Discovery and optimization of a series of benzothiazole phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitors

Article abstract of DOI:10.1021/jm1014605

Phosphoinositide 3-kinase α (PI3K-α) is a lipid kinase that plays a key regulatory role in several cellular processes. The mutation or amplification of this kinase in humans has been implicated in the growth of multiple tumor types. Consequently, PI3K- has become a target of intense research for drug discovery. Our studies began with the identification of benzothiazole compound 1 from a high throughput screen. Extensive SAR studies led to the discovery of sulfonamide 45 as an early lead, based on its in vitro cellular potency. Subsequent modifications of the central pyrimidine ring dramatically improved enzyme and cellular potency and led to the identification of chloropyridine 70. Further arylsulfonamide SAR studies optimized in vitro clearance and led to the identification of 82 as a potent dual inhibitor of PI3K and mTOR. This molecule exhibited potent enzyme and cell activity, low clearance, and high oral bioavailability. In addition, compound 82 demonstrated tumor growth inhibition in U-87 MG, A549, and HCT116 tumor xenograft models.

Full text of DOI:10.1021/jm1014605

ARTICLE
pubs.acs.org/jmc
Discovery and Optimization of a Series of Benzothiazole
Phosphoinositide 3-Kinase (PI3K)/Mammalian Target of Rapamycin
(mTOR) Dual Inhibitors
Noel D. D’Angelo,*,† Tae-Seong Kim,† Kristin Andrews,‡ Shon K. Booker,† Sean Caenepeel,§ Kui Chen,^
Derin D’Amico,† Dan Freeman,§ Jian Jiang,# Longbin Liu,† John D. McCarter,^ Tisha San Miguel,^ Erin L. Mullady,z
Michael Schrag,# Raju Subramanian,# Jin Tang,O Robert C. Wahl,^ Ling Wang,§ Douglas A. Whittington,O
Tian Wu,|| Ning Xi,† Yang Xu,# Peter Yakowec,O Kevin Yang,† Leeanne P. Zalameda,^ Nancy Zhang,§
Paul Hughes,§ and Mark H. Norman†
†
Departments of ^High-Throughput Screening/Molecular Pharmacology, Medicinal Chemistry, ‡Molecular Structure, §Oncology
Research, Pharmaceutics, and #Pharmacokinetics and Drug Metabolism, Amgen Inc.,
One Amgen Center Drive, Thousand Oaks, California 91320-1799, United States
Departments of zHigh-Throughput Screening/Molecular Pharmacology and OMolecular Structure, Amgen Inc.,
360 Binney Street, Cambridge, Massachusetts 02142, United States
S Supporting Information
b
ABSTRACT: Phosphoinositide 3-kinase R (PI3KR) is a lipid
kinase that plays a key regulatory role in several cellular processes.
The mutation or amplification of this kinase in humans has been
implicated in the growth of multiple tumor types. Consequently,
PI3KR has become a target of intense research for drug discovery.
Our studies began with the identification of benzothiazole
compound 1 from a high throughput screen. Extensive SAR
studies led to the discovery of sulfonamide 45 as an early lead, based on its in vitro cellular potency. Subsequent modifications of the
central pyrimidine ring dramatically improved enzyme and cellular potency and led to the identification of chloropyridine 70. Further
arylsulfonamide SAR studies optimized in vitro clearance and led to the identification of 82 as a potentdual inhibitor of PI3K and mTOR.
This molecule exhibited potent enzyme and cell activity, low clearance, and high oral bioavailability. In addition, compound 82
demonstrated tumor growth inhibition in U-87 MG, A549, and HCT116 tumor xenograft models.
’ INTRODUCTION
p110R catalytic domain of PI3K (PIK3CA),10,12-18 (2) the
amplification of the PIK3CA gene,1,19-21 and (3) loss of function
of PTEN.1,22-25 In all three cases, the net result is activation that
leads to increased levels of PIP3 production and the promotion
of cell growth and survival. PI3K signaling subsequently leads to
the activation of downstream targets such as the serine-threo-
nine kinase, PKB, and the mammalian target of rapamycin
(mTOR), which integrates signals from growth factors, nutri-
ents, and cellular engergetics to promote cell growth and
survival.26 Since many human cancers have been found to be
associated with somatic genetic alterations that result in activation
of PI3K signaling, considerable attention has been directed toward
targeting PI3K via ATP-competitive small molecule inhibitors,
either selectively or in conjuction with mTOR inhibition.27-29
Several small-molecule inhibitors of both categories have recently
been reported, and a number of these are currently undergoing
clinical trials, including dual PI3K/mTOR inhibitors such as
2-methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimi-
dazo[4,5-c]quinolin-1-yl]phenyl]propionitrile (BEZ235, Novartis),
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid
kinases that play key regulatory roles in cell proliferation, survival,
and cell translation. The PI3K’s are divided into three classes
based on structural differences and in vitro substrate specificity.1-3
Class I PI3Ks, which utilize both a catalytic and a regulatory
subunit, catalyze the phosphorylation of the 30-OH position of
phosphatidylinositol 4,5-biphosphate (PIP2) to generate phos-
phatidylinositol 3,4,5-triphosphate (PIP3).4,5 The phosphatase
and tensin homologue gene (PTEN) catalyze the depho-
sphorylation process, thereby providing a feedback mechanism
to regulate the amount of PIP3 that is generated.6-9 This
regulation is important for maintaining normal cell growth
because the generation of PIP3 leads to the downstream activa-
tion of serine-threonine kinase Akt (AKT, also known as
protein kinase B or PKB), which in turn results in the promotion
of downstream cellular processes such as cell growth, prolifera-
tion, survival, and metabolism.10,11
The activation of PI3K signaling has been thought to con-
tribute to tumor growth in humans via three distinct mechan-
isms: (1) activating point mutations in the gene that encodes the
Received: November 12, 2010
Published: February 18, 2011
r
2011 American Chemical Society
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Figure 1. X-ray cocrystal of 1 bound to PI3Kγ.
Scheme 1a
a Reagents and conditions: (i) Ac2O, DMAP, DCM, 0 ꢀC to room temperature, 88%; (ii) bis(pinacolato)diboron, Pd(dppf)Cl2/DCM, KOAc, DMSO,
100 ꢀC, 91%; (iii) TFE, EtOH, 45 ꢀC; KHF2, room temperature, 69%; (iv) 2,4-dichloropyrimidine, Pd(PPh3)4, Na2CO3, H2O, 1,4-dioxane, 95 ꢀC, 85%.
Table 1. Synthesis of Carbon, Sulfur, Oxygen, and Nitrogen-
Linked Analogues
N-(4-(N-(3-(3,5-dimethoxyphenylamino)quinoxalin-2-yl)sulfamoyl)
phenyl)-3-methoxy-4-methylbenzamide (XL765, Exelixis), and
2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-
3-pyridinyl}benzenesulfonamide (GSK2126458, GlaxoSmithKline),
as well as selective PI3K inhibitors such as 4-(2-(1H-indazol-4-yl)-
6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]
pyrimidin-4-yl)morpholine dimethanesulfonate (GDC-0941,
Genentech), 5-[2,6-di(4-morpholinyl)-4-pyrimidinyl]-4-(trifluoro-
methyl)-2-pyridinamine (BKM120, Novartis), and N-(3-(benzo-
[c][1,2,5]thiadiazol-5-ylamino)quinoxalin-2-yl)-4-methylbenze-
nesulfonamide (XL147, Sanofi/Exelixis).29-33 Herein, we describe
our initial efforts in this field that led to the discovery and
optimization of a potent series of benzothiazole PI3K/mTOR
dual inhibitors.
We conducted a high-throughput screen and identified 1 as
an initial hit. This compound exhibited good PI3KR enzyme
potency (Ki = 53 nM) and thereby served as a suitable starting
point for our SAR studies, despite its low mTOR activity (Ki >
25 μM). To guide these efforts, we solved the crystal structure
of 1 bound to PI3Kγ, whose structure is likely to closely
mimic that of PI3KR.34,35 As illustrated in Figure 1, the N-
acetylbenzothiazole moiety forms two hydrogen bonds with
the PI3Kγ residue Val882 of the hinge region. In addition, the
pyrimidine ring forms a hydrogen bond with Lys833 and is
adjacent to Asp841 and Tyr867 in the “affinity pocket”. We
postulated that changes to the pyrimidine moiety to engage
these three residues might improve potency. Finally, the
thiomethyl group extends toward the “ribose pocket”, the
region of the kinase that is occupied by the ribose moiety of
ATP. The crystal structure suggests that this pocket could
reagent
PhSH
methoda
R
compd
A
A
A
B
A
B
C
C
SPh
6
BnSH
SBn
OPh
OBn
7
PhOH
8
BnOH
9
PhNH2
NHPh
10
11
12
13
BnNH2
NHBn
PhCH2ZnBr
CH2Ph
PhCH2CH2ZnBr
CH2CH2Ph
a Method A: NaH, DMF or DMSO, 5, 13-25%. Method B: 5, pyridine,
120 ꢀC, microwave irradiation, 300 W, 6-22%. Method C: 5, Pd-
(PPh3)4, THF, 80 ꢀC, 8-11%.
accommodate significantly larger groups, which may also
serve to improve potency.
’ CHEMISTRY
Our first objective was the synthesis of pyrimidine chloride 5,
which was required to make analogues of 1. The synthesis of 5
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Scheme 2a
a Reagents and conditions: (i) NaH, DMF or DMSO, 12-69%; (ii) pyridine, 120 ꢀC, microwave irradiation, 300 W, 5-22%; (iii) Cs2CO3, DMF,
180 ꢀC, microwave irradiation, 180 W, 2%; (iv) NaH, DMF, 5, Pd(OAc)2, Xantphos, 100 ꢀC, microwave irradiation, 100 W, 16-36%.
began with the acylation of commercially available 2-amino-6-
bromobenzothiazole 2 to provide 3 (Scheme 1). Conversion of
3 to boronate 4a, followed by Suzuki coupling with 2,4-dichlo-
ropyrimidine, afforded 5 in three high-yielding steps.36 In addition,
boronic ester 4a was converted into Molander salt 4b, which was
used for subsequent Suzuki couplings.37-39
formation preceded the Suzuki coupling (Scheme 3C). Com-
pounds 57 (Scheme 3D) and 60 (Scheme 3E) were prepared in
a similar manner as 51, with the routes differing only in the manner
in which the sulfonamide was formed. Sulfonamide 57 was
prepared via SNAr chloride displacement of pyrazine 56, while
compound 60 was made by sulfonylation of amine 59 with
4-methoxybenzenesulfonyl chloride. Finally, analogue 63 was
prepared from bromide 61 using the sulfonamamide forma-
tion/Suzuki coupling sequence that was used to prepare 54
(Scheme 3F).
The preparation of analogue 68 required a longer synthetic
sequence, as shown in Scheme 4. Commercially available chlo-
ropyridine 64 was treated with diethyl malonate in the presence of
sodium hydride to introduce carbon functionality at the 2-posi-
tion of the pyridine ring. Subsequent decarboxylation under
acidic conditions provided methylpyridine 65.42 Reduction of
the nitro group afforded amine 66, and then Suzuki coupling with
boronic ester 4a and subsequent sulfonamide formation com-
pleted the synthesis of 68.
Finally, sulfonamide analogues 70-82 were all prepared
from commercially available bromide 69 in two steps
(Table 2). These syntheses involved Suzuki couplings, either
with boronic ester 4a or with Molander salt 4b, and a sulfo-
namide formation step, with the sequence of these steps
varying from compound to compound. This route provided
facile access to compounds in this series, facilitating both the
initial SAR studies and subsequent scale-up efforts of larger
amounts of material for In Vivo studies.
Compound 5 served as a versatile intermediate for the prep-
aration of several analogues, many of which were made via SNAr
displacement of the pyrimidine chloride. As outlined in Table 1,
the sulfur-, oxygen-, and nitrogen-linked phenyl and benzyl
analogues (6-11) were made by base-mediated or microwave-
mediated chloride displacement with the corresponding thiols,
alcohols, and amines. Carbon-linked analogues 12 and 13 were
prepared via Negishi coupling of 5 with benzylzinc bromide
and phenethylzinc bromide, respectively.40 Thiophenyl analogues
14-22 were prepared by the reaction of the corresponding
sodium thiophenoxides with 5. Oxygen-linked analogues 23-35
were prepared by the reaction of the corresponding alcohols
with 5, again with either base-mediated or microwave-mediated
pyrimidine chloride displacement. Finally, nitrogen-linked ana-
logues 36-45 were obtained in a similar manner by the reaction of
5 with the corresponding sulfonamides or, in the case of 36, with
cumylamine (Scheme 2).41 The specific methods used for the
synthesis of analogues 14-45 are summarized in the Supporting
Information.
The syntheses of amide 48 and central ring analogues 51, 54,
57, 60, and 63 are shown in Scheme 3. Commercially available
2-amino-4-chloropyrimidine 46 was converted to amide 47, and
subsequent Suzuki coupling with boronic ester 4a provided 48
(Scheme 3A). Sulfonamide 51 was prepared from commercially
available 2-chloro-4-iodopyridine 49 via a two-step Suzuki cou-
pling/SNAr chloride displacement process (Scheme 3B). By con-
trast, sulfonamide 54 was obtained from commercially available
bromide 52 using a two-step sequence in which the sulfonamide
’ RESULTS AND DISCUSSION
SAR and Lead Generation. On the basis of the X-ray
cocrystal structure of 1, we replaced the thiomethyl group with
thiophenyl (6) and thiobenzyl (7) groups to see how larger
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Scheme 3a
a Reagents and conditions: (i) 4-anisoyl chloride, Et3N, DCM, 5%; (ii) 4a, Pd(PPh3)4, Na2CO3, H2O, 1,4-dioxane, 90 ꢀC, 18-32%; (iii) 4-methoxyben-
zenesulfonamide, NaH, Pd(OAc)2, Xantphos, DMF, 120 ꢀC, microwave irradiation, 100 W, 12%;(iv) 4-methoxybenzenesulfonyl chloride, EtOH, 21%; (v)
4a, Fibercat, Na2CO3, H2O, 1,4-dioxane, 100 ꢀC, microwaveirradiation, 100W, 16%;(vi) 4a, Pd(dppf)Cl2, K2CO3, H2O, DME, 90 ꢀC, 62%;(vii) 4-metho-
xybenzenesulfonamide, NaH, DMSO, 125 ꢀC, 4%; (viii) 4-methoxybenzenesulfonyl chloride, pyridine, pyrrolidine, DCM, 73% (ix) 4-methoxybenzene-
sulfonyl chloride, LiHMDS, THF, -40 ꢀC to room temperature, 8%; (x) 4a, Pd(dppf)Cl2, Na2CO3, H2O, DME, 100 ꢀC, 4%.
substituents would affect enzyme potency (Table 3). These
changes had a negligible effect on PI3KR potency, suggesting
that larger thioaryl groups could be tolerated in the ribose pocket.
Benzyloxy analogue 9 and benzylamine analogue 11 were approxi-
mately 2 times less potent than 7, suggesting that changing the
linker atom from sulfur to oxygen or nitrogen had minimal impact
on enzyme potency with a benzyl group attached. However, with a
phenyl group, the effect of changing the linker atom was more
pronounced. While the phenyloxy analogue 8 was about 2 times
less potent than 6, the aniline analogue 10 was over 100 times less
potent. Finally, carbon-linked analogues 12 and 13 were at least
10 times less potent than 1.
Overall, these results reflect the impact of the linking atom on
enzyme potency. The sulfur and oxygen atoms would position
the benzyl and phenyl groups in the ribose pocket in a manner
that would minimize lone-pair interactions between the linking
atom and the pyrimidine ring. The comparable potencies of
analogues 6-9 suggest that all of these analogues adopt similar
conformations. However, the nitrogen linking atom of com-
pound 10 would likely adopt a planar conformation relative to
the pyrimidine ring and would thereby position the aryl group
differently in the ribose pocket.43 On the basis of the micromolar
potency of 10, this change results in significant steric interference
between the aryl group and the ribose pocket. However, the
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Scheme 4a
a Reagents and conditions: (i) diethyl malonate, NaH, DMF, 40 ꢀC; (ii) HCl, H2O, 110 ꢀC; (iii) Fe, HOAc, H2O, 52%; (iv) 4a, Pd(dppf)Cl2, K2CO3,
H2O, DME, 100 ꢀC, 50%; (v) 4-methoxybenzenesulfonyl chloride, DMAP, pyridine, THF, 65 ꢀC, 53%.
Table 2. Synthesis of Sulfonamide Analogues
Table 3. Linker Atom SARa
compd
R
PI3KR Ki (nM)b
mTOR IC50 (nM)b
methoda
R
compd
1
-SMe
-SPh
-SBn
-OPh
-OBn
53 ( 1
>6000c
>6000c
>6000
>6000
>6000
>6000c
>6000
>6000c
>6000c
6
40 ( 0.4
47 ( 3
A
C
B
B
C
B
C
C
B
C
B
C
C
4-OMe
H
70
71
72
73
74
75
76
77
78
79
80
81
82
7
8
102 ( 4
105 ( 19
6100c
2-CF3
3-CF3
4-CF3
2-Cl
3-Cl
4-Cl
3-tBu
4-tBu
2-F
9
10
11
12
13
-NHPh
-NHBn
85 ( 0.2
605 ( 102
1010c
-CH2Ph
-CH2CH2Ph
a See Experimental Methods for assay details. b Average of two experi-
ments. c Single experiment.
group. However, with the larger methoxy group, the relative
potencies of 20 and 22 suggest that there is more room for larger
substituents at the 4-position than at the 2-position. In addition,
lone pair interactions between the sulfur atom and methoxy
group of 20 could change the orientation of the phenyl group in
the ribose pocket just enough to result in decreased potency. We
also evaluated the compounds listed in Table 7 for their ability to
inhibit Akt phosphorylation in U-87 MG cells. However, none of
the compounds were potent, as the IC50 for all of these
compounds was over 1 μM. Given the similar enzyme potencies
of 6 and 7, we did not expect that extensive SAR studies of 7
would lead to dramatically improved enzyme or cellular activities.
For this reason, further SAR studies on sulfur-linked analogues
were not pursued.
We next examined the optimization of benzyloxypyrimidine 9
(Table 5). Like the sulfur-linked series, we initially examined the
effect of a fluoro, methyl, and methoxy group on potency (23-
28). These analogues had enzyme potencies similar to 9 except
for 25, which was about 5 times more potent. However, the
cellular activity of all of these compounds in the pAKT cellular
assay was also over 1 μM. At this point, it became clear that
simple phenyl substituents were unlikely to significantly affect
enzyme or cellular potencies and that more significant structural
changes would be required.
3-F
4-F
a Method A: (i) 4-methoxybenzenesulfonyl chloride, pyridine, 110 ꢀC,
49%; (ii) 4a, Fibercat, Na2CO3, H2O, 1,4-dioxane, 100 ꢀC, 41%. Method
B: (i) 69, LiHMDS, THF, -78 ꢀC; ArSO2Cl, -78 ꢀC to room temp,
45-99%; (ii) 4b, Pd(dppf)Cl2, Na2CO3, H2O, 1,4-dioxane, 100 ꢀC,
18-62%. Method C: (i) 4a, Pd(PPh3)4, K2CO3, H2O, 1,4-dioxane,
100 ꢀC, 33%; (ii) ArSO2Cl, DMAP, THF, pyridine, 8-69%.
addition of the extra methylene unit in benzyl derivative 11
alleviates this steric encumbrance by extending the aryl group
further into the ribose pocket, resulting in a significant increase in
potency. Finally, the carbon linking atom would orient the aryl
groups to minimize steric interactions with the pyrimidine ring.
On the basis of the potencies of 12 and 13, this results in
conformations that do not bind well to PI3KR.
On the basis of the enzyme activity of 6, we conducted further
SAR studies on the thiophenyl moiety of this compound
(Table 4). In particular, we examined the effect of fluoro, methyl,
and methoxy groups at the 2, 3, and 4 positions. The fluoro and
methyl groups had negligible impact on enzyme potency,
regardless of their positions (14-16 and 17-19, respectively).
However, the larger methoxy group decreased potency at the
2-position (20), maintained potency at the 3-position (21), and
slightly improved potency at the 4-position (22). Overall, these
results suggest that the phenyl group is surrounded by enough
space to tolerate small substituents such as the fluoro or methyl
We replaced the phenyl ring of 9 with a pyridine ring (29), a
change that had a negligible impact on potency. Likewise,
extending the pyridine further out into the ribose pocket (30)
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Table 4. Sulfur-Linker SARa
position (48) resulted in a compound with weak activity (Ki =
3.1 μM). However, replacing this amide group with a sulfonam-
ide (37) restored PI3KR enzyme potency to a similar level
compared with 11. The aryl groups of 11, 36, and 37 all have
similar spatial orientations in the ribose pocket because of the sp3
hybridization of the benzyl carbon for compounds 11 and 36 and
the sulfonamide sulfur for compound 37. These similar orienta-
tions may account for their similar PI3KR enzyme potencies.
However, the amide functionality of 48 (with an sp2 hybridized
carbon) positions the aryl group differently, a change the data
suggest is not well tolerated. In fact, a molecular modeling overlay
of 48 with the X-ray cocrystal of 1 showed significant steric
interactions between the aryl group and the ribose pocket, which
could account for the drop in potency.
The introduction of the sulfonamide group also resulted in a
dramatic improvement in mTOR potency, as 37 was the first
compound in this paper with an mTOR IC50 below 100 nM. In
fact, replacing the gem-dimethyl group of 36 with a sulfonamide
had a much greater impact on mTOR potency than on PI3KR
potency. The change in mTOR potency between these two
compounds is most likely explained by the formation of a
hydrogen bond between the mTOR protein and one or both
of the sulfonamide oxygens. The similar PI3KR enzyme poten-
cies of 36 and 37 suggest that such a hydrogen bond is not
formed in PI3KR nor is it necessary for enzyme activity.
While dimethylbenzylamine 36 was more potent than sulfon-
amide 37 in the PI3KR enzyme assay, its enzyme-to-cell shift was
over 100-fold. By contrast, the enzyme-to-cell shift for 37 was just
over 20-fold. For this reason, we sought to optimize 37, beginning
with the introduction of a methyl group (38) and an ethyl group
(39) off the sulfonamide nitrogen. For both of these analogues, a
modest increase in enzyme potency was observed. However, what
was especially noteworthy among compounds 38 and 39 was the
marked improvement in cell potency relative to 37. The enzyme
potency of compound 38 was virtually unchanged, while the
cellular potency improved by almost 10-fold. A similar gain in
cellular potency was also observed between 2-fluorophenylsulfo-
namides 40 and 41, as well as with 4-methylphenylsulfonamides
44 and 45. In fact, the cellular potency of 41 was over 5 times
more potent than 40 despite the fact that the enzyme potencies of
these compounds were similar. The improvement in cellular
potency for N-methyl analogues 38, 41, and 45 relative to 37,
40, and 44 could be explained by the loss of a hydrogen bond
donor, which could allow for better cell permeability.
compd
R
PI3KR Ki (nM)b
mTOR IC50 (nM)b
6
H
40 ( 0.4
46 ( 5
31 ( 5
44 ( 4
31 ( 7
53 ( 7
59 ( 6
144 ( 19
48 ( 3
18 ( 1
>6000c
>6000c
>6000c
>6000c
>6000c
>6000c
>6000c
>6000c
>6000c
3010 ( 1100
14
15
16
17
18
19
20
21
22
2-F
3-F
4-F
2-Me
3-Me
4-Me
2-OMe
3-OMe
4-OMe
a See Experimental Methods for assay details. b Average of two experi-
ments. c Single experiment.
and subsequently shifting the pyridine nitrogen to the 3-position
(31) had no effect. Interestingly, replacing the pyridine ring with
a phenyl ring (32) improved potency, albeit only by a factor of 3.
However, replacing the pyridine or phenyl ring with a morpho-
line group (33) had no significant impact on potency relative
to 9. Shortening the carbon tether by one unit (34) significantly
reduced enzyme potency to above 1 μM. Likewise, replacing the
aryl or morpholine group of 30-33 with a methoxy group (35)
also reduced potency. The differing potencies of 30-35 suggest
that both a three-carbon tether and an aryl group (or a similar-
shaped substituent like a morpholine group) at the end of this
tether are necessary for preserving enzyme potency relative to 9.
This could be the result of π-π stacking or van der Waals
interactions at the edge of the ribose pocket. These data do not
support the presence of a hydrogen bond in this region, as
pyridine 30 and morpholine 33 have similar potencies (100-
150 nM) as 9 while 32, whose phenyl group would not be
considered a hydrogen bond donor or acceptor, is more potent.
Overall, while these oxygen-linked analogues enabled us to gain a
better understanding of the SAR of this series in the ribose
pocket, they did not result in any significant improvement in
potency relative to 9 (outside of compound 32) or in any cellular
activities with IC50 values of less than 1 μM. As a result of these
findings and the similar enzyme potencies of oxygen linked
analogues 8 and 9, further SAR studies within the oxygen linked
series were not pursued.
The incorporation of the sulfonamide moiety was a key
discovery in our efforts to optimize the enzyme potency of 1.
This change afforded a number of compounds with enzyme
potencies below 100 nM. In addition, the cellular potency and
low enzyme to cell shift data for 38 and 45 were especially
encouraging, as these were among the most potent compounds
prepared thus far.
We began our optimization of the nitrogen-linked series from
benzylamine 11, the more potent of the two initial nitrogen
analogues. Our SAR studies on the sulfur series indicated that
aryl substituents were unlikely to significantly improve enzyme
potency. Likewise, the data from the oxygen series indicated that
extending aryl groups further out into the ribose pocket would
not improve potency. Therefore, within this series, we focused
initially on the region around the nitrogen atom. Along these
lines, we introduced a gem-dimethyl moiety at the benzyl
position of 11, resulting in an improvement in potency of over
3-fold (36, Table 6). Introducing a carbonyl at the benzyl
Because of the improved cellular activity of 45, we next
examined its in vitro and In Vivo pharmacokinetic (PK) profile.
Unfortunately, its in vitro clearance was high in both human and
rat liver microsomes (579 and >399 (μL/min)/mg, respec-
tively). This high in vitro clearance translated into high clearance
(3.7 (L/h)/kg) In Vivo when 45 was administered intravenously
(iv) to rats at a dose of 2 mg/kg. Similarly, compound 38 also
exhibited high in vitro clearance (>399 (μL/min)/mg in both
human and rat liver microsomes) and high In Vivo clearance
(4.6 (L/h)/kg in rats). The high In Vivo clearance of 38 and 45
precluded further In Vivo studies with these molecules.
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Table 5. Oxygen-Linker SARa
while removing all nitrogens from this central ring (60) resulted
in a further loss of activity, especially against mTOR.
The central ring modifications were key discoveries in our
efforts to improve enzyme and cellular potencies. As a result of
these changes, we obtained two compounds with single digit
enzyme potencies and one compound with cellular activity
below 50 nM. We also were able to improve mTOR activity.
The PI3KR and mTOR enzyme potencies of 54 led us to
incorporate the 3,5-disubstituted pyridine ring into future
analogues.
The next phase of our SAR investigations involved the
introduction of substituents at the 2-position of the central
pyridine ring. On the basis of the X-ray crystal structure of 1,
we recognized that only small substituents would be tolerated,
and this influenced our studies on this portion of the molecule
(Table 8). The introduction of cyano, methyl, and chloro groups
had minimal effects on PI3KR and mTOR enzyme potencies.
However, the cyano substitution decreased cellular activity
significantly while the methyl and chloro groups resulted in a
5- to 20-fold improvement in potency. The subnanomolar
enzyme and single-digit nanomolar cellular potencies of 70 were
the best for any compound studied in this series to this point. The
use of the 2-chloro-3-sulfonamidopyridine moiety in PI3KR
inhibitors has been previously reported.33,44-47 However, the
combination of this moiety with the benzothiazole hinge binder
described in this work represents a novel scaffold for a PI3KR
inhibitor. Having optimized the in vitro enzyme and cellular
activities, we focused on improving the PK properties of these
molecules.
We first evaluated the in vitro clearance of 70 in human and rat
liver microsomes (HLM and RLM) and found the latter to be
high, precluding further studies with this molecule (Table 9).
The RLM data for phenylsulfonamide 71 were high as well.
Therefore, we pursued SAR studies on the phenylsulfonamide
portion of the molecule, with the goal of achieving HLM and
RLM clearances below 50 (μL/min)/mg.
All of the analogues shown in Table 9 exhibited similar
enzyme and cellular potencies, indicating that there is enough
room in the ribose pocket to accommodate small groups,
regardless of their position on the phenyl ring (not unlike
the sulfur- and oxygen-linked analogues discussed earlier). In
addition, these analogues were all potent mTOR inhibitors.
However, the HLM and RLM data indicated that metabolic
stability differed significantly. Compounds substituted at the
4-position with electron withdrawing substituents exhibited low
in vitro clearance (values below 20 (μL/min)/mg), while the
corresponding 2- and 3-substituted derivatives exhibited higher
clearance values, as evidenced by the chloro, fluoro, and trifluo-
romethyl compounds. These data suggested that the phenyl
4-position was readily metabolized and that substitution at this
position with an electron withdrawing group was critical
for obtaining metabolic stability. We identified 73, 74, 77, and
82 as the only compounds with HLM and RLM values below
50 (μL/min)/mg.
In order to better understand the potency of the compounds in
Table 9, we next examined an X-ray cocrystal of compound 82
bound to PI3Kγ (Figure 2). The X-ray cocrystal structure of 82
overlaps quite closely with 1, with little movement of the protein
observed. As observed with compound 1, the N-acetylbenzothia-
zole forms two hydrogen bonds with Val882 (Figure 2A).
However, the hydrogen bond with the Lys833 residue was not
retained, despite the proximity of the sulfonamide nitrogen
a See Experimental Methods for assay details. b Average of two experi-
ments. c Single experiment.
To this point, our SAR studies produced compounds with
enzyme potencies under 50 nM and cellular potencies under
400 nM. However, for further In Vivo studies to be pursued, the
cellular potencies and In Vivo clearance would have to be
improved. To address these issues, we investigated changes to
the central pyrimidine ring. In particular, we examined the effect
of removing and/or shifting the central ring nitrogens (Table 7).
Changing the core from pyrimidine 37 to 2,4-disubstituted
pyridine 51 dramatically improved both PI3KR enzyme and
cellular potency. Changing to the isomeric 3,5-disubstituted
pyridine 54 improved enzyme potency to single digit nanomolar
levels and lowered mTOR activity as well. One reason for the
improved enzyme potency of 54 could be the formation of
favorable hydrogen bond interactions between the pyridine
nitrogen and the Asp841 and Tyr867 residues in the affinity
pocket. The introduction of an additional nitrogen (i.e., pyrazine
57) decreased the enzyme and cellular potencies relative to 54,
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Table 6. Nitrogen-Linker SARa
a See Experimental Methods for assay details. b Average of two experiments. c Single experiment.
(3.6 Å) and oxygen atoms (3.3 Å).48 The loss of this hydrogen
bond was offset with the gain of two hydrogen bonds formed via
an ordered water molecule located between the central chloro-
pyridine ring nitrogen and the Tyr867 and Asp841 residues.
These additional hydrogen bonds help explain the gain in enzyme
potency achieved with the chloropyridine central ring. In addition,
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Table 7. Central Ring SARa
a See Experimental Methods for assay details. b Average of two experiments. c Single experiment.
the space-filling model indicates that the arylsulfonamide portion
in the ribose pocket has enough space around it to accommodate
various substituents (e.g., trifluoromethyl, chloro, and tert-butyl) at
the 2-, 3-, and 4-positions (Figure 2B). This explains the relatively
flat SAR exhibited by the arylsulfonamides (Table 9), as well as by
the sulfur analogues (Table 4) and oxygen analogues (Table 5). It
also confirms our earlier hypothesis that the ribose pocket would be
able to accommodate groups significantly larger than the thio-
methyl moiety of 1. By contrast, the pyridine chloro group of 82 fits
into a narrow pocket, confirming (based on the X-ray cocrystal
structure of 1) that only small groups would be tolerated at this
position (Figure 2C).
In addition to be being a potent dual PI3KR/mTOR inhibitor,
compound 82 was also a potent inhibitor of the other class I PI3K
isoforms PI3Kβ (Ki = 2.0 nM), PI3Kγ (Ki = 4.7 nM), and PI3Kδ
(Ki = 1.2 nM). The inhibition of cell proliferation by this
compound was determined using an ATP-based cell viability
assay in PTEN-null U-87 MG glioblastoma and PTEN-null PC-3
prostate carcinoma cell lines. Sulfonamide 82 potently inhibited
cell proliferation in both cell lines, exhibiting IC50 values of
68 nM (U-87 MG) and 47 nM (PC-3).
considered a pan inhibitor of class I PI3K’s with limited selec-
tivity over closely related PIKK’s. The selectivity of compound
82 against a panel of over 350 protein and lipid kinases, including
class I PI3Ks, was examined using the AMBIT KINOMEscan
platform.49 In this panel, the competitive binding of 82 was
measured as a percentage of control (POC) at 1 μM. This
compound demonstrated affinity against class I PI3Ks (POC =
0-1%), a finding consistent with the enzyme data mentioned
above. However, it exhibited moderate activity (POC = 25-
30%) against only two other kinases (CDKL2 and MLCK). For
all other kinases assayed, no competitive binding by 82 was
observed.50
In Vivo Studies. We also evaluated the In Vivo PK profile of
this compound. When 82 was administered intravenously (iv) to
rats, it exhibited a low clearance (0.007 (L/h)/kg), a low volume
of distribution (0.17 L/kg), and a long half-life (19 h). The AUC
exposure was 0.32 mg h/mL. When given orally to rats, it
3
exhibited a bioavailability of 103%.51 The ability of this com-
pound to inhibit HGF-stimulated PI3K signaling was evaluated
in a mouse liver pharmacodynamic (PD) assay. Compound
82 was administered to mice at dosages ranging from 0.03 to
3 mg/kg. Human HGF was injected iv 3 h postdose to induce
PI3K-dependent Akt phosphorylation in the liver. Compound 82
induced significant suppression of PI3K signaling, as indicated by
a dose-dependent decrease in phosphorylated Akt (p-Akt) at
Its selectivity against selected members of phosphatidylinosi-
tol kinase-related kinases (PIKK) was also examined. Compound
82 was a potent inhibitor of the class III PI3K, hVPS34 (Ki =31nM),
and DNA-PK (IC50 = 3 nM), suggesting that 82 could be
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Table 8. Central Ring Substitution SARa
temperature indicated. Reactions run at elevated temperature were
run in an oil bath at the temperature indicated. Microwave reactions
were run in a CEM Discover microwave equipped with PowerMAX
feature using the settings described below. All solvents and reagents
obtained from commercial sources were used without further purifica-
tion. Removal of solvents was conducted by using a rotary evaporator,
and residual solvent was removed from nonvolatile compounds using a
vacuum manifold maintained at approximately 1 Torr. All yields
reported are isolated yields.
Analytical TLC was performed with EMD 0.25 mm silica gel 60 plates
with a 254 nm fluorescent indicator. Plates were developed in a covered
chamber and visualized with ultraviolet light. Flash chromatography
refers to column chromatography as described by Still using EMD silica
gel 60, 230-400 mesh, as the stationary phase.58 Where noted, ISCO
purification refers to a Teledyne ISCO purification system using
RediSep columns in the size noted as well as the solvent system and
gradients noted.
PI3KR Ki
pAKT IC50
(nM)c
mTOR IC50
(nM)b
compd
R
(nM)b
54
63
68
70
H
1.6 ( 0.1
1.4 ( 0.1
1.2 ( 0.1
<1.0
103 ( 54b
11 ( 1
CN
Me
Cl
757c
18c
5c
1.6 ( 0.4
6.0 ( 0.4
2.1 ( 1.0
1H NMR spectra were obtained on a Bruker BioSpin GmbH
a See Experimental Methods for assay details. b Average of two experi-
1
magnetic resonance spectrometer. H NMR spectra are reported as
ments. c Single experiment.
chemical shifts in parts per million (ppm) relative to an internal solvent
reference. Peak multiplicity abbreviations are as follows: s (singlet), br s
(broad singlet), d (doublet), dd (doublet of doublets), t (triplet),
q (quartet), quin (quintet), and m (multiplet).
Analytical HPLC and mass spectrometry were conducted using a
reverse-phase Agilent 1100 series HPLC-mass spectrometer. Purities
for final compounds were measured using UV detection at 254 nm and
are g95.0% unless otherwise noted. A detailed description of the HPLC
methods that were used for analyzing final compounds is included in the
Supporting Information. Elemental analyses (% C, % H, % N) were
obtained from Atlantic Microlab, Inc. in Norcross, GA.
Ser473 (Figure 3A). The EC50 was found to be 399 ng/mL.52 In
addition, in a time course liver study at 3 mg/kg, 82 maximally
inhibited Akt phosphorylation 1 h after dosing and demonstra-
ted significant inhibition for 24 h (Figure 3B). Compound 82
appeared rapidly in plasma with a C1h of 2000 ng/mL and
reached a Cmax of ∼3000 ng/mL at 3-8 h.
We next evaluated whether 82 could inhibit tumor growth in
established mouse xenograft models. In mice bearing U87-MG
(PTEN null) glioblastoma xenografts,53,54 treatment with this
compound, administered orally QD, caused a dose-dependent
reduction in tumor growth with an ED50 of 0.26 mg/kg
(Figure 4A). A similar dose dependency was observed in nude
mice bearing A549 (KRAS mutant) lung adenocarcinoma55,56
and HCT11618,57 (KRAS and PI3KR mutant) colon adenocar-
cinoma xenograft models (Figure 4B and Figure 4C). In all
tumor xenograft studies, sulfonamide 82 was well tolerated in
terms of its effect on body weight. In only one instance (the
10 mg/kg arm of the HCT116 xenograft study) did the body
weight drop below 90% of the starting body weight over the
course of the study. These data suggest that compound 82 could
potentially inhibit tumor growth against a wide range of tumors
with different genetic backgrounds and that its efficacy may not
be restricted to those tumors that are caused by a specific
pathway mutation or mechanism of activation.
N-(6-Bromobenzo[d]thiazol-2-yl)acetamide (3). 6-Bromo-
benzo[d]thiazol-2-amine (51.08 g, 223 mmol) was suspended in
DCM (800 mL), and DMAP (30.71 g, 251 mmol) was added. The
mixture was cooled in an ice-water bath under N2, and acetic anhydride
(23.0 mL, 244 mmol) was added. The mixture was allowed to slowly
warm to room temperature while being stirred overnight under N2. The
mixture was treated with 10% HCl (375 mL), and the layers were
separated. The organic phase was filtered, and the solid was washed with
DCM. The aqueous phase was filtered separately, and the solid collected
from this filtration was washed with water and then collected and
combined with the solid collected from the filtration of the organic
phase. The aqueous phase was extracted with 10:1 DCM/MeOH
(300 mL, 200 mL, 150 mL). These organic extracts were combined
with the filtrate from the filtration of the organic phase and concentrated.
The concentrate was treated with Et2O and filtered. This solid was
washed with Et2O and set aside. The first batch of solid collected (from
the separate filtrations of the organic phase and aqueous phase above)
was also treated with Et2O and filtered. This solid was washed with Et2O,
DCM, and Et2O and then combined with the other batch of solid. The
filtrate from this last filtration was concentrated, treated with Et2O, and
filtered. The solid from this last filtration was washed with Et2O and then
combined with all of the other collected solids to afford 3 (59.97 g, 89%
purity, 88% yield). MS (ESI pos ion) m/z: 271 (M þ Hþ, 79Br), 273 (M
þ Hþ, 81Br). 1H NMR (400 MHz, CDCl3): 12.41 (s, 1H), 8.23 (d, J =
2.0 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.56 (m, 1H), 2.21 (s, 3H).
N-(6-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzo-
[d]thiazol-2-yl)acetamide (4a). N-(6-Bromobenzo[d]thiazol-2-yl)-
acetamide (59.97 g, 221 mmol) and 4,4,5,5-tetramethyl-2-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (76.58 g, 302 mmol)
were suspended in DMSO. KOAc (55.8 mL, 893 mmol) was added,
followed by more DMSO. The total amount of DMSO used was 650 mL.
Then 1,10-bis(diphenylphosphino)ferrocenepalladium(II) dichloride di-
chloromethane complex (12.34 g, 16.9 mmol) was added, followed by a
DMSO (6 mL) rinse. The flask was placed in a preheated oil bath (90 ꢀC),
In conclusion, we have discovered and developed a novel
series of potent PI3K/mTOR inhibitors from an initial hit,
compound 1. Extensive SAR studies resulted in the discovery
of sulfonamide 45. However, high in vitro and In Vivo clearance
necessitated changes to the central pyrimidine ring. This led to
the discovery of chloropyridine 70 and, after aryl sulfonamide
SAR studies focused on optimizing in vitro clearance, compound
82. This compound was the most potent analogue prepared in
this series in terms of enzyme and cellular potencies. In addition,
it also proved to be efficacious when tested in a liver PD assay and
three distinct xenograft models. The biological profile of this
molecule validated the benzothiazole series as a means of
inhibiting tumor growth In Vivo by targeting PI3KR and mTOR.
’ EXPERIMENTAL METHODS
Chemistry. All reactions were conducted in a well-ventilated fume
hood under N2 using a Teflon-coated magnetic stirbar at the
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Table 9. Phenylsulfonamide SARa
compd
R
PI3KR Ki (nM)b
pAKT IC50 (nM)c
HLM/RLM ((μL/min)/mg)c
mTOR IC50 (nM)b
70
71
72
73
74
75
76
77
78
79
80
81
82
4-OMe
H
<1.0
5
42/>399
<14/>399
<14/500
17/36
2.1 ( 1.0
<1.0
1.4 ( 0.1
<1.0
5 ( 1b
2-CF3
3-CF3
4-CF3
2-Cl
3-Cl
4-Cl
3-tBu
4-tBu
2-F
8
<1.0
<1.0
12
<1.0
<1.0
19
<14/<14
<14/>399
16/>399
<14/<14
83/610
1.6 ( 1.0
<1.0
<1.0
4.9 ( 0.3b
<1.0
12
<1.0
<1.0
8.7 ( 0.9b
<1.0
<1.0
13
2.1 ( 0.9
2.1 ( 0.3
1.1 ( 0.6
2.3 ( 0.9
2.0 ( 4.8
<1.0
7
135/460
<14/320
<14/>399
<14/<20
<1.0
9
3-F
1.1 ( 0.5
1.2 ( 0.9
13
4-F
6.3 ( 6.5b
a See Experimental Methods for assay details. b Average of at least two experiments. c Single experiment except where noted.
and the mixture was stirred under argon. After 2 h and 20 min, the mixture
was allowed to cool to room temperature and filtered through a pad of
Celite, which was then washed with MeOH. The filtrate was partially
concentrated and partitioned between H2O (1000 mL) and DCM
(550 mL). The layers were separated, and the aqueous phase was extracted
with DCM (3 ꢀ 450 mL). The organic extracts were combined and
divided into two portions of approximately 1 L each. The first portion
was washed with brine (400 mL). The layers were separated, and the
aqueous phase was extracted with DCM (2 ꢀ 400 mL). These organic
phases were combined and set aside. The second portion was
combined with these organic extracts. These combined organic phases
were dried over Na2SO4, filtered, and concentrated. The resultant
solid was treated with Et2O and filtered. The solid was washed with
Et2O and EtOAc, and the filtrate was set aside. The solid was also
washed with MeOH and again with EtOAc. The filtrates from both
filtrations were combined, concentrated, and purified on silica gel
(DCM f 50:1 f 20:1 DCM/MeOH). The fractions with product
were collected, concentrated, treated with Et2O, and filtered. The
solid was collected and set aside. The filtrate was concentrated and
treated with Et2O and filtered, and the resulting solid was washed with
Et2O and hexanes. This solid was combined with the solid from the
first filtration. The filtrate from this second filtration was concentrated
again. This was treated with 1:1 hexanes/Et2O and filtered. The solid
was washed with 1:1 hexanes/Et2O. This time, the filtrate was con-
centrated while the solid from this filtration was discarded. This
filtrate was combinedwith thesolidcollectedfromthe firsttwo filtrations,
concentrated, washed with hexanes, and dried to afford 4a (73.2 g, 88%
purity, 91% yield). MS (ESI pos ion) m/z: 319 (M þ Hþ). 1H NMR (400
MHz, CDCl3, δ): 10.44 (brs, 1H), 8.32 (s, 1H), 7.90 (d, J = 8.02 Hz, 1H),
7.76 (d, J = 8.22 Hz, 1H), 2.31 (s, 3H), 1.38 (s, 12H).
room temperature for 24 h. The precipitate that formed was collected
using a 0.22 μm PTFE membrane. The solids were washed with EtOH
(2 ꢀ 25 mL), H2O (2 ꢀ 25 mL), and EtOH (1 ꢀ 25 mL). The solids
were dried under a stream of N2 and then at 60 ꢀC and <1 mmHg for
48 h to afford 4b (2.60 g, 69.4% yield). 1H NMR (400 MHz, DMF, δ):
2.28 (s, 3 H), 7.50 (d, J = 7.43 Hz, 1H), 7.58 (d, J = 8.02 Hz, 1H), 7.94 (s,
1H), 11.99 (br s, 1H). 13C NMR (101 MHz, DMF, δ): 22.66, 118.97,
123.6, 130.21, 130.85, 147.24, 156.21, 162.58, 169.17. 19F NMR
(376 MHz, DMF, ref = CFCl3 = 0.00, δ): -140.19 (s, 3 F). Anal.
Calcd for (C9H7BF3KN2OS 0.7H2O) C, H, N.
3
N-(6-(2-Chloropyrimidin-4-yl)benzo[d]thiazol-2-yl)-
acetamide (5). 2,4-Dichloropyrimidine (10.31 g, 69.20 mmol),
N-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]thiazol-2-
yl)acetamide (28.52 g, 89.63 mmol), and Pd(PPh3)4 (6.052 g, 5.237
mmol) were suspended in 1,4-dioxane (300 mL), and aqueous Na2CO3
(2.0 M, 70 mL, 140 mmol) was added. Argon was bubbled through the
solution for about 1 min, and then the flask was fitted with a reflux
condenser and placed in a preheated oil bath (∼95 ꢀC) and stirred under
argon overnight. The mixture was cooled to room temperature, and the
liquid was decanted. The residual solid was treated with 1:1 DCM/
MeOH and filtered through a pad of Celite. This pad was washed with
1:1 DCM/MeOH as well as with some DMF. This filtrate was con-
centrated, combined with the decanted material, and concentrated again.
The concentrated material was treated with DCM and filtered. The solid
was washed with DCM, collected, and dried under high vacuum to afford
5 (17.89 g, 85% yield). MS (ESI pos ion) m/z: 305 (M þ Hþ). 1H NMR
(400 MHz, DMSO-d6, δ): 12.58 (s, 1H), 8.87 (s, 1H), 8.82 (d, J =
5.02 Hz, 1H), 8.28 (d, J = 8.53 Hz, 1H), 8.19 (d, J = 5.52 Hz, 1H), 7.87
(d, J = 8.53 Hz, 1H), 2.23 (s, 3H).
Procedure for the Synthesis of 6-8, 10, 14-22, 24-30, and
37-39. N-(6-(2-(Phenylthio)pyrimidin-4-yl)benzo[d]thiazol-
2-yl)acetamide (6). Thiophenol (106.1 mg, 0.963 mmol) was dis-
solved in DMF (1.0 mL), and sodium hydride (67 mg, 60% in mineral oil,
1.7 mmol) was added. The mixture was stirred under N2 at room
temperature for 1 h, and then 5 (50.4 mg, 0.165 mmol) was added. The
mixturewasstirredatroom temperature for 2.5 h and thenwas treatedwith
Potassium N-(6-(Trifluoroboryl)benzo[d]thiazol-2-yl)-
acetamide (4b). A 125 mL polytetrafluoroethylene (PTFE) beaker
was charged with 4a (4.00 g, 12.6 mmol), 75 mL of EtOH, 25 mL of
TFEm and a stir bar. The slurry was sonicated and heated using a 45 ꢀC
H2O bath until a clear solution was apparent. The solution was treated
with potassium fluoride hydrofluoride (2.45 g, 31.4 mmol) and stirred at
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Figure 2. X-ray cocrystal of 82 bound to PI3Kγ. Parts A and B are from a similar vantage point, while part C views the molecule down the C-Cl bond of
the chloropyridine central ring.
Figure 3. Effect of compound 82 on HGF-mediated AKT phosphorylation (A) at 3 h postdose and (B) in a time course study at 3 mg/kg. Bars represent
the mean ( standard deviation (n = 3). Diamonds represent mean plasma concentration. Asterisk denotes (A) p < 0.05 and (B) p < 0.001 compared with
the vehicle (HGF) group.
H2O and saturated NaHCO3. The aqueous phase was extracted with
DCM, and the organic extracts were combined, concentrated, treated with
MeOH and DCM, and filtered. The filtrate was concentrated and the crude
material was purified on HPLC (10% f 95% MeCN/H2O with 0.1% TFA
over 28 min) to afford 6 (16.0 mg, 25% yield). MS (ESI pos ion) m/z: 379
(M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.55 (s, 1H), 8.67-8.62
(m, 2H), 8.10(d,J=8.53 Hz, 1H), 7.84 (d, J=5.02 Hz, 1H), 7.79 (d, J=8.53
Hz, 1H), 7.69 (dd, J = 6.02, 2.51 Hz, 2H), 7.56-7.51 (m, 3H), 2.23 (s, 3H).
N-(6-(2-(Benzylthio)pyrimidin-4-yl)benzo[d]thiazol-2-yl)
acetamide (7). Using benzylthiol and following the procedure used
to prepare 6 gave 7 in 13% yield. MS (ESI pos ion) m/z: 393 (M þ
Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.50 (s, 1H), 8.85 (s, 1H),
8.68 (d, J = 5.0 Hz, 1H), 8.26 (d, J = 8.5 Hz, 1H), 7.84 (m, 2H), 7.49 (d,
J = 7.0 Hz, 2H), 7.32 (t, J = 7.3 Hz, 2H), 7.25 (t, J = 7.3 Hz, 1H), 4.54 (s,
2H), 2.23 (s, 3H).
N-(6-(2-Phenoxypyrimidin-4-yl)benzo[d]thiazol-2-yl)acet-
amide (8). Using phenol and following the procedure used
to prepare 6 gave 8 in 13% yield. MS (ESI pos ion) m/z: 363 (M þ
Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.54 (s, 1H), 8.79 (s, 1H),
8.67 (d, J = 5.52 Hz, 1H), 8.19 (d, J = 8.53 Hz, 1H), 7.88 (d, J = 5.02 Hz,
1H), 7.84 (d, J = 8.53 Hz, 1H), 7.50-7.44 (m, 2H), 7.32-7.26 (m,
3H), 2.23 (s, 3H).
m/z: 397 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ):12.52(brs, 1H),
8.66-8.59 (m, 2H), 8.06 (d, J = 8.53 Hz, 1H), 7.86 (d, J = 5.02 Hz, 1H),
7.82-7.80 (m, 2H), 7.65 (q, J = 6.86 Hz, 1H), 7.45 (t, J = 8.53 Hz, 1H),
7.36 (t, J = 7.28 Hz, 1H), 2.23 (s, 3 H).
N-(6-(2-(3-Fluorophenylthio)pyrimidin-4-yl)benzo[d]thiazol-
2-yl)acetamide (15). Using 3-fluorothiophenol and following the
procedure used to prepare 6 gave 15 in 42% yield. MS (ESI pos ion)
m/z: 397 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.53 (s, 1H),
8.70-8.64 (m, 2H), 8.11 (d, J = 8.53 Hz, 1H), 7.87 (d, J = 5.02 Hz, 1H),
7.81 (d, J = 8.53 Hz, 1H), 7.64-7.51 (m, 3H), 7.39 (t, J = 8.28 Hz, 1H),
2.23 (s, 3H).
N-(6-(2-(4-Fluorophenylthio)pyrimidin-4-yl)benzo[d]thiazol-
2-yl)acetamide (16). Using 4-fluorothiophenol and following the
procedure used to prepare 6 gave 16 in 34% yield. MS (ESI pos ion)
m/z: 397 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.50 (br s,
1H), 8.68-8.61 (m, 2H), 8.09 (d, J = 8.53 Hz, 1H), 7.84 (d,
J = 5.52 Hz, 1H), 7.80 (d, J = 8.53 Hz, 1H), 7.76-7.69 (m, 2H), 7.38 (t,
J = 8.78 Hz, 2H), 2.23 (s, 3H).
N-(6-(2-(2-Methylphenylthio)pyrimidin-4-yl)benzo[d]thiazol-
2-yl)acetamide (17). Using 2-methylthiophenol and following the
procedure used to prepare 6 gave 17 in 25% yield. MS (ESI pos ion)
m/z: 393 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.53 (s, 1H),
8.64-8.60 (m, 2H), 8.07 (dd, J = 8.61, 1.76 Hz, 1H), 7.82 (d, J =
5.48 Hz, 1H), 7.78 (d, J = 8.61 Hz, 1H), 7.64 (d, J = 7.43 Hz, 1H), 7.48-
7.44 (m, 2H), 7.35-7.29 (m, 1H), 2.38 (s, 3H), 2.23 (s, 3H).
N-(6-(2-(3-Methylphenylthio)pyrimidin-4-yl)benzo[d]thiazol-
2-yl)acetamide (18). Using 3-methylthiophenol and following the
procedure used to prepare 6 gave 18 in 29% yield. MS (ESI pos ion)
m/z: 393 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.53 (s, 1H),
8.67 (s, 1H), 8.64 (d, J = 5.52 Hz, 1H), 8.11 (d, J = 8.53 Hz, 1H), 7.84 (d,
J = 5.52 Hz, 1H), 7.80 (d, J = 9.03 Hz, 1H), 7.54 (s, 1H), 7.49-7.45 (m,
1H), 7.41 (t, J = 7.53 Hz, 1H), 7.36-7.31 (m, 1H), 2.39 (s, 3H),
2.23 (s, 3H).
N-(6-(2-(Phenylamino)pyrimidin-4-yl)benzo[d]thiazol-2-yl)
acetamide (10). Using aniline and DMSO and following the
procedure used to prepare 6 gave 10 in 21% yield. This analogue
was purified by washing the crude material with Et2O and DCM. MS
(ESI pos ion) m/z: 362 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6,
δ): 2.17 (s, 3H), 7.35 (d, J = 5.28 Hz, 1H), 7.54-7.66 (m, 5H), 7.74
(d, J = 8.61 Hz, 1H), 8.20 (d, J = 6.85 Hz, 1H), 8.25 (d, J = 8.61 Hz,
1H), 8.78 (s, 1H).
N-(6-(2-(2-Fluorophenylthio)pyrimidin-4-yl)benzo[d]thiazol-
2-yl)acetamide (14). Using 2-fluorothiophenol and following the
procedure used to prepare 6 gave 14 in 25% yield. MS (ESI pos ion)
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Figure 4. Daily dosing with 82 induced tumor stasis and growth delay in established xenograft models (A) U-87 MG glioblastoma, (B) A549 lung
adenocarcinoma, and (C) HCT116 colon adenocarcinoma. Arrow denotes the first day of dosing. The 10 mpk arm had to be stopped on day 32 in the
HCT116 model because of a drop in body weight to 85% of the initial body weight. (D) Summary of ED50 and AUC (at ED50) values for the three
studies. Asterisk denotes p < 0.0001 compared with the vehicle group.
N-(6-(2-(4-Methylphenylthio)pyrimidin-4-yl)benzo[d]thiazol-
2-yl)acetamide (19). Using 4-methylthiophenol and following the
procedure used to prepare 6 gave 19 in 40% yield. MS (ESI pos ion)
m/z: 393 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.52 (s, 1H),
8.65 (s, 1H), 8.62 (d, J = 5.02 Hz, 1H), 8.11 (d, J = 8.53 Hz, 1H), 7.84-
7.78 (m, 2H), 7.55 (d, J = 7.53 Hz, 2H), 7.34 (d, J = 8.03 Hz, 2H), 2.41
(s, 3H), 2.23 (s, 3H).
following the procedure used to prepare 6 gave 24 in 47% yield.
This compound was purified by washing with Et2O instead of by HPLC.
MS (ESI pos ion) m/z: 391 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6,
δ): 12.55 (br s, 1H), 8.82 (s, 1H), 8.65 (d, J = 5.52 Hz, 1H), 8.24 (d, J =
8.53 Hz, 1H), 7.79 (d, J = 8.53 Hz, 1H), 7.76 (d, J = 5.02 Hz, 1H), 7.35 (s,
1H), 7.33-7.25 (m, 2H), 7.15 (d, J = 6.02 Hz, 1H), 5.47 (s, 2H), 2.33 (s,
3H), 2.20 (s, 3H).
N-(6-(2-(2-Methoxyphenylthio)pyrimidin-4-yl)benzo[d]
thiazol-2-yl)acetamide (20). Using 2-methoxythiophenol and
following the procedure used to prepare 6 gave 20 in 46% yield. MS
(ESI pos ion) m/z: 409 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6,
δ): 12.52 (s, 1H), 8.62 (s, 1H), 8.59 (d, J = 5.52 Hz, 1H), 8.07 (d,
J = 8.53 Hz, 1H), 7.81-7.76 (m, 2H), 7.61 (d, J = 7.53 Hz, 1H), 7.55
(t, J = 7.78 Hz, 1H), 7.20 (d, J = 8.03 Hz, 1H), 7.07 (t, J = 7.53 Hz,
1H), 3.75 (s, 3H), 2.23 (s, 3H).
N-(6-(2-(3-Methoxybenzyloxy)pyrimidin-4-yl)benzo[d]
thiazol-2-yl)acetamide (25). Using (3-methoxyphenyl)methanol
and following the procedure used to prepare 6 gave 25 in 69% yield. This
compound was purified by washing with Et2O instead of by HPLC. MS
(ESI pos ion) m/z: 407 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ):
12.45 (br s, 1H), 8.77 (br s, 1H), 8.63 (d, J = 5.02 Hz, 1H), 8.21 (d,
J = 8.03 Hz, 1H), 7.77-7.69 (m, 2H), 7.31 (t, J = 7.78 Hz, 1H), 7.11-7.06
(m, 2H), 6.90 (d, J = 9.54 Hz, 1H), 5.48 (s, 2H), 3.76 (s, 3H), 2.16 (s, 3H).
N-(6-(2-(4-Fluorobenzyloxy)pyrimidin-4-yl)benzo[d]thiazol-
2-yl)acetamide (26). Using (4-fluorophenyl)methanol and following
the procedure usedtoprepare 6 gave26 in37% yield. This compound was
purified bywashing with Et2O, MeOH, and Et2O instead ofbyHPLC. MS
(ESI pos ion) m/z: 395 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ):
12.51 (s, 1H), 8.88 (s, 1H), 8.68 (d, J = 5.02 Hz, 1H), 8.28 (d, J = 8.53 Hz,
1H), 7.85(d, J =8.53Hz, 1H), 7.78 (d, J =5.02 Hz, 1H), 7.58 (dd, J =7.78,
5.77 Hz, 2H), 7.23 (t, J = 8.78 Hz, 2H), 5.50 (s, 2H), 2.23 (s, 3H).
N-(6-(2-(4-Methylbenzyloxy)pyrimidin-4-yl)benzo[d]thiazol-
2-yl)acetamide (27). Using (4-methylphenyl)methanol and follow-
ing the procedure used to prepare 6 gave 27 in 54% yield. This
compound was purified by washing with Et2O instead of by HPLC.
MS (ESI pos ion) m/z: 391 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6,
δ): 12.50 (br s, 1H), 8.81 (br s, 1H), 8.64 (d, J = 5.02 Hz, 1H), 8.24 (d,
J = 9.03 Hz, 1H), 7.83-7.73 (m, 2H), 7.41 (d, J = 8.03 Hz, 2H), 7.21 (d,
J = 7.53 Hz, 2H), 5.46 (s, 2H), 2.31 (s, 3H), 2.19 (s, 3H).
N-(6-(2-(3-Methoxyphenylthio)pyrimidin-4-yl)benzo[d]
thiazol-2-yl)acetamide (21). Using 3-methoxythiophenol and fol-
lowing the procedure used to prepare 6 gave 21 in 34% yield. MS (ESI pos
1
ion) m/z: 409 (M þ Hþ). H NMR (400 MHz, DMSO-d6, δ): 12.41
(br s, 1H), 8.64-8.58 (m, 2H), 8.10 (d, J = 8.03 Hz, 1H), 7.83 (d, J = 5.02
Hz, 1H), 7.76 (d, J = 8.53 Hz, 1H), 7.44 (t, J = 8.03 Hz, 1H), 7.29-7.22
(m, 2H), 7.10 (d, J = 7.53 Hz, 1H), 3.81 (s, 3H), 2.20 (s, 3H).
N-(6-(2-(4-Methoxyphenylthio)pyrimidin-4-yl)benzo[d]
thiazol-2-yl)acetamide (22). Using 3-methoxythiophenol and
following the procedure used to prepare 6 gave 22 in 38% yield. MS
(ESI pos ion) m/z: 409 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6,
δ): 12.52 (s, 1H), 8.65 (s, 1H), 8.61 (d, J = 5.52 Hz, 1H), 8.11 (d, J =
8.53 Hz, 1H), 7.83-7.78 (m, 2H), 7.58 (d, J = 8.53 Hz, 2H), 7.09 (d,
J = 8.53 Hz, 2H), 3.85 (s, 3H), 2.23 (s, 3H).
N-(6-(2-(3-Methylbenzyloxy)pyrimidin-4-yl)benzo[d]thiazol-
2-yl)acetamide (24). Using (3-methylphenyl)methanol and
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N-(6-(2-(4-Methoxybenzyloxy)pyrimidin-4-yl)benzo[d]
thiazol-2-yl)acetamide (28). Using (4-methoxyphenyl)methanol
and following the procedure used to prepare 6 gave 28 in 31% yield. This
compound was purified by washing with Et2O, MeOH, and Et2O instead
of by HPLC. MS (ESI pos ion) m/z: 407 (M þ Hþ). 1H NMR
(400 MHz, DMSO-d6, δ): 12.50 (br s, 1H), 8.77 (br s, 1H), 8.63 (d, J =
5.02 Hz, 1H), 8.22 (d, J = 8.53 Hz, 1H), 7.73 (d, J = 5.02 Hz, 2H), 7.46
(d, J = 8.03 Hz, 2H), 6.96 (d, J = 8.53 Hz, 2H), 5.43 (s, 2H), 3.76 (s, 3H),
2.16 (s, 3H).
N-(6-(2-(Pyridin-4-ylmethoxy)pyrimidin-4-yl)benzo[d]
thiazol-2-yl)acetamide (29). Using 4-(hydroxymethyl)pyridine
and following the procedure used to prepare 6 gave 29 in 51% yield. This
compound was purified by washing with Et2O instead of by HPLC. MS
(ESI pos ion) m/z: 378 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ):
8.81 (s, 1H) 8.67 (d, J = 5.02 Hz, 1H), 8.59 (d, J = 5.52 Hz, 2H), 8.23 (d,
J = 8.53 Hz, 1H), 7.81-7.76 (m, 2H), 7.49 (d, J = 5.52 Hz, 2H), 5.57 (s,
2H), 2.20 (s, 3H).
8.67 (s, 1H), 8.27 (d, J = 6.53 Hz, 1H), 7.84 (d, J = 7.03 Hz, 1H), 7.77 (s,
1H), 7.60-7.29 (m, 5H), 5.51 (s, 2H), 2.23 (s, 3H).
N-(6-(2-(Benzylamino)pyrimidin-4-yl)benzo[d]thiazol-2-
yl)acetamide (11). Using benzylamine and following the proce-
dure used to prepare 9 gave 11 in 22% yield. MS (ESI pos ion) m/z:
1
376 (M þ Hþ). H NMR (400 MHz, DMSO-d6, δ): 12.48 (s, 1H),
8.73 (s, 1H), 8.36 (d, J = 5.52 Hz, 1H), 8.19 (d, J = 8.53 Hz, 1H), 7.93
(br s, 1H), 7.81 (d, J = 9.03 Hz, 1H), 7.44-7.36 (m, 2H), 7.32 (d, J =
14.56 Hz, 2H), 7.29-7.18 (m, 2H), 4.63 (br s, 2H), 2.23 (s, 3H).
N-(6-(2-(3-Fluorobenzyloxy)pyrimidin-4-yl)benzo[d]thiazol-
2-yl)acetamide (23). Using (3-fluorophenyl)methanol and follow-
ing the procedure used to prepare 9 gave 23 in 22% yield. The mixture
was reheated a second time in the microwave at 120 ꢀC and 300 W with a
5 min ramp time and 10 min run time. MS (ESI pos ion) m/z: 395 (M þ
Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.51 (br s, 1H), 8.87 (s, 1H),
8.68 (d, J = 3.51 Hz, 1H), 8.27 (d, J = 8.03 Hz, 1H), 7.84 (d, J = 8.03 Hz,
1H), 7.78 (d, J = 3.51 Hz, 1H), 7.52-7.29 (m, 3H), 7.22-7.11 (m, 1H),
5.53 (s, 2H), 2.23 (s, 3H).
N-(6-(2-(3-(Pyridin-4-yl)propoxy)pyrimidin-4-yl)benzo[d]
thiazol-2-yl)acetamide (30). Using 3-(pyridin-4-yl)propan-1-ol
and following the procedure used to prepare 6 gave 30 in 18% yield.
This compound was purified by washing with Et2O instead of by HPLC.
MS (ESI pos ion) m/z: 406 (M þ Hþ). 1H NMR (400 MHz, DMSO-
d6, δ): 12.48 (br s, 1H), 8.83 (s, 1H), 8.64 (d, J = 5.52 Hz, 1H), 8.47 (d,
J = 5.52 Hz, 2H), 8.24 (d, J = 8.53 Hz, 1H), 7.84 (d, J = 8.53 Hz, 1H),
7.74 (d, J = 5.02 Hz, 1H), 7.30 (d, J = 5.52 Hz, 2H), 4.43 (t, J = 6.53 Hz,
2H), 2.80 (t, J = 7.78 Hz, 2H), 2.23 (s, 3H), 2.16-2.08 (m, 2H).
N-(6-(2-(4-Methoxyphenylsulfonamido)pyrimidin-4-yl)
benzo[d]thiazol-2-yl)acetamide (37). Using 4-methoxyben-
zenesulfonamide and DMSO and following the procedure used to
prepare 6 gave 37 in 29% yield. The reaction was run at 125 ꢀC
overnight, and following HPLC purification, the material was washed
with Et2O, MeOH, and Et2O to afford the product. MS (ESI pos ion)
m/z: 456 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.53 (s,
1H), 11.66 (br s, 1H), 8.59-8.52 (m, 2H), 8.15 (d, J = 8.53 Hz, 1H),
7.98 (d, J = 8.53 Hz, 2H), 7.85 (d, J = 8.53 Hz, 1H), 7.65 (d, J =
4.52 Hz, 1H), 7.10 (d, J = 9.03 Hz, 2H), 3.81 (s, 3H), 2.24 (s, 3H).
N-(6-(2-(4-Methoxy-N-methylphenylsulfonamido)pyrimidin-
4-yl)benzo[d]thiazol-2-yl)acetamide (38). Using 4-methoxy-N-
methylbenzenesulfonamide and DMSO and following the procedure
used to prepare 6 gave 38 in 25% yield. The reaction was run at 125 ꢀC
overnight. MS (ESI pos ion) m/z: 470 (M þ Hþ). 1H NMR (400 MHz,
DMSO-d6, δ): 12.52 (br s, 1H), 8.64 (d, J = 5.02 Hz, 1H), 8.56 (s, 1H),
8.14 (d, J = 8.53 Hz, 1H), 7.99 (d, J = 8.53 Hz, 2H), 7.84 (d, J = 8.53 Hz,
1H), 7.71 (d, J = 5.02 Hz, 1H), 7.10 (d, J = 9.03 Hz, 2H), 3.82 (s, 3H),
3.69 (s, 3H), 2.23 (s, 3H).
N-(6-(2-(3-(Pyridin-3-yl)propoxy)pyrimidin-4-yl)benzo[d]
thiazol-2-yl)acetamide (31). Using 3-pyridinepropanol and fol-
lowing the procedure used to prepare 9 gave 31 in 18% yield. MS
(ESI pos ion) m/z: 406 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ):
12.51 (s, 1H), 8.86-8.80 (m, 2H), 8.72 (d, J = 5.02 Hz, 1H), 8.64 (d, J =
5.52 Hz, 1H), 8.39 (d, J = 8.03 Hz, 1H), 8.25 (dd, J = 8.53, 1.51 Hz, 1H),
7.91-7.82 (m, 2H), 7.75 (d, J = 5.02 Hz, 1H), 4.47 (t, J = 6.27 Hz, 2H),
2.98 (t, J = 7.53 Hz, 2H), 2.23 (s, 3H), 2.22-2.14 (m, 2H).
N-(6-(2-(3-Phenylpropoxy)pyrimidin-4-yl)benzo[d]thiazol-
2-yl)acetamide (32). Using 3-phenylpropan-1-ol and following the
procedure used to prepare 9 gave 32 in 6% yield. MS (ESI pos ion) m/z:
405 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.50 (s, 1H), 8.83
(s, 1H), 8.64 (d, J = 5.02 Hz, 1H), 8.25 (d, J = 8.53 Hz, 1H), 7.84 (d, J =
8.53 Hz, 1H), 7.74 (d, J = 5.52 Hz, 1H), 7.34-7.24 (m, 4H), 7.23-7.16
(m, 1H), 4.42 (t, J = 6.53 Hz, 2H), 2.78 (t, J = 7.53 Hz, 2H), 2.23 (s, 3H),
2.14-2.04 (m, 2H).
N-(6-(2-(3-Morpholinopropoxy)pyrimidin-4-yl)benzo[d]
thiazol-2-yl)acetamide (33). Using 4-(3-hydroxypropyl)morpho-
line and following the procedure used to prepare 9 gave 33 in 5% yield as
a TFA salt. Before HPLC purification, the crude material was filtered
through silica gel (15:1 DCM/MeOH f 5:1 DCM/2 N ammonia in
MeOH). MS (ESI pos ion) m/z: 414 (M þ Hþ). 1H NMR (400 MHz,
DMSO-d6, δ): 12.52 (br s, 1H), 9.83 (br s, 1H), 8.85 (s, 1H), 8.67 (d, J =
5.52 Hz, 1H), 8.27 (d, J = 8.03 Hz, 1H), 7.86 (d, J = 8.53 Hz, 1H), 7.78
(d, J = 5.02 Hz, 1H), 4.51 (t, J = 5.77 Hz, 2H), 4.05-3.95 (m, 2H),
3.71-3.61 (m, 2H), 3.51 (d, J = 11.04 Hz, 2H), 3.40-3.29 (m, 2H),
3.16-3.05 (m, 2H), 2.30-2.16 (m, 5H).
N-(6-(2-(2-Morpholinoethoxy)pyrimidin-4-yl)benzo[d]
thiazol-2-yl)acetamide (34). Using 4-(2-hydroxyethyl)morpho-
line and following the procedure used to prepare 9 gave 34 in 5% yield as
a TFA salt. MS (ESI pos ion) m/z: 400 (M þ Hþ). 1H NMR (400 MHz,
DMSO-d6, δ): 12.53 (br s, 1H), 9.74 (br s, 1H), 8.87 (s, 1H), 8.71 (d, J =
5.52 Hz, 1H), 8.29 (d, J = 8.53 Hz, 1H), 7.89-7.82 (m, 2H), 4.80 (br s,
2H), 3.99-3.89 (m, 2H), 3.76-3.71 (m, 2H), 3.69-3.64 (m, 2H),
3.23-3.18 (m, 2H), 2.24 (s, 3H). (The additional two methylene
protons could be buried under the H2O peak, which ranges from
3.64-3.35.)
N-(6-(2-(3-Methoxypropoxy)pyrimidin-4-yl)benzo[d]thiazol-
2-yl)acetamide (35). Using 3-methoxypropan-1-ol and following the
procedure used to prepare 9 gave 35 in 7% yield. MS (ESI pos ion) m/z: 359
(M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.50 (s, 1H), 8.85 (s,
1H),8.64(d,J=5.02 Hz, 1H), 8.27 (d, J=9.03 Hz, 1H), 7.85 (d, J=8.53 Hz,
1H), 7.74 (d, J = 5.02 Hz, 1H), 4.46 (t, J = 6.27 Hz, 2H), 3.51 (t, J = 6.27 Hz,
2H), 3.27 (s, 3H), 2.23 (s, 3H), 2.02 (quin, J = 6.40 Hz, 2H).
N-(6-(2-(N-Ethyl-4-methoxyphenylsulfonamido)pyrimidin-
4-yl)benzo[d]thiazol-2-yl)acetamide (39). Using 4-methoxy-N-
ethylbenzenesulfonamide and DMSO and following the procedure used
to prepare 6 gave 39 in 12% yield. The reaction was run at 125 ꢀC
overnight. MS (ESI pos ion) m/z: 484 (M þ Hþ). 1H NMR (400 MHz,
DMSO-d6, δ): 12.51 (br s, 1H), 8.63 (d, J = 5.02 Hz, 1H), 8.48 (s, 1H),
8.08 (d, J = 8.53 Hz, 1H), 7.98 (d, J = 8.53 Hz, 2H), 7.83 (d, J = 8.53 Hz,
1H), 7.68 (d, J = 5.02 Hz, 1H), 7.10 (d, J = 8.53 Hz, 2H), 4.37-4.26 (m,
2H), 3.82 (s, 3H), 2.23 (s, 3H), 1.42 (t, J = 6.53 Hz, 3H).
Procedure for the Synthesis of 9, 11, 23, 31-35, 40, and
42-44. N-(6-(2-(Benzyloxy)pyrimidin-4-yl)benzo[d]thiazol-
2-yl)acetamide (9). Compound 5 (60.8 mg, 0.200 mmol) and
benzyl alcohol (0.20 mL, 1.9 mmol) were suspended in pyridine (0.84
mL) and sealed in a microwave vial. The vial was heated in the CEM
microwave at 120 ꢀC and 300 W with a 5 min ramp time and 20 min run
time. The mixture was cooled to room temperature, concentrated, and
purified on HPLC (10% f 95% MeCN/H2O with 0.1% TFA over 28-
40 min) to afford 9 (4.4 mg, 6% yield). MS (ESI pos ion) m/z: 377 (M þ
Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.51 (br s, 1H), 8.87 (s, 1H),
N-(6-(2-(2-Fluorophenylsulfonamido)pyrimidin-4-yl)benzo-
[d]thiazol-2-yl)acetamide (40). Using 2-fluorobenzenesulfonamide
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and following the procedure used to prepare 9 gave 40 in 17% yield. Before
HPLC purification, the crude material was filtered through silica gel (10:1
DCM/MeOH). After HPLC purification, the material was washed with
and 0.5 M aqueous EDTA (8 mL). The mixture was stirred at room
temperature for 5 min and then was filtered through a Celite pad, which
was washed with H2O, MeOH, DCM, and EtOAc. The layers of the filtrate
were separated, and the aqueous phase was extracted with DCM. The
organic phases were combined, dried over Na2SO4, filtered, concentrated,
and purified on silica gel (30:1 f 20:1 DCM/MeOH). The fractions with
product were collected, concentrated, treated with MeOH, and filtered.
The solid was washed with MeOH, Et2O, and MeOH. The filtrate and
solid were combined, concentrated, treated with DCM, and filtered. The
solidwaswashedwithDCMandMeOHandwasfoundtobelessthan95%
pure by HPLC. The filtrate and solid were combined, concentrated, and
purified on HPLC (10% f 100% MeCN/H2O with 0.1% TFA over
28 min) to afford 13 (26.7 mg, 8% yield). MS (ESI pos ion) m/z: 375 (M
þ Hþ). 1H NMR (400 MHz, CDCl3, δ): 8.80 (d, J = 5.48 Hz, 1H), 8.64
(d, J = 1.37 Hz, 1H), 8.23 (dd, J = 8.61, 1.56 Hz, 1H), 7.91 (d, J = 8.80 Hz,
1H), 7.68 (d, J = 5.48 Hz, 1H), 7.34-7.19 (m, 5H), 3.37-3.53 (m, 2H),
3.19-3.34 (m, 2H), 2.46 (s, 3H).
N-(6-(2-(2-Phenylpropan-2-ylamino)pyrimidin-4-yl)ben-
zo[d]thiazol-2-yl)acetamide (36). A solution of 5 (0.100 g, 0.328
mmol), cumylamine (0.05 mL, 0.4 mmol), and Cs2CO3 (0.2 g, 0.6
mmol) in DMF (1 mL) was heated in the CEM microwave at 180 ꢀC
and 180 W for 20 min. The mixture was diluted with DCM, washed
with H2O three times, dried over Na2SO4, and concentrated. The
residue was purified by HPLC (5% f 100% MeCN/H2O with 0.05%
TFA) to give 36 as an off-white solid (2.5 mg, 2% yield). MS (ESI pos
ion) m/z: 404 (M þ Hþ). 1H NMR (300 MHz, CDCl3, δ): 10.40 (br s,
1H), 8.85 (d, J = 4.82 Hz, 1H), 8.05 (d, J = 6.43 Hz, 1H), 7.78 (dd, J =
7.82, 6.36 Hz, 1H), 7.73-7.70 (m, 2H), 7.57-7.52 (m, 2H), 7.43 (t,
J = 7.67 Hz, 2H), 7.32-7.26 (m, 1H), 7.08 (d, J = 6.58 Hz, 1H), 2.42 (s,
3H), 1.88 (s, 6H).
1
Et2O to afford 40. MS (ESI pos ion) m/z: 444 (M þ Hþ). H NMR
(400MHz, DMSO-d6, δ): 12.54 (br s, 1H), 8.57-8.43 (m, 2H), 8.18 (t, J=
8.28 Hz, 1H), 7.99 (d, J = 7.53 Hz, 1H), 7.80 (d, J = 8.53 Hz, 1H), 7.72-
7.57 (m, 2H), 7.49-7.42 (m, 1H), 7.37 (t, J = 9.03 Hz, 1H), 2.24 (s, 3H).
N-(6-(2-(3-Fluorophenylsulfonamido)pyrimidin-4-yl)benzo-
[d]thiazol-2-yl)acetamide (42). Using 3-fluorobenzenesulfona-
mide and following the procedure used to prepare 9 gave 42 in 6%
yield. Before HPLC purification, the crude material was filtered through
silica gel (10:1 DCM/MeOH). MS (ESI pos ion) m/z: 444 (M þ Hþ).
1H NMR (400 MHz, DMSO-d6, δ): 12.54 (s, 1H), 8.59-8.53 (m, 2H),
8.10 (d, J = 9.03 Hz, 1H), 7.88-7.80 (m, 3H), 7.69-7.61 (m, 2H),
7.53-7.45 (m, 1H), 2.24 (s, 3H).
N-(6-(2-(4-Fluorophenylsulfonamido)pyrimidin-4-yl)benzo-
[d]thiazol-2-yl)acetamide (43). Using 4-fluorobenzenesulfona-
mide and following the procedure used to prepare 9 gave 43 in 7%
yield. Before HPLC purification, the crude material was filtered through
silica gel (10:1 DCM/MeOH). MS (ESI pos ion) m/z: 444 (M þ Hþ).
1H NMR (400 MHz, DMSO-d6, δ): 12.54 (s, 1H), 8.58 (s, 1H), 8.55 (d,
J = 5.02 Hz, 1H), 8.14-8.07 (m, 3H), 7.85 (d, J = 8.53 Hz, 1H), 7.66 (d,
J = 6.02 Hz, 1H), 7.43 (t, J = 8.78 Hz, 2H), 2.24 (s, 3H).
N-(6-(2-(4-Methylphenylsulfonamido)pyrimidin-4-yl)benzo-
[d]thiazol-2-yl)acetamide (44). Using 4-methylbenzenesulfona-
mide and following the procedure used to prepare 9 gave 44 in 13%
yield. Instead of HPLC purification, the crude material was filtered
through silica gel (10:1 DCM/MeOH). This filtrate was concentrated,
treated with Et2O, and filtered. The solid was washed with Et2O, MeOH,
and Et2O to afford 44. MS (ESI pos ion) m/z: 440 (M þ Hþ). 1H NMR
(400 MHz, DMSO-d6, δ): 12.53 (s, 1H), 11.77 (br s, 1H), 8.55 (d, J =
4.52 Hz, 1H), 8.52 (br s, 1H), 8.13 (d, J = 7.53 Hz, 1H), 7.93 (d, J = 8.03
Hz, 2H), 7.84 (d, J = 8.53 Hz, 1H), 7.66-7.60 (m, 1H), 7.39 (d, J = 8.03
Hz, 2H), 2.35 (s, 3H), 2.24 (s, 3H).
N-(6-(2-Benzylpyrimidin-4-yl)benzo[d]thiazol-2-yl)acetamide
(12). N-(6-(2-Chloropyrimidin-4-yl)benzo[d]thiazol-2-yl)acetamide
(5, 59.0 mg, 0.194 mmol) was suspended in THF (1.8 mL) to which
Pd(PPh3)4 (25.6 mg, 0.022 mmol) and benzylzinc bromide (0.5 M solution
in THF, 0.55 mL, 0.275 mmol) were added under argon. The reaction
solution was stirred at room temperature. After 2 h, the mixture was heated to
80 ꢀC, and stirring was continued under argon. After 100 min, additional
Pd(PPh3)4 (21 mg, 0.018 mmol) and benzylzinc bromide (0.5 M solution in
THF, 0.57 mL, 0.285 mmol) were added, and the mixture was stirred. After
another hour, additional benzylzinc bromide (0.5 M solution in THF,
0.73 mL, 0.365 mmol) was added, and stirring was continued at 80 ꢀC.
The mixture was stirred overnight and quenched with saturated ammonium
chloride (1.5 mL) and 0.5 M EDTA (2.5 mL), extracted with 10:1 DCM/
MeOH, and the organic phases were dried over Na2SO4, filtered through
Celite, and concentrated. The crude concentrate was purified on a silica gel
column (20:1 f 5:1 DCM/MeOH), and the product-containing fractions
were collected, concentrated, treated with Et2O and MeOH, and filtered. The
solid was collected and purified on HPLC (10% 95% MeCN/H2Owith0.1%
TFA over 40 min) to provide 12 (8 mg, 11% yield). MS (ESI pos ion) m/z:
361 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.50 (s, 1H), 8.85 (s,
1H), 8.78 (d, J = 5.5 Hz, 1H), 8.29 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 5.5 Hz,
1H), 7.85 (d, J = 8.5 Hz, 1H), 7.38 (m, 2H), 7.31 (m, 2H), 7.21 (m, 1H),
4.28 (s, 2H), 2.23 (s, 3H).
Procedure for the Synthesis of 41 and 45. N-(6-(2-(2-
Fluoro-N-methylphenylsulfonamido)pyrimidin-4-yl)benzo-
[d]thiazol-2-yl)acetamide (41). A microwave vial equipped with a
stir bar was charged with 2-fluoro-N-methylbenzenesulfonamide (0.23 g,
1.2 mmol) in DMF (3 mL). Then NaH (0.12 g, 4.9 mmol) was added to
the mixture and the mixture was allowed to stir for 30 min. Then
Pd(OAc)2 (11 mg, 0.049 mmol), 5 (150 mg, 0.492 mmol), and
Xantphos (10 mg, 0.017 mmol) were added to the mixture. The vial
was capped and placed into a CEM microwave for 10 min at 100 ꢀC,
while 100 W of energy was supplied via Powermax. The mixture was
added to a round-bottomed flask and diluted with H2O (150 mL). The
mixture was allowed to stir overnight. The resulting precipitate was
collected by filtration and washed with hexanes (3 ꢀ 50 mL) and 1:1
hexanes/Et2O (50 mL). Then the crude material was recrystallized from
1:1 MeOH/DCM and then from hexanes to give 41 (35 mg, 16% yield)
1
as a brown solid. MS (ESI pos ion) m/z: 458 (M þ Hþ). H NMR
(400 MHz, DMSO-d6, δ): 12.50 (s, 1H), 8.64 (d, J = 5.02 Hz, 1H), 8.48
(s, 1H), 8.22 (t, J = 7.53 Hz, 1H), 7.99 (d, J = 8.53 Hz, 1H), 7.79 (d, J =
8.53 Hz, 1H), 7.76-7.69 (m, 2H), 7.52-7.39 (m, 2H), 3.70 (s, 3H),
2.23 (s, 3H).
N-(6-(2-(4-Methyl-N-methylphenylsulfonamido)pyrimidin-
4-yl)benzo[d]thiazol-2-yl)acetamide (45). Using N-methyl-4-
toluenesulfonamide and following the procedure used to prepare 41
gave 45 in 36% yield. MS (ESI pos ion) m/z: 454 (M þ Hþ). 1H NMR
(400 MHz, DMSO-d6, δ): 12.52 (s, 1H), 8.64 (s, 1H), 8.48 (s, 1H), 8.10
(d, J = 5.0 Hz, 1H), 7.93 (s, 2H), 7.83 (s, 1H), 7.71 (s, 1H), 7.40 (s, 2H),
3.70 (s, 3H), 2.37 (s, 3H), 2.24 (s, 3H).
N-(4-Chloropyrimidin-2-yl)-4-methoxybenzamide (47). A
mixture of 4-chloropyrimidin-2-amine (46, 0.65 g, 5.0 mmol), triethy-
lamine (1.4 mL, 10 mmol), and 4-anisoyl chloride (0.66 mL, 5.0 mmol)
in DCM (20 mL) was stirred for 16 h at room temperature. Then the
mixture was concentrated and the residue was purified on silica gel (0%
f 8% MeOH in DCM) to afford 47 (70 mg, 5%) as a white solid. MS
(ESI pos ion) m/z: 264 (M þ Hþ). 1H NMR (300 MHz, CDCl3, δ):
N-(6-(2-Phenethylpyrimidin-4-yl)benzo[d]thiazol-2-yl)acet-
amide (13). Compound 5 (277 mg, 0.909 mmol) and Pd(PPh3)4
(160 mg, 0.138 mmol) were suspended in THF (5.0 mL), and
phenethylzinc(II) bromide (0.5 M in THF, 8.3 mL, 4.2 mmol) was added.
The flask was fitted with a reflux condenser and was put in a preheated oil
bath (80 ꢀC) and stirred for 1 h. The mixture was cooled to room
temperature and was treated with saturated ammonium chloride (5 mL)
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8.56 (s, 1H), 8.54 (s, 1H), 8.29 (d, J = 5.70 Hz, 1H), 7.91-7.86 (m, 2H),
7.04-7.00 (m, 2H), 3.91 (s, 3H).
53 (165 mg, 0.481 mmol) in 1,4-dioxane (3 mL) was added 4a (223 mg,
0.701 mmol), Fibercat (24 mg, 20% Pd), and aqueous 2 M Na2CO3
(0.6 mL, 1.2 mmol). A stir bar was added to the vial, and then the vial was
capped and placed into CEM microwave for 10 min at 100 ꢀC, while
100 W of energy was supplied via Powermax. The mixture was allowed to
cool to ambient temperature and diluted with DCM and saturated
NaHCO3. The organic layer was collected by extracting with DCM (3 ꢀ
20 mL). The organic extracts were combined, dried over Na2SO4,
filtered, and concentrated. The mixture was allowed to stand for 2 h, and
the precipitate was collected by filtration. The solid was washed with 1:1
EtOAc/Et2O and then with Et2O to afford 54 (35 mg, 16% yield) as a
tan solid. MS (ESI pos ion) m/z: 455 (M þ Hþ). 1H NMR (400 MHz,
DMSO-d6, δ): 12.42 (s, 1H), 8.59 (s, 1H), 8.24 (s, 2H), 7.86-7.70 (m,
4H), 7.63 (s, 1H), 7.08 (d, J = 5.52 Hz, 2H), 3.79 (s, 3H), 2.22 (s, 3H).
N-(6-(6-Chloropyrazin-2-yl)benzo[d]thiazol-2-yl)acetamide
(56). 2,6-Dichloropyrazine (55, 1.119 g, 7.511 mmol), 4a (3.07 g,
9.65 mmol), Pd(dppf)2Cl2/DCM complex (519.9 mg, 0.637 mmol),
and K2CO3 (2.992 g, 21.65 mmol) were suspended in 1,2-dimethox-
yethane (50 mL), and H2O (15 mL) was added. The flask was fitted with
a reflux condenser and placed in a preheated oil bath (90 ꢀC) and stirred
under N2. After 1.5 h, the mixture was cooled to room temperature and
filtered, and the solid was washed with 1,2-dimethoxyethane. The solid
was set aside, and the filtrate was concentrated, treated with Et2O,
and filtered. The solid from this filtration was washed with Et2O, H2O,
and three cycles of MeOH and Et2O. The solid was collected and dried
to afford 56 (1.954 g, 73% purity, 62% yield). For characterization
purposes, a portion (∼100 mg) was purified on HPLC (10% f 95%
MeCN/H2O with 0.1% TFA over 30 min). The fractions with product
were collected, concentrated, and washed with Et2O, MeOH, and Et2O.
The solid was collected and dried under high vacuum in a H2O bath
(50 ꢀC). MS (ESI pos ion) m/z: 305 (M þ Hþ). 1H NMR (400 MHz,
DMSO-d6, δ): 12.50 (s, 1H), 9.32 (s, 1H), 8.77 (s, 1H), 8.72 (s, 1H),
8.20 (d, J = 8.53 Hz, 1H), 7.86 (d, J = 8.53 Hz, 1H), 2.23 (s, 3H).
N-(6-(6-(4-Methoxyphenylsulfonamido)pyrazin-2-yl)benzo-
[d]thiazol-2-yl)acetamide (57). 4-Methoxybenzenesulfonamide
(185.6 mg, 0.9913 mmol) was dissolved in DMSO (2.0 mL), and
NaH (60% in mineral oil, 54.6 mg, 1.37 mmol) was added. The mixture
was stirred under N2 at room temperature for 1 h, and then 56 (84.8 mg,
0.278 mmol) was added. The flask was put in a preheated oil bath
(125 ꢀC) and was stirred under N2 for 22 h. The mixture was cooled to
room temperature, treated with MeOH, and filtered. The filtrate was
concentrated and purified on HPLC (10% f 95% MeCN/H2O with
0.1% TFA over 40 min) to afford 57 (5.0 mg, 4% yield). MS (ESI pos
ion) m/z: 456 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.51 (s,
1H), 11.54 (s, 1H), 8.87 (s, 1H), 8.47 (s, 1H), 8.22 (s, 1H), 8.05 (d, J =
8.53 Hz, 1H), 7.97 (d, J = 8.53 Hz, 2H), 7.85 (d, J = 8.53 Hz, 1H), 7.13
(d, J = 8.53 Hz, 2H), 3.81 (s, 3H), 2.23 (s, 3H).
N-(4-(2-Acetamidobenzo[d]thiazol-6-yl)pyrimidin-2-yl)-4-
methoxybenzamide (48). To a mixture of 47 (70 mg, 0.27 mmol),
4 (130 mg, 0.40 mmol), and Pd(PPh3)4 (31 mg, 0.027 mmol) under N2
was added aqueous 2 M Na2CO3 (0.5 mL, 1 mmol) and 1,4-dioxane
(3 mL). The flask was placed into a preheated (90 ꢀC) bath, and the
mixture was allowed to stir under an inert atmosphere overnight. The
resulting mixture was concentrated and diluted with DMSO (2 mL) and
purified by HPLC (5% f 95% MeCN in H2O). The fractions with pure
product were concentrated, diluted with DCM, and washed with
aqueous Na2CO3 solution. The organic layer was dried over Na2SO4
and concentrated to give 48 (35 mg, 31%) as a white solid. MS (ESI pos
ion) m/z: 420 (M þ Hþ). 1H NMR (300 MHz, CDCl3, δ): 10.95 (br s,
1H), 8.85 (s, 1H), 8.72 (d, J = 5.26 Hz, 1H), 8.61 (d, J = 1.61 Hz, 1H),
8.16 (dd, J = 8.62, 1.75 Hz, 1H), 7.99-7.92 (m, 2H), 7.84 (d, J =
8.48 Hz, 1H), 7.50 (d, J = 5.41 Hz, 1H), 7.00 (d, J = 8.92 Hz, 2H), 3.90 (s,
3H), 2.33 (s, 3H).
N-(6-(2-Chloropyridin-4-yl)benzo[d]thiazol-2-yl)acetamide
(50). 2-Chloro-4-iodopyridine (49, 500 mg, 2.09 mmol) was dissolved
in 1,4-dioxane (15 mL). Then 4a (0.8 g, 3 mmol), Ph(PPh3)4 (0.3 g,
0.3 mmol), and aqueous 2 M Na2CO3 (2 mL, 4 mmol) were added to
the mixture. The flask was fitted with a reflux condenser, placed into a
preheated (90 ꢀC) bath, and stirred under inert atmosphere for 3 h. The
mixture was allowed to cool to ambient temperature and was diluted
with DCM and saturated NaHCO3. The organic layer was collected by
extracting with DCM (3 ꢀ 20 mL). The organics were combined, dried
over Na2SO4, filtered, and concentrated. The residue was diluted with
Et2O (50 mL) and allowed to stir for 10 min. The precipitate was collected
byfiltration andwashedwithEt2O to afford 50 (0.200 g, 32% yield) as a tan
1
crystalline solid. MS (ESI pos ion) m/z: 304 (M þ Hþ). H NMR
(400 MHz, DMSO-d6, δ): 12.49 (br s, 1H), 8.53 (s, 1H), 8.47 (d, J =
5.09 Hz, 1H), 7.94-7.89 (m, 2H), 7.86-7.79 (m, 2H), 2.22 (s, 3H).
N-(6-(2-(4-Methoxyphenylsulfonamido)pyridin-4-yl)benzo-
[d]thiazol-2-yl)acetamide (51). A microwave vial with a magnetic
stir bar was charged with 4-methoxybenzenesulfonamide (0.25 g,
1.3 mmol) in DMF (3 mL). Sodium hydride (63 mg, 2.6 mmol) was
added, and the mixture was allowed to stir for 15 min. Then 50 (160 mg,
0.528 mmol), Pd(OAc)2 (12 mg, 0.053 mmol), and Xantphos (10 mg,
0.017 mmol) were added to the mixture. The vial was capped and placed
into a CEM microwave for 20 min at 120 ꢀC, while 100 W of energy was
supplied via Powermax. The reaction mixture was then transferred to a
round-bottomed flask and diluted with EtOAc (20 mL) while stirring.
After 20 min, the mixture was filtered and the resulting precipitate was
collected. The precipitate was diluted with DMSO and purified by
HPLC to afford 51 (28 mg, 12% yield) as a tan crystalline solid. MS (ESI
pos ion) m/z: 455 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 7.92
(d, J = 10.04 Hz, 2H), 7.85-7.68 (m, 3H), 7.65-7.54 (m, 2H), 7.46 (s,
1H), 6.96-6.84 (m, 3H), 6.70 (br s, 1H), 3.74 (s, 3H), 2.09 (s, 3H).
N-(5-Bromopyridin-3-yl)-4-methoxybenzenesulfonamide
(53). To a round-bottomed flask was added 5-bromopyridin-3-amine,
52 (400 mg, 2.31 mmol), EtOH (10 mL), and 4-methoxybenzene-1-
sulfonyl chloride (0.96 g, 4.6 mmol). The resulting mixture was allowed
to stir at ambient temperature overnight while under inert atmosphere.
The mixture was diluted with DCM and saturated NaHCO3 and
extracted with DCM (3 ꢀ 25 mL). The organics were combined, dried
over Na2SO4, filtered, concentrated, and purified by ISCO silica gel
chromatography in a gradient of 10% f 30% EtOAc/DCM over 30 min
to afford 53 (0.165 g, 21% yield) as a white crystalline solid. MS (ESI pos
ion) m/z: 343 (M þ Hþ, 79Br), 345 (M þ Hþ, 81Br). 1H NMR
(400 MHz, DMSO-d6, δ): 10.73 (s, 1H), 8.38 (s, 1H), 8.28 (s, 1H), 7.74
(d, J = 8.53 Hz, 2H), 7.67 (s, 1H), 7.11 (d, J = 8.53 Hz, 2H), 3.81 (s, 3H).
N-(6-(5-(4-Methoxyphenylsulfonamido)pyridin-3-yl)benzo-
[d]thiazol-2-yl)acetamide (54). To a microwave vial charged with
N-(6-(3-Aminophenyl)benzo[d]thiazol-2-yl)acetamide (59). A
mixture of 4a (319 mg, 1.00 mmol), 3-bromobenzenamine (58, 0.17 g,
1.0 mmol), Pd(PPh3)4 (1.2 g, 1.0 mmol) in dioxane (2 mL), and aqueous
Na2CO3 solution (2.0 M, 1.0 mL, 2.0 mmol) was heated in the microwave at
130 W and 100 ꢀC for 20 min. Then the mixture was diluted with DCM and
H2O. The organic layer was separated, dried over Na2SO4, and concentrated.
The residue was recrystallized to give 59 (0.050 g, 18%) as a brown solid. MS
(ESI pos ion) m/z: 284 (M þ Hþ).
N-(6-(3-(4-Methoxyphenylsulfonamido)phenyl)benzo[d]
thiazol-2-yl)acetamide (60). To a mixture of 59 (0.030 g,
0.11 mmol) and pyridine (0.03 g, 0.3 mmol) in DCM (2 g) was added
4-methoxybenzene-1-sulfonyl chloride (0.05 g, 0.2 mmol) at room
temperature. The mixture was stirred for 4 h, and then pyrrolidine
(0.02 g, 0.3 mmol) was added. The resulting mixture was concentrated,
diluted with DMSO (2 mL), and purified by HPLC (5% f 95% MeCN/
H2O). The fractions with pure product were collected, concentrated,
diluted with DCM, and washed with aqueous Na2CO3 solution.
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Journal of Medicinal Chemistry
ARTICLE
The organic layer was dried over Na2SO4 and concentrated to give 60
(0.035 g, 73%) as a white solid. MS (ESI pos ion) m/z: 454 (M þ Hþ).
1H NMR (300 MHz, DMSO-d6, δ): 12.39 (s, 1H), 10.26 (s, 1H), 8.12
(d, J = 1.61 Hz, 1H), 7.80 (d, J = 8.62 Hz, 1H), 7.75 (d, J = 8.92 Hz, 2H),
7.55 (dd, J = 8.55, 1.53 Hz, 1H), 7.41-7.31 (m, 3H), 7.10-7.04 (m,
3H), 3.79 (s, 3H), 2.22 (s, 3H).
7.62 (dd, J = 8.28, 1.76 Hz, 1H), 7.21 (d, J = 1.51 Hz, 1H), 5.14 (s, 2H),
2.31 (s, 3H), 2.21 (s, 3H).
N-(6-(5-(4-Methoxyphenylsulfonamido)-6-methylpyridin-
3-yl)benzo[d]thiazol-2-yl)acetamide (68). Compound 67
(76.5 mg, 0.256 mmol), DMAP (31.4 mg, 0.257 mmol), and
4-methoxybenzene-1-sulfonyl chloride (224.7 mg, 1.087 mmol)
were suspended in THF (1.5 mL) and pyridine (1.5 mL). The flask
was fitted with reflux condenser and was placed in a preheated oil
bath (65 ꢀC) and stirred under N2 for 90 min. More DMAP (33.2 mg,
0.272 mmol) and 4-methoxybenzene-1-sulfonyl chloride (163 mg,
0.789 mmol) were added, and stirring continued. After 4 h, more
DMAP (20 mg) and 4-methoxybenzenesulfonyl chloride (85 mg,
0.41 mmol) were added. Stirring was continued for 30 min, and then
the mixture was cooled to room temperature, concentrated, and
purified on silica gel (20:1 f 10:1 DCM/MeOH). The fractions with
product were collected, concentrated, treated with DCM, and filtered. The
solid was washed with DCM and Et2O, collected, and dried under high
vacuum to afford 68 (64.0 mg, 53%). MS (ESI pos ion) m/z: 469 (M þ
Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.43 (s, 1H), 9.82 (s, 1H), 8.64
(d, J = 1.96 Hz, 1H), 8.18 (d, J = 1.56 Hz, 1H), 7.81 (d, J = 8.41 Hz, 1H),
7.63 (d, J = 6.65 Hz, 2H), 7.62-7.56 (m, 2H), 7.11 (d, J = 8.80 Hz, 2H),
3.83 (s, 3H), 2.22 (s, 3H), 2.19 (s, 3H).
N-(6-(6-Chloro-5-(4-methoxyphenylsulfonamido)pyridin-
3-yl)benzo[d]thiazol-2-yl)acetamide (70). A solution of 4-meth-
oxybenzene-1-sulfonyl chloride (1 g, 5 mmol) and 3-amino-5-bromo-2-
chloropyridine, 69 (0.45 g, 2.2 mmol), in pyridine (15 mL) was heated in a
microwave vial at 100 ꢀC for 20 min. The mixture was then concentrated
and the residue was purified by silica gel column chromatography to afford
the intermediate N-(5-bromo-2-chloropyridin-3-yl)-4-methoxybenzene-
sulfonamide (0.4 g, 49%).
Then N-(5-bromo-2-chloropyridin-3-yl)-4-methoxybenzenesulfona-
mide (0.15 g, 0.4 mmol), 4a (0.18 g, 0.57 mmol), and Fibercat were
mixed with 10% Na2CO3 (1 mL) and 1,4-dioxane (3 mL). The mixture
was heated at 100 ꢀC for 12 min. The mixture was filtered, and the filtrate
was concentrated and washed with EtOAc. The collected solid was
recrystallized in MeOH to afford 70 (0.08 g, 41% yield) as a white solid.
MS (ESI pos ion) m/z: 489 (M þ Hþ). 1H NMR (300 MHz, CD3OD,
δ): 8.44 (d, J = 2.34 Hz, 1H), 8.17 (d, J = 2.34 Hz, 1H), 8.11 (d, J =
1.61 Hz, 1H), 7.90-7.81 (m, 1H), 7.78-7.70 (m, 2H), 7.66 (dd, J =
8.48, 1.90 Hz, 1H), 7.07-6.97 (m, 2H), 3.85 (s, 3H), 2.29 (s, 3H). Anal.
N-(5-Bromo-2-cyanopyridin-3-yl)-4-methoxybenzenesul-
fonamide (62). To a 100 mL round-bottomed flask was added
3-amino-5-bromopicolinonitrile 61 (0.7 g, 4 mmol) in 20 mL of THF.
The solution was cooled to -40 ꢀC and treated with LiHMDS (1.0 M in
hexanes, 1 mL, 1 mmol) dropwise. The mixture was stirred for 10 min at
the same temperature, and then 4-methoxybenzenesulfonyl chloride
(0.9 g, 4 mmol) was added. The ice bath was removed, and the mixture
was stirred at room temperature for 2 h. The mixture was then treated
with brine and EtOAc, and the aqueous phase was extracted with EtOAc
(2ꢀ). The combined organic phases were washed with brine, dried over
MgSO4, and concentrated. Purification by ISCO chromatography
provided 62 (0.1 g, 8% yield) as a white solid. MS (ESI pos ion) m/z:
368 (M þ Hþ, 79Br), 370 (M þ Hþ, 81Br). 1H NMR (400 MHz, CDCl3,
δ): 8.45 (d, J = 1.76 Hz, 1H), 8.30 (d, J = 1.76 Hz, 1H), 7.80 (d, J = 8.80
Hz, 2H), 7.06 (s, 1H), 7.00 (d, J = 8.80 Hz, 2H), 3.88 (s, 3H).
N-(6-(6-Cyano-5-(4-methoxyphenylsulfonamido)pyridin-
3-yl)benzo[d]thiazol-2-yl)acetamide (63). To a 100 mL round-
bottomed flask were added 62 (100 mg, 0.272 mmol) and 4a (108 mg,
0.339 mmol) in DME (10 mL). Argon was bubbled in for 2 min, and
then aqueous Na2CO3 (2 M, 5 mL, 10 mmol) was added, followed by
Pd(dppf)Cl2 (80 mg, 0.11 mmol). The mixture was heated at 100 ꢀC for
2 h and then was cooled to room temperature. The mixture was diluted
with EtOAc (200 mL), causing precipitation. The suspension was fil-
tered, and the filtrate was concentrated and purified via ISCO chroma-
tographic purification (5% f 20% MeOH/DCM) to afford 63 (5 mg,
4% yield) as a brown solid. MS (ESI pos ion) m/z: 478 (M - H). 1H
NMR (400 MHz, DMSO-d6, δ): 8.11 (d, J = 1.56 Hz, 1H), 8.05 (d, J =
1.96 Hz, 1H), 7.81 (d, J = 8.41 Hz, 1H), 7.78 (d, J = 1.96 Hz, 1H), 7.71
(d, J = 8.80 Hz, 2H), 7.48 (dd, J = 8.31, 1.66 Hz, 1H), 6.95 (d, J = 8.80
Hz, 2H), 3.75 (s, 3H), 2.22 (s, 3H).
5-Bromo-2-methylpyridin-3-amine (66). 5-Bromo-2-methyl-
3-nitropyridine40 (1.808 g, 8.331 mmol) was suspended in glacial acetic
acid (16 mL) and H2O (4 mL), and iron powder (1.411 g, 25.27 mmol)
was added in portions over 5 min. The mixture was stirred under N2 at
room temperature for 70 min, using a water bath to cool the reaction
flask. Then the mixture was diluted with EtOAc (20 mL) and the
suspension was poured into 5 N NaOH (50 mL). The resulting emul-
sion was filtered through a pad of Celite, which was washed with H2O
and EtOAc. The layers were separated, and the aqueous phase was
extracted with EtOAc (2 ꢀ 50 mL). The organic extracts and phases
were combined, dried over Na2SO4, filtered, concentrated, and dried
under high vacuum to afford 66 (1.86 g, 95% pure at 254 nm; 44% pure
at 215 nm). Yield (based on purity at 215 nm): 52%. MS (ESI pos ion)
m/z: 187 (M þ Hþ, 79Br), 189 (M þ Hþ, 81Br).
Calcd for (C21H17ClN4O4S2 0.1DCM) C, H, N.
3
Procedure for the Synthesis of 72, 73, 75, 78, and 80.
N-(6-(6-Chloro-5-(2-chlorophenylsulfonamido)pyridin-3-yl)
benzo[d]thiazol-2-yl)acetamide (75). In a 20 mL sealed tube,
compound 69 (200 mg, 0.964 mmol) was dissolved in THF (2.0 mL).
The solution was cooled to -78 ꢀC, and LiHMDS (1.0 M in THF,
2.9 mL, 2.9 mmol) was added via syringe. The mixture was stirred at
-78 ꢀC for 10 min, and then 2-chlorobenzenesulfonyl chloride (203 mg,
0.964 mmol) was added. The dry ice bath was removed, and the mixture
was allowed to warm to room temperature and was stirred for 17 h. Then
the mixture was treated with ammonium chloride. To the mixture was
added 2 N HCl to bring the aqueous phase to neutral pH. The mixture
was extracted with ethyl acetate, and the organic phase was washed with
H2O and brine, dried over Na2SO4, filtered, and concentrated. The
crude material was purified via ISCO silica gel chromatography (0-50%
A/B, where A = DCM/MeOH (90:10) and B = DCM). This afforded
the intermediate N-(5-bromo-2-chloropyridin-3-yl)-2-chlorobenzene-
sulfonamide (200 mg, 54% yield) as a tan solid.
N-(6-(5-Amino-6-methylpyridin-3-yl)benzo[d]thiazol-2-yl)
acetamide (67). Compound 66 (224.9 mg, 1.202 mmol), compound
4a (413.7 mg, 1.300 mmol), K2CO3 (549.4 mg, 3.975 mmol), and
Pd(dppf)Cl2/DCM complex (108.7 mg, 0.133 mmol) were suspended
in DME (5.0 mL) and H2O (1.25 mL). The flask was fitted with a reflux
condenser, and argon was bubbled through for about 15 s. The flask was
placed in a preheated oil bath (100 ꢀC) and stirred under argon for
80 min. The mixture was cooled to room temperature, and the aqueous
phase was removed via pipet. The mixture was then concentrated,
treated with MeOH, and filtered. The solid was washed with MeOH,
H2O, MeOH, and Et2O. The solid was collected and dried under high
vacuum to afford 67 (179.7 mg, 50% yield). MS (ESI pos ion) m/z: 299
(M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.37 (br s, 1H), 8.18
(d, J = 1.00 Hz, 1H), 8.01 (d, J = 2.01 Hz, 1H), 7.79 (d, J = 8.53 Hz, 1H),
In a 20 mL sealed tube, dioxane (0.500 mL) was added, the solvent
was purged with N2 for 5 min, and the tube was sealed. Then N-(5-
bromo-2-chloropyridin-3-yl)-2-chlorobenzenesulfonamide (75 mg,
0.196 mmol), 4b (64 mg, 0.22 mmol), and Na2CO3 (42 mg, 0.39 mmol)
were added, and the flask was again purged with N2 and sealed. Finally,
Pd(dppf)Cl2 (7.2 mg, 0.0098 mmol) was added, and the flask was
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purged with N2, sealed, and heated to 100 ꢀC while the mixture was
stirred for 1 h. The mixture was cooled to room temperature, concen-
trated, and extracted with DCM. A few drops of 2 N HCl were added,
and the organic phase was washed with H2O (2ꢀ), dried over Na2SO4,
filtered, and concentrated. The crude material was purified via ISCO
silica gel chromatography (0% f 100% A/B, where A = DCM/MeOH
(90:10) and B = DCM) to afford 75 (23 mg, 93% purity, 24% yield) as a
red solid. MS (ESI pos ion) m/z: 493 (M þ Hþ). 1H NMR (400 MHz,
DMSO-d6, δ): 12.44 (s, 1H), 10.68 (br s, 1H), 8.62 (d, J = 2.15 Hz, 1H),
8.32 (d, J = 1.56 Hz, 1H), 8.02 (d, J = 2.35 Hz, 1H), 7.95 (dd, J = 7.92,
1.27 Hz, 1H), 7.83 (d, J = 8.41 Hz, 1H), 7.72-7.64 (m, 3H), 7.52-7.48
(m, 1H), 2.22 (s, 3H). Analytical HPLC: 94.5%.
N-(6-(6-Chloro-5-(2-(trifluoromethyl)phenylsulfonamido)
pyridin-3-yl)benzo[d]thiazol-2-yl)acetamide (72). Using 2-
(trifluoromethyl)benzene-1-sulfonyl chloride and following the proce-
dure used to prepare 75 gave 72 in 45% yield for the sulfonylation step
and 62% yield for the Suzuki step. In addition, for the Suzuki coupling,
microwave irradiation at 140 ꢀC for 5 min was used. The final
compound was purified by MPLC (Teledine ISCO Combiflash
Companion). The crude residue was taken up in minimal DCM/
MeOH and absorbed onto a minimal amount of silica gel, which was
then packed into a 25 g custom loading cartridge and passed through a
Redi-Sep prepacked silica gel column (80 g) using 99:1 DCM/MeOH
f 95:5 DCM/MeOH. The resulting solid was triturated from MeOH
and collected by vacuum filtration to afford 72 (72 mg, 62% yield) as a
colorless amorphous solid. MS (ESI pos ion) m/z: 527 (M þ Hþ). 1H
NMR (400 MHz, DMSO-d6, δ): 12.45 (s, 1H), 10.60 (br s, 1H), 8.66
(d, J = 2.25 Hz, 1H), 8.07 (d, J = 2.35 Hz, 1H), 8.05-7.98 (m, 2H),
7.81-7.92 (m, 3H), 7.71 (dd, J = 8.46, 1.91 Hz, 1H), 2.22 (s, 3H).
N-(6-(6-Chloro-5-(3-(trifluoromethyl)phenylsulfonamido)
pyridin-3-yl)benzo[d]thiazol-2-yl)acetamide (73). This ma-
terial was prepared using an analogous procedure as was used for the
preparation of 75. For the Suzuki step, microwave heating at 100 ꢀC with
100 W of power was used for 10 min. In addition, Cs2CO3 was used
instead of Na2CO3. MS (ESI pos ion) m/z: 527 (M þ Hþ). 1H NMR
(400 MHz, DMSO-d6, δ): 12.39 (s, 1H), 8.08-7.99 (m, 3H), 7.91-
7.62 (m, 5H), 7.46 (d, J = 7.53 Hz, 1H), 2.21 (s, 3H).
N-(6-(5-(3-tert-Butylphenylsulfonamido)-6-chloropyridin-
3-yl)benzo[d]thiazol-2-yl)acetamide (78). Using 3-(tert-butyl)
benzene-1-sulfonyl chloride and following the procedure used to pre-
pare 75 gave 78 in 99% yield for the sulfonylation step and 18% yield for
the Suzuki step. For the Suzuki step, microwave heating at 100 ꢀC with
100 W of power was used for 10 min. In addition, Cs2CO3 was used
instead of Na2CO3. MS (ESI pos ion) m/z: 515 (M þ Hþ). 1H NMR
(400 MHz, DMSO-d6, δ): 12.46 (s, 1H), 10.37 (br s, 1H), 8.58 (s, 1H),
8.27 (s, 1H), 7.92 (s, 1H), 7.82 (d, J = 8.03 Hz, 1H), 7.74-7.67 (m, 2H),
7.64 (d, J = 8.03 Hz, 1H), 7.59 (d, J = 7.03 Hz, 1H), 7.54-7.46 (m, 1H),
2.22 (s, 3H), 1.22 (s, 9H).
N-(6-(6-Chloro-5-(2-fluorophenylsulfonamido)pyridin-3-yl)
benzo[d]thiazol-2-yl)acetamide (80). Using 2-fluorobenzene-1-
sulfonyl chloride and following the procedure used to prepare 75 gave
80 in 87% yield for the sulfonylation step and 61% yield for the Suzuki
step. For the Suzuki coupling, microwave irradiation at 140 ꢀC for 5 min
was used. The crude material from the Suzuki step was purified by
MPLC (Teledine ISCO Combiflash Companion) following the proce-
dure outlined for compound 72. The final compound was purified via
trituration from DCM and filtration. MS (ESI pos ion) m/z: 477 (M þ
Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.45 (s, 1H), 10.79 (br s,
1H), 8.65 (d, J = 2.05 Hz, 1H), 8.36 (d, J = 1.66 Hz, 1H), 8.09 (s, 1H),
7.85 (d, J = 8.41 Hz, 1H), 7.81-7.69 (m, 3H), 7.47 (dd, J = 10.03,
8.46 Hz, 1H), 7.40-7.32 (m, 1H), 2.24 (s, 3H).
compound 4a (1.73 g, 5.45 mmol), Pd(PPh3)4 (0.836 g, 0.723 mmol), and
K2CO3 (2.20 g, 15.9 mmol) were suspended in 1,4-dioxane (25 mL), and
H2O (2.5 mL) was added. Argon was bubbled through the suspension for
30 s, and the flask was fitted with a reflux condenser and was placed in a
preheated oil bath (100 ꢀC). The mixture was stirred under an inert
atmosphere. The mixture was cooled to room temperature after 3 h and
thenwas diluted with DCM andsaturatedNaHCO3. The organic layer was
extracted with DCM (3 ꢀ 30 mL), and the organic phases were combined,
dried over Na2SO4, filtered, and concentrated. The residue was diluted
with EtOAc and the precipitate was collected by filtration and washed with
hexanes to give the intermediate N-(6-(5-amino-6-chloropyridin-3-
yl)benzo[d]thiazol-2-yl)acetamide (0.500 g, 32.5% yield) as a brown
1
crystalline solid. MS (ESI pos ion) m/z: 319 (M þ Hþ). H NMR
(400MHz, DMSO-d6, δ):12.41(br s, 1H), 8.24(s,1H), 7.94 (s, 1H), 7.81
(d, J = 8.53 Hz, 1H), 7.65 (d, J = 8.03 Hz, 1H), 7.42 (s, 1H), 5.66 (s, 2H),
2.22 (s, 3H).
Then a 50 mL round-bottomed flask equipped with stir bar was
charged with N-(6-(5-amino-6-chloropyridin-3-yl)benzo[d]thiazol-2-
yl)acetamide (50 mg, 0.16 mmol) and THF (1.5 mL). Benzenesulfonyl
chloride (0.30 mL, 2.4 mmol), DMAP (1.1 mg, 0.0094 mmol), and
pyridine (1.0 mL, 13 mmol) were added. The mixture was allowed to stir
under an inert atmosphere at ambient temperature until LCMS analysis
indicated that the product was present. The mixture was diluted with
DCM and saturated NaHCO3 and then was extracted with DCM (3 ꢀ
25 mL). The organics were combined, dried over Na2SO4, filtered,
concentrated, and purified by ISCO silica gel chromatography in a
gradient of 10% f 50% EtOAc/DCM over 35 min to afford 71 (50 mg,
69% yield) as a white crystalline solid. MS (ESI pos ion) m/z: 459 (M þ
1
Hþ). H NMR (400 MHz, DMSO-d6, δ): 12.46 (s, 1H), 10.44 (br s,
1H), 8.61 (d, J = 2.01 Hz, 1H), 8.32 (d, J = 1.51 Hz, 1H), 7.99 (d, J =
2.51 Hz, 1H), 7.84 (d, J = 8.53 Hz, 1H), 7.79-7.74 (m, 2H), 7.70-7.64
(m, 2H), 7.62-7.56 (m, 2H), 2.23 (s, 3H).
N-(6-(6-Chloro-5-(4-(trifluoromethyl)phenylsulfonamido)
pyridin-3-yl)benzo[d]thiazol-2-yl)acetamide (74). Using 4-
(trifluoromethyl)benzenesulfonyl chloride and following the procedure
used to prepare 71 gave 74 in 8% yield. To isolate the crude material,
H2O was added, and the mixture was stirred at room temperature for 1 h.
The resultant precipitate was collected by filtration and purified by ISCO
silica gel chromatography in a gradient of 1% f 10% MeOH/DCM over
35 min. MS (ESI pos ion) m/z: 527 (M þ Hþ). 1H NMR (400 MHz,
DMSO-d6, δ): 12.42 (br s, 1H), 8.54 (d, J = 1.51 Hz, 1H), 8.24 (d, J =
1.51 Hz, 1H), 8.01-7.99 (m, 5H), 7.81 (d, J = 8.53 Hz, 1H), 7.65 (dd,
J = 8.53, 1.51 Hz, 1H), 2.22 (s, 3H).
N-(6-(6-Chloro-5-(3-chlorophenylsulfonamido)pyridin-3-
yl)benzo[d]thiazol-2-yl)acetamide (76). Using 3-chlorobenze-
nesulfonyl chloride and following the procedure used to prepare 71
gave 76 in 19% yield. MS (ESI pos ion) m/z: 493 (M þ Hþ). 1H NMR
(400 MHz, DMSO-d6, δ): 12.46 (s, 1H), 10.64 (br s, 1H), 8.34 (s, 1H),
8.02 (d, J = 2.01 Hz, 1H), 7.84 (d, J = 8.53 Hz, 1H), 7.81-7.75 (m, 2H),
7.74-7.67 (m, 2H), 7.65-7.59 (m, 1H), 2.23 (s, 3H).
N-(6-(6-Chloro-5-(4-chlorophenylsulfonamido)pyridin-3-
yl)benzo[d]thiazol-2-yl)acetamide (77). Using 4-chlorobenze-
nesulfonyl chloride and following the procedure used to prepare 71
gave 77 in 24% yield. The following modifications were made: The
sulfonylation reaction was run using only pyridine as the solvent, and the
final compound was purified by ISCO silica gel chromatography using a
gradient of 0% f 10% MeOH/DCM over 30 min. MS (ESI pos ion)
m/z: 493 (M þ Hþ). 1H NMR (400 MHz, DMSO-d6, δ): 12.46 (s, 1H),
10.57 (br s, 1H), 8.63 (s, 1H), 8.33 (s, 1H), 8.02 (s, 1H), 7.84 (d, J =
8.03 Hz, 1H), 7.79-7.64 (m, 5H), 2.23 (s, 3H).
N-(6-(6-Chloro-5-(4-tert-butylphenylsulfonamido)pyridin-
3-yl)benzo[d]thiazol-2-yl)acetamide (79). Using 4-tert-butyl-
benzenesulfonyl chloride and following the procedure used to prepare
71 gave 79 in 16% yield. MS (ESI pos ion) m/z: 515 (M þ Hþ). 1H
Procedure for the Synthesis of 71, 74, 76, 77, 79, 81, and
82. N-(6-(6-Chloro-5-(phenylsulfonamido)pyridin-3-yl)benzo-
[d]thiazol-2-yl)acetamide (71). Compound 69 (1.00 g, 4.82 mmol),
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Journal of Medicinal Chemistry
ARTICLE
NMR (400 MHz, DMSO-d6, δ): 12.39 (s, 1H), 7.94 (br s, 1H), 7.78-
7.70 (m, 2H), 7.68 (d, J = 8.53 Hz, 2H), 7.63 (br s, 1H), 7.45-7.37 (m,
3H), 2.21 (s, 3H), 1.26 (s, 9H).
mTOR LanthaScreen Assay. The mTOR LanthaScreen is a TR-
FRET assay measuring the phosphorylation of mTOR’s substrate
4EBP1. The 384-well compound plates were prepared by Amgen’s
sample bank containing 1 μL of compound per well starting at 5 mM and
diluted 1:2 across the row, resulting in a 22-well serial dilution. Then
24 μL of assay buffer (Invitrogen, PV4794) with 2 mM DTT was added
to the compound plate in rows 1-24 using the VELOCITY11 VPREP
384 ST, resulting in a DMSO concentration of 4%. The compound plate
was mixed, and 2.5 μL of serially diluted compound or controls was
added to the assay plate (Costar, 3658).
The assay was conducted on the PerkinElmer FlexDrop PLUS. A
5 μL mix of 800 nM GFP-4E-BP1 (Invitrogen, PV4759) and 20 μM
ATP (Amgen) was added to rows 1-24. Then 2.5 μL of 0.6 μg/mL of
recombinant mTOR (Amgen, N-terminal GST-tagged mTOR fragment
spanning amino acids 1360-2549) was added to rows 1-23. An
amount of 2.5 μL of assay buffer was added to row 24 for the low
control. The final concentration of the compounds was 50 μM serially
diluted to 23.84 pM in 1% DMSO. The final high control had 1%
DMSO, and the low control was a no enzyme control with a concentra-
tion of 1% DMSO. The final concentrations of the assay reagents were
400 nM GFP-4E-BP1, 10 μM of ATP, and 0.15 μg/mL mTOR enzyme.
Compound, enzyme, and substrate were incubated for 90 min. At this
point, 10 μL of stop solution was added (20 mM Tris, pH 7.5
(Invitrogen, 15567-027), 0.02% sodium azide (Teknova, S0208),
0.01% NP-40 (Roche, 11754599001), 20 mM EDTA (Invitrogen,
15575-038), and 4 nM Tb-anti-p4E-BP1 (Invitrogen, PV4758)) for a
final concentration of 2 nM Tb-anti-p4E-BP1. The mixtures were then
incubated for 60 min.
N-(6-(6-Chloro-5-(3-fluorophenylsulfonamido)pyridin-3-
yl)benzo[d]thiazol-2-yl)acetamide (81). Using 3-fluorobenze-
nesulfonyl chloride and following the procedure used to prepare 71
gave 81 in 40% yield. MS (ESI pos ion) m/z: 477 (M þ Hþ). 1H NMR
(400 MHz, DMSO-d6, δ): 12.46 (s, 1H), 10.62 (br s, 1H), 8.62 (d, J =
1.51 Hz, 1H), 8.34 (s, 1H), 8.02 (d, J = 1.51 Hz, 1H), 7.84 (d, J = 8.53 Hz,
1H), 7.71 (d, J = 8.03 Hz, 1H), 7.67-7.57 (m, 4H), 2.22 (s, 3H).
N-(6-(6-Chloro-5-(4-fluorophenylsulfonamido)pyridin-3-
yl)benzo[d]thiazol-2-yl)acetamide (82). Using 4-fluorobenze-
nesulfonyl chloride and following the procedure used to prepare 71
gave 82 in 57% yield. The following modifications were made: The
sulfonylation reaction was run using only pyridine as the solvent, and
crude product was collected by treating the mixture with H2O and
filtering the resulting suspension. The solid was washed with H2O and
EtOAc and recrystallized from MeOH to afford 82 as a white powder.
MS (ESI pos ion) m/z: 477 (M þ Hþ). 1H NMR (300 MHz, DMSO-d6,
δ): 12.47 (s, 1H), 10.50 (br s, 1H), 8.64 (d, J = 2.34 Hz, 1H), 8.35 (d, J =
1.61 Hz, 1H), 8.04 (d, J = 2.34 Hz, 1H), 7.88-7.78 (m, 3H), 7.72 (dd,
J = 8.48, 1.90 Hz, 1H), 7.48-7.40 (m, 2H), 2.23 (s, 3H).
In Vitro Assays. PI3K Enzyme Assay. Human p110-R, -β, and-δ
with N-terminal poly-His tags were coexpressed with p85-R in a Sf9
baculovirus expression system. p110-δ/p85 heterodimers were purified by
sequential Ni-NTA, Q-HP, Superdex-100 chromatography. A truncated
form of PI3Kγ encompassing residues 114-1102, N-terminally labeled
with poly-His tag, was expressed with baculovirus in Hi5 insect cells and
purified by sequential Ni-NTA, Superdex-200, Q-HP chromatography.
The inhibition of PI3K activity was determined using a modified in
vitro AlphaScreen assay.59 From a starting concentration of 5 mM, test
compounds were serially diluted 1:2 across 384-well Greiner clear
polypropylene plates (supplier) to yield 22 test concentrations. Col-
umns 23 and 24 contained only DMSO and were designated for positive
and negative controls. Source plates were replicated into 384-well
Optiplates (PerkinElmer, Waltham, MA), 0.5 μL/well, to make assay-
ready plates. PI3K enzyme was diluted in enzyme reaction buffer
(50 mM Tris-HCl, pH 7, 14 mM MgCl2, 2 mM sodium cholate, 100 mM
NaCl, and 2 mM DTT) to give 2ꢀ working solutions. PI3KR and PI3Kδ
were diluted to 1.6 nM. PI3Kβ was diluted to 0.8 nM, and PI3Kγ was
diluted to a final concentration of 15 nM. 2ꢀ substrate solutions
containing 10 μM PI(4,5)P2 (Echelon Biosciences, Salt Lake City,
UT) were generated containing either 20 μM ATP, used in the assays
testing PI3KR and PI3Kβ, or 8 μM ATP used in the assays testing PI3Kγ
and PI3Kδ. By use of a 384-well dispensing Multidrop (Titertek,
Huntsville, AL), 10 μL of 2ꢀ enzyme reaction mixture and 10 μL of
the appropriate substrate solution were then added to columns 1-24 of
the 384-well containing serially diluted test compound. Plates were then
incubated at room temperature for 20 min.
The plates were read on the PerkinElmer EnVision 2103 multilable
reader using the excitation filter 340 nm and the emission filters 520 and
495 nm. The ratio of 520 nm/495 nm was calculated, and the POC data
were analyzed to report the IC50 IP for the phosphorylation of 4EBP1.
IC50 values were determined by Genedata Screener software using an
Amgen designed fit strategy.
DNA-PK LanthaScreen Enzyme Assay. IC50 values for the
inhibition of the DNA-PK were measured using a LanthaScreen TR-
FRET enzyme assay. The enzyme assay was carried out in 384-well
polystyrene low volume plates. All reagents were diluted in kinase buffer
(50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35,
and 1 mM DTT). The 3-fold, 10-point serial compound dilutions (4ꢀ)
were prepared in kinase buffer (20% DMSO) with a top concentration of
12 μM and aliquoted into assay plates in 2.5 μL/well volumes. A 4ꢀ
substrate working stock solution containing 44 μM ATP, 1600 nM
fluorescein-p53 [Ser15] peptide (Invitrogen, PV5132), and 10 μg/mL
sheared calf thymus DNA (Invitrogen, 15633-019) was prepared in
kinase buffer and aliquoted into the assay plate in 2.5 μL/well volumes.
The reaction was initiated by adding 5 μL/well of a 2ꢀ enzyme working
stock solution containing 2 U/μL of DNA-PK enzyme (Promega,
9PIV581) prepared in kinase buffer. The enzyme reaction was carried
out for 30 min before being terminated by adding 10 μL/well of a 2ꢀ
termination solution containing 20 mM EDTA and 4 nM Tb-anti-
phospho-p53(S15) Ab (Invitrogen, PV5130) solution. The assay plate
was then incubated at room temperature for 1 h and read on a Tecan
Safire II. IC50 values were determined by Genedata Screener software
using an Amgen designed fit strategy.
hVPS34 LanthaScreen Enzyme Assay. IC50 values for the
inhibition of hVPS34 were measured using a fluorescence-based im-
munoassay for the detection of ADP (Adapta universal kinase assay kit,
Invitrogen, PV5099). The enzyme assay was carried out in 384-well
polystyrene low volume plates. All reagents were diluted in kinase buffer
(50 mM HEPES (pH 7.5), 0.1% CHAPS, 1 mM EGTA, 2 mM MnCl2,
2 mM DTT). The 3-fold, 10-point serial compound dilutions (4ꢀ) were
prepared in kinase buffer (4% DMSO) with a top concentration
of 36 μM and aliquoted into assay plates in 2.5 μL/well volumes.
The reaction was subsequently terminated by the addition of 10 μL/
well of the donor bead solution composed of 40 nM biotinylated-IP4
(Echelon Biosciences, Salt Lake City, UT), 80 μg/mL streptavadin-
donor beads diluted in AlphaScreen reaction buffer (10 mM Tris-HCl,
pH 7.5, 150 mM NaCl, 0.10% Tween 20, and 30 mM EDTA). The plates
were incubated at room temperature in the dark for 30 min. Then 10 μL
of the acceptor bead solution, containing 40 nM PIP3-binding protein
(Echelon Biosciences, Salt Lake City, UT) and 80 μg/mL anti-GST-
acceptor beads diluted in R screen reaction buffer, was then added to
each well. The plates were then incubated in the dark for an additional
1.5 h prior to being read on an Envision multimode plate reader
(PerkinElmer, Waltham, MA) with a 680 nm excitation filter and a
520-620 nm emission filter. IC50 determinations were analyzed using
the Genedata Screener (ALDI) high content data analysis program
nonlinear regression Hill equations.
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ARTICLE
A 4ꢀ enzyme working solution was prepared by diluting hVPS34
(Invitrogen, PV5126) in kinase buffer to 650 μg/mL and aliquoted into
assay plates in 2.5 μL/well volumes. The reaction was initiated by adding
5 μL/well of a 2ꢀ substrate working stock solution containing 200 μM
phosphatidylinositol/phosphatidylserine (Invitrogen, PV5122) and
20 μM ATP. The enzyme reaction was carried out at room temperature
for 30 min before being terminated with 5 μL/well of kinase quench
buffer containing 30 mM EDTA, 6 nM ADP-Ab, and 15 nM ADP-tracer.
The assay plate was then incubated at room temperature for 30 min and
read on a Tecan Safire II. IC50 values were determined by Genedata
Screener software using an Amgen designed fit strategy.
Akt (Ser 473) Phosphorylation Cell Based Assay. U-87 MG
cells diluted to 0.25 million cells/mL were plated at 20 μL per well in
384-well white tissue culture plates. The plates were then incubated
overnight at 37 ꢀC at 5% CO2 in MEM supplemented with 10% FBS,
nonessential amino acids, and L-glutamine (all purchased from Gibco,
Carlsbad, CA).
The 384-well compound plates were prepared containing 1 μL of
compound per well at a starting concentration of 5 mM and diluted 1:2
to produce a 22-well serial dilution. Then 39 μL of growth medium was
added to the compound plate in rows 1-22 using the PerkinElmer
FlexDrop PLUS, resulting in a DMSO concentration of 2.5%. The cell
plates and diluted compound plates were put onto the VELOCITY11
VPREP 384 ST where the compound plate was mixed, and 5 μL of
serially diluted compound or controls was added to the cell plate. The
final concentration of the compounds was 25 μM serially diluted to
11.9 pM in 0.5% DMSO. The cell plates were then incubated with
compound for 2 h at 37 ꢀC, 5% CO2. After 2 h, the medium in the cell
plates was aspirated using the BioTek ELx405HT plate washer
(Winooski, VT), removing most of the medium and compound without
disturbing the adherent U-87 MG cells.
Akt (Ser 473) phosphorylation was determined using a SureFire Akt
(Ser 473) phosphorylation (TGR BioSciences, Adelaide, Austalia) and
IgG detection kits (PerkinElmer, Waltham, MA). An amount of 5 μL of
1ꢀ lysis buffer was added to each well using the PerkinElmer FlexDrop
PLUS. The plates were then incubated at room temperature on a shaker
for 10 min. The AlphaScreen reaction was then prepared under low light
conditions (subdued or green light) containing p-Akt(Ser 473) reaction
buffer, dilution buffer, activation buffer, acceptor beads, and donor beads
at a ratio of 40:20:10:1:1, respectively. The AlphaScreen reaction was
added to the cell lysate at 6 μL per well using the PerkinElmer FlexDrop
PLUS. The plates were placed in a humid environment to reduce edge
effects and incubated overnight at room temperature with restricted air
flow in the dark. The plates were read on the PerkinElmer EnVision
2103 multilable reader using the standard AlphaScreen readout. IC50
values were then determined by Genedata Screener software using an
Amgen designed fit strategy.
For the liver PD assay, mice were dosed orally (via gavage) with
compound 82 at different doses as indicated. Three hours after dosing,
0.2 mL of recombinant human HGF (0.06 mg/mL) was administrated
through the tail of each mouse (12 μg/mouse). After 5 min, animals
were anesthetized and sacrificed and blood samples were collected and
placed in heparinized tubes. Blood samples were then centrifuged to
obtain the plasma for PK analysis. Liver tissues were harvested and fast-
frozen in liquid nitrogen for analysis of Akt (Ser 473) phosphorylation.
For Akt (Ser 473) phosphorylation analysis, each liver was homo-
genized in 1 mL of ice-cold MSD lysis buffer (containing 150 mM NaCl,
20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton-X-100,
1/10 protease inhibitor tablet (Roche, catalog no. 1836170), 20 μL of
phosphatase inhibitor I (Sigma-Aldrich, Inc., catalog no. P-2850), 20 μL
of phosphatase inhibitor II (Sigma-Aldrich, Inc., catalog no. P-5726) and
40 mM NaF) with GenoGrinder 2000 (BT&C Inc., product no.
SP2100-115) at 350 strokes/min for 2 min at room temperature. The
lysate was centrifuged (2ꢀ) in a 1.5 mL Eppendorf microcentrifuge tube
at 14 000 rpm for 15 min at 4 ꢀC. The supernatant was precleared with
Protein A/G beads (PIERCE, product no. 20421) in an Eppendorf
microcentrifuge tube at 4 ꢀC for 1 h. The beads were centrifuged at
14 000 rpm for 15 min at 4 ꢀC, and the supernatant was transferred to a
clean Eppendorf microcentrifuge tube. The protein concentration of
each sample was determined using Bio-Rad DC protein assay (Bio-Rad,
catalog no. 500-0116) and normalizedtoafinal concentration of 1 mg/mL.
The level of phosphorylated Akt(S473) and total Akt in mouse liver
lysates were detected using Meso Scale Discovery phospho-Akt(S473)
(catalog no. K151CAD-2) and total Akt (catalog no. K151CBD-2)
whole cell lysate kits. pAkt(S473) MSD plate and total Akt MSD plate
were blocked with 150 μL of MSD blocking solution (containing 3%
bovine serum albumin, 50 mM Tris, pH 7.5, 150 mM NaCl, and 0.02%
Tween-20) and incubated at room temperature for 1 h. The plates were
then washed (4ꢀ) with 300 μL of MSD Tris wash buffer (containing
50 mM Tris, pH 7.5, 150 mM NaCl, and 0.02% Tween-20). An amount
of 25 μL (1mg / mL) of liver lysate was dispensed to each well of the
MSD plate. The plate was incubated with shaking in Eppendorf
thermonixer R (Eppendorf, product no. 22670107) at 600 rpm at room
temperature for 2 h. The plate was then washed four times with 300 μL
of MSD Tris wash buffer. An amount of 25 μL (1.5 μg/mL) of anti-
pAkt(S473) or antitotal Akt detection antibody solution was added to
each well of the MSD plate. The plate was incubated with shaking in
Eppendorf Thermomixer R at 600 rpm at room temperature for 1 h. The
plate was then washed (4ꢀ) with 300 μL of MSD Tris wash buffer. An
amount of 150 μL of 1ꢀ MSD read buffer was added to each well of the
MSD plate. The intensity of pAkt(S473) or total Akt signal was
measured using MSD sector imager 6000.
The raw phosphorylated Akt(S473) values were divided by the ratio
of the mean of total Akt in each group in order to adjust the values for
phosphorylated Akt(S473) data to account for varying total Akt levels in
individual tissue samples. All results were expressed as the mean (
standard error. Statistical analysis was performed by analysis of variance
(ANOVA) followed by Bonferroni-Dunn post hoc test.
Xenograft Studies. Mice were injected subcutaneously with
0.2 mL of tumor cell suspension in medium mixed with 1:1 ratio of
matrigel (BD Bioscience), which contains 5 ꢀ 106 U-87 MG cells
(ATCC), 5 ꢀ 106 A549 cells (ATCC), or 2 ꢀ 106 HCT116 cells
(ATCC). Subconfluent cells were harvested prior to injection. Ten to
thirteen days after tumor cell inoculation, when the tumor volume had
reached ∼200 mm3, animals were divided randomly into test groups
(each with 10 mice), and the daily oral administration of 82 at indicated
doses began and continued to the end of each study. Tumor volumes and
body weights were recorded at intervals of 3 or 4 days. Tumor volume
was calculated as length ꢀ width ꢀ width/2 and is in mm3. Results are
expressed as the mean ( standard error. The data were statistically
analyzed with ANOVA or ANOVA followed by RM-ANOVA for
U-87 MG and PC3 Cell Viability Assay. U-87 MG and PC-3
tumor cells were seeded at a density of 3000 cells/well (U-87 MG-
growth medium containing MEM supplemented with 10% FBS, 1ꢀ
nonessential amino acids, and 1ꢀ L-glutamine (all purchased from
Invitrogen); PC-3 growth medium containing RPMI supplemented
with 10% FBS and 1ꢀ L-glutamine (all purchased from Invitrogen))
in 96-well tissue culture plates and subsequently incubated overnight at
37 ꢀC and 5% CO2 to allow cells to adhere to the plate. Ten-point 1:3
serial dilutions of compound 82 were prepared in DMSO and added to
cells, covering a dose range of 10 μM to 500 pM. ATPlite one-step
viability assays (Perkin-Elmer, 6016731) were carried out following 72 h
of compound treatment. IC50 IP values were calculated with XLfit
(IDBS software), applying a four-parameter logistic model for curve
fitting. Mean IC50 IP values are reported from a minimum of two
experiments.
In Vivo Assays. PD Assay. Four- to five-week old female CD1
nude mice (Charles Rivers Laboratories) were used in all In Vivo studies.
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repeated measurements (JMP 7). Mice were euthanized with CO2
asphyxiation, and final plasma samples were harvested for PK analysis.
All experiments were conducted in accordance with institutional guide-
lines, which include mandates for sacrificing mice when tumors exceed
size thresholds.
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ing Phosphoinositide 3-Kinase: Moving towards Therapy. Biochim.
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Phosphoinositide 3-Kinase Pathway in Cancer. Nat. Rev. Drug Discovery
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Crystallography: Determination of p110γ Crystal Struc-
tures. Human p110γ(144-1102) was expressed, purified, and
crystallized according to published procedures.60 Inhibitor com-
plexes were obtained by soaking apo crystals overnight in cryo
solutions (mother liquor plus 20% glycerol) containing 1 mM com-
pound. Crystals were then flash-frozen in liquid nitrogen prior to data
collection. Diffraction data for p110γ þ 1 were collected at the
Advanced Photon Source, beamline 31-ID using λ = 0.9793 Å and a
MAR 165 mm CCD detector. Diffraction data for p110γ þ 82 were
collected on a FR-E rotating anode X-ray source equipped with an
RAXIS IVþþ detector. Data were reduced using the HKL software
suite,61 and the structures were solved by molecular replacement
using apo human p110γ as a search model (PDB code 1E8Y).
Structures were refined using REFMAC,62,63 and model building
was performed with COOT.64
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’ ASSOCIATED CONTENT
S
Supporting Information. Synthetic methods used for
b
analogues 14-45, X-ray data for 1 and 82, enzyme selectivity
data for 82, analytical HPLC methods, and PD data for com-
pound 73. This material is available free of charge via the Internet
at http://pubs.acs.org.
’ AUTHOR INFORMATION
(13) Jimenez, C.; Jones, D. R.; Rodriguez-Viciana, P.; Gonzalez-
Garcia, A.; Leonardo, E.; Wennstrom, S.; von Kobbe, C.; Toran, J. L.; R.-
Borlado, L.; Calvo, V.; Copin, S. G.; Albar, J. P.; Gaspar, M. L.; Diez, E.;
Marcos, M. A. R.; Downward, J.; Martinez-A., C.; Merida, I.; Carrera,
A. C. Identification and Characterization of a New Oncogene Derived
from the Regulatory Subunit of Phosphoinositide 3-Kinase. EMBO J.
1998, 17, 743–753.
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W.; Feldman, R. A. Expression of a Mutated Form of the p85R
Regulatory Subunit of Phosphatidylinositol 3-Kinase in a Hodgkin’s
Lymphona-Derived Cell Line (CO). Leukemia 2002, 16, 894–901.
(15) Samuels, Y.; Wang, Z.; Bardelli, A.; Silliman, N.; Ptak, J.; Szabo,
S.; Yan, H.; Gazdar, A.; Powell, S. M.; Riggins, G. J.; Willson, J. K. V.;
Markowitz, S.; Kinzler, K. W.; Vogelstein, B.; Velculescu, V. E. High
Frequency of Mutations of the PIK3CA Gene in Human Cancers.
Science 2004, 304, 554.
Corresponding Author
*Telephone: 805-313-6550. Fax: 805-480-3016. E-mail: dangelo@
amgen.com.
’ ACKNOWLEDGMENT
We thank Teresa Burgess, Randall Hungate, Richard Kendall,
Robert Radinsky, and Terry Rosen for their support and direc-
tion of this work. We also thank Yunxin Bo, Derin D’Amico,
Lillian Liao, Philip Olivieri, and Emily Peterson for their con-
tributions of specific compounds and intermediates to this
manuscript. Finally, we thank Paul Andrews for his support with
the cellular assay.
’ ABBREVIATIONS USED
(16) Isakoff, S. J.; Engelman, J. A.; Irie, H. Y.; Luo, J.; Brachmann,
S. M.; Pearline, R. V.; Cantley, L. C.; Brugge, J. S. Breast Cancer-
Associated PIK3CA Mutations Are Oncogenic in Mammary Epithelial
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DeLong, L.; Cheong, I.; Rago, C.; Huso, D. L.; Lengauer, C.; Kinzler, K. W.;
Vogelstein, B.; Velculescu, V. E. Mutant PIK3CA Promotes Cell Growth
and Invasion of Human Cancer Cells. Cancer Cell 2005, 7, 561–573.
(19) Shayesteh, L.; Lu, Y.; Kuo, W. L.; Baldocchi, R.; Godfrey, T.;
Collins, C.; Pinkel, D.; Powell, B.; Mills, G. B.; Gray, J. W. PIK3CA Is
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Ac, acetyl; AKT, serine-threonine kinase Akt; DCM, dichloro-
methane; DMAP, 4-dimethylaminopyridine; DME, 1,2-dimeth-
oxyethane; DMF, N,N-dimethylformamide; DMSO, dimethyl-
sulfoxide; EC, effective concentration; ED, effective dose; HGF,
hepatocyte growth factor; HLM, human liver microsomes; iv,
intravenous; mTOR, mammalian target of rapamycin; PD, phar-
macodynamic; PIP2, phosphatidylinositol 4,5-biphosphate;PIP3,
phosphatidylinositol 3,4,5-triphosphate; PI3K, phosphoinositide
3-kinase; PK, pharmacokinetic; PTEN, phosphatase and tensin
homologue gene; RLM, rat liver microsomes; SAR, structure-
activity relationship; TFA, trifluoroacetic acid; TFE, trifluoroetha-
nol; THF, tetrahydrofuran; Xantphos, 4,5-bis(diphenylphosphino)-
9,9-dimethylxanthene
(20) Borlado, L. R.; Redondo, C.; Alvarez, B.; Jimenez, C.; Criado,
L. M.; Flores, J.; Marcos, M. A. R.; Martinez-A, C.; Balomenos, D.;
Carrera, A. C. Increased Phosphoinositide 3-Kinase Activity Induces a
Lymphoproliferative Disorder and Contributes to Tumor Generation
In Vivo. FASEB J. 2000, 14, 895–903.
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Journal of Medicinal Chemistry
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’ NOTE ADDED AFTER ASAP PUBLICATION
After this paper was published online February 18, 2011, three
authors were added to the author list (Derin D'Amico, Longbin
Liu, and Kevin Yang). The revised version was published March
1, 2011.
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dx.doi.org/10.1021/jm1014605 |J. Med. Chem. 2011, 54, 1789–1811