Antiproliferative and protein kinase binding activities of some N 6, 5'-bis-ureido 5'-amino-5'-deoxyadenosine derivatives

Article abstract of DOI:10.1080/15257770903044432

Two novel N6,5'-bis-ureido 5'-amino-5'-deoxyadenosine derivatives are shown to inhibit tumor cell growth in the NCI 60 human tumor cell panel. Compounds 2c and 2d exhibited GI50 values of 1-6 M in 35 and 14 cell lines, respectively. Compound 2c was shown

Full text of DOI:10.1080/15257770903044432

Nucleosides, Nucleotides and Nucleic Acids, 28:394–407, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903044432
ANTIPROLIFERATIVE AND PROTEIN KINASE BINDING ACTIVITIES
OF SOME N6, 5ꢀ-BIS-UREIDO 5ꢀ-AMINO-5ꢀ-DEOXYADENOSINE
DERIVATIVES
Matt A. Peterson, Marcelio Oliveira, and Michael A. Christiansen
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah, USA
Two novel N6,5 ꢁ-bis-ureido 5ꢁ-amino-5ꢁ-deoxyadenosine derivatives are shown to inhibit tumor
2
cell growth in the NCI 60 human tumor cell panel. Compounds 2c and 2d exhibited GI50 values of
1–6 µM in 35 and 14 cell lines, respectively. Compound 2c was shown to selectively inhibit binding
of protein kinases to immobilized ATP-binding site ligands via a competitive binding assay (11 of
353 protein kinases inhibited by ≥30% at 10 µM compound concentration). Enzyme inhibition
assays revealed modest inhibition for PAK4 and FMS (21 and 17%, respectively). A brief SAR
study suggests that a 2 ꢁ-O-TBDMS is necessary for antiproliferative activity.
Keywords protein kinase inhibitors; bis-ureidoadenosine derivatives; antiproliferative
activity
INTRODUCTION
Interest in N 6-ureidoadenosine or 3ꢁ(5ꢁ)-ureido-3ꢁ(5ꢁ)-deoxyadenosine
derivatives has been stimulated by the unique biological and physical
properties exhibited by these compounds.[1–7] Various 3ꢁ-ureido derivatives
have exhibited low- to submicromolar binding affinities for mutant human
A3(AR) receptors, an effect not observed with wild type A3(AR), thus
offering orthogonal and selective activation of the reengineered receptor
via an organ-targeted “neoceptor” approach.[1] Select N 6-ureido-purine and
-adenosine derivatives were prepared as early as the 1970’s as potential
antiproliferative agents,[2] and the N 6-carbamoyl moiety continues to be
exploited as a versatile means of linking such useful functionalities to
adenosine as fluorescent chromophores,[3] duplex stabilizing aryl groups,[4]
Received 29 January 2009; accepted 20 March 2009.
This article is dedicated to Professor Morris J. Robins on the occasion of his 70th birthday.
Generous support from the BYU Cancer Research Center is gratefully acknowledged. The NCI is
also thanked for performance of cytotoxicity evaluations.
Address correspondence to Matt A. Peterson, Department of Chemistry and Biochemistry, Brigham
Young University, Provo, Utah 84602. E-mail: matt peterson@byu.edu
394

Some N6,5ꢁ-Bis-ureido 5ꢁ-Amino-5ꢁ-deoxyadenosine Derivatives
395
FIGURE 1 Putative HIV integrase inhibitors.
and reporter groups for spin-labeling studies.[5]5ꢁ-Nitrosoureidoadenosine
and other 5ꢁ-nitrosoureidonucleoside derivatives were prepared as potential
antileukemia agents in the late 1970s. Negligible activities were reported for
these compounds against murine L1210, and a 5ꢁ-N -methylureidoadenosine
derivative was also ineffective against this cell line at concentrations as high
as 60 µM.[6]
Recently, we reported the synthesis and biological activities of some
3ꢁ-carboxymethyl-3ꢁ-deoxyadenosine derivatives designed to bind to the
active site of HIV integrase (compounds 2a–h, Figure 1).[7] A key in-
termediate in the synthesis of compounds 2a–h was compound 1 which
was prepared via stereospecific reduction of 2ꢁ-O-(tert-butyldimethylsilyl)-3ꢁ-
deoxy-3ꢁ-[(ethoxycarbonyl)methylene]adenosine following the procedure
of Robins et al.[8–9] Evaluation of compounds 2b–h in HIV replication or
HIV integrase assays revealed that 2b–h were ineffective as inhibitors of
HIV replication, and 3ꢁ-endprocessing and strand transfer activities of HIV
integrase were not inhibited at the concentrations tested. Compounds 2c,
2d, 2g, and 2h did however show promising activities (IC50 = 21.9–58.5 µM)
against MT2 cells in vitro. These activities prompted an evaluation of
compounds 2c, 2d, 2g, and 2h in the NCI 60 human tumor cell line panel,
and the results of such screening along with protein kinase binding activities
are presented in this article.
RESULTS AND DISCUSSION
Compounds 2c, 2d, 2g, and 2h were submitted to the National Cancer
Institute Developmental Therapeutics Program for screening against the
NCI 60 panel of human tumor cells in vitro.[10] Results of the single dose

396
M. A. Peterson et al.
SCHEME 1 Reagents and conditions: (a) PhN
(c) p-NO2C6H4OCONHCH3, Na2CO3, EtOAc, 4 hours; (d) TFA, CH2Cl2, H2O, 4 hours.
C
O, CH2Cl2, 5 d; (b) H2/Pd–C, EtOAc, 12 hours;
(10 µM) initial screen are illustrated in Table 1. Compounds 2c and 2d were
superior to lactones 2g and 2h in the single dose growth inhibition assay.
Only one cell line (renal cancer RXF393) was inhibited to any appreciable
extent by 2g and 2h. In contrast, numerous cell lines were inhibited by
2c and 2d. To determine whether the inferior antiproliferative activities
exhibited by compounds 2g and 2h reflected a structural requirement
for the 2ꢁ-O-TBDMS, compounds 4a and 4b were prepared and tested
(Scheme 1). The antiproliferative activities for 2g(h) and 4a(b) suggests
that the 2ꢁ-O-TBDMS is a minimal requirement for anticancer activity
(Table 2).
On the basis of the results from the single dose assay, compounds 2c
and 2d were selected for multi-dose testing (Tables 3 and 4). In the multi-
dose assays, compounds 2c and 2d exhibited low µM inhibition of all six
leukemias tested. Numerous additional cell lines from the other subclasses
were also inhibited at the low µM level. Compound 2d appeared somewhat
more toxic than 2c toward the leukemia cell lines, and LC50 values for 2d
ranged from 9.43–59.0 µM for leukemias SR, HL-60(TB), and RPMI-8226.
In contrast, LC50 values for compound 2c were >100 µM for all leukemias
except RPMI-8226. LC50 values for RPMI-8226 were identical for both 2c and
2d (59.0 µM). Compound 2c was more cytotoxic than 2d against all cell lines
tested (16 cell lines showed LC50 values <100 µM for 2c compared to only
three cell lines for compound 2d), but a majority of cell lines for 2c showed
LC50 values >100.0 µM. Compound 2c also exhibited greater efficacy in cell
growth inhibition than 2d, with 35 cell lines having GI50 values ≤6 µM for
2c compared to only 14 cell lines inhibited at similar levels for compound
2d.
A COMPARE[11] analysis of the GI50 data for compound 2c suggested
that protein kinases might be molecular targets for this compound. Accord-
ingly, compound 2c was screened against a commercially available panel
of 353 protein kinases (KinomeScan, Ambit Biosciences, San Diego, CA,
USA),[12] and the binding affinities of compound 2c for these kinases
were determined. The KinomeScan assay is a competitive binding assay
involving phage-display of protein kinases and immobilized ATP-binding

Some N6,5ꢁ-Bis-ureido 5ꢁ-Amino-5ꢁ-deoxyadenosine Derivatives
397
TABLE 1 Results of single dose growth inhibition assay (GI Percent at 10 µM)a
Cell Line
2c
2d
2g
2h
Cell Line
2c
2d
2g
2h
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI–8226
SR
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
50
—
92
94
83
81
70
105
90
59
33
103
100
98
106
94
110
100
103
104
103
97
45
59
50
11
28
−33
87
93
85
98
90
16
53
94
63
34
−41
16
0
−11
−75
−56
37
SNB-75
U251
−7
−15
96
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
22
64
79
63
76
92
91
48
80
2
21
60
95
103
98
120
102
94
71
99
51
80
61
100
65
94
−37
8
19
44
20
41
91
95
106
96
103
106
92
92
106
97
96
102
HOP-62
HOP-92
−34
87
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
41
106
105
100
112
107
48
79
18
66
90
101 Renal Cancer
114
106
786–0
A498
76
71
69
48
46
12
105
100
101
101
98
94
95
ACHN
CAKI-1
RXF393
SN12C
TK-10
43 −100
104
106
100
91
109
106
105
110
93
98
92
110
82
43
100
62
17
56
27
43
71
−13
−45
−68
−29 −23
17
61
17
118
101
79
113
99
64
12
44
57
7
−7
HT29
KM12
SW620
−26
UO-31
17
106 Breast Cancer
24
106
BT-549
HS578T
MCF7
MDA-MB-231/ATCC
MDA-MB-435
NCI/ADR-RES
T-47D
82
44
18
51
54
86
27
26
4
3
15
8
54
−34
104
111
92
123
109
102
103
100
105
98
99
102
97
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
50
55
67
54
84
58
88
80
19
12
34
−4
8
26
6
34
93
97
107
91
118
106
122
107
63
100
105
70
107
92
110 Prostate Cancer
125
99
DU-145
PC-3
63
47
25
−21
109
97
110
100
aGrowth inhibition percent calculated as:
[(Ti-TZ)/C-TZ)] × 100 for Ti ≥ Tz
[(Ti-Tz)/Tz)] × 100 for Ti < Tz
Where Tz = absorbance at t = 0; Ti = absorbance at t = 48 h (10 µM test compound);
C = absorbance of control at t = 48 h.
site ligands.[13] Binding of phage-displayed protein kinases to immobilized
ATP-binding site ligands was inhibited by compound 2c by ≥30% for 11 of
the 353 protein kinases evaluated. A majority of the kinases were unaffected
by 2c and binding inhibition of ≤10% was observed for >225 kinases (Figure
2A). Such selectivity is a desirable property suggestive of the potential
utility of derivatives of compound 2c in therapeutic applications.[14] Kinases
for which binding was inhibited by ≥30% included EGFR, TYK2, FLT3,
CSNK2A2, PAK3, MARK3, BTK, IKK-α, CSNK1G2, RPS6KA1, and BMPR1B,

398
M. A. Peterson et al.
TABLE 2 Results of single dose growth inhibition assay (GI Percent at 10 µM)a
Cell Line
4a
4b
Cell Line
4a
4h
Leukemia
CCRF-CEM
HL-60(TB)
K-562
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
95
84
90
103
87
100
79
60
—
91
119
84
85
60
99
123
91
89
66
MOLT-4
RPMI-8226
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
HOP-62
HOP-92
90
SNB-75
U251
94
86
103
102
99
14
109
87
101
101
104
99
113
96
71
99
95
95
101
92
Ovarian Cancer
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal Cancer
786-0
A498
ACHN
CAKI-1
RXF393
SN12C
TK-10
UO-31
Breast Cancer
BT-549
HS578T
MCF7
MDA-MB-231/ATCC
MDA-MB-468
T-47D
Prostate Cancer
DU-145
PC-3
89
90
109
100
79
85
92
102
105
77
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW620
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
105
87
105
73
97
95
106
103
99
55
114
96
96
81
98
99
90
91
78
89
—
97
89
98
134
74
152
98
72
102
85
107
100
75
—
93
91
90
98
78
100
76
89
104
92
86
99
97
108
117
100
94
107
92
105
88
94
71
95
85
aGrowth inhibition percent calculated as:
[(Ti-Tz)/C-Tz)] × 100 for Ti ≥ Tz
[(Ti-Tz)/Tz)] × 100 for Ti < Tz
Where Tz = absorbance at t = 0; Ti = absorbance at t = 48 h (10 µM test compound);
C = absorbance of control at t = 48 h.
each of which has been implicated in various forms of cancer (Figure
2B). Binding inhibition was greatest for BMPR1B (or ALK6), a recently
implicated modulator of estrogen receptor positive breast cancer.[15,16] The
selective inhibition of binding of ALK6 compared to other members of the
ALK family of protein kinases suggests that 2c might be a useful probe for
elucidating the role played by ALK6 in BMP-mediated signaling (Figure
2C).[17] Selective inhibition of binding was also observed for other protein
kinase families (e.g., p38 and PAK kinase families; Figure 3).
Compound 2c was also screened for its ability to inhibit a panel of
cancer-related protein kinases (Figure 4). Activities for several of the protein

Some N6,5ꢁ-Bis-ureido 5ꢁ-Amino-5ꢁ-deoxyadenosine Derivatives
399
TABLE 3 Results of multi dose growth inhibition assay for compound 2c (µM)a
Cell Line
GI50
TGI
LC50
Cell Line
GI50
TGI
LC50
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
6.69
88.6 > 100.0
> 100.0
32.9 > 100.0
23.3 > 100.0
6.53
27.0
92.5
3.01
3.59
2.39
1.09
2.23
—
5.73 > 100.0 > 100.0
5.19 > 100.0 > 100.0
29.0 > 100.0 > 100.0
4.56 > 100.0 > 100.0
4.57
59.0
SNB-75
U251
7.07 > 100.0
4.69
20.9
76.0
Non-Small Cell Lung Cancer
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
A549/ATCC
EKVX
4.18
19.2
79.1
3.85
4.59
18.0
17.2
79.2
91.3
17.7 > 100.0 > 100.0
HOP-62
8.96
26.4
—
73.1
41.2
1.23 > 100.0 > 100.0
31.1 > 100.0 > 100.0
HOP-92
< 0.01
NCI-H226
NCI-H23
> 109.0 > 100.0 > 100.0
33.3 > 100.0 > 100.0
4.92
77.2 > 100.0
21.0 > 100.0 > 100.0
NCI-H322M > 100.0 > 100.0 > 100.0 Renal Cancer
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
5.54
4.36
2.5 > 100.0
85.7 > 100.0
786-0
A498
2.00
3.34
5.21
17.1 > 100.0
17.9
ACHN
CAKI-1
RXF393
SN12C
TK-10
UO-31
8.55 > 100.0 > 100.0
29.7 > 100.0 > 100.0
2.01
9.10 > 100.0 > 100.0
12.4 40.5 > 100.0
12.1 29.5 71.7
3.84 > 100.0 > 100.0
> 100.0 > 100.0 > 100.0
4.63
19.5
3.20
16.1
45.6
8.50 > 100.0 > 100.0
4.20 > 100.0 > 100.0
HT29
KM12
SW620
3.95
4.80
20.9 > 100.0 Breast Cancer
28.4 > 100.0
MCF7
3.42
45.1 > 100.0
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
NCI/ADR-RES
MDA-MB-231/ATCC
HS578T
MDA-MB-435
BT-549
> 100.0 > 100.0 > 100.0
5.46 > 100.0 > 100.0
10.3 > 100.0 > 100.0
3.96
3.60
41.3 > 100.0
53.6 > 100.0
2.51
5.42
6.85
11.6
33.1 > 100.0
20.2 48.7
7.86
6.21 > 100.0 > 100.0
> 100.0 > 100.0 > 100.0
2.55 > 100.0 > 100.0
T-47D
4.34 > 100.0 > 100.0 Prostate Cancer
5.68 > 100.0 > 100.0
> 100.0 > 100.0 > 100.0
PC-3
DU-145
2.25
4.97
4.85
19.4
12.5
78.4
aGI50 = concentration at which cell growth is inhibited by 50%; TGI = concentration required to
achieve total growth inhibition; LC50 = concentration required to achieve 50% reduction in measured
protein after 48 h test period. TGI signifies a cytostatic effec; LC50 signifies a cytotoxic effect.
[(Ti-Tz)/C-Tz)] × 100 = 50 for GI50
Ti = Tz for TGI
[(Ti-Tz)/Tz)] × 100 = −50 for LC50
Where Tz = absorbance at t = 0; Ti = absorbance at t = 48 h; C = absorbance of control at t = 48 h.
kinases were modestly enhanced at 20 µM compound concentration and
two of the kinases (FMS and PAK4) were inhibited.
CONCLUSIONS
Novel N 6,5ꢁ-bis-ureido 5ꢁ-amino-5ꢁ-deoxyadenosine derivatives 2c and 2d
inhibited proliferation of several human tumor cell lines in vitro. GI50 values

400
M. A. Peterson et al.
TABLE 4 Results of multi dose growth inhibition assay for compound 2d (µM)a
Cell Line
GI50
TGI
LC50
Cell Line
GI50
TGI
LC50
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
6.37 > 100.0 > 100.0
1.81 4.34 16.4
3.12 > 100.0 > 100.0
8.29 > 100.0 > 100.0
9.09 > 100.0 > 100.0
22.3 > 100.0 > 100.0
> 100.0 > 100.0 > 100.0
12.7 > 100.0 > 100.0
5.66 > 100.0 > 100.0
2.23
1.58
1.27
2.23 > 100.0
1.58
1.27
59.0
9.43
SNB-75
U251
Non-Small Cell Lung Cancer
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
A549/ATCC
EKVX
HOP-62
9.35 > 100.0 > 100.0
26.4 > 100.0 > 100.0
24.9 > 100.0 > 100.0
3.72
65.7 > 100.0
7.11 > 100.0 > 100.0
53.0 > 100.0 > 100.0
38.2 > 100.0 > 100.0
9.02 > 100.0 > 100.0
52.7 > 100.0 > 100.0
HOP-92
2.71
24.3 > 100.0
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
41.9 > 100.0 > 100.0
> 100.0 > 100.0 > 100.0
> 100.0 > 100.0 > 100.0 Renal Cancer
7.49 > 100.0 > 100.0
11.1 > 100.0 > 100.0
786-0
A498
9.01 > 100.0 > 100.0
3.87
40.3 > 100.0
ACHN
CAKI-1
RXF393
SN12C
TK-10
UO-31
14.4 > 100.0 > 100.0
53.8 > 100.0 > 100.0
12.3 > 100.0 > 100.0
30.6 > 100.0 > 100.0
4.20 > 100.0 > 100.0
6.47 > 100.0 > 100.0
5.37 > 100.0 > 100.0
9.74
38.0 > 100.0
85.3 > 100.0 > 100.0
20.5 > 100.0 > 100.0
7.79 > 100.0 > 100.0
HT29
KM12
23.9 > 100.0 > 100.0 Breast Cancer
SW620
> 100.0 > 100.0 > 100.0
MCF7
5.59 > 100.0 > 100.0
> 100.0 > 100.0 > 100.0
12.3 > 100.0 > 100.0
5.79 > 100.0 > 100.0
10.9 > 100.0 > 100.0
29.0 > 100.0 > 100.0
13.9 > 100.0 > 100.0
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
NCI/ADR-RES
MDA-MB-231/ATCC
HS578T
MDA-MB-435
BT-549
7.30 > 100.0 > 100.0
14.1 > 100.0 > 100.0
15.2 > 100.0 > 100.0
14.9
83.1 > 100.0
7.77 > 100.0 > 100.0
5.81 > 100.0 > 100.0 Prostate Cancer
22.6 > 100.0 > 100.0
41.9 > 100.0 > 100.0
T-47D
PC-3
DU-145
—
—
—
1.66 > 100.0 > 100.0
aGI50 = concentration at which cell growth is inhibited by 50%; TGI = concentration required to
achieve total growth inhibition; LC50 = concentration required to achieve 50% reduction in measured
protein after 48 h test period. TGI signifies a cytostatic effec; LC50 signifies a cytotoxic effect.
[(Ti-Tz)/C-Tz)] × 100 = 50 for GI50
Ti = Tz for TGI
[(Ti-Tz)/Tz)] × 100 = −50 for LC50
Where Tz = absorbance at t = 0; Ti = absorbance at t = 48 h; C = absorbance of control at t = 48 h.
in the low (1–6) µM region were exhibited in over half of the NCI 60
panel for compound 2c. A competitive binding assay suggests that 2c may
bind to the ATP-binding site of several protein kinases. ALK6, a recently
implicated modulator of estrogen positive breast cancer, was inhibited most
significantly in this assay. Inhibition of phosphorylation of FMS and PAK4,
two cancer-related protein kinases, was modest (17% and 21%, respectively)

Some N6,5ꢁ-Bis-ureido 5ꢁ-Amino-5ꢁ-deoxyadenosine Derivatives
401
FIGURE 2 Inhibition of binding of protein kinases to immobilized ATP-binding site ligands by
compound 2c (10 µM). (A) Results of 353 member KinomeScan competitive binding assay; (B) Protein
kinases inhibited by ≥30%; (C) Selective inhibition of binding of ALK6.
suggesting that the primary mechanism of antiproliferative activity may not
be attributable to inhibition of these two enzymes. A minimal requirement
for antiproliferative activity appears to be the presence of the sterically bulky
2ꢁ-O-TBDMS group. Future SAR studies will focus on the impact of the N6
and 5ꢁ-ureido groups.

402
M. A. Peterson et al.
FIGURE 3 Inhibition of binding of protein kinases to immobilized ATP-binding site ligands by
compound 2c (10 µM). (A) Selective inhibition of p38 kinase family; (B) Selective inhibition of PAK
kinase family.
Compound Preparation
5ꢁ-Deoxy-2ꢁ,3ꢁ-O-isopropylidene-5ꢁ-[(N-methylcarbamoyl)amino]-N6-
(N-phenylcarbamoyl)adenosine (4a)
To a flame dried flask containing compound 3 (454 mg, 1.37 mmol)
was added phenylisocyanate (190 mg, 1.6 mmol) in CH2Cl2 (16 mL). The
resulting solution was stirred protected from moisture until TLC indicated
that starting material had been consumed (5 days). The crude solution
was added directly to a flash chromatography column and eluted (50 →
75%EtOAc/hexanes → 10%MeOH/EtOAc). Appropriate fractions were
pooled and volatiles were evaporated under reduced pressure to give
5ꢁ-azido-5ꢁ-deoxy-2ꢁ,3ꢁ-O-isopropylidene-N6-(N -phenylcarbamoyl) adenosine
1
(491 mg, 79%): H NMR (CDCl3, 500 MHz) δ 11.71 (s, 1H), 8.66 (s, 1H),
8.25 (s, 1H), 8.21 (s, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.39 (t, J = 8.0 Hz,
2H), 7.15 (t, J = 7.3 Hz, 1H), 6.19 (d, J = 2.5 Hz, 1H), 5.44 (dd, J = 6.3,
2.3 Hz, 1H), 5.07 (dd, J = 6.0, 3.5 Hz, 1H), 4.43 (dd, J = 9.0, 5.0 Hz,
1H), 3.63 (dd, J = 9.5, 4.8 Hz, 2H), 1.65 (s, 3H), 1.42 (s, 3H); 13C NMR
(CDCl3, 125 MHz) δ 151.2, 151.1, 150.2, 149.9, 142.1, 137.9, 129.1, 124.1,
121.1, 120.4, 115.1, 90.7, 85.3, 84.1, 81.8, 52.3, 27.2, 25.4; MS (FAB) m/z
452.17923 (MH+ [C20H22N9O4] = 452.17948).
A solution of 5ꢁ-azido-5ꢁ-deoxy-2ꢁ,3ꢁ-O-isopropylidene-N6-(N -phenylcar-
bamoyl)adenosine (70 mg, 0.16 mmol) and 10% Pd–C (40 mg) in EtOAc
(10 mL) was vigorously stirred for 15 hours under an atmosphere of H2

Some N6,5ꢁ-Bis-ureido 5ꢁ-Amino-5ꢁ-deoxyadenosine Derivatives
403
FIGURE 4 Percent activity change in cancer-related protein kinases treated with 2c (20 µM).

404
M. A. Peterson et al.
(balloon pressures). p-Nitrophenyl N -methylcarbamate (43 mg, 0.22 mmol)
and anhydrous Na2CO3 (45 mg, 0.43 mmol) were added, and the resulting
mixture was stirred for 4 hours under N2. Solids were removed via filtration
(celite/EtOAc), and volatiles were evaporated under reduced pressure. The
crude residue was chromatographed (5 → 10% MeOH/CH2Cl2) to give 4a
1
(65 mg, 87%): H NMR (CDCl3, 500 MHz) δ 12.10 (s, 1H), 9.79 (s, 1H),
8.69 (s, 1H), 8.68 (s, 1H), 7.56 (dd, J = 8.8, 0.8 Hz, 2H), 7.40 (t, J = 8.0 Hz,
2H), 7.20 (t, J = 8.0 Hz, 1H), 6.15 (d, J = 4.0 Hz, 1H), 5.84 (m, 1H), 5.27
(dd, J = 6.3, 3.8 Hz, 1H), 4.98 (dd, J = 6.3, 2.3 Hz, 1H), 4.77 (m, 1H), 4.52
(dd, J = 6.8, 2.8 Hz, 1H), 3.74 (ddd, J = 13.8, 7.4, 4.1 Hz, 1H), 3.38 (dt, J =
3.8, 14.8 Hz, 1H), 2.56 (d, J = 4.5 Hz, 3H), 1.65 (s, 3H), 1.40 (s, 3H); 13C
NMR (CDCl3, 125 MHz) δ 159.2, 152.6, 150.9, 150.34, 150.30, 143.3, 137.2,
129.2, 124.9, 121.5, 121.2, 114.6, 91.6, 85.9, 83.9, 81.6, 41.8, 27.4, 26.9, 25.4;
MS (FAB) m/z 483.2099 (MH+ [C22H27N8O5] = 483.2099).
5ꢁ-Deoxy-5ꢁ-[(N-methylcarbamoyl)amino]-N6-(N-phenylcarbamoyl)adenosine
(4b)
A solution of 4a (10 mg, 0.021 mmol), TFA (100 µL), and H2O (25 µL)
in CH2Cl2 (500 µL) was vigorously stirred at ambient temperature until TLC
indicated complete conversion to baseline product (4 hours). Volatiles were
removed under reduced pressure (≤25◦C) and the crude was purified via
flash chromatography (EtOAc/iPrOH/H2O, 4:1:2) to give 4b (7 mg, 75%):
1H NMR (CDCl3, 500 MHz) δ 11.75 (s, 1H), 10.21 (s, 1H), 8.71 (s, 1H),
8.70 (s, 1H), 7.61 (d, J = 7.5 Hz, 2H), 7.35 (t, J = 7.8 Hz, 2H), 7.08 (t, J =
7.5 Hz, 1H), 6.10 (s, 1H), 5.97 (d, J = 6.0 Hz, 1H), 5.80 (s, 1H), 4.67 (t, J =
5.5 Hz, 1H), 4.09 (dd, J = 3.5, 5.0 Hz, 1H), 3.94–3.91 (m, 1H), 3.41 (dd,
J = 14.3, 4.3 Hz, 1H), 3.26 (dd, J = 14.3, 6.3 Hz, 1H), 2.49 (s, 3H; overlaps
with DMSO); 13C NMR (CDCl3, 125 MHz) δ 159.1, 151.4, 151.2, 151.1, 150.2,
143.4, 138.9, 129.4, 123.7, 121.1, 119.9, 87.9, 84.9, 73.6, 71.6, 42.2, 26.8; MS
(FAB) m/z 443.17757 (MH+ [C19H23N8O5] = 443.17859).
KinomeScan Competitive Binding Assay
This assay is based on ligand-affinity/protein kinase phage display and
was employed essentially as described by Lockhart and co-workers.[13] In
this assay, protein kinases are cloned into T7 bacteriophage which express
the kinase fusion proteins on the phage capsid. T7 kinase-tagged phage are
then screened for binding to ATP-binding-site ligands that have been immo-
bilized on a solid support. Phage are screened for binding to the anchored
ligands both in the presence of test compound and in its absence (control).
Elution of the bound phage by free ligand (ATP-binding site ligand that
is not immobilized on a solid support) followed by determination of the
phage titre provides a reliable measure of the ability of test compounds
to block binding of target kinases to resin-bound ATP-binding-site ligands.

Some N6,5ꢁ-Bis-ureido 5ꢁ-Amino-5ꢁ-deoxyadenosine Derivatives
405
This method has allowed rapid mapping of small molecule interactions with
ATP-binding sites across a broad cross-section of disease related protein
kinases and has been validated as a reliable tool for identifying ligands
with strong affinities for ATP-binding sites in numerous protein kinases.[13]
Analyses were performed in duplicate and the values reported in Figure
2A–C are averages for the two experiments.
Kinases screened in this assay included: AAK1, ABL1, ABL1(E255K),
ABL1(F317I), ABL1(F317L), ABL1(H396P), ABL1(M351T), ABL1
(Q252H), ABL1(T315I), ABL1(Y253F), ABL2, ACVR1, ACVR1B,
ACVR2A, ACVR2B, ACVRL1, ADCK3, ADCK4, AKT1, AKT2, AKT3, ALK,
AMPK-alpha1, AMPK-alpha2, ANKK1, ARK5, AURKA, AURKB, AURKC,
AXL, BIKE, BLK, BMPR1A, BMPR1B, BMPR2, BMX, BRAF, BRAF(V600E),
BRSK1, BRSK2, BTK, CAMK1, CAMK1D, CAMK1G, CAMK2A, CAMK2B,
CAMK2D, CAMK2G,
CAMK4, CAMKK1, CAMKK2, CDC2L1, CDC2L2, CDK11, CDK2, CDK3,
CDK5, CDK7, CDK8, CDK9, CDKL2, CHEK1, CHEK2, CIT, CLK1, CLK2,
CLK3, CLK4, CSF1R, CSK, CSNK1A1L, CSNK1D, CSNK1E, CSNK1G1,
CSNK1G2, CSNK1G3, CSNK2A1, CSNK2A2, DAPK1, DAPK2, DAPK3,
DCAMKL1, DCAMKL2, DCAMKL3, DDR1, DDR2, DLK, DMPK, DMPK2,
DRAK1, DRAK2, DYRK1B, EGFR, EGFR(E746-A750del), EGFR(G719C),
EGFR(G719S), EGFR(L747-E749del, A750P), EGFR(L747-S752del,
P753S), EGFR(L747-T751del,Sins), EGFR(L858R), EGFR(L861Q),
EGFR(S752-I759del), EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6,
EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, ERBB2, ERBB4,
ERK1, ERK2, ERK3, ERK4, ERK5, ERK8, FER, FES, FGFR1, FGFR2,
FGFR3, FGFR3(G697C), FGFR4, FGR, FLT1, FLT3, FLT3(D835H),
FLT3(D835Y), FLT3(ITD), FLT3(K663Q), FLT3(N841I), FLT4, FRK, FYN,
GAK, GCN2(Kin.Dom.2,S808G), GSK3A,
GSK3B, HCK, HIPK1, IGF1R, IKK-alpha, IKK-beta, IKK-epsilon,
INSR, INSRR, IRAK3, ITK, JAK1 (Kin.Dom.1), JAK1(Kin.Dom.2),
JAK2(Kin.Dom.2), JAK3(Kin.Dom.2), JNK1, JNK2, JNK3, KIT, KIT
(D816V), KIT(V559D), KIT(V559D,T670I), KIT(V559D,V654A), LATS1,
LATS2, LCK, LIMK1, LIMK2, LKB1, LOK, LTK, LYN, MAP3K3, MAP3K4,
MAP3K5, MAP4K1, MAP4K2, MAP4K3, MAP4K4, MAP4K5, MAPKAPK2,
MAPKAPK5, MARK1, MARK2, MARK3, MARK4, MEK1, MEK2, MEK3,
MEK4, MEK6, MELK, MERTK, MET, MINK, MKNK1, MKNK2, MLCK,
MLK1, MLK2, MLK3, MRCKA,
MRCKB, MST1, MST1R, MST2, MST3, MST4, MUSK, MYLK, MYLK2,
MYO3A, MYO3B, NDR2, NEK1, NEK2, NEK5, NEK6, NEK7, NEK9, NLK,
p38-alpha, p38-beta, p38-delta, p38-gamma, PAK1, PAK2, PAK3, PAK4,
PAK6, PAK7/PAK5, PCTK1, PCTK2, PCTK3, PDGFRA, PDGFRB, PDPK1,
PFTAIRE2, PFTK1, PHKG1, PHKG2, PIK3C2B, PIK3CA, PIK3CA(E545K),
PIK3CB, PIK3CD, PIK3CG, PIM1, PIM2, PIM3, PIP5K1A, PIP5K2B,
PKAC-alpha, PKAC-beta, PKMYT1, PKN1, PKN2, PLK1, PLK3, PLK4,

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M. A. Peterson et al.
PRKCD, PRKCE, PRKCH, PRKCQ, PRKD1, PRKD2, PRKD3, PRKG1,
PRKG2, PRKR, PRKX, PTK2, PTK2B, PTK6, RAF1, RET, RET(M918T),
RET(V804L), RET(V804M), RIOK1, RIOK2, RIOK3, RIPK1, RIPK2,
RIPK4, ROCK2, ROS1, RPS6KA1(Kin.Dom.1), RPS6KA1 (Kin.Dom.2),
RPS6KA2(Kin.Dom.1), RPS6KA2(Kin.Dom.2), RPS6KA3(Kin.Dom.1),
RPS6KA4
(Kin.Dom.1), RPS6KA4(Kin.Dom.2), RPS6KA5(Kin.Dom.1), RPS6KA5
(Kin.Dom.2), RPS6KA6 (Kin.Dom.1), RPS6KA6(Kin.Dom.2), SgK085,
SgK110, SLK, SNARK, SNF1LK, SNF1LK2, SRC, SRMS, SRPK1, SRPK2,
SRPK3, STK16, STK33, STK35, STK36, SYK, TAK1, TAOK1, TAOK3, TEC,
TESK1, TGFBR1, TGFBR2, TIE1, TIE2, TLK1, TLK2, TNIK, TNK1, TNK2,
TNNI3K, TRKA, TRKB, TRKC, TSSK1, TTK, TXK, TYK2(Kin.Dom.1),
TYK2(Kin.Dom.2), TYRO3, ULK1, ULK2, ULK3, VEGFR2, WEE1, WEE2,
YANK2, YANK3, YES, YSK1, ZAK, ZAP70.
Kinase Inhibition Assay
The various protein kinase targets employed in the kinase inhibition
assay were cloned, expressed, and purified in-house at SignalChem Inc.
(Richmond, BC, Canada) using proprietary methods. Quality control test-
ing is routinely performed on each of the SignalChem targets to ensure
compliance to acceptable standards. Protein substrates employed in the
target profiling process were synthesized internally. 33P-ATP was purchased
from PerkinElmer. All other materials were of standard grade. Compound
2c was supplied to SignalChem in a powder form. It was reconstituted in
DMSO to form a stock solution which was then diluted with 10% DMSO to
form a working stock solution (100 µM) that was then profiled against the
various protein kinase targets. Protein kinase assays were performed in trip-
licate at ambient temperature for 20–40 minutes (depending on the target)
in a final volume of 25 µl according to the following assay reaction recipe:
Component 1: 5 µl of diluted active protein kinase target (∼10–40 nM
final protein concentration in the assay); Component 2: 5 µl of stock
solution of substrate (1–5 µg of peptide or protein substrate); Component
3: 5 µl of kinase assay buffer or protein kinase activator in kinase assay
buffer; Component 4: 5 µl of Compound 2c (100 µM stock solution) or
10% DMSO; Component 5: 5 µl of 33P-ATP (25 µM stock solution, 0.8 µCi).
The assay was initiated by the addition of 33P-ATP and the reaction
mixture incubated at ambient temperature for 20–40 minutes, depending
on the protein kinase target. After the incubation period, the assay was
terminated by spotting 10 µl of the reaction mixture onto a Millipore
Multiscreen plate (Billerica, MA, USA). The Millipore Multiscreen plate was
washed 3 times for approximately 15 minutes each in a 1% phosphoric acid
solution. The radioactivity on the P81 plate was counted in the presence
of scintillation fluid in a Trilux scintillation counter. Blank control, which

Some N6,5ꢁ-Bis-ureido 5ꢁ-Amino-5ꢁ-deoxyadenosine Derivatives
407
included all the assay components except the addition of the appropriate
substrate (replaced with equal volume of assay dilution buffer), was set up
for each protein kinase target. The corrected activity for each protein kinase
target was determined by removing the blank control value. Activity of the
50 kinase targets in the presence of compound 2c is shown in Figure 4.
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