Pyrazolobenzodiazepines: Part I. Synthesis and SAR of a potent class of kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.08.079

A novel series of pyrazolobenzodiazepines 3 has been identified as potent inhibitors of cyclin-dependent kinase 2 (CDK2). Their synthesis and structure-activity relationships (SAR) are described. Representative compounds from this class reversibly inhibit CDK2 activity in vitro, and block cell cycle progression in human tumor cell lines. Further exploration has revealed that this class of compounds inhibits several kinases that play critical roles in cancer cell growth and division as well as tumor angiogenesis. Together, these properties suggest a compelling basis for their use as antitumor agents.

Full text of DOI:10.1016/j.bmcl.2010.08.079

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5984–5987
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazolobenzodiazepines: Part I. Synthesis and SAR of a potent class
of kinase inhibitors
Jin-Jun Liu a, , Irena Daniewski a, Qingjie Ding a, Brian Higgins b, Grace Ju b, Kenneth Kolinsky b,
⇑
Fred Konzelmann a, Christine Lukacs a, Giacomo Pizzolato a, Pamela Rossman a, Amy Swain a,
Kshitij Thakkar a, Chung-Chen Wei a, Dorota Miklowski a, Hong Yang b, Xuefeng Yin b, Peter M. Wovkulich a,
⇑
a Discovery Chemistry, Hoffmann-La Roche Inc., 340 Kingsland St. Nutley, NJ 07110, USA
b Discovery Oncology, Hoffmann-La Roche Inc., 340 Kingsland St. Nutley, NJ 07110, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2010
Revised 13 August 2010
Accepted 17 August 2010
Available online 21 August 2010
A novel series of pyrazolobenzodiazepines 3 has been identified as potent inhibitors of cyclin-dependent
kinase 2 (CDK2). Their synthesis and structure–activity relationships (SAR) are described. Representative
compounds from this class reversibly inhibit CDK2 activity in vitro, and block cell cycle progression in
human tumor cell lines. Further exploration has revealed that this class of compounds inhibits several
kinases that play critical roles in cancer cell growth and division as well as tumor angiogenesis. Together,
these properties suggest a compelling basis for their use as antitumor agents.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Pyrazolobenzodiazepines
Kinase inhibitors
CDK2
Antitumor agents
Cyclin dependent kinases are critical components utilized by
cells in the progression through the cell cycle. Their aberrant con-
trol in a wide range tumor types has suggested a potential for ther-
apeutic intervention and has been the subject of numerous studies
and reviews.1–4 While multiple avenues of exploration existed
from the modulation of the various CDK activities, our initial
excursion began with a focus on finding inhibitors of cyclin-depen-
dent kinase 2 (CDK2).5,6
was directed towards the exploration of the substitution on the
two phenyl rings of 2, with typical groups like methyl, nitro, acetyl
and so on. In general, substitutions at positions other than at 7 or 20
provided no improvements (data not shown).
The dramatic loss of activity by simple alkylation of the amino-
pyrazole nitrogens provided confirmation that these atoms were
making key contributions to the binding. Further SAR exploration
was then concentrated on manipulation of R1, R2, and R3 groups
in structure 3 (Fig. 2). This effort led to a series of novel, potent pyr-
azolobenzodiazepine derived multi kinase inhibitors, which had
superior cellular activities compared to compound 2.
A high throughput screen identified the aminopyrazole com-
pound 1 (Fig. 1) as a moderate CDK2 inhibitor (CDK2-cyclin E
IC50 = 2
lM) which had weak activity in the cell proliferation
(MTT) assays (SW480, IC50 = 6
l
M; HCT116 IC50 = 16 M). As a pre-
l
caution against false negatives in the high throughput screen, a dif-
ferently formatted follow-up screen was conducted on structurally
related substances in the screening inventory. One of the more inter-
esting hits identified was compound 2, a clonazepam analog pre-
pared during an earlier program on valium-librium analogs.7–11
This compound was found to be devoid of any CNS activity.
Despite the good enzymatic potency and interesting selectivity
of compound 2, it possessed modest cellular activity (SW480
IC50 = 6 lM) and unattractive pharmacokinetic properties that pro-
H
N
N
NH
CF3
NH2
N
N
O2N
N
Cl
N
H
S
1
2
hibited further development. Herein, we describe the optimization
of compound 2 with an emphasis on improving cellular potency
and ultimately demonstrating in vivo efficacy. The initial SAR work
CDK2-cyclin E 2 µM
CDK1-cyclin B 10 µM
CDK4-cyclin D1 > 30 µM
SW480 6 µM
CDK2-cyclin E 0.07 µM
CDK1-cyclin B 0.5 µM
CDK4-cyclin D1 10 µM
SW480 6 µM
⇑
Corresponding author. Tel.: +1 973 235 2851; fax: +1 973 235 7122.
E-mail address: jin-jun.liu@roche.com (J.-J. Liu).
Figure 1. Structure and biological activity of 1 and 2.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.079

J.-J. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5984–5987
5985
1
Table 1
H
N
N
Inhibition of CDK2 and cell proliferation (IC50
pyrazole ring
,
l
M) of analogs substituted at the
9
NH
H
N
N
NH 2
R1
3
N
H
N
R2
NH
R1
N
N
O2N
7
R3
Cl
N
O2N
2'
2
3
5'
3
Cl
Figure 2. Optimization plan.
Compd
R1
H
Me
iPr
–CH2OH
–CF3
–CONH2
Ph
–CH2CH2Ph
2-Pyrrolyl
3-Pyridinyl
4-Pyridinyl
CDK2a,c
SW480b,c
The general synthetic approach for the preparation of
pyrazolobenzodiazepines 3 is shown in Scheme 1. The starting
1,4-benzodiazepines 4, which can be easily prepared from substi-
tuted 2-amino-benzophenones according to the known meth-
ods,7–11 were converted to the corresponding thiolactam 5 by the
reaction with Lawesson’s reagent. The thiolactam 5 was a more
useful and adaptable intermediate for analog generation than the
lactam 4, due to its greater reactivity. When R1 was hydrogen, final
construction of the pyrazole ring system was accomplished by
formylation [Me2NCH(OEt)2] of 5 to form enamine 6, followed by
reaction with hydrazine. In all other examples pyrazole ring forma-
tion required piperidine mediated coupling of 5 with an aldehyde
(R1CHO) to form 7, followed by reaction of 7 with hydrazine to
form the dihydropyrazole, and oxidation (air, DMSO, 150 °C) to af-
ford the pyrazole, 3.
2
0.070
0.035*
0.010
6.00
0.16
0.64
0.24
N/A
3a
3b
3c
3d
3e
3f
3g
3h
3i
**
***
****
0.238
0.232
0.258
0.210
5.000
0.020
0.300
0.050
N/A
18.00
15.00
5.30
26.00
12.00
3j
a
IC50 values were determined with at least two replicates at each concentration
except the following: *n = 52, SD = 0.012; **n = 2, SD = 0.031; ***n = 3, SD = 0.039;
****n = 2, SD = 0.053.
b
Assay done once in duplicate wells.
Assay details see Ref. 6.
c
The structure–activity relationship of R1 was explored, keeping
R2 and R3 fixed as shown in Table 1. These data show that only
small hydrophobic groups were tolerated at R1. Four analogs were
more potent than 2 against CDK2 (3a, 3b, 3h, and 3j).6 The methyl
(3a), 2-pyrrolyl (3h) and the 4-pyridinyl (3j) produced modest
improvements in potency, while the isopropyl substitution (3b)
provided the greatest improvement (7ꢀ). Hydroxymethyl (3c), trif-
louromethyl (3d), amide (3e), phenyl (3f), phenylethyl (3g), and
3-pyridinyl (3i) analogs showed no improvement over hydrogen.
In a SW480 cellular assay, three analogs (3a, 3b and 3c) were found
to show potent inhibition of proliferation. Interestingly, the methyl
analog 3a is more potent than isopropyl analog 3b although it’s
less active in the CDK2 assay.
An X-ray crystal structure of compound 3a complexed with
CDK2 is shown in Figure 3. The pyrazolobenzodiazepine core occu-
pies the same site as the adenosine of ATP, and makes three critical
H-bonds to the hinge region. N1 of the pyrazole accepts a hydrogen
bond from the backbone Leu83 NH. The pyrazole N2H forms a
hydrogen bond with the backbone carbonyl of Glu81 and the dia-
zepine N4H forms a hydrogen bond with the Leu83 carbonyl. The
methyl substituent attached to the pyrazole ring is buried and
H
N
S
O
H
N
a
R2
N
R2
N
R3
R3
5
4
S
H
N
For R1 = H
b
R2
NMe2
N
5
c
H
N
N
NH
R1
R3
R2
N
6
R3
H
N
S
e, f
R1-CHO
d
3
R1
R2
N
5
R3
7
Scheme 1. Reagents and conditions: (a) Lawesson’s Reagent, dimethoxyethane/75 °C; (b) Me2NCH(OEt)2/THF/rt; (c) Anhydrous hydrazine/CH2Cl2/rt; (d) R1CHO/piperidine/
dimethoxyethane/rt; (e) Hydrazine/DMSO/rt; (f) air oxidation, DMSO, 150 °C.

5986
J.-J. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5984–5987
Replacement of either R2 or both R2 and R3 with hydrogens re-
sulted in a potency drop (3k, 3l vs 3a) of about threefold in the
CDK2 assay and a 10- to 25-fold drop in the cellular assay. Other
electron-withdrawing groups at the 7-position are also tolerated
(3s, 3v, 3w, 3x, and 3y). On the other hand, electron-donating
groups tended to decrease the potency in both the CDK2 and cellu-
lar assays (3t and 3u). In general, fluoro was better than chloro in
both assays (3l vs 3m, 3q vs 3r).
A limited round of selectivity profiling was conducted with a
few of the compounds to assess interactions with other kinase tar-
gets; the results are shown in Table 3. Compounds 3a, 3s, 3u and
3v all had weaker activity against CDK1, CDK4 and FAK.
We also tested this set of compounds against receptor tyrosine
kinases involved in angiogenesis, such as KDR, FGFr, PDGFr and
EGFr.13–17 Of particular interest, compounds 3u and 3v showed good
potency against KDR, FGFr, PDGFr and EGFr suggesting an additional
therapeutic modality for this class of compounds (Table 4).
Compounds 3u and 3v were then tested in panel of five human
tumor cell lines to confirm cellular activities. As shown in Table 5,
both analogs demonstrated strong antiproliferative activities when
tested in cells. The IC50 values ranged from 0.3 to 1.5 lM. Subse-
Figure 3. Crystal structure of compound 3a bound to CDK2. Selected nearby protein
residues are shown. Hydrogen bonding interactions with the protein are indicted as
dashed black lines. The figure was prepared using PyMOL.
quent evaluation in additional cell lines (data not shown) showed
activity against a wide range of cancer cell types. However, more
detailed studies on the effects of compound 3u on the cell cycle
(FACS analysis) revealed that its antiproliferative activities may
not directly correlate with effects on CDK2.18
points towards the face of the gatekeeper phenylalanine (Phe80);
this space could easily accommodate an isopropyl (most potent
compound 3b) or a small aromatic ring but not large substitutions.
The chlorophenyl ring is oriented 80 degrees from the core, allow-
ing the chlorine to point toward a mostly hydrophobic dimple cre-
ated by Ala144, Leu134, and Asn132. The nitro substituent is
solvent exposed and other water mediated contacts to the protein.
Finally, the diazepine N is involved in a hydrogen bonding network
to Asp145 via a series of bridging water molecules.12
Table 3
Kinase inhibition profile of selected analogs (IC50, l
M)a
Compd
CDK2b
CDK1b
CDK4b
FAK
Srca
3a
3s
3u
3v
0.035
0.016
0.088
0.038
0.310
0.139
0.382
0.352
0.252
0.520
0.303
0.213
6.900
1.200
0.150
0.180
>60
5.400
1.840
0.930
We next focused on the modification of R2 and R3 groups on the
aromatic rings with R1 as methyl. The results are shown in Table 2.
a
IC50 values were determined with at least two replicates at each concentration.
Cyclin E1, D1, and A1 were used in the CDK assays as the CDK partners for CDK2,
b
Table 2
Inhibition of CDK2 and cell proliferation (IC50
groups
,
l
M) of analogs with various R2 and R3
CDK4, and CDK1 respectively.
N
H
N
NH
Me
Table 4
Kinase inhibition profile of selected analogs (IC50, lM)
R2
N
KDRb
FGFrc
PDGFrd
EGFre
7
Compd
R3
*
*
3a
3s
3u
3v
0.072 0.015
0.039 0.008
0.100
0.067
0.050 0.008*
0.045 0.014*
0.083
0.051
0.015 0.002*
0.023
>10
3
10.900
0.989
0.726
0.034 0.006*
0.033 0.005*
Compd
R2
R3
CDK2a,c
SW480b,c
a
IC50 values were determined with at least two replicates at each concentration
except the following: *n = 2.
*
3a
3k
3l
3m
3n
3o
3p
3q
3r
NO2
H
H
H
F
F
Cl
Cl
Cl
CN
OMe
NH2
AcNH
NH2CO
Cl
H
Cl
F
H
F
H
Cl
F
0.035
0.115
0.105
0.064
0.093
0.040
0.490
0.440
0.190
0.016
0.320
0.088
0.16
1.50
3.80
0.40
2.20
0.70
5.50
3.60
3.00
0.11
7.00
0.50
0.70
0.21
0.20
1.10
b
With 300
lM ATP.
c
d
e
With 10
With 2.3
With 0.5
lM ATP.
M ATP.
M ATP.
l
l
Table 5
Cellular activities of selected analogs (IC50
**
3s
3t
F
,
l
M)a
RKO
Cl
Cl
Cl
F
Cl
Cl
3u
3v
3w
3x
3y
Compd H460ab
HCT116
MDA-MB435
SW480
0.500
0.038***
0.017
0.024
3u
3v
1.071 (n = 6,
SD = 0.210)
0.550
1.018 (n = 5,
SD = 0.124)
0.460
0.310 0.950 (n = 2,
SD = 0.050)
0.793 0.794 (n = 3,
SD = 0.074)
MeSO2NH–
Me2NSO2NH–
0.046
0.701
a
IC50 values were determined with at least two replicates at each concentration
except the following: *n = 52, SD = 0.012; **n = 2, SD = 0.007; ***n = 3, SD = 0.011.
a
Values represent the mean of duplicates wells.
The tissue origins are: lung: H460a; colon: HCT116, RKO, SW480; breast: MDA-
b
b
Assay done once in duplicate wells.
Assay details see Ref. 6.
c
MB435.

J.-J. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5984–5987
5987
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100
0
Acknowledgments
Vehicle bid
The authors would like to thank Mr. Gino Sasso for 1H NMR
spectral analysis and NOE studies, Mr. Vance Bell, Mr. Richard
Szypula, Ms. Theresa Burchfield, and Mr. Michael Lanyi for mass
and IR spectral analysis. We thank members of the Discovery
Oncology In Vivo Section for assistance with tumor efficacy
studies.
3.125 mg/kg bid
1.56 mg/kg bid
0.78 mg/kg bid
References and notes
1. While the initial rationale for developing a CDK2 selective inhibitor inspired
the undertaking of this work, the continuing examination of the cell cycle has
revealed that the roles of the CDKs are more complex. Reviews on the more
recent aspects may be found in: (a) Lapenna, S.; Giordano, A. Nat. Rev. Drug Disc.
2009, 8, 547; (b) Satyanarayana, A.; Kaldis, P. Oncogene 2009, 28, 2925; (c)
Johnsson, M.; Persson, J. L. Anti-Cancer Agents Med. Chem. 2009, 9, 153; (d)
Fischer, P. M. Cancer Drug Des. Discovery 2008, 253.
2. Kong, N.; Fotouhi, N.; Wovkulich, P.; Roberts, J. Drugs Fututre 2003, 28, 881.
3. Meijer, L.; Raymond, E. Acc. Chem. Res. 2003, 36, 417.
4. Vassilev, L. T. Cell Cycle 2006, 5, 2555.
18 20 22 24 26 28 30 32 34 36 38 40 42
Days Post-Tumor Cell Implant
Average % Weight Change
15.0
10.0
5.0
5. Liu, J. J.; Dermatakis, A.; Lukacs, C.; Konzelmann, F.; Chen, Y.; Kammlott, U.;
Depinto, W.; Yang, H.; Yin, X.; Chen, Y.; Schutt, A.; Simcox, M. E.; Luk, K. C.
Bioorg. Med. Chem. Lett. 2003, 13, 2465.
Vehicle
6. Ding, Q.; Liu, J. J.; Pizzolato, G.; Wei, C. C.; Wovkulich, P. U.S. Patent 6,440,959,
2002.
7. Sternbach, L. H.; Fryer, R. I.; Keller, O.; Metlesics, W.; Sach, G.; Steiger, N. J. Med.
Chem. 1963, 6, 261.
3.125mg/kg
1.56mg/kg
0.78mg/kg
0.0
8. Sternbach, L. H.; Saucy, G.; Smith, F. A.; Mueller, M.; Lee, J. Helv. Chim. Acta
1963, 46, 1720.
9. Fryer, R. I.; Brust, B.; Earley, J.; Sternbach, L. H. J. Med. Chem. 1964, 7, 386.
10. Sternbach, L. H.; Fryer, R. I.; Metlesics, W.; Sach, G.; Stempel, A. J. Org. Chem.
1962, 27, 3781.
-5.0
20 22 24 26 28 30 32 34 36 38 40 42
Days Post-Tumor Cell Implant
Figure 4. Efficacy and body weight changes in SW480 tumor-bearing nude mice
treated with 3u by oral administration. Compound was administered per os (po)
using a 1-cc syringe and 18-gauge gavage needle (0.2 ml/animal; all groups were
treated twice-daily (b.i.d.), 8 h apart, 7x/week for a total of 20 days. Treatments
ended on day 41 post-implant.
11. (a) Sternbach, L. H.; Fryer, R. I.; Metlesics, W.; Reeder, E.; Sach, G.; Saucy, G.;
Stempel, A. J. Org. Chem. 1962, 27, 3788; (b) Walser, A.; Fryer, R. I.. In Chemistry
of Heterocyclic Compounds; Chichester: United Kingdom, 1991; Vol. 50
. pp 431–543, 545–629, 631–848, 849–946.
12. Crystals of the CDK2:3a complex were grown at 4 °C by the vapor diffusion
method. CDK2(1–298) at 12 mg/mL was mixed with a five molar excess of
compound and equilibrated against 10% PEG 3350, 0.2 M ammonium fluoride,
0.1 M Tris pH 8.5. 0.2% DTT was added to the reservoir after mixing of the drop.
Cryoprotectant was the same as the reservoir with 20% PEG 3350 and the
addition of 15% ethylene glycol. X-ray data was collected at beamline X8C at
Brookhaven National Laboratories. Data was processed to 2.0 Å with the HKL
package (Otwinowski, Z., Minor, W. Methods Enzymol. 1997, 276, 307). The
structure was determined by molecular replacement using AmoRe (Navaza, J.
Acta Crystallogr. 1994, A50, 157) and was refined with CNX (CNX v.99.0-BETA,
Molecular Simulations Inc.) to an R-factor/Rfree of 0.196/0.243. The structure
has been deposited to the PDB with the code 3LE6.
Based on its superior overall profile in the kinase and cellular
assays, and PK properties,18 compound 3u was selected for
in vivo antitumor studies. Compound 3u was administered orally
for 20 days to tumor-bearing mice (SW480) at doses of 3.125,
1.56 and 0.78 mg/kg (b.i.d.). The compound was well tolerated,
with no overt signs of drug related toxicity (ca. 9% body weight
gain over the course of the study) and produced significant inhibi-
tion of tumor growth (Fig. 4). At the highest dose, 3.125 mg/kg,
tumor growth inhibition was 93% (%T/C = 7, p 6 0.001), while the
mid dose (1.56 mg/kg) produced 70% tumor growth inhibition
(%T/C = 30, p = 0.021). Compound 3u has also shown significant
antitumor activity in additional xenograft models (H460, LOX,
LoVo and A549) as well as angiogenesis models.17,18
In summary, starting from pyrazolobenzodiazepine 2, as a CDK2
inhibitor with modest cellular activities, we developed this novel
template into a very interesting series of potent multikinase inhib-
itors. Further optimization of this pyrazolobenzodiazepine series
with a focus on improving their dual activities as of anti-tumor
and anti-angiogenesis agents will be described in subsequent
publications.
13. Carmeliet, P.; Jain, R. K. Nature 2000, 407, 249.
14. Tonini, T.; Rossi, F.; Claudio, P. P. Oncogene 2003, 22, 6549.
15. Risau, W. Nature 1997, 386, 671.
16. Folkman, J. Nat. Med. 1995, 1, 27.
17. Muruganandham, M.; Lupu, M.; Dyke, J. P.; Matei, C.; Linn, M.; Packman, K.;
Kolinsky, K.; Higgins, B.; Koutcher, J. A. Mol. Cancer Ther. 2006, 5, 1950.
18. Higgins, B.; Kolinsky, K.; Yang, H.; Tovar, C.; Carvaial, D.; Yin, X.; Bachynsky, M.;
Margolis, R.; Nevins, T.; Geng, W.; Lamb, M.; Linn, M.; Liu, J. J.; Wovkulich, P.;
Packman, K.; Ju, G., 17th AACR-NCI-EORTC Int Conf Mol Targets Cancer Ther.
2005, Abst B217. In summary, this poster presented data which showed that
the antiproliferative activity correlated with a block in M phase in the cell cycle
and subsequent apoptosis, activities which would not be expected for a pure
CDK2 inhibitor. A more detailed analysis of the biological properties of the
related pyrazolobenzodiazepine, R1530, will be presented in future
publications. Pharmacokinteic data, as reported in the poster, was obtained
in female C57 BL6 mice (n = 3) after a 10 mg/kg IV dose and a 100 mg/kg PO
dose. IV: AUC = 14809 ng h/mL, CL = 11.2 ml/min/kg, Vdss = 3.5 L/kg, T1/2 = 2.9 h,
PO: AUC = 263289 ng h/mL, Cmax = 26400 ng/mL, Tmax = 4 h, T1/2 = 2.7 h.