1-substituted (dibenzo[ b,d ]thiophen-4-yl)-2-morpholino-4 h -chromen-4-ones endowed with dual DNA-PK/PI3-K inhibitory activity

Article abstract of DOI:10.1021/jm400915j

Analogues of (dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one (NU7441), a potent inhibitor of DNA-dependent protein kinase (DNA-PK; IC ₅₀ = 42 ± 2 nM), have been synthesized in which water-solubilizing groups [NHCO(CH₂)_nNR¹R ², where n = 1 or 2 and the moiety R¹R²N was derived from a library of primary and secondary amines, e.g., morpholine] were placed at the 1-position. Several of the newly synthesized compounds exhibited high potency against DNA-PK and potentiated the cytotoxicity of ionizing radiation (IR) in vitro 10-fold or more (e.g., 2-(4-ethylpiperazin-1-yl)-N-(4- (2-morpholino-4-oxo-4H-chromen-8-yl)dibenzo[b,d]thio-phen-1-yl)acetamide, 39; DNA-PK IC₅₀ = 5.0 ± 1 nM, IR dose modification ratio = 13). Furthermore, 39 was shown to potentiate not only IR in vitro but also DNA-inducing cytotoxic anticancer agents, both in vitro and in vivo. Counter-screening against other members of the phosphatidylinositol 3-kinase (PI-3K) related kinase (PIKK) family unexpectedly revealed that some of the compounds were potent mixed DNA-PK and PI-3K inhibitors.

Full text of DOI:10.1021/jm400915j

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Article
1-Substituted Dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-
chromen-4-ones Endowed With Dual DNA-PK/PI3-K Inhibitory Activity
Celine Cano, Bernard Thomas Golding, ian hardcastle, nicola curtin, julia bardos, Keith Menear,
Marc Hummersone, Mark Frigerio, Pia Thommes, Attila Ting, Graeme c m Smith, David R. Newell,
Kerry Shea, Kappusamy Saravanan, Chris Bailey, caroline richardson, and Roger John Griffin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400915j • Publication Date (Web): 15 Jul 2013
Downloaded from http://pubs.acs.org on July 18, 2013
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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1ꢀSubstituted Dibenzo[b,d]thiophenꢀ4ꢀyl)ꢀ2ꢀmorpholinoꢀ4Hꢀchromenꢀ4ꢀones
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Endowed With Dual DNAꢀPK/PI3ꢀK Inhibitory Activity
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Céline Cano,*a Kappusamy Saravanan ,a Chris Bailey,b Julia Bardos,b Nicola J. Curtin,c Mark
Frigerio,b Bernard T. Golding,a Ian R. Hardcastle,a Marc G. Hummersone,b Keith A. Menear,b David R.
Newell,c Caroline J. Richardson,b K. Shea,b,d Graeme C. M. Smith,b,d Pia Thommes,b,d Attilla Tingd and
Roger J. Griffin.*a
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a Newcastle Cancer Centre, Northern Institute for Cancer Research, School of Chemistry, Bedson
Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, United Kingdom. b KuDOS
Pharmaceuticals, Ltd., 410 Cambridge Science Park, Milton Road, Cambridge CB4 0PE, United
Kingdom. c Newcastle Cancer Centre, Northern Institute for Cancer Research, Newcastle University,
Newcastle upon Tyne, NE2 4HH, United Kingdom. d AstraZeneca, Oncology Innovative Medicines,
Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK
RECEIVED DATE
Abstract
Analogues of (dibenzo[b,d]thiophenꢀ4ꢀyl)ꢀ2ꢀmorpholinoꢀ4Hꢀchromenꢀ4ꢀone (NU7441), a potent
inhibitor of DNAꢀdependent protein kinase (DNAꢀPK; IC50 = 42 ± 2 nM), have been synthesized in
which waterꢀsolubilizing groups [NHCO(CH2)nNR1R2, where n = 1 or 2 and the moiety R1R2N was
derived from a library of primary and secondary amines, e.g. morpholine] were placed at the 1ꢀposition.
Several of the newly synthesized compounds exhibited high potency against DNAꢀPK and potentiated
the cytotoxicity of ionizing radiation (IR) in vitro 10ꢀfold or more (e.g. 2ꢀ(4ꢀethylpiperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀ
(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thioꢀphenꢀ1ꢀyl)acetamide, 39; DNAꢀPK IC50 = 5.0
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± 1 nM, IR Dose Modification Ratio = 13). Furthermore, 39 was shown to potentiate not only IR in
vitro but also DNA ꢀinducing cytotoxic anticancer agents, both in vitro and in vivo. Counterꢀscreening
against other members of the phosphatidylinositol 3ꢀkinase (PIꢀ3K) related kinase (PIKK) family
unexpectedly revealed that some of the compounds were potent mixed DNAꢀPK and PIꢀ3K inhibitors.
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Introduction
Repair of potentially lethal DNA doubleꢀstrand breaks (DSBs) within mammalian cells is a key
component of the DNA damageꢀresponse (DDR) pathway and arises principally via the processes of
homologous recombination (HR) and nonꢀhomologous end joining (NHEJ).1ꢀ3 The detection, signaling
and repair of DSBs by HR and NHEJ are coordinated by members of an atypical class of serineꢀ
threonine kinases, collectively termed the phosphatidylinositol 3ꢀkinase (PIꢀ3K) related kinase (PIKK)
family.4ꢀ6 Paradoxically, while essential for cell survival, DNA DSB repair may also constitute a
mechanism of resistance to DNAꢀdamaging cancer therapeutics. DNAꢀrepair inhibitors are thus of
increasing interest as chemoꢀ and radioꢀsensitizing agents in cancer treatment.7ꢀ9 Pharmacological
modulation of DNA repair has also gained considerable impetus more recently as a consequence of the
synthetic lethality of PARPꢀ1 inhibitors in BRCA1 and BRCA2 deficient tumors, suggesting that DNAꢀ
repair inhibitors may have single agent anticancer activity in tumors harboring DDR defects.10
The DNAꢀdependent protein kinase (DNAꢀPK), a multicomponent PIKK family member comprising
a catalytic subunit (DNAꢀPKcs) and two Ku subunits, plays an essential role in NHEJꢀmediated DSB
repair.11,12 Importantly, tumor cell lines defective in DSB repair as a consequence of compromised
DNAꢀPK activity are highly sensitive to topoisomerase II inhibitors and ionizing radiation (IR),13,14
whereas overꢀexpression of DNAꢀPKcs confers resistance to these agents by enhancing DSB repair.15,16
Taken together, these data provide compelling evidence that DNAꢀPK is an attractive therapeutic target
for the modulation of DNA DSB repair in cancer therapy, and preliminary results with inhibitors have
proved encouraging.17ꢀ20
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S
O
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N
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1
2
Our interest in the development of smallꢀmolecule DNAꢀPK modulators stemmed from the
observation that the PIꢀ3K inhibitor LY294002 (2ꢀmorpholinoꢀ8ꢀphenylꢀ4Hꢀchromenꢀ4ꢀone, 1)21 also
exhibits albeit modest ATPꢀcompetitive inhibition of DNAꢀPK (Ki = 6 ꢀM).22 Extensive structureꢀ
activity relationship (SAR) studies conducted with 1 were directed towards the development of potent
and selective DNAꢀPK inhibitors. For this chemotype, the 2ꢀmorpholinoꢀ4Hꢀchromenꢀ4ꢀone moiety is
connected at the 8ꢀposition to an aryl or heteroaryl ring, which may be substituted or unsubstituted, with
a dibenzofuranꢀ4ꢀyl or dibenzothiophenꢀ4ꢀyl group proving especially favorable.23ꢀ26 These studies
culminated in the identification of NU7441 (2), which combined potent DNAꢀPK inhibition (IC50 = 42
± 2 nM) with good selectivity over other PIKKs as well as PIꢀ3K family members.24 Sensitization of
human tumor cell lines to IR and etoposide in vitro and in vivo was also demonstrated with 2,27 although
that further biological studies were impeded by formulation problems arising from the poor aqueous
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solubility of the chromenone derivative.
A
B
Figure 1. Homology model of the ATPꢀbinding site of DNAꢀPK with NU7441 (2) docked and
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minimized. A; Ball and stick model indicating putative Hꢀbond interactions (dashed lines). B; Protein
surface colored by residue properties (red – acidic, blue – basic, dark green – hydrophobic and cyan –
polar). The homology model of DNAꢀPK was derived from the known Xꢀray crystal structure of PI3Kγ
(RCSB protein databank: PDB ID: 1E7V), employing the DNAꢀPK sequence from SwissꢀPort (ID:
PRKDC_DICDI). The model of 2 was constructed using the Maestro molecular modeling program,
minimized using the OPLS_2005 force field in Macromodel and docked using Glide. Macromodel,
Glide and Maestro were licensed from Schrödinger, LGG (www.schrodinger.com).
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In the absence of a high resolution crystal structure of DNAꢀPK to direct inhibitor design, we have
utilized a homology model of the ATPꢀbinding domain derived from the crystal structure of PIꢀ3Kγ.28 In
accordance with this model (Figure 1A), the chromenone carbonyl and morpholine oxygens of 2 serve
as hydrogen bond acceptors to anchor the inhibitor within the ATPꢀbinding site of DNAꢀPK.
Importantly, the model predicts that groups introduced at the dibenzothiophene 1ꢀposition of 2 will be
directed out of the binding pocket into bulk solvent (Figure 1B, red arrow). In this paper we describe the
synthesis and biological evaluation of a series of derivatives of 2 bearing polar substituents at the
dibenzothiophene 1ꢀposition, in the expectation that this would improve the physicochemical properties
of the DNAꢀPK inhibitors without compromising potency. Surprisingly, the potent DNAꢀPK inhibitory
activity arising from this structural modification was accompanied by a concomitant increase in activity
against other PIꢀ3K family members, resulting in the identification of a new series of potent dual DNAꢀ
PK/PIꢀ3K inhibitors.
Scheme 1a Synthesis of dibenzothiopheneꢀchromenone derivatives bearing 2ꢀaminoacetyl (Series A) or
3ꢀaminopropionyl (Series B) substituents at the dibenzothiophene 1ꢀposition
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a
o
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Reagents and conditions: (a) i) nꢀBuLi, THF, reflux; ii) MeMgBr, O2, 25 C, 39%; (b) MeI, K2CO3,
acetone, reflux, 96%; (c) HNO3, AcOH, 25 oC, 99%; (d) pyridine hydrochloride, 150 °C, 41%; (e) triflic
anhydride, NEt3, DCM, 0 °C, 98%; (f) bis(pinacolato)diboron, KOAc, PdCl2(dppf), dppf, dioxane, 95
°C, 66%; (g) 2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl trifluoromethanesulfonate, PdCl2(dppf), Cs2CO3,
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o
THF, 85 °C, 46%; (h) Zn, AcOH, 25 C, 95%; (j) i) chloroacetyl chloride or bromopropionyl chloride,
NEt3, DMA, 25 oC; ii) HNR1R2, DMA, 25 oC.
Chemistry
The structures of all compounds synthesized and evaluated for biological activity are recorded in Tables
1 and 2. Intermediates required for the synthesis of chromenꢀ4ꢀone DNAꢀPK inhibitors were prepared
following standard procedures. The preparation of 4ꢀhydroxydibenzothiophene 6 was achieved by
reaction of 4ꢀlithiodibenzothiophene with dioxygen in the presence of methylmagnesium bromide.29
Nitration of 6 gave appreciable orthoꢀnitration; this was avoided by prior methylation of the hydroxyl
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group of 6 to afford 7, with subsequent nitration occurring exclusively at the 1ꢀposition to give 8. Oꢀ
Demethylation of 8 to furnish 9, followed by reaction with triflic anhydride/triethylamine gave the
required triflate 10, which was converted into the corresponding boronate 11 by treatment with
bis(pinacolato)diboron as described previously.30 Nitroboronate ester 11 was coupled to 2ꢀmorpholinoꢀ
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4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl trifluoromethanesulfonate via SuzukiꢀMiyaura methodology to give 12, and
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reduction to the required amine 13 proceeded in excellent yield using zinc in acetic acid (Scheme 1).
Scheme 2a Synthesis of dibenzothiopheneꢀchromenone derivatives bearing 2ꢀaminoethoxy (Series C) or
2ꢀoxoacetamido (Series D) substituents at the dibenzothiophene 1ꢀposition
a Reagents and conditions: (a) i) HBF4, tꢀbutyl nitrite, 0 oC; ii) Cu(NO3)2, Cu2O, H2O, 25 oC, 76%; (b)
1,2ꢀdibromoethane, K2CO3, DMF, 25 oC; (c) HNR1R2, DMF, 25 oC; (d) i) BrCH2CO2Me, K2CO3, DMF,
60 °C, 41%; ii) NaOH, MeOH, 60 oC; (e) HBTU, HOBt, HNR1R2, DMF, 25 oC.
Library Syntheses: The effect of substitution at the 1ꢀposition of the dibenzothiophenꢀ4ꢀyl moiety was
investigated through the preparation of focused libraries, employing a solution multipleꢀparallel
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Journal of Medicinal Chemistry
approach. Acylation of arylamine 13 with chloroacetyl chloride or 3ꢀbromopropionyl chloride, followed
by treatment with a range of amines (Scheme 1), enabled access to compound libraries with a methylene
(Series A; 14ꢀ51) or ethylene (Series B; 52ꢀ86) spacer separating the amide from the sideꢀchain amine
group, following purification by semiꢀpreparative HPLC (Table 1). The synthesis of small compound
libraries where the sideꢀchain group at the dibenzothiophene 1ꢀposition was attached via an ether
linkage required the 1ꢀhydroxydibenzothiopheneꢀchromenone derivative 87, which was readily
accessible from 13 (Scheme 2). Subsequent alkylation or acylation of 87 with 1.2ꢀdibromoethane or
methyl bromoacetate, respectively, followed by treatment with the appropriate amines under standard
conditions, gave the target 2ꢀaminoethoxy (Series C; 89ꢀ102) and 2ꢀoxoacetamido (Series D; 104ꢀ113,
Scheme 2 and Table 2).
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Table 1. Chemical structures and biological activities for inhibitors bearing a dibenzothiophenꢀ1ꢀyl
acylamino motif (Series A and B).
DMR in HeLa
DNAꢀPK
Inhibition
(IC50 nM)a
DNAꢀPK
No Structure Inhibition
(IC50 nM)a
DMR in HeLa cells
at 2 Gy IR
cells
R
No Structure
at 2 Gy IR
0.1
0.5 ꢀM
0.1 ꢀM
0.5 ꢀM
ꢀM
11 ± 4
21 ± 4
12.8
10.4
3.9
1.2
52, 41
37 ± 8
6.1
5
2
14
15
A
A
52
53
B
B
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24 ± 6
12 ± 4
7, 6
5
1.4
2.4
1.1
68, 37
28, 37
35, 26
1.5
6.6
0.8
1
16
17
18
A
A
A
54
55
56
B
B
B
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7.9
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ꢀ
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A
B
8, 25
6.2
1.5
1.6
1.1
16, 10
26, 22
0.9
1.4
0.9
1.2
20
21
A
A
57
58
B
B
16, 29
55, 111
114, 51
84, 38
7.6
3.9
6.8
1.7
1.3
1.4
59, 41
17, 11
11, 9
2.6
8.4
7.2
1.1
3.1
1.7
22
23
24
A
A
A
59
60
61
B
B
B
84, 100
27, 12
3.7
9
1.2
2.5
16, 12
10, 8
5.7
1.4
1.8
1.1
25
26
A
A
62
63
B
B
31, 12
38, 14
4.7
7.7
1.4
1.7
16, 6
7, 10
1.9
4.7
1.3
2
27
28
A
A
64
65
B
B
97, 61
9.6
5.7
1.3
1.1
74, 33
3.3
5.1
1
29
30
A
A
66
67
B
B
151, 57
78, 106
1.2
4, 9
8.2
1.3
1.5
19, 9
9.3
1.6
2.4
1
31
32
A
A
68
69
B
B
29, 8
14.1
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21, 7
1.2
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6, 12
0.9
1.1
33
A
70
B
3
4
5
6
7
8
66, 43
21, 7
5, 7
2.4
5.9
4.8
1.1
1.2
1.4
13, 35
8, 10
1.2
5.9
1.4
1.3
2.3
0.9
34
35
36
A
A
A
71
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B
B
B
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23, 28
10, 6
6, 7
7.7
3.2
2.2
1.6
4.0
19, 34
29, 32
33, 69
3.5
1
1.3
1.1
1.4
37
38
39
A
A
A
74
75
76
B
B
B
5.0 ± 1
13.0
4.1
25, 7
9.9
4.9
1.8
1
44, 24
4.4
3.8
1.5
1.5
40
41
A
A
77
78
B
B
59, 15
30, 170
6, 10
8.8
ꢀ
2
ꢀ
ꢀ
ꢀ
ꢀ
42
ꢀ
A
A
ꢀ
B
B
ꢀ
18, 31
7.1
1.6
79
10, 11
60, 30
5.9
5.9
1.7
1.2
10, 37
62, 49
1.3
1.7
1.1
1.2
43
44
A
A
80
81
B
B
5 ± 2
8.4
3.3
2
29, 42
42, 54
1.2
1.1
1
45
46
A
A
82
83
B
B
30, 52
1.2
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24, 13
7.4
2
9, 13
1.4
0.9
47
A
84
B
1
2
3
4
5
6
7
8
9
22, 40
56, 18
8, 10
1.5
7.4
5.5
1.4
1
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
48
49
50
A
A
A
ꢀ
ꢀ
B
B
B
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12, 15
2.6
1.1
85
9, 19
12.9
1.7
37, 62
3.6
1.2
51
A
86
B
a
IC50 values were determined at concentrations in the range 0.001ꢀ10 ꢀM. Values are the mean ± the
standard deviation or the values for at least two separate determinations.
Table 2. Chemical structures and biological activities for inhibitors bearing a dibenzothiophenꢀ1ꢀ
yloxy motif (Series C and D).
DNAꢀPK
Inhibition
(IC50 nM)a
DMR in HeLa cells
at 2 Gy IR
DNAꢀPK
No Structure Inhibition
(IC50 nM)a
DMR in HeLa cells
at 2 Gy IR
R
No Structure
0.5 ꢀM
6.4
0.1 ꢀM
1.5
0.5 ꢀM
0.1 ꢀM
8, 33
20, 47
7, 13
ꢀ
ꢀ
ꢀ
89
90
91
C
C
C
ꢀ
D
D
D
3.5
1.4
21, 19
31, 29
5.1
1.8
1.4
1.1
104
105
5.1
3.1
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Journal of Medicinal Chemistry
10, 10
27, 22
3.6
ꢀ
1.5
ꢀ
47, 44
ꢀ
2
ꢀ
1.1
ꢀ
92
93
C
C
106
D
D
1
2
3
4
ꢀ
5
6
7
8
9
9, 7
5, 16
10, 29
10.1
ꢀ
1.4
ꢀ
ꢀ
ꢀ
ꢀ
2.1
1
ꢀ
94
95
96
C
C
C
D
D
D
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
53, 76
244, 61
1.4
0.9
107
108
3.2
1.1
2, 2
7.4
2.4
16, 26
1.6
1
97
C
109
D
10, 8
ꢀ
3.3
ꢀ
1.8
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
98
C
C
D
D
ꢀ
10, 20
1.1
1
110
4, 2
3, 4
1
0.9
1.6
ꢀ
ꢀ
ꢀ
ꢀ
99
C
C
D
D
6.3
16, 16
3.1
1.3
100
111
2, 3
3, 3
10.5
7.7
2.9
1.4
9, 6
6.1
1.4
1.5
0.9
101
102
C
C
112
113
D
D
16, 11
45
a
IC50 values were determined at concentrations in the range 0.001ꢀ10 ꢀM. Values are the mean ± the
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
standard deviation or the values for at least two separate determinations.
Results and Discussion
The major overall objective of the studies described in this paper was to improve the physicochemical
properties of 2 without compromising biological activity, with a view to facilitating a more extensive
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biological evaluation of this interesting class of DNAꢀPK inhibitor. On the basis of homology modeling
that predicted tolerance to substitution at the dibenzothiophene 1ꢀposition of 2 (Figure 1), focused
compound libraries (AꢀD) were synthesized, where a polar functionality was linked to the
dibenzothiophene via an amide or ether group. Members of the four compound libraries arising from
this empirical approach, and deemed sufficiently pure by HPLC (> 95%), were initially screened for
DNAꢀPK inhibitory activity (IC50) utilizing an established biochemical assay (Tables 1 and 2).30
Potency at least comparable with the parent chromenone 2 (DNAꢀPK, IC50 = 30 nM) was observed for
the majority of derivatives across the four series AꢀD, supporting the proposal that the dibenzothiophene
1ꢀposition is tolerant to substitution. Notably, a number of compounds (Series A/B ꢀ 18, 31, 36, 38, 39,
45; Series C/D ꢀ 97, 99ꢀ102) were at least 5ꢀfold more potent than 2 as DNAꢀPK inhibitors. However,
there were no obvious differences in activity between series A and the homologous series B with
otherwise identical structures, or for the more limited number of compounds synthesized where the 1ꢀ
acetamido linker was replaced by a 1ꢀalkoxy (series C) or 1ꢀacetyloxy (series D) linker.
1
2
3
4
5
6
7
8
9
10
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13
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15
16
17
18
19
20
21
22
23
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26
27
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29
30
31
32
33
34
35
36
37
38
39
40
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42
43
44
45
46
47
48
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50
51
52
53
54
55
56
57
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59
60
Having established that modifications at the dibenzothiophene 1ꢀposition were generally tolerated
without detriment to potency against DNAꢀPK compared with 2, cellular activity was assessed in the
HeLa cervical carcinoma cell line employing a radiosensitization survival assay.31 Briefly, cells were
exposed to 2 Gy of IR in the absence or presence of a defined concentration (0.1 or 0.5 ꢀM) of DNAꢀ
PK inhibitor, and the number of surviving cells was determined by a colonyꢀforming assay. The IR
Dose Modification Ratio (DMR) thereby derived is defined as the ratio of the number of cells that
survive a single 2 Gy dose of ionizing radiation, to those that survive a single dose of 2 Gy in
combination with a given concentration of DNAꢀPK inhibitor. The DMR assay represents a rapid
measure of the ability of the compound to modulate cell survival following the induction of DNA
damage. Assuming that the potentiation of IRꢀinduced cytotoxicity arises as a consequence of DNAꢀPK
inhibition, the DMR values reflect both potency against DNAꢀPK and the cell permeability and cellular
retention of a particular compound. Importantly, there was little intrinsic cytotoxicity observed at the
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Journal of Medicinal Chemistry
concentration of inhibitors employed, and the data obtained are thus a measure of radiosensitizsation
rather than the combined cytotoxic effect of the DNAꢀPK inhibitor and IR.
1
2
3
4
5
6
7
8
9
As shown in Tables 1 and 2, at an inhibitor concentration of 0.5 ꢀM, seven compounds from series A
generated DMR values of ≥10 in combination with IR, namely 14, 15, 29, 32, 39, 40, and 51, whereas
none of the homologous chromenones in series B gave comparable DMR values. Moreover, only 94 and
101 from the etherꢀlinked series C and D afforded DMR values of ≥10. This implied that an aminoacyl
group at the dibenzothiophene 1ꢀposition was generally preferred over an ether substituent as the linker
for attachment of the polar substituent, possibly as a consequence of favorable cellular uptake and/or
retention of the DNAꢀPK inhibitors. Within series A, compound 39 emerged as of particular interest by
exhibiting DMR values of 13.0 and 4.0 at 0.5 ꢀM and 0.1 ꢀM, respectively, in tandem with potent
DNAꢀPK inhibitory activity (IC50 = 5 nM), and this chromenone was selected for further studies.
Perhaps more importantly, 39 exhibited low intrinsic cytotoxicity, as reflected by the > 50ꢀfold
difference between the concentration of compound alone that inhibited HeLa cell survival by 50% (>25
ꢁM) and that necessary to reduce survival of irradiated cells by 50% (0.5 ꢁM).
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1
2
3
Table 3. Biological and physicochemical properties of representative DNAꢀPK inhibitors.a
4
5
6
7
Assay
Compound
2
17
39
45
97
8
9
Enzyme
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
42
±
2
12
±
4
5.0
±
1
5.2
±
1.8
2.2, 2.1
DNAꢀPK IC50 (nM)
212, 339 426
±
97
0.4
0.7
136
4.0
13
±
17
109
2.1
14
±
22 166
±
70
pDNAꢀPK EC50 (nM)
Cellular
2.2
2.8
±
±
0.2 2.4
0.1 7.4
±
±
0.4
2
±
0.3
11
2.3, 2.4
DMR (0.1 ꢁM DNAꢀPK inhibitor)
DMR (0.5 ꢁM DNAꢀPK inhibitor)
±
3
±
±
9.6
±
ꢀ
2.5
>4.3
3.05
2.7
LogD (pH = 7.4)
14, 19
>10
5
>20
>30
>33
ꢀ
hERG IC50 (ꢁM)
<0.3, <0.2
161 ±
103b 154, 194
Solubility at pH 7.4 (ꢁM)
Other
Human plasma protein binding (% Free)
0.04, 0.17
3.6
6.2, 3.6
> 10
9.8, 15 0.85, 0.49
CYP450 inhibition (ꢀM)c
ꢀ
>10
>10
ꢀ
aData are individual values or the mean ± the standard deviation.
bAmorphous material (crystalline solubility at pH7.4 buffer = 6.0 ꢀM)
cTested in CYP 3A4, 2D6, 2C9, 2C19 and 1A2
Table 3 summarizes the biological and physicochemical properties of 39 in comparison with 2 and
representative library members 17, 45, and 97. The promising biological activity of 39 was
accompanied by better drugꢀlike properties compared to 2, and acceptable plasma protein binding,
combined with weak activity against hERG and a panel of CYP450 enzymes (Table 3). To assess kinase
selectivity, 39 was screened against a diverse panel of 131 kinases (MRC Protein Phosphorylation Unit,
University of Dundee) at a single concentration of 1 ꢀM (data not shown). Only one kinase (Aurora B –
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Journal of Medicinal Chemistry
44% inhibition) was inhibited by > 30% at this concentration and the selective inhibition profile of 39
was remarkably similar to that observed for the parent dibenzothiophene derivative 2 in the same screen.
To establish whether the potent DNAꢀPK inhibitory activity of 39 in the biochemical assay translated
into cellular enzyme inhibition, the ability of the compound to inhibit DNAꢀPK autophosphorylation of
S2056 was determined in HeLa cells as described previously, employing a polyclonal antibody specific
for DNAꢀPK phosphoꢀS2056.31 The cellular potency of 39 as a DNAꢀPK inhibitor (EC50 = 136 nM) was
approximately 25ꢀfold lower than for the cellꢀfree assay (IC50 = 5 nM), and comparable differences were
observed for 2, 17, 45, and 97 (Table 3). This disparity may either relate to concentration of ATP in the
cellular (~ 5 mM) versus DNAꢀPK assay (50 ꢁM) or reflect differences in cell permeability and/or
sequestration of the compounds by intracellular proteins.
1
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60
The relationship between cellular DNAꢀPK inhibition by 39 and radiopotentation was investigated
further employing a human glioma cell line model. MO59J cells are DNAꢀPK deficient through a lack
of functional DNAꢀPKcs, whereas their MO59JꢀFus1 counterparts have been modified by chromosome
transfer to reꢀexpress human DNAꢀPKcs.32 Significant DNAꢀPK autophosphorylation at S2056 was
observed in MO59JꢀFus1 cells following exposure to 10 Gy IR, and this was completely abrogated by
39 (1.0 ꢀM) (Figure 2). By contrast, the corresponding phosphoprotein was not detectable in the DNAꢀ
PK deficient MO59J cells, confirming that the cellular activity of DNAꢀPK following exposure to IR is
attenuated by 39.
Figure 2. Inhibition of IRꢀinduced DNAꢀPK autophosphorylation (p2056) by compound 39 in MO59Jꢀ
Fusꢀ1 cells. Data presented are from a single representative experiment.
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Further evidence to support the mechanism of action of 39 was adduced using the H2AX
1
2
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4
5
6
7
8
9
phosphorylation assay. Generation of DNA DSBs following exposure of cells to IR manifests as nuclear
γH2AX foci, which accumulate rapidly at sites of DSBs and stalled replication forks, and can be
determined by immunofluorescence microscopy.27 Within 30 minutes of a single exposure to 5 Gy IR,
the nuclei of HeLa cells exhibited high levels of γH2AX foci, which subsequently declined as a result of
DSB repair (Figure 3). However, cellular γH2AX foci were observed to persist and continue to
accumulate in the presence of 39 (1.0 ꢀM). These data are supportive of the proposed mechanism of
action of 39, namely that by inhibiting DNAꢀPK activity, repair of DSB will also be compromised, and
are also consistent with data generated previously with the parent compound 2.27
A.
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B.
3
2
1
0
Vehicle
1ꢀM 39
0
2
4
6
8
10 12 14 16 18 20 22 24
time post ꢀ IR (h)
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Figure 3. Repair of radiation induced DNA damage in Hela cells is inhibited by compound 39. A)
Immunostaining with antibodies against γH2Ax as a time course after treatment with 5 Gy of ionizing
radiation; B) Quantification of immunostaining shown in panel A. Data presented are from a single
representative experiment.
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Preliminary in vitro radiosensitization studies with 39 were also very promising. Thus, exposure of
FaDu head and neck squamous cell carcinoma (HNSCC) cells to IR in the presence of 39 (0.1 ꢀM)
enhanced cytotoxicity, with clonogenic survival being significantly reduced at all doses of IR studied,
and an approximately 3ꢀfold sensitization being observed at the therapeutically relevant dose of 2 Gy
(Figure 4). The choice of the FaDU HNSCC tumor cell line for this study is noteworthy, as radiotherapy
combined with chemotherapy is the standard treatment regimen for HNSCC.
100
Vehicle
39 (100 nM)
75
50
25
0
0
0.5
1
2
4
6
IR [Gy]
Figure 4. Compound 39 potentiates IRꢀinduced cytotoxicity at 100 nM in a clonogenic survival assay
with FaDu cells. Data are the mean ± the standard deviation.
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Although 39 was found to show negligible inhibitory activity against a panel of 131 representative
kinases, PIKK and PI3K family members were not included in this initial screen, and hence the
chromenone was evaluated inꢀhouse against these kinases (Table 4). As expected, and in common with
1 and 2, the 1ꢀsubstituted chromenone derivative 39 was essentially inactive against ATM and ATR
(IC50 > 10 ꢀM) and exhibited only micromolar activity against mTOR. By contrast, and unexpectedly,
39 proved to be an extremely potent PIꢀ3K inhibitor, with low to subꢀnanomolar activity being observed
against PIꢀ3Kα, PIꢀ3Kβ and PIꢀ3Kδ. A comparison of the PIꢀ3K inhibitory activity of 39 with the
parent dibenzothiopheneꢀchromenone derivative 2, clearly implicates the 1ꢀacylamino substituent on the
dibenzothiophene of 39 as responsible for conferring activity against PIꢀ3K family members through
favorable interactions within the ATPꢀbinding domain.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
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51
52
53
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57
58
59
60
Table 4. Inhibitory activity of chromenones 1, 2 and 39 against PIKK and PI3ꢀK family members.a
Kinase
Inhibitory activity (IC50 ꢀM)
1
2
39
DNAꢀPK
1.4
> 10b
> 10
2.8
0.3
0.04
> 10
> 10
2.4
0.005
> 10
> 10
10
ATM
ATR
mTOR
PIꢀ3Kα
0.13
0.004
0.27
3.02
0.22
0.016
0.22
0.03
0.0005
0.59
PIꢀ3Kβ
PIꢀ3Kγ
PIꢀ3Kδ
<0.0001
aValues are the means of at least two determinations, and were measured as described in reference 23.
bNo inhibitory activity observed at 10 ꢀM.
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The precise nature of the additional binding interactions made by the 1ꢀsubstituent of 39 has not been
elucidated. However, docking and minimization of 39 within the homology model of the DNAꢀPK
ATPꢀbinding domain, indicates that the 3ꢀ(4ꢀethylpiperazinꢀ1ꢀyl)propanoylamino substituent on the
dibenzothiophene group of 39 is readily accommodated, with the terminal Nꢀethylpiperazinyl group
being located within a polar region (Figure 5A). Interestingly, an overlay of the best docking pose of 39
within the ATPꢀbinding pockets of DNAꢀPK and PIꢀ3Kγ, reveals that the 1ꢀsubstituent is located within
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a region of structural similarity for both kinases as Aillustrated in Figure 5B. In this model, the 1ꢀ
substituent is readily accommodated in a common solvent channel, and this may accounts for the high
potency of 39 against both DNAꢀPK and PIꢀ3K.
A
B
Figure 5. A Homology model of the ATPꢀbinding site of DNAꢀPK with 39 docked and minimized.
Protein surface colored by residue properties (red – acidic, blue – basic, dark green – hydrophobic and
cyan – polar). B Crystal structure of PIꢀ3Kα with the surface colored to indicate differences between
DNAꢀPK and PIꢀ3Kα (redꢀmost dissimilar, and blue most similar). The homology model of DNAꢀPK
was derived from the known Xꢀray crystal structure of PI3Kγ (RCSB protein databank: PDB ID: 1E7V),
employing the DNAꢀPK sequence from SwissꢀPort (ID: PRKDC_DICDI). The model of 39 was
constructed using the Maestro molecular modeling program, minimized using the OPLS_2005 force
field in Macromodel and docked using Glide. Macromodel, Glide and Maestro were licensed from
Schrödinger, LGG (www.schrodinger.com).
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1
2
3
4
Conclusions
5
6
7
8
9
The pivotal role of DNAꢀPK in DNA DSB repair renders this kinase of considerable interest in cancer
chemotherapy, both in the context of tumor chemoꢀ and radioꢀsensitization by modulation of DNAꢀPK
activity, and potentially as a singleꢀagent therapeutic target for tumors harboring appropriate DNA
repair defects. Preliminary biological investigations with NU7441 (2), the prototypic chromenoneꢀ
derived DNAꢀPK inhibitor, were encouraging, prompting a lead optimization campaign with the goal of
improving physicochemical properties to facilitate in vivo studies. The introduction of polar substituents
at the dibenzothiophene 1ꢀposition of 2, guided by homology modeling of the DNAꢀPK ATPꢀbinding
domain, was evaluated employing an empirical compound library approach. Chromenone 39 emerged as
a potent cell permeable DNAꢀPK inhibitor (IC50 = 5 nM), demonstrating significant in vitro tumor cell
radiosensitization at subꢀmicromolar concentrations, and combining improved drugꢀlike properties with
excellent selectivity in a broad kinase screen. A major objective of improving aqueous amorphous
solubility compared with 2 was also achieved with 39, albeit that the lower solubility of the crystalline
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60
compound (6.0 ꢀM) compared with the amorphous material (161
± 103 ꢀM) presented subsequent
formulation challenges. More detailed validation studies with 39 established that the compound inhibits
DNAꢀPK activity in intact cells, retards DNA DSB repair, and sensitizes human cervical and head and
neck cancer cell lines to IR.
The discovery that 39 is also a highly potent inhibitor of several PIꢀ3K family members was
unexpected, although a retrospective comparison of the ATPꢀbinding domain of PIꢀ3Kγ with that of the
homology model of DNAꢀPK indicates a putative common binding pocket. Screening of other
compound library members has since confirmed that dual DNAꢀPK/PIꢀ3K inhibition is shared by other
library members, and appears to be a common facet of this structural class (data not shown). The role of
PIꢀ3K in promoting tumor growth and survival is very well established, and a number of smallꢀmolecule
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Journal of Medicinal Chemistry
PIꢀ3K inhibitors are currently in advanced clinical trial for cancer as well as other diseases.33 In this
context, it is perhaps surprising that 39 was essentially devoid of intrinsic growthꢀinhibitory activity in
the HeLa and FaDu tumor cell lines utilized for the radiosensitization studies. However, a more
comprehensive assessment of the activity of 39 (designated the house code KUꢀ0060648) against a
panel of human cancer cell lines has since been conducted, and substantial singleꢀagent growth
inhibition has been observed, albeit in a cell lineꢀspecific manner. The results of these studies have been
published elsewhere.34 Encouragingly, the use of isogenically paired DNAꢀPKcsꢀproficient and –
deficient tumor cell lines with comparable levels of PIꢀ3K activity, confirmed that potentiation of
etoposide and doxorubicin cytotoxicity arises predominantly via DNAꢀPK inhibition.34
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60
In summary, our studies have resulted in the identification of a new chemotype exhibiting potent dual
DNAꢀPK/PIꢀ3K inhibition as exemplified by the 1ꢀsubstituted dibenzothiopheneꢀchromenone derivative
39. Subsequent pharmacokinetic and efficacy studies have demonstrated that 39 is orally bioavailable,
effectively inhibits DNAꢀPK and PIꢀ3K in tumors, and potentiates the activity of DSBꢀinducing
chemotherapeutic anticancer drugs both in vitro and in vivo.34
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Experimental Section
1
2
3
4
5
6
7
8
9
Solvents were purified and stored according to standard procedures. Petrol refers to that fraction of
hexanes boiling in the range 40ꢀ60 °C. Melting points were obtained on a Stuart Scientific SMP3
apparatus and are uncorrected. TLC was performed with Merck 60 F254 silica gel plates. ‘Flash’
column chromatography was conducted under medium pressure on silica (Merck silica gel 40ꢀ63 ꢁm).
HPLC purification were performed on Gilson LC instruments, with a 15 min gradient of 0.1% aqueous
TFA and 10ꢀ97% acetonitrile, at a flow rate of 6 mL/min, using as the stationary phase a Jones
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55
56
57
58
59
60
Chromatography Genesis 4ꢁ C18 column, 10 mm
x 250 mm, and peak acquisition based on UV
detection at 254 nm. Solutionꢀphase palladiumꢀmediated coupling reactions were conducted in
1
13
Greenhouse reactors (Radley’s Ltd., U.K.) under an argon atmosphere. H NMR and C NMRspectra
were recorded either on a Bruker Spectrospin AC 300E spectrometer (300 MHz for 1H, 75 MHz for 13C)
1
or a Bruker AMX (500MHz for H, 125 MHz for 13C) using CDCl3 as solvent, unless indicated
otherwise. LCMS was carried out on either a Micromass Platform instrument operating in positive and
negative ion electrospray mode, employing a 50
Symmetry) and a 15 min gradient elution of 0.05% formic acid and methanol (10ꢀ90%), or on a
Finnegan LCQ instrument in positive ion mode with a Phenomenex 5ꢀ Luna C18 column, 4.6 mm 50
x 4.6 mm C18 column (Supelco Discovery or Waters
x
mm and an 8 min gradient of 0.1% aqueous formic acid and acetonitrile (5ꢀ98%), with a flow rate of 2
mL/min. IR spectra were recorded on a BioꢀRad FTS 3000MX diamond ATR. Analytical purity of
compounds was determined using Waters XTerra RP18, 5 ꢁm (4.6 × 150 mm) column at 1 mL/min
using either 0.1% aqueous ammonia and acetonitrile or 0.1% aqueous formic acid and acetonitrile with
a gradient of 5ꢀ100% over 15 minutes and all library compounds were found to be ≥ 95% pure.
Accurate masses were measured using a Finnigan MAT 95 XP or a Finnigan MAT 900 XLT by the
EPSRC National Mass Spectrometry Service Centre, Swansea, SA2 8PP.
Compounds 6 to 11 were synthesized as described in reference 19.
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Page 23 of 49
Journal of Medicinal Chemistry
2ꢀMorpholinoꢀ8ꢀ(1ꢀnitrodibenzo[b,d]thiophenꢀ4ꢀyl)ꢀ4Hꢀchromenꢀ4ꢀone (12). In a Schlenk tube,
4,4,5,5ꢀtetramethylꢀ2ꢀ(1ꢀnitrodibenzo[b,d]thiophenꢀ4ꢀyl)ꢀ1,3,2ꢀdioxaborolane 11 (550 mg, 1.55 mmol)
and cesium carbonate (1.52 g, 1.55 mmol) were mixed in THF (4 mL) and degassed. Concurrently, 2ꢀ
morpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl trifluoromethanesulfonate26 (645 mg; 1.70 mmol) and PdCl2dppf
(62 mg; 0.08 mmol) were suspended in THF (4 mL) and degassed. The solutions were mixed together in
the Schlenk tube, stirred and heated at 80 °C for 18 h. Upon cooling, DCM (20 mL) was added. The
solution was washed with water (20 mL), dried over magnesium sulfate and concentrated under reduced
pressure. The residue was purified by flash chromatography using AcOEt/MeOH (9:1) as eluent. After
evaporation, the product (327 mg, 46%) was obtained as a yellow solid: Rf = 0.57 (AcOEt/MeOH 9:1);
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
mp: 179ꢀ183 °C; IR (cmꢀ1) 3095, 2980, 2858, 1709, 1681, 1619, 1559, 1404; H NMR, (300 MHz,
CDCl3)
δ 2.99ꢀ3.02 (4H, m, 2 x CH2Nꢀmorpholine), 3.41ꢀ3.47 (4H, m, 2 x CH2Oꢀmorpholine), 5.43
(1H, s, Hꢀ3), 7.39ꢀ7.48 (3H, m, HꢀAr), 7.75 (1H, d, J = 7.5 Hz, HꢀAr), 7.82 (1H, d, J = 8.1 Hz, HꢀAr),
8.05 (1H, d, J = 7.5 Hz, HꢀAr), 8.25 (1H, dd, J = 1.8 and 8.1 Hz, HꢀAr); 13C NMR, (75 MHz, CDCl3)
δ
45.1 (2
x CH2Nꢀmorpholine), 66.2 (2 x CH2Oꢀmorpholine), 87.6 (Cꢀ3), 120.7, 123.0, 124.4, 125.2,
127.1, 127.4, 127.5, 128.7, 128.9, 129.8, 131.7, 133.3, 138.9, 140.6, 143.4, 146.7, 150.9, 162.6, 176.7.
8ꢀ(1ꢀAminodibenzo[b,d]thiophenꢀ4ꢀyl)ꢀ2ꢀmorpholinoꢀ4Hꢀchromenꢀ4ꢀone (13). Zinc powder (317
mg, 4.84 mmol) was added to 2ꢀmorpholinoꢀ8ꢀ(1ꢀnitrodibenzo[b,d]thiophenꢀ4ꢀyl)ꢀ4Hꢀchromenꢀ4ꢀone
12 (222 mg, 0.48 mmol) in AcOH (8 mL) and stirred at room temperature overnight. The reaction
mixture was filtered through Celite and washed successively with methanol (4
x 35 mL) and DCM (4 x
35 mL). The combined organic layers were evaporated under reduced pressure and the residue was
purified by flash chromatography using AcOEt/MeOH (9:1) as eluent. After evaporation, the product
(197 mg, 95%) was obtained as a white solid: Rf = 0.51 (AcOEt/MeOH 9:1); mp: 103ꢀ105 °C; IR (cmꢀ1)
1
2925, 2857, 1616, 1558, 1507, 1408, 1435, 1359, 1246, 1119, 1020, 978, 852; H NMR, (300 MHz,
CDCl3)
δ 3.08ꢀ3.11 (4H, m, 2 x CH2Nꢀmorpholine), 3.31ꢀ3.38 (4H, m, 2 x CH2Oꢀmorpholine), 5.67
(1H, s, Hꢀ3), 6.88 (1H, d, J = 7.8 Hz, HꢀAr), 7.25 (1H, d, J = 7.8 Hz, HꢀAr), 7.28ꢀ7.48 (3H, m, HꢀAr),
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Journal of Medicinal Chemistry
Page 24 of 49
7.71 (1H, dd, J = 1.8 and 7.5 Hz, HꢀAr), 7.77 (1H, dd, J = 1.8 and 8.1 Hz, HꢀAr), 8.24 (1H, td, J = 1.5
and 7.8 Hz, HꢀAr); 13C NMR, (75 MHz, CDCl3)
45.0 (2 x CH2Nꢀmorpholine), 66.1 (2 x CH2Oꢀ
1
2
3
4
δ
5
6
7
8
morpholine), 87.1 (Cꢀ3), 113.1, 121.9, 122.9, 123.7, 124.9, 125.1, 125.8, 125.9, 128.9, 129.3, 134.1,
135.9, 138.9, 141.7, 144.5, 151.3, 162.7, 177.8; LCMS m/z 427.2 ([M+H]+).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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SolutionꢀPhase Library Synthesis of 2ꢀaminoꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamides (14ꢀ51) : Method I. General Procedure. To 8ꢀ(1ꢀ
aminodibenzo[b,d]thiophenꢀ4ꢀyl)ꢀ2ꢀmorpholinoꢀ4Hꢀchromenꢀ4ꢀone 13 (1 mol equiv.) in dry DMA (19
mL), were added triethylamine (2.2 mol equiv.) and chloroacetyl chloride (1 mol equiv.) and the
reaction mixture was stirred at room temperature for 18 h. Aliquots (0. 5 mL) of the resulting solution
were added to each of 38 tubes in a Greenhouse workstation, with each tube containing a different
amine (3 equiv.). The reaction mixtures were stirred in parallel at room temperature for 18 h. After
dilution with 1.5 mL of methanol, the product was purified by semiꢀpreparative HPLC.
2ꢀMorpholinoꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide
(14). Compound 14 was prepared from morpholine (16 mg, 0.19 mmol) according to Method I and was
1
obtained in 97% purity: LCMS m/z 556.4 ([M+H]+); mp: 165ꢀ167 °C; H NMR, (300 MHz, CDCl3)
δ
3.18ꢀ3.20 (4H, m, 2
x
CH2Nꢀmorpholine), 3.25ꢀ3.27 (4H, m, 2
x
CH2Nꢀmorpholine), 3.52ꢀ3.54 (4H, m,
CH2Oꢀmorpholine), 6.59 (1H, s, Hꢀ
2
x
CH2Oꢀmorpholine), 3.82 (2H, s, NCH2), 4.01ꢀ4.03 (4H, m, 2 x
3), 7.54ꢀ7.58 (4H, m, HꢀAr), 7.85ꢀ7.87 (2H, m, HꢀAr), 8.04 (1H, d, J = 8.1 Hz, HꢀAr), 8.29 (1H, d, J =
7.9 Hz, HꢀAr), 8.44ꢀ8.46 (1H, m, HꢀAr), 10.27 (1H, s, NH).
Nꢀ(4ꢀ(2ꢀMorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀyl)ꢀ2ꢀ(piperidinꢀ1ꢀ
yl)acetamide (15). Compound 15 was prepared from piperidine (16 mg, 0.19 mmol) according to
Method I and was obtained in 96% purity: LCMS m/z 554.5 ([M+H]+); mp: 187ꢀ189 °C; 1H NMR, (300
MHz, CDCl3)
δ 1.95ꢀ1.97 (6H, m, CH2ꢀpiperidine), 3.09ꢀ3.42 (14H, m, CH2ꢀmorpholine and CH2ꢀ
piperidine), 4.10 (2H, s, NCH2), 6.15 (1H, s, Hꢀ3), 7.37ꢀ7.40 (3H, m, HꢀAr), 7.47ꢀ7.50 (2H, m, HꢀAr),
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Page 25 of 49
Journal of Medicinal Chemistry
7.70ꢀ7.72 (2H, m, HꢀAr), 8.19 (1H, dd, J = 8.1 and 1.6 Hz, HꢀAr), 8.26ꢀ8.28 (1H, m, HꢀAr), 10.80 (1H,
s, NH).
1
2
3
4
5
6
7
8
Nꢀ(4ꢀ(2ꢀMorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀyl)ꢀ2ꢀ(piperazinꢀ1ꢀ
yl)acetamide (16). Compound 16 was prepared from piperazine (16 mg, 0.19 mmol) according to
Method I and was obtained in 100% purity: LCMS m/z 555.4 ([M+H]+).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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2ꢀ(4ꢀMethylpiperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀ
yl)acetamide (17). Compound 17 was prepared from 1ꢀmethylꢀpiperazine (19 mg, 0.19 mmol)
according to Method I and was obtained in 95% purity: LCMS m/z 569.4 ([M+H]+).
2ꢀ(Bis(2ꢀhydroxyethyl)amino)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (18). Compound 18 was prepared from diethanolamine (20
mg, 0.19 mmol) according to Method I and was obtained in 95% purity: LCMS m/z 574.4 ([M+H]+).
2ꢀ(Bis(2ꢀmethoxyethyl)amino)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (19). Compound 19 was prepared from bis(2ꢀ
methoxyethyl)amine (25 mg, 0.19 mmol) according to Method I and was obtained in 98% purity:
LCMS m/z 602.4 ([M+H]+).
2ꢀ(2ꢀHydroxyethylamino)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀ
yl)acetamide (20). Compound 20 was prepared from ethanolamine (16 mg, 0.12 mmol) according to
Method I and was obtained in 98% purity: LCMS m/z 530.4 ([M+H]+).
2ꢀ(2ꢀAminoethylamino)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀ
yl)acetamide (21). Compound 21 was prepared from ethylenediamine (11 mg, 0.19 mmol) according to
Method I and was obtained in 95% purity: LCMS m/z 529.4 ([M+H]+).
2ꢀ(4ꢀMethylꢀ1,4ꢀdiazepanꢀ1ꢀyl)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (22). Compound 22 was prepared from 1ꢀ
methylhomopiperazine (21 mg, 0.19 mmol) according to Method I and was obtained in 98% purity:
LCMS m/z 583.5 ([M+H]+).
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2ꢀ(Azepanꢀ1ꢀyl)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀ
1
2
3
4
5
6
7
8
9
yl)acetamide (23). Compound 23 was prepared from hexamethyleneimine (19 mg, 0.19 mmol)
according to Method I and was obtained in 95% purity: LCMS m/z 568.5 ([M+H]+).
2ꢀ(4ꢀMethoxybenzylamino)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ
1ꢀyl)acetamide (24). Compound 24 was prepared from 4ꢀmethoxybenzylamine (26 mg, 0.19 mmol)
according to Method I and was obtained in 97% purity: LCMS m/z 606.4 ([M+H]+).
2ꢀ(Diethylamino)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀ
yl)acetamide (25). Compound 25 was prepared from diethylamine (14 mg, 0.19 mmol) according to
Method I and was obtained in 96% purity: LCMS m/z 542.5 ([M+H]+).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
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31
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33
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35
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2ꢀAminoꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (26).
Compound 26 was prepared from ammonia (7N solution in methanol) (3.2 mg, 0.19 mmol) according to
Method I and was obtained in 96% purity: LCMS m/z 486.4 ([M+H]+).
Nꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀyl)ꢀ2ꢀ(2ꢀ
morpholinoethylamino)acetamide
(27).
Compound
27
was
prepared
from
4ꢀ(2ꢀ
aminoethyl)morpholine (24 mg, 0.19 mmol) according to Method I and was obtained in 95% purity:
LCMS m/z 599.4 ([M+H]+).
Nꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀyl)ꢀ2ꢀ((tetrahydrofuranꢀ2ꢀ
yl)methylamino)acetamide (28). Compound 28 was prepared from tetrahydrofurfurylamine (19 mg,
0.19 mmol) according to Method I and was obtained in 95% purity: LCMS m/z 570.4 ([M+H]+).
Nꢀ(4ꢀ(2ꢀMorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀyl)ꢀ2ꢀ(2ꢀ(piperidinꢀ1ꢀ
yl)ethylamino)acetamide (29). Compound 29 was prepared from 1ꢀ(2ꢀaminoethyl)piperidine (24 mg,
0.19 mmol) according to Method I and was obtained in 96% purity: LCMS m/z 597.5 ([M+H]+).
2ꢀ((2ꢀ(Dimethylamino)ethyl)(methyl)amino)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (30). Compound 30 was prepared from N,N,N’ꢀ
trimethylethylenediamine (19 mg, 0.19 mmol) according to Method I and was obtained in 95% purity:
LCMS m/z 571.4 ([M+H]+).
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Journal of Medicinal Chemistry
2ꢀ((2S,6R)ꢀ2,6ꢀdimethylmorpholino)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
1
2
3
4
5
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (31). Compound 31 was prepared from cisꢀ2,6ꢀ
dimethylmorpholine (22 mg, 0.19 mmol) according to Method I and was obtained in 97% purity: LCMS
6
7
8
9
m/z 584.5 ([M+H]+); mp: 152ꢀ154 °C; 1H NMR, (300 MHz, CDCl3)
2.50ꢀ2.52 (2H, m, CH2Nꢀcisꢀdimethylmorpholine), 3.15ꢀ3.17 (4H, m, 2
(2H, m, CH2Nꢀcisꢀdimethylmorpholine), 3.44ꢀ3.45 (4H, m, 2 CH2Oꢀmorpholine), 3.77ꢀ3.78 (2H, m,
δ
1.21 (6H, d, J = 6.2 Hz, 2
x CH3),
x
CH2Nꢀmorpholine), 3.27ꢀ3.29
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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x
CHꢀcisꢀdimethylmorpholine), 3.96 (2H, br s, NCH2), 6.40 (1H, s, Hꢀ3), 7.47ꢀ7.51 (4H, m, HꢀAr), 7.74ꢀ
7.77 (2H, m, HꢀAr), 7.90 (1H, d, J = 8.1 Hz, HꢀAr), 8.20 (1H, d, J = 6.4 Hz, HꢀAr), 8.32 (1H, d, J = 6.4
Hz, HꢀAr), 10.26 (1H, s, NH).
2ꢀ(Ethyl(pyridinꢀ4ꢀylmethyl)amino)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (32). Compound 32 was prepared from Nꢀ(4ꢀ
pyridylmethyl)ethylamine (26 mg, 0.19 mmol) according to Method I and was obtained in 95% purity:
LCMS m/z 605.5 ([M+H]+).
Nꢀ(4ꢀ(2ꢀMorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀyl)ꢀ2ꢀ(2ꢀ(2ꢀ
oxoimidazolidinꢀ1ꢀyl)ethylamino)acetamide (33). Compound 33 was prepared from 1ꢀ(2ꢀaminoethyl)ꢀ
2ꢀimidazolidinone (24 mg, 0.19 mmol) according to Method I and was obtained in 99% purity: LCMS
m/z 598.5 ([M+H]+).
2ꢀ(2ꢀ(1ꢀMethylpyrrolidinꢀ2ꢀyl)ethylamino)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (34). Compound 34 was prepared from 2ꢀ(2ꢀaminoethyl)ꢀ1ꢀ
methylpyrrolidine (24 mg, 0.19 mmol) according to Method I and was obtained in 96% purity: LCMS
m/z 597.5 ([M+H]+).
2ꢀ(2ꢀMethoxyethylamino)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀ
yl)acetamide (35). Compound 35 was prepared from 2ꢀmethoxyethylamine (14 mg, 0.19 mmol)
according to Method I and was obtained in 95% purity: LCMS m/z 544.4 ([M+H]+).
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2ꢀ(4ꢀAcetylpiperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀ
yl)acetamide (36). Compound 36 was prepared from 1ꢀacetylpiperazine (24 mg, 0.19 mmol) according
to Method I and was obtained in 95% purity: LCMS m/z 597.6 ([M+H]+).
1
2
3
4
5
6
7
8
2ꢀ(4ꢀ(2ꢀMethoxyethyl)piperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
9
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (37). Compound 37 was prepared from 1ꢀ(2ꢀmethoxyethyl)ꢀ
piperazine (27 mg, 0.19 mmol) according to Method I and was obtained in 96% purity: LCMS m/z
613.6 ([M+H]+).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
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59
60
2ꢀ(4ꢀ(2ꢀ(2ꢀHydroxyethoxy)ethyl)piperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (38). Compound 38 was prepared from 1ꢀ
hydroxyethylethoxypiperazine (33 mg, 0.19 mmol) according to Method I and was obtained in 98%
purity: LCMS m/z 643.6 ([M+H]+).
2ꢀ(4ꢀEthylpiperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀ
yl)acetamide (39). Compound 39 was prepared from 1ꢀethylpiperazine (21 mg, 0.19 mmol) according
to Method I and was obtained in 95% purity: LCMS m/z 583.6 ([M+H]+). 1H NMR (500 MHz, CDCl3)
δ 1.15 (3H, t, J = 7.2 Hz, CH3), 2.53 (2H, m, CH2CH3), 2.70 (4H, br. s, 2 x CH2Nꢀpiperidine), 2.90 (4H,
br. s, 2 x CH2Nꢀmorpholine), 3.08ꢀ3.13 (4H, m, 2 x CH2Nꢀpiperidine), 3.49ꢀ3.54 (4H, m, 2 x CH2Oꢀ
morpholine), 3.41 (2H, s, CH2), 5.50 (1H, s, Hꢀ3), 7.45ꢀ7.54 (4H, m, 4 x HꢀAr), 7.74 (1H, dd, J = 1.7
and 7.4 Hz, HꢀAr), 7.85ꢀ7.89 (1H, m, HꢀAr), 8.27 (1H, dd, J = 1.7 and 7.9 Hz, HꢀAr), 8.46 (1H, d, J =
8.2 Hz, HꢀAr), 8.55ꢀ8.58 (1H, m, HꢀAr), 10.17 (1H, s, NH); 13C NMR (125 MHz, CDCl3) δ 12.0 (CH3),
44.5 (2 x CH2Nꢀmorpholine), 52.5 (2 x CH2Nꢀpiperidine), 52.8 (2 x CH2Nꢀpiperidine), 54.0, 62.7, 65.8
(2 x CH2Oꢀmorpholine), 87.0 (Cꢀ3), 118.2, 123.2, 123.6, 123.6, 124.8, 124.9, 125.7, 126.0, 126.6,
127.3, 128.2, 128.4, 133.6, 134.2, 139.4, 141.0, 150.7, 162.2, 168.9.
Nꢀ(4ꢀ(2ꢀMorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀyl)ꢀ2ꢀ(4ꢀ(pyridinꢀ2ꢀ
yl)piperazinꢀ1ꢀyl)acetamide (40). Compound 40 was prepared from 1ꢀpyridinꢀ2ꢀylꢀpiperazine (31 mg,
0.19 mmol) according to Method I and was obtained in 95% purity: LCMS m/z 632.6 ([M+H]+); mp:
171ꢀ174 °C; 1H NMR, (300 MHz, CDCl3) δ 3.02ꢀ3.05 (4H, m, 2
x
CH2Nꢀpiperazine), 3.15ꢀ3.17 (4H, m,
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x CH2Nꢀmorpholine), 3.45ꢀ3.47 (4H, m, 2 x CH2Nꢀpiperazine), 3.50ꢀ3.52 (4H, m, 2 x CH2Oꢀ
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morpholine), 3.89 (2H, br s, NCH2), 6.32 (1H, s, Hꢀ3), 6.88ꢀ6.91 (2H, m, HꢀAr), 7.44ꢀ7.51 (4H, m, Hꢀ
Ar), 7.76ꢀ7.80 (3H, m, HꢀAr), 8.22ꢀ8.25 (3H, m, HꢀAr), 8.38ꢀ8.40 (1H, m, HꢀAr), 9.90 (1H, s, NH).
2ꢀ(4ꢀ(4ꢀFluorophenyl)piperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (41). Compound 41 was prepared from 1ꢀ(4ꢀ
fluorophenyl)piperazine (34 mg, 0.19 mmol) according to Method I and was obtained in 99% purity:
LCMS m/z 649.6 ([M+H]+).
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2ꢀ(4ꢀ(3ꢀ(Isopropylamino)ꢀ2ꢀoxopropyl)piperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ
8ꢀyl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (42). Compound 42 was prepared from Nꢀisopropylꢀ1ꢀ
piperazineacetamide (35 mg, 0.19 mmol) according to Method I and was obtained in 95% purity: LCMS
m/z 654.5 ([M+H]+).
2ꢀ(4ꢀ(2ꢀ(Dimethylamino)ethyl)piperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (43). Compound 43 was prepared from 1ꢀ(2ꢀ
dimethylaminoethyl)piperazine (30 mg, 0.19 mmol) according to Method I and was obtained in 95%
purity: LCMS m/z 626.4 ([M+H]+).
2ꢀ((1ꢀEthylpyrrolidinꢀ2ꢀyl)methylamino)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (44). Compound 44 was prepared from 2ꢀ(aminomethyl)ꢀ1ꢀ
ethylpyrrolidine (24 mg, 0.19 mmol) according to Method I and was obtained in 97% purity: LCMS m/z
597.4 ([M+H]+).
2ꢀ(4ꢀ(2ꢀHydroxyethyl)piperazinꢀ1ꢀyl)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (45). Compound 45 was prepared from 1ꢀ(2ꢀ
hydroxyethyl)piperazine (24 mg, 0.19 mmol) according to Method I and was obtained in 97% purity:
LCMS m/z 599.4 ([M+H]+).
2ꢀ(4ꢀ(2ꢀHydroxyethyl)ꢀ1,4ꢀdiazepanꢀ1ꢀyl)ꢀNꢀ(4ꢀ(2ꢀmorpholinoꢀ4ꢀoxoꢀ4Hꢀchromenꢀ8ꢀ
yl)dibenzo[b,d]thiophenꢀ1ꢀyl)acetamide (46). Compound 46 was prepared from 2ꢀ(1,4ꢀdiazepanꢀ1ꢀ
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