisomer either by flash column chromatography on silica
gel or by reversed phase HPLC.11
Figure 2B depicts an overlay of docked poses of compounds
9 and 1 in the homology model of PI3K beta, as viewed from
above the plane of the thiazolopyrimidinone core. The inter-
actions between the compounds and the binding site are the
same as described above; however, for visual clarity, parts of the
P-loop including the residue Trp-787 are not shown in Figure 2B.
Since the above-mentioned alkylation is hindered by any sub-
stitution at the 2-position, two methods that are complemen-
tary in many cases have been developed to introduce a variety
of functional groups (R2 in Figure 1). For example, compound
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20 was prepared via method A. Hydrolysis of compound 16 in
aqueous NaOH with gentle heat (65−75 °C) afforded amino-
amido thiazole 4, which was treated with propionyl chloride
followed by ring closure under basic conditions to provide
compound 20. Compound 18 was first prepared in a similar
alkylation procedure to that of 16 but in low yield.12 It was later
prepared in large scale using method B.13 4-Amino-2-(4-
morpholinyl)-1,3-thiazole-5-carbonitrile (5), prepared follow-
ing the literature procedures,14 was mixed with acetyl chloride
and DMAP in pyridine at 80 °C overnight to afford compound
6. Alkylation of compound 6 with 1-(bromomethyl)-2-methyl-
3-(trifluoromethyl)benzene in the presence of K2CO3 in DMF
at 90 °C gave compound 7. Subsequent cyclization of 7 under
mild oxidative conditions with sodium perborate provided
compound 18 in good yield.
To determine if the SAR of this series was consistent with
the modeling results, and tracked with the previously reported
imidazopyrimidinone and triazolopyrimidinone series, we
initially explored substitutions at the benzylic group at the
N1-position while maintaining the critical morpholine-hinge
interaction. Indeed, preference for substitution at the 2- or
3-position was similar to that of imidazo- and triazolopyr-
imidinones. Compared to unsubstituted benzyl analogue 8,
2-Me and 3-Cl analogues (9 vs 8 and 14 vs 8) increased
potency by 8- and 4-fold, respectively. In agreement with our
prediction based on the overlay of compounds 9 and 1 in a
PI3K-beta homology model, 2,3-disubstituted analogues such
as 16 and 17 were single-digit nM PI3K-beta inhibitors. In
addition, the thiophene analogue was about 5-fold less potent
than the benzene analogue with a similar substitution pattern
(13 vs 12), while both alpha-Me benzyl analogue 10 and
phenethyl analogue 11 were slightly less potent than 8.
Replacement of the benzene ring with a polar heteroaryl
group such as 2-pyridine or 3-pyridine was not tolerated;
neither was the polar substitution on the benzene ring, such as
CN, CO2H, NO2, etc. (structures or data not shown).
This series has also demonstrated good to excellent isoform
selectivity versus the alpha and gamma enzymes, that is especi-
ally profound in more potent analogues such as compounds 16
and 17, although selectivity versus the delta enzyme was a more
modest 10- to 20-fold in most cases (Table 1).
These SAR results support our hypothesis of how a PI3K-
beta selective inhibitor might interact with the enzyme in a
PI3K-beta homology model, in which the substituted benzene
group could induce a lipophilic pocket formed by Met-779 and
Trp-787.
Compounds with substitution at the 2-position of the thia-
zolopyrimidinone ring were prepared, since the model
suggested available space and possible H-bond interactions
with flexible residues such as Met-779, Lys-805, Asp-923, and
Asp-937 in this region. The robustness of the synthesis of this
chemical series has allowed us to explore a variety of functional
groups at the 2-position to explore SAR with great detail and in
depth comprehension.
As demonstrated in Table 2, various substituents on the
pyrimidinone ring, in combination with our best R1 groups,
provided compounds with improved enzymatic potency,
without erosion of selectivity versus the other PI3K isoforms.
For example, the addition of a methyl group at the 2-position of
compounds 16 (PI3K-beta IC50 of 0.003 μM) and 17 (PI3K-
beta IC50 of 0.003 μM) led to extremely potent compounds 18
and 19 (PI3K-beta IC50 values of 0.0006 and 0.0005 μM,
respectively). While small alkyl groups increased potency
Table 1. SAR of R1 and Biochemical Activity in PI3K
Isoforms15,16
PI3K enzyme IC50 (μM)
no.
R1
R1′
α
β
δ
γ
8
Ph
H
H
Me
H
H
H
H
H
H
H
7.9
2.5
0.32
2.0
0.25
0.50
1.0
6.3
2.5
9
(2-Me)Ph
Ph
0.025
0.40
10
11
12
13
14
15
16
17
>4.0
>2.0
2.5
5.0
Bn
0.79
3.2
(2-Cl)Ph
2-Cl-3-thieno
(3-Cl)Ph
(2,3-diCl)Ph
(2-Me,3-CF3)Ph
(2-Me,3-Cl)Ph
0.16
>0.79
2.5
7.9
3.2
0.79
4.0
>3.2
1.0
0.079
0.010
0.003
0.003
0.32
0.079
0.063
0.040
>0.79
1.3
4.0
1.6
2.5
2.5
Table 2. SAR of R2 and Biochemical Activity in PI3K
Isoforms18
PI3K enzyme IC50 (μM)
no.
R1
R2
α
β
δ
γ
16 2-Me,3-CF3
17 2-Me,3-Cl
H
H
4.0
2.5
0.003
0.063
0.040
0.020
0.010
0.013
0.016
0.50
1.6
2.5
0.003
18 2-Me,3-CF3 Me
19 2-Me,3-Cl Me
2.5
0.0006
0.0005
0.0005
0.0005
0.050
0.79
1.0
2.0
20 2-Me,3-CF3 Et
21 2-Me,3-CF3 c-Pr
22 2-Me,3-CF3 Ph
23 2-Me,3-CF3 Bn
>4.0
1.3
1.3
1.0
>3.2
2.5
4.0
0.003
0.063
0.003
0.16
2.0
24 2-Me,3-CF3 CH2OH
25 2-Me,3-CF3 CH2NH2
0.63
>2.5
0.50
50
1.6
0.0002
0.003
0.25
>7.9
0.25
>6.3
0.40
>3.2
2.5
26 2-Me,3-Cl
27 2-Me,3-Cl
28 2-Me,3-Cl
29 2-Me,3-Cl
30 2-Me,3-Cl
31 2-Me,3-Cl
32 2-Me,3-Cl
CH2OH
OH
0.0001
0.008
0.002
0.50
OMe
0.0003
0.005
0.010
0.050
0.010
0.002
0.003
SH
2.0
SMe
2.0
0.0003
0.00005
0.0002
SCH2-CO2H
NH2
0.40
0.25
0.16
0.50
(20 and 21 vs 16) and Bn was tolerated (23), a bulky group
such as Ph reduced potency by 16-fold (22 vs 16). Polar
functional groups were well-tolerated, and some of them
increased potency significantly. For example, 2-CH2NH2,
2-OH, and 2-SH analogues were as potent (25 and 27) as or
slightly less potent (29) than 16, while 2-OMe, 2-SMe, and
2-NH2 analogues (28, 30, and 32) were 10-fold more potent than
16. Compound 31 was found to be the most potent inhibitor in
this series.17
A select set of potent inhibitors was tested in the PTEN-
deficient MDA-MB-468 breast cell line to assess the inhibition of
downstream phosphorylation of AKT and antiproliferative activity
under anchorage-independent conditions.19 As expected, they
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a c
−
were very potent in both assays. In Table 3, Compound 26 was a
particularly potent inhibitor with EC50 value of 6 nM and gIC50
value of 8 nM in pAKT and growth inhibition assays, respectively.
Table 4. Mouse PK
Profile of Compounds 18, 21, and 26
d
no.
T1/2
Vdss
Cl
PO DNAUC
F%
18
21
26
1.20
0.75
1.90
2.80
2.60
3.35
29.6
48.6
29.4
278
192
49
54
21
a
Table 3. Cellular Activity
264
a
b
The data are the average of two animals (male CD mice). Units: T1/2, h;
c
Vdss, L/kg; Cl, mL/(min kg); DNAUC, ng/(h mL mg kg). iv: studies
were carried out at 0.3 mg/kg dose. Based on noncrossover studies.
d
selectivity, oral exposure in mice, and the ease of synthesis,
we selected compound 18 for in vivo pharmacodynamic and
efficacy studies.
Compound 18 was evaluated in a PTEN-deficient PC-3
prostate carcinoma xenograft mouse model.23 As shown in
Figure 4, compound 18 demonstrated a time dependent
PI3Kβ IC50
(μM)
MDA-MB-468 (μM)
pAKT
EC50
proliferation
gIC50
no.
R1
2-Me,3-CF3 Me
2-Me,3-Cl Me
R2
18
19
20
21
26
28
32
0.0006
0.0005
0.0005
0.0005
0.0001
0.0003
0.0002
0.024
0.019
0.016
0.009
0.006
0.006
0.003
0.103
0.032
0.023
0.043
0.008
0.021
0.026
2-Me,3-CF3 Et
2-Me,3-CF3 c-Pr
2-Me,3-Cl
2-Me,3-Cl
2-Me,3-Cl
CH2OH
OMe
NH2
a
IC50 values given are means of at least two experiments
The thiazolopyrimidinone series, represented by compound
18, is selective over the other PI3K isoforms in class I
lipid kinases, and also some class II, III, and IV lipid kinases
available in house, including C2B, VSP34, FRAP1 (mTOR),
and DNA-PK, as shown in Figure 3. Furthermore, compound
Figure 4. Pharmacodynamic effect of compound 18 in the PC-3
xenograft mouse model. Numbers above the bars indicate the percent
inhibition of phosphor AKT/total AKT relative to vehicle.
inhibition of pAKT with similar levels of pAKT inhibition
observed from 1 to 6 h (53% to 64% with 100 mg/kg and 63%
to 72% with 300 mg/kg). By 8 h, the levels of inhibition were
reduced at both the 100 and 300 mg/kg doses (46% and 48%,
respectively) with further reduction by 10 h (33% and 22%,
respectively) and a return to baseline by 24 h. The changes in
pAKT levels were consistent with the drug concentrations in
both blood and tumor samples with similar compound levels
from 1 to 6 h, with a decrease at 8 h followed by a further
decrease at 10 and 24 h. Higher levels (1.2- to 2-fold) of
compound were observed at the 300 mg/kg dose compared to
the 100 mg/kg dose, resulting in overall slightly greater levels of
pAKT inhibition with the 300 mg/kg dose compared to the
100 mg/kg dose. The blood concentrations of compound 18 at
both doses at the 10 h time point exceeded EC50 in the pAKT
assay and gIC50 in the growth inhibition assay.
To further establish the antitumor activity in an in vivo
setting, compound 18 was administered once-a-day orally for
21 consecutive days in the same xenograft model (Figure 5). At
both 100 and 300 mg/kg doses, complete tumor growth
inhibition relative to vehicle treated mice was observed with no
effect on body weight. The pharmacodynamic and efficacy data
from the PC-3 xenograft model (Figures 4 and 5) indicate that
approximately 63% to 24% inhibition of pAKTser473 from 1 to 10
h, respectively, results in tumor growth inhibition. These data do
not, however, rule out the possibility that other unknown activities
of PI3K beta may be contributing to effects on tumor growth.
In summary, we have designed a novel series of thiazolopyri-
midinone PI3K-beta inhibitors based on our previous findings
Figure 3. Selectivity profile of compound 18 vs other lipid kinases.
18 was tested against more than 30 protein kinases for dose
response curves and was found to be inactive (IC50 > 10 μM).
At 10 μM concentration, compound 18 showed <30%
inhibition against a panel of 290 kinases screened.20 The
clean selectivity profile of compound 18 provided us with a
high level of confidence that any biological effects observed
with this tool compound would be directly associated with
PI3K-beta inhibition.
Mouse pharmacokinetic (PK) studies (All animal studies
were conducted after review by the GSK Institutional Animal
Care and Use Committee and in accordance with the GSK
Policy on the Care, Welfare and Treatment of Laboratory
Animals.) were carried out with three potent inhibitors (see
Table 4),21 and they showed low to moderate clearance.22 To
enable in vivo biological evaluations, all three compounds were
subjected to high dose (100 mg/kg) oral suspension mouse PK
studies. We observed good oral exposure of all three, measured
by DNAUC. Considering overall profile including potency,
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2008, 105, 13057−13062. (b) Jia, S.; Liu, Z.; Zhang, S.; Liu, P.; Zhang,
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R.; Schaber, M. D.; Schulz, M.; Spengler, M. D.; Tedesco, R.; Xie, R.;
Zeng, J. J.; Rivero, R. A. Synthesis and structure-activity relationships
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isoform selective phosphatidylinositol 3-kinase inhibitors. Bioorg. Med.
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(6) Clearance of the evaluated analogues exceeded the hepatic blood
flow of mouse or rat.
Figure 5. Effect of compound 18 on tumor growth in the PC-3
xenograft mouse model.
of how to best optimize potency and selectivity. Many
compounds demonstrated subnanomolar potency against the
beta enzyme, and several compounds have achieved nano-
molar potency in cell-based assays (21, 26, 28, 31). After further
profiling, compound 18 emerged as an excellent tool molecule for
in vivo studies, as it is potent and selective and displays good oral
exposure in mouse. To the best of our knowledge, these studies
constitute the first time that an in vivo pharmacodynamic effect
and efficacy have been demonstrated with an orally bioavailable
small molecule PI3K-beta selective inhibitor in a PTEN-deficient
xenograft model, a significant milestone toward validating PI3K-
beta as a potential target for treatment of proliferative disorders.
ASSOCIATED CONTENT
* Supporting Information
■
S
Biological assays, biological data, and experimental procedures.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(7) Knight, Z. A.; Gonzalez, B.; Feldman, M. E.; Zunder, E. R.;
Goldenberg, D. D.; William, O.; Loewith, R.; Stokoe, D.; Balla, A.;
Toth, B.; Balla, T.; Weiss, W. A.; Williams, R. L.; Shokat, K. M. A
pharmacological map of the PI3-K family defines a role for p110α in
insulin signaling. Cell 2006, 125, 733−747 and references therein.
(8) The methyl group at the 2-position greatly improved the potency
of both compounds 1 and 2 compared to their unmethylated
counterparts. See ref 54.
AUTHOR INFORMATION
Corresponding Author
*E-mail: hong.2.lin@gsk.com.
■
Notes
The authors declare no competing financial interest.
(9) Reid, W.; Kuhnt, D. Thiazolsynthesen mit N-Cyanimidsaur-
̈
eestern oder 3-Cyanisoharnstoffen und Thioglycolsaurederivaten.
Liebigs Ann. Chem. 1986, 4, 780−784.
̈
ACKNOWLEDGMENTS
The authors thank Dr. Minghui Wang for his help with structural
confirmation with NMR techniques throughout the program.
■
(10) Depending on the scale, the reaction can last for 1−3 days, or it
can be carried out under microwave irradiation at 200 °C for 30 min to
1 h.
ABBREVIATIONS
(11) Some analogues were prepared with an additional 1 equiv of
MgiPrCl in this step to improve the ratio of the desired regioisomer.
The following is the minor regioisomer, whose carbonyl group has a
distinctive IR absorption at 1630 cm−1, while the carbonyl group of the
major isomer has IR absorption at 1610 cm−1.
■
PI3K, phosphoinositide 3-kinase; PTEN, phosphotase and
tensin homologue; DNAUC, dose-normalized area under the
curve
REFERENCES
■
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morpholinyl)-1,3-thiazole-5-carboxamide with acetic anhydride in
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ACS Medicinal Chemistry Letters
Letter
acetic acid. See experimental details in this patent: Lin, H.; Luengo, J.
I.; Ralph, R. A.; Schulz, M. J.; Xie, R.; Zeng, J. PCT Int. Appl. (2010),
WO2010135504 A1 20101125.
(13) Compound 18 prepared from the alkylation method was
identical to that prepared from method B illustrated in Scheme 1. This
result supports the structural assignment of compound 16 and all
others prepared from the alkylation method.
(14) Thomae, D.; Perspicace, E.; Xu, Z.; Henryon, D.; Schneider, S.;
Hesse, S.; Kirsh, G.; Seck, P. One-pot synthesis of new 2,4,5-trisubstituted
1,3-thiazoles and 1,3-selenazoles. Tetrahedron 2009, 65, 2982−2988.
(15) Inhibition of PI3K-isoforms was measured using a continuous
read time-resolved fluorescence resonance energy transfer displace-
ment assay: Gray, A.; Olsson, H.; Batty, I. H.; Priganica, L.; Downes,
C. P. Nonradioactive methods for the assay of phosphoinositide 3-
kinase and phosphoinositide phosphatase and selective detection of
signaling lipids in cell and tissue extracts. Anal. Biochem. 2003, 313,
234−245.
(16) Compounds were routinely tested only once in the PI3K
bundle; replicate analyses were done only by special requests, as the
potency of the pharmacological standards (which were run on every
plate) was consistently reproducible.
(17) We hypothesized that the extreme potency of compound 31
might come from the interaction of its extended acid group with
catalytic Lys805, which appears to have reasonable flexibility.
(18) The synthesis of compounds 25 and 27−32 can be found in this
patent: Lin, H.; Luengo, J. I.; Ralph, R. A.; Schulz, M. J.; Xie, R.; Zeng,
J. PCT Int. Appl. (2010), WO2010135504 A1 20101125.
(19) See notes 19 and 21 in ref 54.
(20) See single shot results of compound 18 from Reaction Biology
Corporation (RBC) in the Supporting Information.
(21) Xiang, H.; McSurdy-Freed, J.; Moorthy, G. S.; Hugger, E.;
Bambal, R.; Han, C.; Ferrer, S.; Gargallo, D.; Davis, C. B. Preclinical
drug metabolism and pharmacokinetic evaluation of GW844520, a
novel anti-malarial mitochondrial electron transport inhibitor. J.
Pharm. Sci. 2006, 95, 2657−72.
(22) Both compounds 18 and 26 were subjected to dog and monkey
PK studies and showed high clearance in these two species. The
clearance values of all three compounds in Table 4 exceeded the rate
of rat hepatic flow as well.
(23) Female nude mice bearing PC-3 prostate carcinoma xenografts
were administered compound 18 at 100 or 300 mg/kg and euthanized
using carbon dioxide at the indicated time points. Vehicle-treated mice
were euthanized at the 2 h time point. Blood was collected for
compound concentration determination. The tumor was excised; half
was snap frozen in liquid nitrogen for subsequent compound
concentration determination, and the other half was immediately
processed by Medicon in 1 mL of Meso-Scale Discovery (MSD) lysis
buffer with protease inhibitors (Roche complete protease cocktail,
catalog # 04 693 116 001) and phosphatase inhibitors (Sigma, catalog
# P2850 and P-5726). After centrifugation, the supernatant was serially
diluted and the ratio of phosphoAKT (Ser473) to total AKT was
measured using MSD Multi-Spot assay plates (whole cell lysate kit:
Phospho(ser473), Total AKT Assay, catalog # K15100D-3).
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dx.doi.org/10.1021/ml300045b | ACS Med. Chem. Lett. 2012, 3, 524−529