Improvement of the synthesis and pharmacokinetic properties of chromenotriazolopyrimidine MDM2-p53 protein-protein inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.11.027

Human murine double minute 2 (MDM2) is a negative regulator of p53, which plays an important role in cell cycle and apoptosis. We report several optimizations to the synthesis of the chromenotriazolopyrimidine series of MDM2-p53 protein-protein interaction inhibitors. Additionally, the in vitro and in vivo stability, pharmacokinetic properties and solubility were improved through N-substitution.

Full text of DOI:10.1016/j.bmcl.2010.11.027

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2752–2755
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Improvement of the synthesis and pharmacokinetic properties
of chromenotriazolopyrimidine MDM2-p53 protein-protein inhibitors
Hilary P. Beck a, , Michael DeGraffenreid a, Brian Fox a, John G. Allen b, Yosup Rew a, Stephen Schneider c,
⇑
Anne Y. Saiki d, Dongyin Yu d, Jonathan D. Oliner d, Kevin Salyers e, Qiuping Ye f, Steven Olson a
a Chemistry Research & Discovery, Amgen Inc., 1120 Veterans Blvd, South San Francisco, CA 94080, USA
b Chemistry Research & Discovery, Amgen Inc., One Amgen Center Drive Thousand Oaks, CA 91320, USA
c Chemistry Research & Discovery, Amgen Inc., 360 Binney Street Cambridge, MA 02142, USA
d Oncology Research, Amgen Inc., One Amgen Center Drive Thousand Oaks, CA 91320, USA
e PKDM, Amgen Inc., One Amgen Center Drive Thousand Oaks, CA 91320, USA
f PKDM, Amgen Inc., 1120 Veterans Blvd, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Human murine double minute 2 (MDM2) is a negative regulator of p53, which plays an important role in
cell cycle and apoptosis. We report several optimizations to the synthesis of the chromenotriazolopyrim-
idine series of MDM2-p53 protein-protein interaction inhibitors. Additionally, the in vitro and in vivo sta-
bility, pharmacokinetic properties and solubility were improved through N-substitution.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 7 October 2010
Revised 1 November 2010
Accepted 3 November 2010
Available online 10 November 2010
Keywords:
Oncology
Protein–protein interaction
Pharmacokinetics
The tumor suppressor p53 is responsible for the transcription of
several genes controlling cell cycle arrest and apoptosis and is
known to play an important role in regulating tumor formation
and proliferation.1 The oncoprotein MDM2 directly blocks p53
activity and promotes the degradation of p53.2 MDM2 is often over
expressed in cancer and its expression has been correlated with
accelerated disease progression.3 Inhibition of the MDM2-p53 pro-
tein-protein interaction, and the resulting p53 activation, has been
proposed as a novel oncology therapeutic strategy.4 The chro-
menotriazolopyrimidine MDM2 inhibitor 1 demonstrated p53
dependent inhibition of HCT116 cancer cell proliferation (Fig. 1).5
Although exhibiting moderate cellular antiproliferative activity, 1
displayed poor pharmacokinetic (PK) properties. In rat in vivo
experiments,6 1 exhibited moderate-to-high clearance (2.4 L/h/
kg) and an oral bioavailability of only 23%. In addition, 1 suffered
make several improvements to the synthesis (Scheme 1). For
example, in the initial condensation reaction, adjustments to the
workup procedure improved the yield of enone 2 from 65% to
90% yield. By slowly adding the reaction mixture to a rapidly
stirring solution of ice water, we were able to obtain a free flowing
off-white powder of enone 2. Employing the finely powdered en-
one 2 in the subsequent melt reaction with aminotriazole resulted
in an improvement of the yield for phenol 3 from 41% to 86%. The
previous procedure for the condensation of 3 employed 1.3 equiv
of bromobenzaldehyde at 160 °C and led to a 1:4 ratio of 4/5 in
20% yield. Since the desired diastereomer 4 was the minor product
in this transformation, we sought to further optimize this reaction.
It was found that decreasing the reaction temperature to 140 °C
and increasing the equivalents of bromobenzaldehyde to 3.0
from low solubility in PBS (<1 l
g/mL).7 Herein we report our
Me
N
efforts to improve the PK properties within this series of MDM2
inhibitors.
With the aim of rapidly exploring the SAR and improving the
physicochemical and PK properties of 1, a more robust and scalable
synthetic route to this series of inhibitors was required. The origi-
nal synthesis provided 1 in an overall yield of 1%.5 We were able to
HTRF IC50 = 1.4 uM
SJSA-1 p21 IC50 = 32 uM
N
N
PK (rat)
N
O
MRT (hr) 2.4
Vdss (L/kg) 9.9
CL (L/hr/kg) 2.4
F (%) 23
solubility (µg/mL)
PBS <1
Br
1
Br
⇑
Corresponding author. Tel.: +1 650 244 2190.
E-mail address: hbeck@amgen.com (H.P. Beck).
Figure 1. Potency and PK properties of 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.027

H. P. Beck et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2752–2755
2753
H
O
N
N
N
N
N
a
b
O
OH
4
5
OH
N
OH
+
4
N
O
a
b
N
Me
2
3
6
Br
Br
O
Br
Br
H
H
N
Scheme 2. Chemical instability of 4. Reagents and conditions: (a) DMSO, rt, 12 h;
(b) dioxane, 45 °C, 5 min.
N
N
N
N
c
d
N
N
N
O
1
resulted in a 10:1 ratio of 4/5 in 75% yield. Utilizing the optimized
conditions, we were able to obtain the desired chromenotriazolo-
pyrimidine core 4 in 53% overall yield. After the requisite N-alkyl-
ation, chiral separation provided the active enantiomer 1.8
In order to evaluate the metabolic stability of 1, it was incu-
bated with rat liver microsomes (RLM) and after 30 min <5% of 1
remained.9 Furthermore, the plasma concentration of 1 in vivo rap-
idly declined with time (Fig. 2).6 Interestingly, after intravenous
administration of 1 in rats, measureable concentrations of 4/5 were
found in the plasma. Following an oral dose of 1, quantities of 4/5
were also observed in the rat plasma (Fig. 3). We hypothesized that
oxidative N-demethylation and tautomeric isomerization was
responsible for the rapid biotransformation of 1 to 4/5 in vitro
and in vivo.
Br
5
Br
4
Br
Br
Scheme 1. Optimized synthesis of 1. Reagents and conditions: (a) 4-bromobenz-
aldehyde (1.0 equiv), KOH (3.6 equiv), EtOH, rt, 16 h, 90%; (b) 1,2,4-triazol-5-amine
(1.0 equiv), neat, 160 °C, 30 min, 86%; (c) 4-bromobenzaldehyde (3.0 equiv), neat,
140 °C, 10 min, 75%; (d) MeI (2.0 equiv), Cs2CO3 (1.9 equiv), rt, 30 min, 57%.
Intravenous
Rat 1-Compd 1
Rat 1-Compd 4/5
Rat 2-Compd 1
Rat 2-Compd 4/5
Rat 3-Compd 1
Rat 3-Compd 4/5
1000
100
10
In order to test our N-demethylation hypothesis, we sought to
improve the metabolic stability of 1 by installing N-alkyl substitu-
ents that would block the metabolic soft-spot.10 The X-ray co-crys-
tal structure of 4 bound to MDM2 revealed the N–H makes no
Table 1
N-alkylation conditions
R
N
R
N
N
N
N
N
RX
N
N
4
O
O
S
1
DMF,
0-25 oC,
18 hr
0
5
10
15
Time (hr)
Figure 2. In vivo conversion of 1 to 4/5 in rats following an intravenous dose of 1.6
Br
Br
Br
Br
7a
8a
, R =
7b
, R =
S
, R =
8b
, R =
Entry Base
Equiv of
base
RX
Equiv of Ratio a:b,a
Oral
RX
1.5
1.5
yield of ab
Rat 1-Compd 1
Rat 1-Compd 4/5
Rat 2-Compd 1
Rat 2-Compd 4/5
Rat 3-Compd 1
Rat 3-Compd 4/5
1000
1
2
Pyridine
1.0
1.0
NR
Cl
Cl
S
2,6-
Lutidine
S
NR
3
4
5
6
7
8
9
DMAP
1.0
1.0
1.5
NR
Cl
Cl
Cl
Cl
Cl
Cl
Cl
S
S
S
S
S
S
S
100
10
1
Imidazole
1.5
NR
Morpholine 1.0
1.5
NR
TMG
Cs2CO3
DBU
1.0
1.5
1.5
0.9
1.5
1:1.3
1:1
1.25
1.25
1.25
1:1.3
1.9:1, 20%
DBU
10
11
iPr2NEt
DBU
2.0
1.0
Allylbr
Allylbr
2.0
2.0
1:2.8
2.7:1, 35%
0
5
10
15
DMF was degassed with Ar (g) for 10 min before use.
Time (hr)
a
Ratio of a:b determined by LCMS.
b
Figure 3. In vivo conversion of 1 to 4/5 in rats following an oral dose of 1.6
Isolated yield.

2754
H. P. Beck et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2752–2755
Table 2
H
Et
R
a
Potencies and RLM stability of N-alkyl analogs
N
N
N
4
9
HTRFa
IC50
SJSA-1 p21b
RLMc
%
remaining
HO
HO
O
Compd
R@
N
(
lM)
IC50
(
lM)
O
c
b
1d
Me
Et
1.4 0.78
32
<5
8
9
2.4 0.06
1.8 0.23
>50
>50
<5
35
8a
10
O2
S
11
Br
d
S
Br
10
11
12
13
0.43 0.007
0.51 0.02
0.51 0.12
4.4 0.15
19
19
11
>90
27
OH
CO2H
7a
12
SO2Me
NHBoc
NH3TFA
TFA
26
H
N
BocHN
EtO2C
H2N
g
f
e
m
m
m
j
>50
44
4
15
14
13
14
15
3.7 0.17
>50
>25
>90
66
H
HO2C
H2NOC
NC
h
k
i
N
n
7.5 0.007
n
4
16-18
19-23
24
25
19
20d
21
22
0.91 0.06
0.48 0.15
0.65 0.04
0.76 0.07
>50
38
87
>90
83
CO2H
l
HO
CO2H
m = 3
n = 1, 3, and 4
26
CO2H
46
Scheme 3. Preparation of N-alkyl compounds 9–26. Reagents and conditions: (a)
iodoethane (2.0 equiv), Cs2CO3 (2.0 equiv), DMF, 25 °C, 1 h, 38%; (b) 9-BBN
(6.0 equiv), THF, 25 °C, 2 h; then 3 N NaOH/30% H2O2 (1:1), 30 min, 80%; (c) KBr
(1.0 equiv), TEMPO (1.1 equiv), NaOCl (1.3 equiv), acetone/5% aq NaHCO3 (4:1), 0 °C,
30 min, 2%; (d) oxone, alumina, H2O, CHCl3, 50 °C, 10 h, 38%; (e) 3-(Boc-amino)pro-
pyl bromide (1.0 equiv), Cs2CO3 (1.0 equiv), DMF, 18 h, 8%; (f) TFA (15 equiv), DCM,
25 °C, 30 min, 90%; (g) formaldehyde (2.0 equiv), NaBH3CN (4.0 equiv), MeOH, 4 Å
sieves, 25 °C, 8 h, 47%; (h) n = 1 ethyl iodoacetate (1.2 equiv) , Cs2CO3 (1.0 equiv),
DMF, 0–25 °C, 30 min, 67%; n = 3 ethylbromobutyrate (2.0 equiv), DBU (1.0 equiv),
DMF, 0–25 °C, 30 min, 67%; n = 4 ethylbromovalerate (1.2 equiv), DBU (1.0 equiv),
DMF, 0–25 °C, 30 min, 31%; (i) n = 1 LiOH (26 equiv), THF/MeOH (1:1), 25 °C, 1 h,
42%; n = 3 LiOH (5 equiv), THF/MeOH (1:1), 25 °C, 24 h, 40%; n = 4 LiOH (10 equiv),
THF/MeOH (1:1), 25 °C, 2 h, 35%; (j) SOCl2 (1.3 equiv), THF, reflux, 30 min; then
NH3OH (25 equiv), DCM, 25 °C, 1 h, 65%; (k) POCl3 (1.0 equiv), DMF, 100 °C, 1 h,
54%; (l) MeMgBr (2.5 equiv), THF, 0–25 °C, 18 h, 50%.
20
73
CO2H
CO2H
23d
0.35 0.02
12
87
24
25
0.54 0.001
0.73 0.04
26
22
13
CONH2
>50
CN
OH
26
0.37 0.06
14
36
a
Values reported as mean of at least two experiments, followed by standard
deviation, see Ref. 15.
b
See Ref. 16.
See Ref. 9.
c
d
Compound 1, 20, and 23 are single enantiomers, other compounds are racemic
mixtures.
contacts with the protein.5 Thus, we predicted that N-substitution
would project into solvent and would not have a detrimental im-
pact on potency of this series. A reliable method for N-alkylation
of 4 was required in order to prepare several N-alkyl analogs.
Unfortunately, 4 readily epimerizes to a 1:1 mixture of 4/5 upon
standing at room temperature in DMSO (Scheme 2).11 Further-
more, 4 undergoes facile oxidation to 6 in the presence of oxophilic
solvents (i.e., dioxane, DMA or DMF) and mild temperature (45 °C)
within 5 min. Thus, N-alkylation reactions would require both pre-
cautions to remove oxygen and short reaction time in order to min-
imize undesired side-products.
First we explored alkylation of 4 using chlorodimethylsulfide.
Weak bases (Table 1, entries 1–5) resulted in no reaction while
the use of TMG (tetramethylguandine) provided a 1:1.3 ratio of
7a:7b (Table 1, entry 6).12 Using 1.5 equiv of DBU or Cs2CO3, affor-
ded a 1:1.3 and 1:1 ratio of 7a:7b, respectively (Table 1, entries 7
and 8). Reducing the equivalents of DBU to 0.9 provided a 1.9:1 ra-
tio of 7a:7b with a 20% isolated yield of the desired 7a (Table 1, en-
try 9). Alternatively, allylation of 4 with allyl bromide employing
iPr2NEt provide a 1:2.8 ratio of 8a:8b (Table 1, entry 10). DBU pro-
vided the most favorable result, giving a 2.7:1 ratio of 8a:8b and a
35% isolated yield of the desired 8a (Table 1, entry 11).
were hydrolyzed to provide acids 19–23. Acid 19 was further con-
verted into amide 24 and nitrile 25. Ester 16 was also used to pre-
pare tertiary alcohol 26.
The binding and cellular potencies for the N-alkyl analogs are
shown in Table 2.14 Several N-alkyl groups were tolerated and a
few analogs exhibited increased stability in the presence of RLM.
Small groups, such as allyl 8a and ethyl 9, maintained potency
but exhibited high turnover in the presence of microsomes. While,
the addition of polarity to the terminal alkyl position enhanced the
MDM2 affinity (i.e., 10, 12, and 24–26), these compounds lacked
improved intrinsic clearance. Propyl carbamate 13 and amines
14–15 showed increased stability in the presence of RLM, but these
compounds suffered from reduced potency. On the other hand,
acids 11, and 19–23 maintained potency and showed improved
stability in the presence of RLM.
Since acids 1917 and 2218 were prepared in reasonable yield, po-
tent in the biochemical assay, and stable to RLM, the racemates
were separated using chiral chromoatography.19 The single enanti-
omers 20 and 23 were found to be more potent and more metabol-
ically stable than 1. Also, 20 and 23 exhibited improved solubility
With the optimized N-alkylation reaction conditions, we rapidly
prepared several N-alkyl analogs as shown in Scheme 3. Alkylation
of 4 with iodoethane afforded the desired 9 in 38% yield. N-allyl
analog 8a was converted to the alcohol 10 using hydroboration-
oxidation conditions. The oxidation of 10 to provide acid 11 proved
difficult. Many conditions were explored,13 but only TEMPO oxida-
tion gave the acid 11. Sulfide 7a could be readily oxidized to the
desired sulfone 12 in 38% yield. N-alkylation of 4 with 3-(Boc-
amino)propyl bromide provided 13, which was converted to 14.
Amine 14 was further transformed into dimethylamine 15.
N-alkylation of 4 with several bromoesters afforded 16–18 which
Table 3
PK and solubility properties of 20 and 23
Compd
In vivo PKa
Solubilityb
PBS (lg/mL)
MRT (iv, h)
Vdss (iv,
CL (iv,
F (po, %)
L/kg)
L/h/kg)
20
23
0.46
4.1
2.6
1.7
5.7
0.32
14
54
132
6.1
a
Dosed iv 0.5 mg/kg in 100% DMSO; po 2.0 mg/kg in 0.5% methylcellulose/1%
Tween 80/98.5% water.
b
See Ref. 7.

H. P. Beck et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2752–2755
2755
7. PBS = phosphate buffer saline; for experimental details see Tan, H.; Semin, D.;
Wacker, M.; Cheetham, J. J. Assoc. Lab. Autom. 2005, 10, 364.
8. For chiral separation procedure see Ref. 5.
in PBS. We characterized these analogs in rat in vivo PK experi-
ments (Table 3). Acid 20 exhibited high clearance of 5.7 L/h/kg.
However, 23 demonstrated low clearance (0.32 L/h/kg) and had
modest oral bioavailability (F 54%).
In conclusion, by blocking the metabolic soft-spot we were able
to substantially improve the PK properties of the chromenotriazol-
opyrimidine series of MDM2–p53 inhibitors while maintaining
both the biochemical and cellular potencies. Further in vivo phar-
macodynamic experiments of this series will be reported in due
course.
9. Compound (1 lM) was incubated with liver microsomes (0.25 mg/mL) and
NADPH (1 mM) in PBS for 30 min.
10. For example of incorporation of water-solubilizing moieties to improve
properties of MDM2 inhibitors see: Leonard, K.; Marugan, J. J.; Raboisson, P.;
Calvo, R.; Gushue, J. M.; Koblish, H. K.; Lattanze, J.; Zhao, S.; Cummings, M. D.;
Player, M. R.; Maroney, A. C.; Lu, T. Bioorg. Med. Chem. Lett. 2006, 16, 3463.
11. For hypothesized mechanism of epimerization see Ref. 5.
12. Stronger bases such as LiH resulted in O-alkylation.
N
N
N
N
O
S
Acknowledgements
The authors thank Dr. Michael Bartberger for helpful discus-
sions and Dr. Manuel Ventura for chiral separations.
Br
Br
References and notes
13. Both m-CPBA and Dess-Martin periodinate failed to provide any acid 11.
14. All biologically active compounds were characterized by 1H NMR, and their
purity was determined to be >95% by reverse phase HPLC; 1H NMR (Bruker
either 400 or 500 MHz), HPLC (A: 0.1% TFA in H2O, B: 0.1% TFA in CH3CN, 10–
100% B in 15 min.
15. Kane, S. A.; Fleener, C. A.; Zhang, Y. S.; Davis, L. J.; Musselman, A. L.; Huang, P. S.
Anal. Biochem. 2000, 278, 29.
16. mRNA p21 levels in SJSA-1 cells with 10% FBS were measured using Taqman;
see Ref. 5 for experimental details; the standard deviation for benchmark
compound 1.0, (n = 34).
1. (a) Vazquez, A.; Bond, E. E.; Levine, A. J.; Bond, G. L. Nat. Rev. Drug Disc. 2008, 7,
979; (b) Fridman, J. S.; Lowe, S. W. Oncogene 2003, 22, 9030.
2. (a) Haupt, Y.; Maya, R.; Kazaz, A.; Oren, M. Nature 1997, 387, 296; (b) Kubbutat,
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17. 1H NMR of 19 (400 MHz, MeOH) d ppm 4.79 (s, 1H) 4.84 (s, 1H) 5.62 (s, 1H)
6.06 (s, 1H) 6.75–6.84 (m, 3H) 6.96–7.03 (m, 2H) 7.06–7.12 (m, 3H) 7.12–7.18
(m, 2H) 7.20–7.28 (m, 1H) 7.51 (dd, J = 7.83, 1.57 Hz, 1H) 7.70 (s, 1H).
18. 1H NMR of 21 (400 MHz, chloroform-d) d ppm 1.56–1.67 (m, 4H) 2.37 (t,
J = 6.65 Hz, 2H) 4.21 (t, J = 7.53 Hz, 2H) 5.50 (s, 1H) 5.84 (s, 1H) 6.79 (dq,
J = 8.90, 4.47 Hz, 4H) 6.98 (dd, J = 8.22, 1.17 Hz, 1H) 7.13–7.20 (m, 3H) 7.22–
7.26 (m, 2H) 7.34 (ddd, J = 8.22, 7.34, 1.47 Hz, 1H) 7.48 (dd, J = 7.83, 1.57 Hz,
1H) 8.03 (br s, 1H).
3. (a) Rayburn, E.; Zhang, R.; He, J.; Wang, H. Curr. Cancer Drug Targets 2005, 5, 27;
(b) Polsky, D.; Bastin, B. C.; Hazan, C.; Melzer, K.; Pack, J.; Houghton, A.; Busam,
K.; Cordon-Cardo, C.; Osman, I. Cancer Res. 2001, 61, 7642; (c) See 2d and 2e.
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2009, 49, 223; (b) Hu, C.-Q.; Hu, Y.-Z. Curr. Med. Chem. 2008, 15, 1720; (c) Patel,
S.; Player, M. R. Expert Opin. Invest. Drugs 2008, 17, 1865.
5. Allen, J. G.; Bourbeau, M. P.; Wohlhieter, G. E.; Bartberger, M. D.; Michelsen, K.;
Hungate, R.; Gadwood, R. C.; Gaston, R. D.; Evans, B.; Mann, L. W.; Matison, M.
E.; Schneider, S.; Huang, X.; Yu, D.; Andrews, P. S.; Reichelt, A.; Long, A. M.;
Yakowec, P.; Yang, E. Y.; Lee, T. A.; Oliner, J. D. J. Med. Chem. 2009, 52, 7044.
6. Iv 2.0 mg/kg dose in DMSO; po 5.0 mg/kg dose in 2% HPMC, 1% Tween 80, pH
2.2 witsh HCl.
19. For 20: OD-H column with SFC [24 g/min MeOH/0.2% diethylamine + 46 g/min
CO2]; for 23: separated at ester 18 250 Â 30 mm OD-H column with SFC [20 g/
min MeOH/0.2% diethylamine + 60 g/min CO2].