Synthesis and structure-activity relationships of imidazo[1,2-a]pyrimidin- 5(1H)-ones as a novel series of beta isoform selective phosphatidylinositol 3-kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.01.092

A series of PI3K-beta selective inhibitors, imidazo[1,2-a]-pyrimidin-5(1H)- ones, has been rationally designed based on the docking model of the more potent R enantiomer of TGX-221, identified by a chiral separation, in a PI3K-beta homology model. Synthesis and SAR of this novel chemotype are described. Several compounds in the series demonstrated potent growth inhibition in a PTEN-deficient breast cancer cell line MDA-MB-468 under anchorage independent conditions.

Full text of DOI:10.1016/j.bmcl.2012.01.092

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2230–2234
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationships
of imidazo[1,2-a]pyrimidin-5(1H)-ones as a novel series of beta
isoform selective phosphatidylinositol 3-kinase inhibitors
Hong Lin a, , Karl Erhard a, Mary Ann Hardwicke b, Juan I. Luengo a, James F. Mack a,
⇑
Jeanelle McSurdy-Freed c, Ramona Plant b, Kaushik Raha d, Cynthia M. Rominger b, Robert M. Sanchez a,
Michael D. Schaber e, Mark J. Schulz a, Michael D. Spengler c, Rosanna Tedesco a, Ren Xie a, Jin J. Zeng a,
Ralph A. Rivero a
a Cancer Metabolism Chemistry, GlaxoSmithKline, 1250 South Collegeville Road, PO Box 5089, Collegeville, PA 19426-0989, United States
b Cancer Metabolism Biology, GlaxoSmithKline, 1250 South Collegeville Road, PO Box 5089, Collegeville, PA 19426-0989, United States
c Cancer Metabolism DMPK, GlaxoSmithKline, 1250 South Collegeville Road, PO Box 5089, Collegeville, PA 19426-0989, United States
d Platform Technology Sciences – Computational Chemistry, GlaxoSmithKline, 1250 South Collegeville Road, PO Box 5089, Collegeville, PA 19426-0989, United States
e Platform Technology Sciences – Screening & Compound Profiling, GlaxoSmithKline, 1250 South Collegeville Road, PO Box 5089, Collegeville, PA 19426-0989, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of PI3K-beta selective inhibitors, imidazo[1,2-a]-pyrimidin-5(1H)-ones, has been rationally
designed based on the docking model of the more potent R enantiomer of TGX-221, identified by a chiral
separation, in a PI3K-beta homology model. Synthesis and SAR of this novel chemotype are described.
Several compounds in the series demonstrated potent growth inhibition in a PTEN-deficient breast cancer
cell line MDA-MB-468 under anchorage independent conditions.
Received 27 December 2011
Revised 23 January 2012
Accepted 24 January 2012
Available online 4 February 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
PI3K-beta inhibitor
PTEN-null
Phosphatidylinositol 3-kinase
Homology model
Structure–activity relationship
The phosphatidylinositol 3-kinases (PI3K) are a family of lipid
kinases involved in the regulation of cell survival, metabolism and
proliferation.1 Dysregulation of the PI3K signaling pathway plays
an important role in the etiology of oncogenic transformation,
and aberrant pathway activation occurs frequently through inacti-
vation of the tumor suppressor protein PTEN.2 Loss of PTEN protein
as measured by immunohistochemical staining of PTEN has been
observed in approximately 40% of glioblastoma, 50% of prostate,
57% of endometrial cancers, in addition to a number of other tumor
types, including melanoma and breast cancers.3 Preclinical studies
have shown that, in a PTEN-loss context, tissue-specific deletion of
the PI3K-beta isoform in the prostate specifically reduces PI3K sig-
naling and blocks the formation of aggressive prostate tumors.4 Gi-
ven the implication of PI3K-beta activity on the progression of these
PTEN-deficient tumors, we initiated a program to identify PI3K-
beta selective inhibitors that might be efficacious in the treatment
of cancers driven by loss of PTEN.
A series of 4H-pyrido[1,2-a]pyrimidin-4-ones, exemplified by
TGX-221 (Fig. 1), was reported as selective PI3K-beta inhibitors
and investigated as potential antithrombotic agents.5 TGX-221
contains a chiral center with an aniline moiety, and, since all pub-
lished work had been conducted with racemic material, we sought
first to elucidate the stereochemical preference of the molecule in
its interaction with PI3K-beta. TGX-221 was thus prepared follow-
ing the published procedure, and the two enantiomers were sepa-
rated by chiral chromatography.6 Interestingly, the R-enantiomer
O
O
N
Me
Me
N
N
chiral
separation
N
N
N
O
O
Me
N
H
Me
N
H
*
TGX-221-R
PI3Kβ IC50 0.006 μM
TGX-221
⇑
Corresponding author.
E-mail address: hong.2.lin@gsk.com (H. Lin).
Figure 1. Preferred configuration of TGX-221.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.092

H. Lin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2230–2234
2231
Figure 2. (A) Refined model of TGX-221-R in PI3K-beta homology model; (B) Overlay of TGX-221-R and compound 7.
(TGX-221-R) turned out to be 100-fold more potent as a PI3K-beta
inhibitor than the S-enantiomer (IC50 values of 0.006 vs 0.80 M,
attractive chemotype as the simple substituted benzyl group can
replace the aniline of TGX-221-R to play a crucial role in inducing
the Met-779-Trp-787 pocket without the need of a chiral center. As
shown in Figure 2B, we anticipated that the core template in 7
would maintain the key H-bond interactions with the hinge-region
Val-854, thus directing the side chain benzyl group to interact with
the P-loop. However, the ortho-methylbenzyl group of compound 7
could not extend into the induced-pocket as deeply as the aniline
group of TGX-221-R unless a small lipophilic substitution at the
meta-position was added in compound 7. We then predicted that
the analogs containing a 2,3-disubstituted benzyl group should
show an ideal balance of potency and PI3K-beta selectivity.
The synthetic route used to prepare imidazo[1,2-a]pyrimidin-
5(1H)-ones is illustrated in Scheme 1. 2-Aminoimidazoles, pur-
chased or prepared as described in the literature,9 were condensed
with dimethyl malonate and sodium ethoxide in refluxing ethanol
to provide imidazopyrimidine-dione II.10 Heating of dione II in
POCl3 at 80 °C provided the key intermediate dichloride III.11 This
dichloride III was hydrolyzed to IV with sodium hydroxide at
100 °C. Analogs V were prepared via alkylation of IV with benzyl
bromides in the presence of K2CO3 in DMF. Finally, microwave as-
sisted direct displacement of the chlorine by nitrogen nucleophiles
or a Suzuki coupling reaction at 150 °C gave final compounds VI.12
Initially we explored substitutions at a benzylic group at N(1)
while maintaining the critical morpholine (R3) hinge-binder. (Ta-
ble 1). The compounds obtained had inhibitory potencies against
l
respectively), suggesting that the aniline side chain plays an
important role in the interaction with the active site of the kinase.
Following up on this observation, TGX-221-R was docked into a
homology model of the PI3K-beta enzyme, which we built based
on the available public and unpublished in-house crystal struc-
tures of PI3Kc .
and PI3Ka 7
The docking exercise revealed several interactions that are typ-
ical of ATP-dependent lipid kinase inhibitors with the enzyme. As
illustrated in Figure 2, the morpholine-oxygen accepts an H-bond
from the hinge region Val-854 while the pyrido-pyrimidinone tem-
plate core interacts in the central pocket lined by Met-926 and Ile
residues 803, 851 and 936 from the N and C-terminal of the kinase
domain.
The carbonyl group of TGX-221-R was initially modeled as
interacting with the catalytic Lys-805. However, based on public
and in-house structural information, the carbonyl group was even-
tually modeled as interacting with the back-pocket Tyr-839, via a
network of H-bonds involving a bridge water molecule (Fig. 2A).
Additionally, literature evidence suggested that the aromatic ani-
line ring of TGX-221-R 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 (see Fig. 2B), similar to what
was described by Knight et al. to explain the selectivity of a panel
of PI3K inhibitors.8 We speculated that such an induced-fit pocket
in the P-loop was an important contributor to the beta-isoform
selectivity of TGX-221-R over other isoforms. Based on this
hypothesis, further refinement of the PI3K-beta model suggested
PI3K-beta ranging from IC50 values of 0.003–0.40 lM, with a clear
preference for substitution at the 2- or 3-position for the mono
substituted derivatives. The key finding was the strong preference
for 2,3-disubstitution (12, 13, 14), with all three analogs demon-
strating potent IC50 values of <20 nM. The SAR results are
that imidazo[1,2-a]-pyrimidin-5(1H)-one with
a
substituted
enzyl group at N1-position, such as compound 7, would be an
O
Cl
HN
O
R2
R2
R2
a
N
b
N
c
N
I
H2N
N
N
N
H
O
Cl
N
III
II
O
O
R2
R2
N
N
R2
d
e
N
N
N
Cl
N
R3
N
N
Cl
N
H
R1
R1
VI
IV
V
Scheme 1. Synthesis of imidazo[1,2-a]pyrimidin-5(1H)-ones. Reagents and conditions: (a) Dimethyl malonate, NaOEt, EtOH, reflux (56–100%); (b) POCl3, 80 °C (10–60%); (c)
1 N NaOH, reflux (50–80%); (d) R-Br, K2CO3, DMF, RT (32–72%); (e) morpholine or thiomorpholine, EtOH or 1,4-dioxane, microwave, 150 °C, 45 min to 1 h (40–90%); for
compound 30, 2-(1-cyclohexen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, PdCl2(dppf), Na2CO3, microwave, 150 °C, 30 min, 85%.

2232
H. Lin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2230–2234
Table 1
Table 3
SAR of R1 and biochemical activity20 against PI3K isoforms
Modifications on the core structure
O
O
Y
5
X
N
N
N
N
N
N
N
R1
O
O
R1
#
R1
PI3K enzyme IC50 (lM)
#
R1
X/Y
PI3K enzyme IC50 (lM)
a
b
d
c
a
b
d
c
1
2
3
4
5
6
7
8
9
10
11
12
13
14
(2-F)Ph
(3-F)Ph
(4-F)Ph
(2-Cl)Ph
(3-Cl)Ph
(4-Cl)Ph
(2-Me)Ph
(3-Me)Ph
(3-CF3)Ph
(3,4-DiCl)Ph
(3-Cl, 4-F)Ph
(2,3-DiCl)Ph
3.2
5.0
5.0
1.6
>4.0
>4.0
4.0
4.0
6.3
>1.3
3.2
0.50
0.63
1.0
0.40
1.0
6.3
12
23
24
25
2,3-DiCl
2,3-DiCl
2,3-DiCl
CH/N
CF/N
N/CH
N/CH
0.50
0.79
>4.0
>4.0
0.013
0.025
0.079
0.032
0.016
0.031
0.63
1.3
1.3
>10
>7.9
0.10
0.25
0.40
0.63
>6.3
>4.0
—
>6.3
>6.3
5.0
0.080
0.025
0.40
—
0.20
1.3
2-Me,3-CF3
0.40
0.040
0.063
0.10
0.20
0.25
0.80
10
>6.3
>6.3
>6.3
1.3
3.2
3.2
stitution at the 2 and 3-positions of the imidazole ring, including
fusing a phenyl ring to the imidazole (18–22) seemed to provide
no advantage over the corresponding unsubstituted imidazole ana-
logs 12, 13 and 14.
In addition, substitution at the 5-position and an isosteric series
of imidazo[1,2-c]pyrimidin-5(1H)-ones were considered. As shown
in Table 3, 5-F compound 2313 is well-tolerated compared to 12,
while the compound (not shown) with Cl at 5-position is inactive.
The isosteric compounds 24 and 2514 appear to be less potent
against all four PI3K isoforms but slightly more selective compared
to compounds 12 and 14, respectively.
0.032
0.040
0.013
0.003
0.003
0.063
0.13
0.016
0.040
0.020
a-Naphthyl
(2-Me,3-CF3)Ph
Table 2
SAR of R2 and biochemical activity in PI3K isoforms
Due to the importance of the hinge-binder (R3) to the overall
pharmacophore of this inhibitor chemotype, only subtle changes
to this region were explored (see Table 4). Interestingly, addition
of a methyl group to the 2-position of the morpholine (26) seemed
to have little effect compared to the unsubstituted compound 12;
however, separation of the enantiomers revealed a significant pref-
erence for the slower eluting enantiomer 26b with improved po-
tency and selectivity profile (beta selectivity over alpha)
compared with the faster eluting enantiomer 26a. This observation
was repeated with compounds 28, 28a and 28b.15 The activity for
the first compound, a racemic mixture, was not much different
O
3
R2
2
N
N
N
N
O
R1
PI3K enzyme IC50 (lM)
#
R1
R2
a
b
d
c
12
14
15
16
17
18
19
20
21
22
(2,3-DiCl)Ph
(2-Me,3-CF3)Ph
(2,3-DiCl)Ph
(2-Me,3-CF3)Ph
(2-Me,3-CF3)Ph
(2,3-DiCl)Ph
2,3-H,H
2,3-H,H
2-Me
2-Me
2-Et
0.50
1.0
0.80
2.0
1.6
>13
1.6
>4.0
>4.0
>4.0
0.013
0.003
0.002
0.001
0.002
0.008
0.013
0.006
0.004
0.032
0.016
0.020
0.016
0.008
0.016
0.10
0.080
0.020
0.050
0.080
1.3
3.2
1.6
1.0
1.6
>10
2.5
4.0
7.9
1.6
Table 4
2,3-fusedPh
2,3-di-Me
SAR of R3 and biochemical activity in PI3K isoforms
(2,3-DiCl)Ph
(2-Me,3-CF3)Ph
O
N
2,3-di-Me
a-Naphthyl
2,3-fusedPh
2,3-fusedPh
(2-Me,3-CF3)Ph
N
R3
N
R1
consistent with the model shown in Figure 2B. Most importantly,
most of the analogs demonstrated good to excellent isoform selec-
tivity of 10 to 1000-fold versus the alpha and gamma enzymes,
with a modest three to eight fold selectivity versus the delta-
enzyme.
Compounds with substitution at 2- and/or 3-positions of the
imidazole ring were prepared, since the model suggested available
space at this region. As demonstrated in Table 2, the substituents
on the imidazole ring, particularly at the 2-position and in combi-
nation with our best R1 groups, provided compounds with im-
proved enzymatic potency, without erosion of selectivity versus
the other PI3K-isoforms. For example, the addition of a methyl at
#
R1
R3
PI3K enzyme IC50 (lM)
a
b
d
c
12
26
26a
26b
27
2,3-DiCl
2,3-DiCl
2,3-DiCl
2,3-DiCl
Morph
2-Me Morph
2-Me Morph(E1)
2-Me Morph(E2)
2,2-Di-Me Morph
2-Me Morph
2-(S)Me Morph
2-(R)Me Morph
2-HOCH2 Morph
4-DHP
0.50
1.6
0.013
0.013
0.10
0.016
0.016
0.25
1.3
0.40
>1.0
0.32
1.6
1.3
3.2
0.40
2.5
>6.3
>6.3
1.6
0.80
0.16
0.63
0.79
>2.0
0.40
1.0
>4.0
>3.9
>3.2
0.002
1.0
0.013
0.25
2,3-DiCl
28
2-Me,3-CF3
2-Me,3-CF3
2-Me,3-CF3
2,3-DiCl
2-Me,3-CF3
2-Me,3-CF3
2,3-DiCl
0.006
0.025
0.001
0.020
0.013
0.32
0.013
0.16
0.003
0.16
0.040
1.3
28a
28b
29
30
31a
32
4-THP
Thiomorpholine
2.0
0.63
the 2-position of compound 12 (PI3Kb IC50 of 0.013
pound 14 (PI3Kb IC50 of 0.003 M), led to compounds 15 and 16
with PI3Kb IC50 values of 0.002 and 0.001 M, respectively. Disub-
lM) and com-
a
l
Compound 31 was prepared by hydrogenation of compound 30 catalyzed by 5%
Pd/C in methanol, 42%.
l

H. Lin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2230–2234
2233
Table 5
Cellular activity in MDA-MB-468 breast cancer cell linea
O
N
R2
N
R3
N
R1
#
R1
R2
R3
PI3Kb
IC50
MDA-MB-468 (
l
M)
l
M
pAKT
IC50
Growth
EC50
12
14
16
28b
2,3-DiCl
H
H
Me
H
4-Morph
4-Morph
4-Morph
2-(R)Me Morph
0.013
0.003
0.001
0.001
0.11
1.1
2-Me,3-CF3
2-Me,3-CF3
2-Me,3-CF3
0.090
0.078
0.19
0.72
0.14
0.36
a
IC50 values given are means of at least two experiments.
a]pyrimidin-5(1H)-ones as potent and selective PI3K-beta inhibi-
tors. SAR exploration of multiple regions of the molecule led to
the identification of several very potent and selective inhibitors
with good growth inhibition in a PTEN-deficient cancer cell line.
This study has successfully revealed the clear SAR that is responsi-
ble for PI3K-beta isoform selectivity and provided an excellent tool
compound 16 for understanding PI3K-beta biology at a cellular
level. Work is in progress to develop a suitable tool molecule for
in vivo target validation based on these findings.
O
N
Me
Compound 16
N
N
N
α β δ γ
μ
PI3K / / / IC50 ( M) 2.0/0.001/0.008/1.0
pAKT IC50 (μM) 0.078
growth inhib. EC50 (μM) 0.14
O
Me
CF3
Figure 3. An excellent tool molecule.
Acknowledgments
The authors thank Dr. Minghui Wang for NMR determination of
the structure of compound 15; thank Mr. Dean Phelps for VCD
analysis to determine the absolute configurations of TGX-221-R,
compounds 26a, 26b, 28a and 28b; and thank Dr. Joel Greshock
for his valuable input on PTEN-deficient tumors.
than the unsubstituted analog 14; however, compound 28b, the R-
enantiomer was significantly more potent than the S-enantiomer
28a.16 Removing the chiral center by adding a second methyl to
the 2-position (27) resulted in approximately a two-order of mag-
nitude loss of potency compared with the unsubstituted analog 12.
2-Hydroxymethylmorpholine (compound 29) is tolerated but less
potent, while 2-aminomethylmorpholine analog is inactive (com-
pound not shown). The morpholino-nitrogen cannot be adequately
replaced by a carbon as evidenced by the significant loss in activity
(100-fold) with tetrahydropyran (THP) compound 31; however,
replacing the morpholine with a dihydropyran (DHP) (30), resulted
in a more modest reduction of potency of less than 10-fold. Finally,
the importance of the morpholino-oxygen in accepting the hydro-
gen bond from the hinge valine-854 is well illustrated by the loss
in potency seen with compound 32 where the morpholine is re-
placed by a thiomorpholine.
Demonstrating activity in a cellular context is an important step
in the development of intracellular enzyme inhibitors. In this case,
a PTEN-deficient breast cancer cell line, MDA-MB-46817 was se-
lected to develop a mechanistic cell-based assay measuring the
inhibition of phosphorylation of AKT.18 A good correlation between
the PI3K-beta enzyme IC50 values and the pAKT inhibition IC50 val-
ues was observed (Table 5).
References and notes
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centrations under anchorage independent conditions.19 Compound
16 (see Fig. 3) was a particularly potent inhibitor of cell growth
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with an EC50 of 0.14 lM.
In summary, based on the observed docking pose of TGX-221-R
in a homology model, we designed a novel series of imidazo[1,2-
5. (a) Knight, Z. A.; Chiang, G. G.; Alaimo, P. J.; Kenski, D. M.; Ho, C. B.; Coan, K.;
Abraham, R. T.; Shokat, K. M. Bioorg. Med. Chem. Lett. 2004, 12, 4749; (b) Jackson
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2234
H. Lin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2230–2234
Kenche, V.; Anderson, K. E.; Dopheide, S. M.; Yuan, Y.; Sturgeon, S. A.;
17. The absolute configurations of compounds 28a and 28b were assigned by
vibrational circular dichroism (VCD) analysis technique.
Prabaharan, H.; Thompson, P. E.; Smith, G. D.; Shepard, P. R.; Daniele, N.;
Kulkarni, S.; Abbott, B.; Saylik, D.; Jones, C.; Lu, L.; Giuliano, S.; Hughan, S. C.;
Angus, J. A.; Robertson, A. D.; Salem, H. H. Nat. Med. 2005, 11, 507.
6. Chiral separation was carried out on the mixture of enantiomers under the
following conditions: Chiralpak AS-H 4.6 Â 150 mm; C = H3CN (0.1% isopropyl-
amine); D = IPA (0.1% Isopropylamine); 90% C + 10% D; 1.0 ml/min; UV 254 nm:
RT 3.7 min E1, 49.2%; RT 5.7 min E2 TGX-221-R, 50%. The purity of ee is greater
than 99%. The absolute configuration was determined by vibrational circular
dichroism (VCD) analysis technique.
7. Huang, C.-H.; Mandelker, D.; Schmidt-Kittler, Y. S.; Velculescu, V. E.; Kinzler, K.
W.; Vogelstein, B.; Gabelli, S. B.; Amzel, L. M. Science 2007, 318, 1744–1748.
8. 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.;
Roger, L.; Williams, R. L.; Shokat, K. M. Cell 2006, 125, 733–747.
9. Little, T. L.; Webber, S. E. J. Org. Chem. 1994, 59, 7299–7305.
10. The regio-chemistry of the R2 group was determined with compound 15 using
2D NMR techniques such as COSY, HMQC and HMBC. The indicated
regioisomer was the only one isolated.
18. Breast cancer cells were plated, incubated for approximately 16–20 h and then
treated with compound for 30 min. Final DMSO concentration on all cells was
0.15%. The cells were washed with Tris buffer and lysed in MesoScale Discovery
(MSD) lysis buffer containing protease and phosphatase inhibitors (included in
MSD kit). MSD Ser473-AKT duplex plates (Cat # MS6000) were used according
to the manufacturer’s instructions and plates were read on a SECTOR™ Imager
6000 using MSD Workbench software. For analysis of the Ser473-pAKT
concentration response curves, the data was normalized using the
corresponding total AKT value (sum of pAKT and AKT signal) and plotted as
the percent of the DMSO-treated control values. The data was fit in Graphpad
Prism version 4 for Windows (Graphpad Software, San Diego, California).
19. Torbett, N. E.; Luna-Moran, A.; Knight, Z. A.; Houk, A.; Moasser, M.; Weiss, W.;
Shokat, K. M.; Stokoe, D. Biochem. J. 2008, 415, 97–110.
20. For the tumor cell growth assay, breast cancer cell lines were plated under
anchorage independent conditions. To generate anchorage independent
growth conditions, a 0.6% agar/media + 10% fetal bovine serum (FBS) solution
was made to generate a bottom agar layer in the plates to prevent cell
attachment. After solidification, a cell solution in 0.3% agar/media + 10% FBS
was added to the plates. After the cell layer solidified, media + 10% FBS was
11. Monochloro compound IV was readily separable via trituration.
12. The order of the alkylation step (d) and morpholine replacement step (e) can be
switched to explore R1 SAR more efficiently.
added to the top of the cells.
A 0.3% Brij 35 (Sigma B4184) solution in
13. Inhibition of PI3K-isoforms was measured using a continuous read time-
resolved fluorescence resonance energy transfer displacement assay: Gray, A.;
Olsson, H.; Batty, I. H.; Priganica, L.; Downes, C. P. Anal. Biochem. 2003, 313,
234–245.
media + 10% FBS was added as a background control. The cells were incubated
for approximately 16–20 h, treated with compound and the plates incubated
for 6 days. One plate of cells was processed at the time of compound addition
to quantify the starting number of cells. The final concentration of DMSO in all
14. Compound 23 was prepared shown in the following scheme.
O
N
O
N
O
F
F
N
N
N
a
b
N
N
N
Cl
Cl
N
N
O
Cl
Cl
Cl
Cl
Cl
Cl
23
Reagents and conditions: (a) Selectfluor, DCM/MeOH, RT overnight, 33% based on
recovered starting material; (b) morpholine, 160 °C, microwave, 45 min, 1,4-diox-
ane, 77%.
15. Compounds 24 and 25 were synthesized by the alkylation of compound 33
(prepared according to the procedures in PCT Int. Appl., 2008113469, 25 Sep
2008), followed by direct replacement of chloride with morpholine under the
conditions (d) and (e) described in Scheme 1.
wells was 0.15%. Following the 6-day incubation, Alamar Blue (Invitrogen
#DAL1100) was added to the cells, incubated for 6 h and the plates read on a
Spectramax (Gemini EM)) at 530 nm (excitation) and 590 nm (emission). For
analysis of cell growth inhibition dose response curves, the data was plotted as
the percent of the DMSO-treated control samples. Values from wells containing
0.3% Brij 35 were subtracted from all samples for background correction. The
data was fit in Excel with a four parameter logistical fit using XLfit from ID
Business solutions (IDBS, model 205).
O
N
N
N
H
Cl
33
16. Compounds 28a and 28b were prepared by chiral separation under the
following conditions: Analytical chiral HPLC; Chiralpak AS-H 4.6 Â 150 mm;
CH3OH/Isopropylamine—100:0.1; 1 ml/min; UV 254 nm; RT E1 3.1 min 49.5%;
E2 3.5 min 49.9%.