Discovery of potent and selective spiroindolinone MDM2 inhibitor, RO8994, for cancer therapy

Article abstract of DOI:10.1016/j.bmc.2014.05.072

The field of small-molecule inhibitors of protein-protein interactions is rapidly advancing and the specific area of inhibitors of the p53/MDM2 interaction is a prime example. Several groups have published on this topic and multiple compounds are in various stages of clinical development. Building on the strength of the discovery of RG7112, a Nutlin imidazoline-based compound, and RG7388, a pyrrolidine-based compound, we have developed additional scaffolds that provide opportunities for future development. Here, we report the discovery and optimization of a highly potent and selective series of spiroindolinone small-molecule MDM2 inhibitors, culminating in RO8994.

Full text of DOI:10.1016/j.bmc.2014.05.072

Bioorganic & Medicinal Chemistry 22 (2014) 4001–4009
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of potent and selective spiroindolinone MDM2 inhibitor,
RO8994, for cancer therapy
a
a
a
a
a
a
Zhuming Zhang a, , Qingjie Ding , Jin-Jun Liu , Jing Zhang , Nan Jiang , Xin-Jie Chu , David Bartkovitz ,
⇑
Kin-Chun Luk a, Cheryl Janson b, Christian Tovar c, Zoran M. Filipovic c, Brian Higgins c, Kelli Glenn d,
Kathryn Packman c, Lyubomir T. Vassilev c, Bradford Graves b,
⇑
a Discovery Chemistry, Roche Research Center, Hoffmann-La Roche, Inc., 340 Kingsland Street, Nutley, NJ 07110, United States
b Discovery Technologies, Roche Research Center, Hoffmann-La Roche, Inc., 340 Kingsland Street, Nutley, NJ 07110, United States
c Discovery Oncology, Roche Research Center, Hoffmann-La Roche, Inc., 340 Kingsland Street, Nutley, NJ 07110, United States
d Non-Clinical Development, Roche Research Center, Hoffmann-La Roche, Inc., 340 Kingsland Street, Nutley, NJ 07110, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The field of small-molecule inhibitors of protein–protein interactions is rapidly advancing and the spe-
cific area of inhibitors of the p53/MDM2 interaction is a prime example. Several groups have published
on this topic and multiple compounds are in various stages of clinical development. Building on the
strength of the discovery of RG7112, a Nutlin imidazoline-based compound, and RG7388, a pyrroli-
dine-based compound, we have developed additional scaffolds that provide opportunities for future
development. Here, we report the discovery and optimization of a highly potent and selective series of
spiroindolinone small-molecule MDM2 inhibitors, culminating in RO8994.
Received 10 April 2014
Revised 23 May 2014
Accepted 30 May 2014
Available online 11 June 2014
Keywords:
MDM2
p53
Ó 2014 Elsevier Ltd. All rights reserved.
Protein–protein interaction
Apoptosis
Cancer
1. Introduction
and is progressing in clinical studies.14 Nevertheless, identification
of candidates from distinctive chemical classes may also be desir-
Tumor suppressor p53 is a potent transcription factor that plays
a central role in guarding the integrity of cell genome.1–4 In
non-stressed cells, p53 is tightly controlled by its master negative
regulator MDM2.5 In about 50% of human cancers, p53 remains
wild-type but its function is impaired by other mechanisms, such
as overexpression of MDM2.6–9 Thus, MDM2 has been deemed a
valid target for cancer therapy.10 The early structure of a p53 pep-
tide bound to MDM2 showed that the interactions were mediated
by a limited set of amino acids and provided a reasonable expecta-
tion that small molecules could successfully interfere with the
p53-MDM2 interaction.11 The discovery of Nutlins verified this
approach.12 An advanced member of the Nutlin family of mole-
cules, 1 (RG7112, Fig. 1), is in clinical evaluation.13 Internal efforts
identified a novel pyrrolidine compound, 2 (RG7388, Fig. 1), which
was found to display superior in vitro potency and in vivo efficacy
able to enhance the chances for ultimate success in the clinic.
Since the discovery of Nutlins, several groups have identified
additional compound classes and a few have even progressed into
clinical development.15 The most notable one has been a spiroind-
olinone-3,30-pyrrolidine MI-219 series reported by Ding et al.16
Just recently, this group published their latest findings in which
the original stereochemistry was found to be unstable and con-
verted over time to a more stable diastereomeric configuration
and the most potent compound 3 (MI-888, Fig. 1) was found to
achieve tumor regression in mice bearing SJSA-1 xenograft model
at a relatively high dose.17,18 Prior to their work, we also demon-
strated that stereochemistry of the pyrrolidine core structure in
which the two aryl rings (‘A’ and ‘B’) adopt a ‘Trans’ orientation
as shown in 2 (RG7388, Fig. 1) was important for optimal binding
to MDM2.14 Guided by X-ray co-crystal structures, we further
explored the pyrrolidine analogues by combining the key struc-
tural elements of 2 (RG7388) and the spiroindolinone core struc-
ture of 3 (MI-888).18,19 Here we report our work leading to the
⇑
Corresponding authors. Tel.: +1 215 628 7076; fax: +1 215 540 4612 (Z.Z.);
tel.: +1 973 680 8039 (B.G.).
discovery of
a potent and highly efficacious compound 4
(RO8994) with promising potential for clinical development
E-mail addresses: zzhang85@its.jnj.com (Z. Zhang), bjgraves101@gmail.com
(B. Graves).
(Fig. 1).19
http://dx.doi.org/10.1016/j.bmc.2014.05.072
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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Z. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 4001–4009
Phe19
O
advantage of the knowledge gained from the development of 2
OH
O
(RG7388, Fig. 1).14,19
Leu26
Several factors were taken into consideration for our explora-
tion of scaffold 8. First, the relative spatial orientation of hydropho-
Leu26
Cl
C
Cl
B
O
Phe19
N
bic groups in scaffold
8 could be differentiated in relation
O
F
NH
NH
to scaffold 7 and 9 in terms of binding to MDM2 due to the
pseudorotation propensity of pyrrolidine core structure. The five-
membered pyrrolidine ring is more flexible than the corresponding
six- and seven-membered ring systems and is known to exhibit
two predominant puckering modes.25–27 It was further demon-
strated that the stereochemical configuration of the pyrrolidine
core structure in which the two aryl rings (‘A’ and ‘B’) adopt a
‘Trans’ orientation was very important for optimal binding as
shown in compound 2 (RG7388, Scheme 2).14 Second, the use of
a substituted para-benzoic acid group (‘C’ in Fig. 1 and Scheme 2)
as the R2 group proved to be critical for RG7388 in lead optimiza-
tion of the scaffold 10 as it stabilized the molecule metabolically
and significantly improved cellular potency/selectivity and PK pro-
files.14 Third, the architecture of spiroindolinone-3,30-pyrrolidine
series was initially reported by Ding et al. as shown in compound
12 (MI-219).16 However, the stereochemical configuration appears
to be suboptimal based on the observation by multiple groups that
an aromatic ring is preferred in the Leu26 pocket.24,28 Consistent
with these observations, this group recently disclosed their latest
findings that original ‘Cis’ stereochemistry was found to be unsta-
ble and isomerized into a more stable ‘Trans’ configuration as
N
O
B
A
N
CN
Cl
Trp23
A
N
F
Cl
O
S
Trp23
1 (RG7112)
2 (RG7388)
NH2
O
O
OH
Leu26
C
O
O
Cl
Cl
F
NH
NH
Phe19
O
Cl
F
NH
B
NH
B
O
A
O
NH
A
NH
Cl
3 (MI-888)
4 (RO8994)
Trp23
shown in
3 3 displayed improved
(MI-888).17,29 Compound
Figure 1. Chemical structures and binding modes of p53–MDM2 inhibitors.
potency and PK over 12 and achieved complete and durable tumor
regression in a dose of 100 mg/kg.18 However, we envisioned the
presence of an aromatic ring as R2 (‘C’ in Fig. 1 and Scheme 2)
would be important and thus expanded our exploration into the
analogues 11 (Scheme 2) driven by the impressive in vitro and
in vivo potency of 2 (RG7388).14
2. Results and discussion
Compared to the identification of Nutlins by high throughput
screenings,12 the discoveries of 2 (RG7388) and 4 (RO8994) were
driven by the structure-based de novo design and information based
approach. Our initial design started with the indolinone compound
To keep the optimal binding mode to MDM2, the stereochemi-
cal configuration of the pyrrolidine in 2 (RG7388) was preserved in
analogues 11. Since the substituted para-benzoic acid (‘C’) group
was considered to be a critical element in compound 2, optimiza-
tion focused on the phenyl group as the side chain (‘C’) to diverge
from compound 3. Variations in groups R and R0 were explored for
their effects on potency and pharmacological properties. As the R
group on the para-position points to solvent, different polar or sol-
ubilizing groups with electron withdrawing (R = COOH, CONH2)
and electron donating properties (R = OCH2CH2OH) were selected
and combined with small R0 groups at the 2- or 3-position to min-
imize metabolic liability, enhance cellular potency, and maximize
PK parameters. The impact of a fluorine substituent (R3 = F) at
the 4-, 5- or 7-position of the 6-chlorooxindole on potency was also
examined.
5, which was confirmed to be a validated hit with an IC50 of 3.9 lM
in a biochemical binding assay (Fig. 2).20 In the crystal structure, the
6-chlorooxindole (‘A’) fills the deep, narrow Trp23 pocket. Only the
(S)-configuration of compound 5 is active, which places the 3-chlo-
rophenyl (‘B’) in the Leu26 pocket but the binding to Phe19 pocket is
suboptimal. As a strategy to optimize compound 5, rigidifying the
structure 6 by cyclizing between the 3,30 positions with a six- or
seven-membered core ring led to the spiroindolinone scaffolds 7
and 9 (Fig. 2 and Scheme 1).21,22 This was successful in enhancing
the binding affinities but unfavorable physicochemical or pharma-
cological properties rendered these compounds difficult to pro-
gress.23,24 Thus, we shifted our exploration to the five-membered
spiroindolinone scaffold 8 (Scheme 1) while simultaneously taking
The synthesis of analogs in scaffold 8 is outlined in Scheme 3.29
The preparation of the desired stereochemical configuration in the
pyrrolidine core structure 8 was problematic prior to our work. AgF
mediated 1,3-dipolar cycloaddition reaction of 3-benzylidene oxin-
dole 13 with imine 14 led mostly to spiroindolinone 15 with pre-
dominant ‘Cis’ configuration between the aryl rings (‘A’ and ‘B’)
likely due to the pre-established ‘Cis’ or (E)-configuration in
13.30–32 Other diastereomers were also observed but only trace
amounts of the desired spiroindolinone 16 with ‘Trans’ configura-
tion was observed. By contrast, the ‘Trans’ configuration was
generated for the pyrrolidine compound 2 (RG7388) series under
similar reaction conditions as the results of the predominant ‘Trans’
Cl
O
B
N
(S)
O
A
N
H
Cl
configuration in (Z)-a
-cyanostilbene.14 The formation of spiroindo-
5
line 15 is clearly kinetically favored.17,29,33 After some explorations,
it was found that treatment of 15 with DBU under heating condi-
tions could afford key intermediate 16. The isomerization from
15 to 16 involves the inversion of two stereocenters and occurs
most likely via a retro-Mannich reaction followed by a ring-closure
Figure 2. X-ray structure of compound 5 bound to MDM2. The 6-chlorooxindole
ring fills the Trp23 pocket. The 3-chloro-phenyl occupies the Leu26 pocket. This
structure has been deposited at the PDB (code 4LWT).

Z. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 4001–4009
4003
Cl
B
Cl
Cl
O
B
B
Grafting a ring
6-membered
NH
R1
R1
R1
A
O
A
A
O
O
N
H
N
H
N
H
Cl
Cl
Cl
6
7
7-membered
5-membered
R2
NH
Cl
O
Cl
B
O
F
NH
B
NH
R1
O
R1
O
A
R3
Cl
A
NH
N
H
Cl
8
9
Scheme 1. Variations of spiroindolinone scaffolds as p53–MDM2 inhibitors.
O
OH
2
R
1
1
2
R'
3
C
C
R2
NH
O
3
O
O
Cl
Cl
F
F
O
NH
Cl
F
NH
Stereochemical
configuration
Optimization
of R1, R2
NH
NH
B
B
NH
B
5
R1
4
CN
CN
O
A
A
R3
Cl
A
F
F
NH
Cl
Cl
7
2 (RG7388)
11
10
OH
R = COOH, CONH2, OCH2CH2OH
R' = H, F, OMe
OH
HO
R3 = H, F
O
Cl
O
F
Cl
Cl
NH
NH
NH
NH
Isomerization
& Derivatization
B
B
O
F
O
A
A
NH
12 (MI-219)
Scheme 2. The evolution of MDM2 inhibitors from RG7388, 3 (MI-888) to analogues 11.
NH
Cl
3 (MI-888)
Mannich reaction.29 Our studies indicated that the stereochemical
configuration in compound 16 could be thermodynamically pre-
ferred albeit kinetically less favorable. The analogs in scaffold 8
were synthesized initially as a racemic mixture from which the
two chiral enantiomers can be separated by chiral SFC. The prepa-
ration of analogues in scaffold 8 was later optimized by internal
process research and the method can be amenable to the produc-
tion of multihundred gram scale.29
The biochemical binding and cellular antiproliferative activity
of analogs 4 and 18–27 are summarized in Table 1. In the biochem-
ical HTRF binding assay, all are highly active with IC50 <20 nM
(Fig. 3 and Table 1) and compare favorably with 2 (RG7388).
Compound 18 (IC50 = 4 nM) is >900-fold more potent than its enan-
tiomer (180, IC50 = 3741 nM) (SI Table S1). The absolute stereo-
chemical configuration and binding mode of the more active
enantiomer was confirmed by determination of the crystal struc-
ture of compound 20 bound to MDM2 (Fig. 4). The 6-chlorooxin-
dole occupies the Trp23 pocket with the NH forming a hydrogen
bond with protein backbone. The neopentyl group is located in
the Phe19 pocket. The 3-chloro-2-fluorophenyl ring is buried in
the Leu26 pocket, forming a stacking interaction with the
His96 residue on MDM2. The pyrrolidine carbonyl forms a hydro-
p–p
gen bond with NH of the His96 side chain.
By inspection of available co-crystal structures, it appears that
an aromatic moiety is favored over an aliphatic group in the
Leu26 pocket of MDM2 in spite of pseudosymmetric spatial rela-
tionship of Leu26 and Phe19 pockets.24,28 The presence of His96
residue in the Leu26 pocket is an important structural feature
for the differentiation of these two pockets in terms of designing
novel MDM2 inhibitors (Fig. 4). The interaction with His96 resi-
due could be a key factor for the high MDM2 binding affinities
of these compounds and also explain the lack of binding to
MDMX.24,28 The mismatch of aromatic and aliphatic elements in
the Leu26 and Phe19 pockets also appears to be a common motif
between non-peptide small molecules and peptide inhibitors.
Further exploration and elaboration in these areas will have
important implications for the design of MDMX or MDM2/MDMX
dual inhibitors.24,34,35

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Z. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 4001–4009
Racemic
Racemic
Cl
O
Cl
O
F
F
Cl
O
F
O
B
O
O
N
N
NH
B
B
R1
OH
b
a
R1
O
R3
+
R3
Cl
O
A
A
R1
R3
Cl
A
NH
NH
N
Cl
H
13
O
14
15
Racemic
Racemic
R2
NH
R2
NH
O
O
F
Cl
O
F
Cl
Cl
Cl
F
F
OH
NH
O
Figure 4. X-ray structure of spiroindolinone 20 bound to MDM2. This structure has
NH
NH
NH
B
B
B
B
been deposited at the PDB (code 4LWU).
R1
O
R1
O
R1
R1
O
d,e
c
+
O
R3
Cl
A
R3
Cl
A
R3
Cl
R3
Cl
A
A
NH
NH
NH
NH
In the cellular proliferation (MTT) assays, compounds 4 and 18–
24 displayed potent anti-proliferative activities with average MTT
IC50 ranging from 0.01 to 0.19 lM against three wild-type p53
8'
8
17
16
human cancer cell lines (SJSA1, RKO, HCT116). Selectivity, defined
as the ratio of average IC50 values derived from mutant (SW480 &
MDA-MB-435) and wild-type p53 cell lines, ranged from 51- to
882-fold (Table 1). Cellular potency and selectivity of the better
compounds track well with those for 2 (RG7388). In spite of the
Scheme 3. The racemic synthesis of designed analogues in spiroindolinone scaffold
8. Reagents and conditions: (a) AgF, NEt3, DCM, rt; (b) DBU, tBuOH, reflux; (c) TFA,
i
DCM, rt; (d) NH2R2, Ph2C(@O)Cl, Pr2NEt, DCM, rt; (e) chiral SFC separation 11.
Table 1
In vitro activity of spiroindolinone analogues 18–27, 4 and 2 (RG7388) in HTRF binding assays and MTT proliferation assays with human cancer cell linesa
Compound
18
19
20
21
22
4
23
24
25
26
27
2 (RG7388)
HTRF IC50
(
(l
l
M)
M)
0.004
0.15
89
0.005
0.01
882
0.005
0.09
150
0.005
0.06
82
0.007
0.13
67
0.005
0.02
312
0.008
0.19
57
0.005
0.01
782
0.006
0.09
132
0.006
0.25
51
0.018
0.69
12
0.006
0.03
344
b
MTT IC50
Selectivityc
a
b
c
IC50 was determined by one experiment performed in duplicate.
Average IC50 of three wt-p53 cancer cell lines (SJSA1, RKO, HCT116).
Ratio of average IC50 of two mutant p53 cell lines (SW480, MDA-MB-435) and average IC50 of three wild-type p53 cell lines (as above).
O
OH
2
O
NH2
2
HO
F
O
2
3
V
V
V
W
3
W
W
3
O
O
O
Cl
Cl
Cl
Cl
Cl
Cl
F
F
NH
NH
NH
NH
NH
NH
O
O
O
NH
NH
NH
18 V = OMe, W = H
19 V = H, W = OMe
20 V = H, W = H
23 V = H, W = H
24 V = H, W = OMe
21 V = OMe, W = H
22 V = H, W = F
4 V = H, W = OMe (RO8994)
NH2
3
NH2
NH2
O
O
O
O
3
O
O
O
O
Cl
Cl
Cl
F
F
F
NH
NH
NH
NH
NH
NH
F
5
O
O
O
F
4
NH
NH
NH
7
F
Cl
Cl
Cl
25
26
27 (Racemic)
Figure 3. The chemical structures of spiroindolinone analogues in scaffold 8.

Z. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 4001–4009
4005
Table 2
The human liver microsomal stability and mean PK parameters of compounds 19, 20, 22, 4, 24, 26 and RG7388 in C57 male mice by single oral and iv dosing
Compound
19
20
22
4
24
26
2 (RG7388)
HLM_CLa (mL/min/kg)
PO Dose (mg/kg)
12.0
25
0.1
0.8
5
34.6
11.1
21
7.5
50
3.0
8.3
5
3.5
10.4
63
7.4
50
3.4
13.1
5
3.4
15.3
75
7.5
25
5.1c
25
1.4
4.3
5
6.7
6.6
55
9.3
50
2.6
8.7
5
2.6
8.8
45
4.3
50
1.3
9.9
5
10.3
1.6
80
PO AUC/PO dose (
l
g⁄h/mL/mg/kg)
3.7
5.8
0.64
5.8
7.1
92
PO Cmax g/mL)
(l
iv dose (mg/kg)
CL (mL/min/kg)
T1/2 (h)
Fb (%)
a
b
c
For HLM_CL: low clearance < 6.5, 6.5 < medium clearance < 35, high clearance > 35.
Oral bioavailability.
In vitro human hepatocyte clearance rat.
difference in polar groups at the para position, compounds 19, 4,
and 24 showed excellent cellular potency and selectivity that are
at least as good as those observed for 2. All three compounds pos-
sess a 3-methoxy group on the phenyl ring (as does 2). Compounds
with no methoxy group (20 and 23) or with a 2-methoxy (18 and
21) were clearly less potent and less selective, recapitulating the
preference for the 3-methoxy substitution observed with the opti-
mization resulting in 2.14 Similarly, 3-fluoro (compound 22) was
not as active or selective. Compound 19 displayed a cellular MTT
IC50 of 0.01 lM and a selectivity of 882, indicating that replace-
ment of 4-chloro-2-fluorophenyl in 2 with 6-chlorooxindole led
to enhanced cellular potency and selectivity presumably due to
the formation of hydrogen bond between 6-chlorooxindole and
protein backbone. Mono-fluoro substitution on the 4- or 5-position
of 6-chlorooxindole led to a loss in cellular potency/selectivity
(compare compounds 25, 26 to corresponding 4, 20). Substitution
with fluorine at the 7-position is clearly detrimental and led to a
substantial loss in cellular potency/selectivity (compare 27 to 4).
Compounds 19, 20, 22, 4, 24, 26 were selected for in vivo phar-
macokinetic (PK) evaluation based on their parameters in human
liver microsomal stability or cellular potency/selectivity (Table 2).
Compound 19 showed suboptimal oral bioavailability and a high
clearance rate despite its impressive cellular potency and identical
side chain with 2. By contrast, compounds 20, 22, 4 showed
excellent PK properties, with dose-normalized exposures that are
ꢀ2.5-fold higher than 2. It is noteworthy that the only structural
difference between 4 and 19 is the terminal carboxamide versus
carboxylic acid. Thus, in spite of the many parallel trends between
this spiroindolinone series 8 and the RG7388 series 10, there are
subtle differences. Within the carboxamide series in 11, the pres-
ence of 3-methoxy in compound 4 led to not only higher cellular
potency and selectivity (as mentioned above) but also a higher oral
bioavailability over compound 20. This trend was also observed in
optimization of the RG7388 series. However, despite a similar
clearance rate in in vivo PK studies, the oral bioavailability of 24
is much lower than that of 4. The discrepancy could be explained
by their difference in physicochemical properties such as perme-
ability (data not shown). The 5-position of 6-chlorooxindole was
considered to be a potential metabolic hotspot for oxidative
Figure 6. Apoptosis in SJSA cells treated with compound 4. Cells were incubated for
48 h.
Figure 7. The oral in vivo efficacy profile of 4 (RO8994) in the SJSA-1 human
osteosarcoma xenograft model in nude mice. Tumor growth inhibition was
observed at 1.56 mg/kg qd and tumor regression was observed at 6.25 mg/kg qd.
hydroxylation. It was hypothesized that blocking this site could
potentially improve metabolic liability. However, 5-fluoro
substitution offers no noticeable improvement in PK parameters
(compare 26 to 20). Thus, the data so far suggest that the 6-chloro-
oxindole group is optimal for these analogs in scaffold 8.
While not the most potent or selective in the series, compound
4 was chosen for further study based on the balance of its excellent
in vitro activities and in vivo pharmacological profile. From in vitro
mechanistic studies, compound 4 induced dose-dependent up-
regulation of p53 target genes and apoptosis in wild-type p53
cancer cells, consistent with its non-genotoxic mechanism of
p53 activation (Figs. 5 and 6, respectively).10 Compound 4 also
displayed remarkable tumor growth inhibition in the wild-type
p53, MDM2-amplified SJSA-1 osteosarcoma tumor xenograft
Figure 5. Weston blot analysis of p53 activation induced by compound 4 (RO8994)
in SJSA osteosarcoma tumor tissue.

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Z. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 4001–4009
model (Fig. 7)—exhibiting significant (>60%) tumor growth inhibi-
tion at the low dose of 1.56 mg/kg, tumor stasis at 3.125 mg/kg and
regression at 6.25 mg/kg. This would appear to be at least as good
as the efficacy results with 2 (RG7388) and significantly more effi-
cacious than 3 (MI-888).14,18
was stirred at room temperature for 20 h. The mixture was concen-
trated. The residue was partitioned between ethyl acetate and
water. The organic layer was separated, washed with water, brine,
dried over Na2SO4, then concentrated. The residue was purified by
chromatography (3–20% of EtOAc in CH2Cl2) to give a white solid
(yield 0.12 g, 54%).
Step (B): To a solution of the white solid from Step (A) (0.1 g,
0.16 mmol) in tetrahydrofuran (20 mL) was added an aqueous
solution (1 N) of NaOH (8 mL, 8 mmol) and methanol (8 mL). The
reaction mixture was heated at 80 °C for 1 h, and then cooled to
room temperature. The ‘pH’ of the mixture was adjusted to 5–6
by aqueous HCl solution, then concentrated to a small volume.
The residue was partitioned between ethyl acetate and water.
The organic layer was separated, and the aqueous layer was
extracted with ethyl acetate twice. The combined organic extract
was washed with water, brine, dried over MgSO4, and concentrated
to give the racemic product as an off white solid (75 mg, 77%).
Step (C): The racemic product from Step (B) was separated by
chiral SFC chromatography (prep OJ, or AD, or OD, 35 °C at
100 bar, eluting with 20–40% methanol in carbon dioxide) to
provide pure two enantiomers (>95% ee).
3. Conclusion
In summary, our investigation of 5-membered spiroindolinones
8 led to the identification of a highly potent and selective
p53-MDM2 inhibitor 4 (RO8994). The synthesis of compound 4
has been optimized for large scale and the compound has also dem-
onstrated acceptable toxicity profiles in both rodent and non-rodent
dose range finding studies (data not shown).29 Like 2 (RG7388),
compound 4 (RO8994) represents a new generation of p53–MDM2
antagonists with marked improvement in both in vitro and in vivo
pharmacological properties for potential clinical development. The
further exploration of additional chemical series on the basis of
RO8994 has led to the identification of two new potent MDM2
RO5353 and RO2468.36 Our studies also highlighted a subtle differ-
ence of MDM2 inhibitors in mimicking Leu26 and Phe19 residue of
p53. The mismatch of Leu26 and Phe19 binding groups between
non-peptide small molecules and peptide inhibitors appears to be
a common motif and further exploration of structural difference
would have implications in design of MDMX or dual MDM2/MDMX
non-peptide small-molecule inhibitors.
4.3. Chiral 4-{[(20S,30R,40S,50R)-6-chloro-40-(3-chloro-2-fluoro-
phenyl)-20-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-
3,30-pyrrolidine]-50-carbonyl]-amino}-2-methoxy-benzoic acid
(18)
A light yellow solid (40 mg, 36%). 1H NMR (300 MHz, DMSO-d6)
d 10.46 (s, 1H), 10.27 (s, 1H), 7.69 (d, J = 8.53 Hz, 1H), 7.63 (d,
J = 8.03 Hz, 1H), 7.51–7.60 (m, 2H), 7.36 (t, J = 7.09 Hz, 1H), 7.10–
7.20 (m, 1H), 7.04 (dd, J = 1.81, 8.00 Hz, 1H), 6.97 (d, J = 8.45 Hz,
1H), 6.68 (d, J = 1.81 Hz, 1H), 4.72 (d, J = 9.36 Hz, 1H), 4.56 (d,
J = 9.36 Hz, 1H), 3.84 (d, J = 9.36 Hz, 1H), 3.75 (s, 3H), 1.25 (dd,
J = 9.51, 13.74 Hz, 1H), 0.82 (s, 9H), 0.72–0.78 (m, 1H) ppm; 13C
NMR (101 MHz, DMSO-d6) d 177.0, 172.1, 166.4, 159.6, 156.8,
154.3, 143.4, 142.9, 132.4, 129.2, 128.3, 126.0, 125.9, 125.0,
124.9, 121.4, 119.4, 119.2, 115.1, 110.0, 109.3, 102.2, 65.5, 64.8,
64.5, 55.6, 47.9, 43.3, 30.0, 29.7 ppm; HRMS (ES+) m/z calcd for
4. Experimental section
4.1. General chemistry
All commercial reagents and anhydrous solvents were purchased
and used without further purification, unless otherwise specified.
Mass samples were analyzed on a MicroMass ZQ, ZMD, Quattro LC,
or Quattro II mass spectrometer operated in a single MS mode with
electrospray ionization. High resolution mass spectra (HRMS) were
measured on a Bruker Daltonics APEXII 3 Tesla Fourier transform
mass spectrometer. 1H NMR spectra (d, ppm) were recorded using
either a Bruker Avance 400 (400 MHz) or a Bruker Avance II-300
(300 MHz) instrument. 1H and 13C NMR chemical shifts are reported
in parts per million(d) relativeto the 1H and 13C signals in the solvent
(CDCl3: d 7.26, 77.16 ppm; DMSO-d6: d 2.50, 39.52 ppm). Column
chromatography was performed using an ISCO Combiflash or glass
column packed with Merck silica gel 60 (0.040–0.063 mm). All com-
pounds for biological assays were >95% pure by HPLC (Column: Zor-
C
31H30Cl2FN3O5+H [(M+H)+]: 614.1620, found: 614.1617.
4.4. Chiral 4-{[(20R,30S,40R,50S)-6-chloro-40-(3-chloro-2-fluoro-
phenyl)-20-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-
3,30-pyrrolidine]-50-carbonyl]-amino}-2-methoxy-benzoic acid
(180)
A light yellow solid (39 mg, 35%). 1H NMR (300 MHz, DMSO-d6) d
10.49 (s, 1H), 10.30 (s, 1H), 7.72 (d, J = 8.53 Hz, 1H), 7.65 (d,
J = 8.03 Hz, 1H), 7.56–7.63 (m, 2H), 7.39 (t, J = 7.09 Hz, 1H), 7.18 (t,
J = 7.91 Hz, 1H), 7.07 (dd, J = 1.76, 8.03 Hz, 1H), 7.01 (dd, J = 1.51,
8.53 Hz, 1H), 6.71 (d, J = 1.76 Hz, 1H), 4.75 (d, J = 9.36 Hz, 1H), 4.60
(d, J = 9.36 Hz, 1H), 3.87 (d, J = 9.36 Hz, 1H), 3.79 (s, 3H), 1.29 (dd,
J = 9.66, 13.74 Hz, 1H), 0.85 (s, 9H), 0.79 (d, J = 13.74 Hz, 1H) ppm;
13C NMR (101 MHz, DMSO-d6) d 177.0, 172.1, 166.4, 159.6, 156.8,
154.3, 143.4, 142.9, 132.4, 129.2, 128.3, 126.1, 125.1, 124.9, 121.4,
119.4, 119.2, 115.1, 110.0, 109.3, 102.2, 65.5, 64.9, 64.6, 55.6, 47.9,
43.3, 30.0, 29.7 ppm; HRMS (ES+) m/z calcd for C31H30Cl2FN3O5+H
[(M+H)+]: 614.1620, found: 614.1614.
bax SB-CN, 3.5
l
m, 120 A, 150 mm ꢁ 4.6 mm i.d.; mobile phases:
35% Acetonitrile and 65% water plus 0.2% H3PO4 over 30 min; col-
umn temperature: room temperature; detector setting: UV at
220 nm; flow rate: 1.0 mL/min).
Synthesis of racemic 17 from intermediates 13 and 14
(Scheme 3) either has been reported recently29 or described in SI
section (R3 = H, F; R1 = CH2C(CH3)3).
4.2. General procedures for preparation of compounds 18, 180
and 19 from racemic 17 (R3 = H, R1 = CH2C(CH3)3)
Step (A): To a solution of racemic (20S,30R,40S,50R)-6-chloro-40-(3-
chloro-2-fluoro-phenyl)-20-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-
spiro[indole-3,30-pyrrolidine]-50-carboxylic acid trifluoroacetic acid
salt 17 (R3 = H, R1 = CH2C(CH3)3, Ref. 29) (0.2 g, 0.36 mmol) in
dichloromethane (10 mL) was added diisopropylethylamine
(0.18 g, 1.4 mmol), diphenylphosphinic chloride (Aldrich) (0.25 g,
1.1 mmol), respectively. The mixture was stirred at room tempera-
ture for 0.5 h, then methyl 4-amino-2-methoxybenzoate (Acros)
(0.077 g, 0.43 mmol) or methyl 4-amino-3-methoxy-benzoate
(Ark Pharm) (0.16 g, 0.9 mmol) was added. The reaction mixture
4.5. Chiral 4-{[(20S,30R,40S,50R)-6-chloro-40-(3-chloro-2-fluoro-
phenyl)-20-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-
3,30-pyrrolidine]-50-carbonyl]-amino}-3-methoxy-benzoic acid
(19)
A white solid (57 mg, 37%). 1H NMR (400 MHz, DMSO-d6) d
10.79 (s, 1H), 10.56 (br s, 1H), 8.43 (d, J = 8.53 Hz, 1H), 7.72 (d,
J = 8.03 Hz, 1H), 7.56–7.67 (m, 3H), 7.38 (t, J = 7.15 Hz, 1H), 7.18

Z. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 4001–4009
4007
(t, J = 7.78 Hz, 1H), 7.03 (d, J = 8.03 Hz, 1H), 6.72 (s, 1H), 4.69 (d,
J = 9.29 Hz, 1H), 4.50 (d, J = 9.29 Hz, 1H), 3.93 (s, 3H), 3.87 (d,
J = 9.85 Hz, 1H), 1.32 (dd, J = 9.85, 13.49 Hz, 1H), 0.93 (s, 9H), 0.79
(d, J = 13.49 Hz, 1H) ppm; 13C NMR (101 MHz, DMSO-d6) d 176.9,
172.1, 167.0, 156.8, 154.3, 147.5, 143.4, 132.4, 131.4, 129.0,
128.4, 126.6, 125.7, 125.6, 125.3, 124.8, 122.7, 121.4, 119.2,
117.0, 110.9, 109.3, 65.9, 65.0, 55.7, 48.8, 42.7, 30.0, 29.6 ppm;
13C NMR (101 MHz, DMSO-d6) d 176.9, 172.1, 167.0, 156.8, 154.3,
147.5, 143.4, 132.4, 131.4, 129.0, 128.4, 126.7, 126.6, 125.7,
125.6, 125.3, 124.8, 124.8, 122.7, 121.4, 119.3, 119.1, 117.0,
110.9, 109.3, 65.9, 65.1, 65.0, 55.7, 48.8, 42.7, 30.0, 29.6 ppm;
HRMS (ES+) m/z calcd for C31H30Cl2FN3O5+H [(M+H)+]: 614.1619,
2H), 7.55 (br s, 2H), 7.45 (br s, 1H), 7.38 (t, J = 7.09 Hz, 1H), 7.16 (t,
J = 8.00 Hz, 1H), 7.06 (dd, J = 1.66, 8.15 Hz, 1H), 7.00 (d, J = 8.76 Hz,
1H), 6.69 (d, J = 1.66 Hz, 1H), 4.69–4.78 (m, 1H), 4.57 (d, J = 9.66 Hz,
1H), 3.85 (s, 3H), 3.81–3.90 (m, 1H), 3.46–3.57 (m, 1H), 1.22–1.31
(m, 1H), 0.84 (s, 9H), 0.79 (d, J = 13.89 Hz, 1H) ppm; HRMS (ES+)
m/z calcd for
613.1779.
C
31H30Cl2FN4O4+H [(M+H)+]: 613.1779, found:
4.9. Chiral (20S,30R,40S,50R)-6-chloro-40-(3-chloro-2-fluoro-phenyl)-
20-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,30-
pyrrolidine]-50-carboxylic acid (4-cyano-2-fluoro-phenyl)-
amide (22)
found: 614.1606. [
a]
20 = ꢂ54.0° (c 0.08, MeOH).
D
An off white solid (49 mg, 33%). 1H NMR (400 MHz, DMSO-d6) d
10.54 (d, J = 2.26 Hz, 1H), 10.47 (s, 1H), 8.38 (t, J = 8.16 Hz, 1H), 7.97
(br s, 1H), 7.80 (dd, J = 1.63, 11.92 Hz, 1H), 7.73 (d, J = 8.53 Hz, 1H),
7.70 (d, J = 8.03 Hz, 1H), 7.60 (t, J = 7.28 Hz, 1H), 7.43 (br s, 1H),
7.35–7.41 (m, 1H), 7.17 (t, J = 8.03 Hz, 1H), 7.04 (dd, J = 1.76,
8.03 Hz, 1H), 6.68 (d, J = 1.76 Hz, 1H), 4.68–4.74 (m, 1H), 4.53 (d,
J = 9.60 Hz, 1H), 3.87–3.95 (m, 1H), 3.72–3.81 (m, 1H), 1.29 (dd,
J = 9.60, 13.87 Hz, 1H), 0.86 (s, 9H), 0.75 (d, J = 13.87 Hz, 1H)
ppm; HRMS (ES+) m/z calcd for C30H28Cl2F2N4O3+H [(M+H)+]:
601.1580, found: 601.1575.
4.6. General procedures for preparation of compounds 20–22, 4
from racemic 17 (R3 = H, R1 = CH2C(CH3)3)
Step (A): In a manner similar to the method described in general
procedure (A) for compound 18, 180 and 19, racemic
(20S,30R,40S,50R)-6-chloro-40-(3-chloro-2-fluoro-phenyl)-20-(2,2-
dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,30-pyrrol-
idine]-50-carboxylic acid trifluoroacetic acid salt 17 (R3 = H, R1 = CH2C
(CH3)3) (2.94 g, 5.07 mmol) was reacted with diisopropylethyl-
amine (5.25 g, 40.6 mmol), diphenylphosphinic chloride (4.8 g,
20.3 mmol), then corresponding optionally substituted 4-amino-
benzonitrile (2.4 g, 20.3 mmol) to give the coupled product as an
off white foam (Yield 1 g, 34%).
Step (B): To a solution of the above coupled product from (A)
(0.35 g, 0.62 mmol) in DMSO (7 mL) at 0 °C was added an aqueous
solution (30% Aldrich) of H2O2 (1.05 g, 9.3 mmol), followed by the
addition of aqueous solution (1 N) of NaOH (3 mL, 3 mmol) drop-
wise. The reaction mixture was stirred at 0 °C for 1 h. The mixture
was partitioned between ethyl acetate and saturated aqueous
Na2SO3 solution. The organic layer was separated, washed with
water, brine, dried over MgSO4, and concentrated. The residue
was purified by chromatography (50–100% EtOAc in DCM) to give
the racemic product as a white solid (yield 0.26 g, 72%).
4.10. Chiral (20S,30R,40S,50R)-6-chloro-40-(3-chloro-2-fluoro-
phenyl)-20-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro
[indole-3,30-pyrrolidine]-50-carboxylic acid (4-carbamoyl-
2-methoxy-phenyl-phenyl)-amide (4)
An off white solid (68 mg, 31%). 1H NMR (300 MHz, DMSO-d6) d
10.73 (br s, 1H), 10.50 (br s, 1H), 8.37 (d, J = 8.28 Hz, 1H), 7.96 (br s,
1H), 7.74 (d, J = 7.78 Hz, 1H), 7.58–7.67 (m, 2H), 7.54 (d, J = 8.28 Hz,
1H), 7.35–7.43 (m, 1H), 7.32 (br s, 1H), 7.19 (t, J = 7.78 Hz, 1H),
7.01–7.07 (m, 1H), 6.71 (br s, 1H), 4.65–4.73 (m, 1H), 4.50 (d,
J = 9.29 Hz, 1H), 3.93 (s, 3H), 3.86 (d, J = 9.80 Hz, 1H), 3.74 (t,
J = 10.54 Hz, 1H), 1.32 (dd, J = 9.80, 13.60 Hz, 1H), 1.28–1.37 (m,
1H), 0.94 (br s, 9H), 0.79 (d, J = 13.60 Hz, 1H) ppm; 13C NMR
(101 MHz, DMSO-d6) d 176.9, 171.9, 167.3, 156.8, 154.3, 147.4,
143.4, 132.4, 129.9, 129.0, 128.4, 126.7, 126.6, 125.8, 125.3,
124.8, 121.4, 120.4, 119.3, 119.1, 116.8, 109.8, 109.2, 65.9, 65.0,
55.7, 48.8, 42.7, 30.0, 29.6 ppm; HRMS (ES+) m/z calcd for C31H31
Step (C): The above racemic product from (B) was separated by
chiral SFC chromatography (prep OJ, or AD, or OD, 35 °C at 100 bar,
eluting with 20–40% methanol in carbon dioxide) to provide pure
two enantiomers (>95% ee).
Cl2FN4O4+H [(M+H)+]: 613.1779, found: 613.1779. [
(c 0.07, MeOH).
a]
20 = ꢂ45.1°
D
4.7. Chiral (20S,30R,40S,50R)-6-chloro-40-(3-chloro-2-fluoro-phenyl)-
20-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,
30-pyrrolidine]-50-carboxylic acid (4-carbamoyl-phenyl)-amide (20)
4.11. General procedures for preparation of compounds 23, 24
from racemic 17 (R3 = H, R1 = CH2C(CH3)3)
A white solid (0.134 g, 25%). 1H NMR (400 MHz, DMSO-d6) d
10.51 (s, 1H), 10.26 (s, 1H), 7.91 (br s, 1H), 7.90 (d, J = 8.78 Hz, 2H),
7.65 (d, J = 8.78 Hz, 2H), 7.57–7.61 (m, 2H), 7.35–7.40 (m, 1H), 7.29
(br s, 1H), 7.17 (t, J = 8.03 Hz, 1H), 7.07 (dd, J = 1.89, 8.03 Hz, 1H),
6.72 (d, J = 1.89 Hz, 1H), 4.74 (t, J = 8.41 Hz, 1H), 4.58 (d,
J = 9.72 Hz, 1H), 3.89 (t, J = 8.41 Hz, 1H), 3.56 (br s, 1H), 1.30 (dd,
J = 9.72, 13.74 Hz, 1H), 0.86 (s, 9H), 0.80 (d, J = 13.74 Hz, 1H) ppm;
13C NMR (101 MHz, DMSO-d6) d 177.0, 171.9, 167.3, 156.8, 154.3,
143.4, 140.8, 132.5, 129.1, 128.6, 128.2, 126.2, 126.0, 125.9, 125.1,
124.9, 121.4, 119.4, 119.2, 118.0, 109.3, 65.5, 64.9, 64.6, 48.1, 43.2,
30.0, 29.7 ppm; HRMS (ES+) m/z calcd for C30H29Cl2FN4O3+H
Step (A): To a solution of racemic (20S,30R,40S,50R)-6-chloro-40-(3-
chloro-2-fluoro-phenyl)-20-(2,2-dimethyl-propyl)-2-oxo-1,2-dihy-
dro-spiro[indole-3,30-pyrrolidine]-50-carboxylic acid trifluoroacetic
acid salt 17 (R3 = H, R1 = CH2C(CH3)3) (0.4 g, 0.69 mmol) in
dichloromethane (9 mL) was added diisopropylethylamine
(0.46 g, 3.6 mmol), diphenylphosphinic chloride (Aldrich) (0.34 g,
1.42 mmol) respectively. The mixture was stirred at room temper-
ature for 8 min, then 2-(4-aminophenoxy)ethanol (0.16 g,
1.1 mmol) or 4-[2-(tert-butyl-dimethyl-silanyloxy)-ethoxy]-2-
methoxy-phenylamine (prepared in SI section) (0.32 g, 1.1 mmol)
was added. The reaction mixture was stirred at room temperature
for 72 h. The mixture was concentrated. The residue was dissolved
into tetrahydrofuran (9 mL), and an aqueous solution (1 N) of HCl
(1 mL) was added. The reaction mixture was stirred at room tem-
perature for 2 h, then concentrated. The residue was partitioned
between ethyl acetate and aqueous saturated NaHCO3 solution.
The organic layer was separated, and aqueous layer was extracted
with ethyl acetate twice. The combined organic extract was
washed with water, brine, dried over Na2SO4, then concentrated.
[(M+H)+]: 583.1674, found: 583.1674. [
a
]
D
20 = ꢂ21.8° (c 0.11, MeOH).
4.8. Chiral (20S,30R,40S,50R)-6-chloro-40-(3-chloro-2-fluoro-phenyl)-20-
(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,
30-pyrrolidine]-50-carboxylic acid (4-carbamoyl-3-methoxy-
phenyl)-amide (21)
An off white solid (10 mg, 14%). 1H NMR (300 MHz, DMSO-d6) d
10.47 (s, 1H), 10.27 (s, 1H), 7.85 (d, J = 8.76 Hz, 1H), 7.62–7.66 (m,

4008
Z. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 4001–4009
The residue was purified by chromatography (0–50% of EtOAc in
CH2Cl2) to give the racemic product as an off white solid (0.18 g,
41%).
Step (B): The above racemic product from (A) was separated by
chiral SFC chromatography (prep OJ, or AD, or OD, 35 °C at 100 bar,
eluting with 20–40% methanol in carbon dioxide) to provide pure
two enantiomers (>95% ee).
dropwise. The reaction mixture was stirred at 0 °C for 1 h. The
mixture was partitioned between ethyl acetate and saturated
aqueous Na2SO3 solution. The organic layer was separated, washed
with water, brine, dried over MgSO4, and concentrated. The residue
was triturated with dichloromethane and hexanes to give the
product as a white solid (yield 0.1 g, 97%).
Step (C) The above racemic product from (B) (0.11 g) was sepa-
rated by chiral SFC chromatography (prep OJ, or AD, or OD, 35 °C at
100 bar, eluting with 20–40% methanol in carbon dioxide) to pro-
vide pure two enantiomers (>95% ee).
4.12. Chiral (20S,30R,40S,50R)-6-chloro-40-(3-chloro-2-fluoro-phen
yl)-20-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,
30-pyrrolidine]-50-carboxylic acid [4-(2-hydroxy-ethoxy)-phenyl]-
amide (23)
4.15. Chiral (20S,30R,40S,50R)-6-chloro-40-(3-chloro-2-fluoro-
phenyl)-20-(2,2-dimethyl-propyl)-4-fluoro-2-oxo-1,2-dihydro-
spiro[indole-3,30-pyrrolidine]-50-carboxylic acid (4-carbamoyl-
2-methoxy-phenyl)-amide (25)
An off white solid (38 mg, 41%). 1H NMR (400 MHz, DMSO-d6) d
10.47 (s, 1H), 9.92 (s, 1H), 7.63 (d, J = 8.03 Hz, 1H), 7.58 (t,
J = 6.90 Hz, 1H), 7.46 (d, J = 9.04 Hz, 2H), 7.36 (t, J = 7.03 Hz, 1H),
7.12–7.19 (m, 1H), 7.05 (dd, J = 1.76, 8.03 Hz, 1H), 6.90 (d,
J = 9.04 Hz, 2H), 6.69 (d, J = 1.76 Hz, 1H), 4.84 (t, J = 5.52 Hz, 1H),
4.65 (d, J = 9.29 Hz, 1H), 4.51 (d, J = 9.54 Hz, 1H), 3.94 (t,
J = 5.02 Hz, 2H), 3.83 (d, J = 8.28 Hz, 1H), 3.69 (q, J = 5.10 Hz, 2H),
3.51 (br s, 1H), 1.27 (dd, J = 9.66, 13.93 Hz, 1H), 0.84 (s, 9H), 0.76
(d, J = 13.93 Hz, 1H) ppm; 13C NMR (101 MHz, DMSO-d6) d 177.0,
170.8, 156.8, 154.8, 154.3, 143.4, 132.4, 131.5, 129.1, 128.3,
126.3, 126.2, 126.0, 125.0, 124.9, 124.8, 121.4, 120.2, 119.3,
119.1, 114.6, 109.3, 69.7, 65.6, 64.9, 64.6, 59.6, 48.4, 43.2, 40.4,
30.0, 29.7 ppm; HRMS (ES+) m/z calcd for C31H32Cl2FN3O4+H
[(M+H)+]: 600.1827, found: 600.1824.
A white solid (56 mg, 31%). 1H NMR (400 MHz, DMSO-d6) d
10.81 (s, 1H), 10.60 (s, 1H), 8.32 (d, J = 8.28 Hz, 1H), 7.94 (br s,
1H), 7.57–7.65 (m, 3H), 7.51 (dd, J = 1.38, 8.28 Hz, 1H), 7.41 (t,
J = 7.03 Hz, 1H), 7.31 (br s, 1H), 7.16–7.24 (m, 1H), 7.00 (dd,
J = 2.01, 9.54 Hz, 1H), 6.56 (dd, J = 2.01, 8.53 Hz, 1H), 4.91 (d,
J = 8.53 Hz, 1H), 4.69–4.78 (m, 1H), 4.20 (t, J = 10.52 Hz, 1H), 3.91
(s, 3H), 3.79–3.88 (m, 1H), 1.37 (dd, J = 10.52, 13.93 Hz, 1H),
0.91–0.98 (m, 11H), 0.84 (d, J = 13.93 Hz, 1H) ppm; 13C NMR
(101 MHz, DMSO-d6) d 176.0, 172.0, 167.2, 163.4, 160.9, 156.9,
154.4, 147.5, 145.3, 145.2, 130.4, 130.3, 129.6, 129.4, 129.3,
128.6, 126.2, 126.1, 124.9, 120.4, 119.3, 119.1, 118.1, 109.9, 66.9,
64.6, 60.8, 55.8, 45.3, 43.0, 30.2, 29.5 ppm; HRMS (ES+) m/z calcd
for C31H30Cl2F2N4O4+H [(M+H)+]: 631.1685, found: 631.1683.
4.13. Chiral (20S,30R,40S,50R)-6-chloro-40-(3-chloro-2-fluoro-phen
yl)-20-(2,2-dimethyl-propyl)-2-oxo-1,2-dihydro-spiro[indole-3,
30-pyrrolidine]-50-carboxylic acid [4-(2-hydroxy-ethoxy)-2-
methoxy-phenyl]-amide (24)
4.16. Chiral (20S,30R,40S,50R)-6-chloro-40-(3-chloro-2-fluoro-
phenyl)-20-(2,2-dimethyl-propyl)-5-fluoro-2-oxo-1,2-dihydro-
spiro[indole-3,30-pyrrolidine]-50-carboxylic acid (4-carbamoyl-
phenyl)-amide (26)
An off white solid (58 mg, 39%). 1H NMR (300 MHz, DMSO-d6) d
10.46 (s, 1H), 10.35 (s, 1H), 8.17 (d, J = 8.53 Hz, 1H), 7.69 (d,
J = 8.53 Hz, 1H), 7.60 (t, J = 6.84 Hz, 1H), 7.37 (t, J = 6.84 Hz, 1H),
7.16 (t, J = 8.10 Hz, 1H), 7.01 (dd, J = 2.00, 8.10 Hz, 1H), 6.64–6.70
(m, 2H), 6.48 (dd, J = 2.00, 8.91 Hz, 1H), 4.84 (t, J = 5.34 Hz, 1H),
4.56–4.64 (m, 1H), 4.41 (d, J = 9.60 Hz, 1H), 3.96 (t, J = 4.89 Hz,
2H), 3.82 (s, 3H), 3.73–3.81 (m, 1H), 3.61–3.72 (m, 3H), 1.28 (dd,
J = 9.60, 13.88 Hz, 1H), 0.90 (s, 9H), 0.75 (d, J = 13.88 Hz, 1H)
ppm; 13C NMR (75 MHz, DMSO-d6) d 176.9, 170.7, 157.2, 155.1,
153.9, 149.0, 143.4, 132.4, 128.9, 128.4, 126.9, 126.8, 125.8,
125.2, 124.8, 124.7, 121.4, 121.0, 119.3, 119.1, 118.5, 109.2,
104.7, 99.3, 69.8, 66.0, 65.2, 65.0, 59.6, 55.6, 49.1, 42.7, 30.0,
29.6 ppm; HRMS (ES+) m/z Calcd for C32H34Cl2FN3O5+H [(M+H)+]:
An off white solid (43 mg, 29%). 1H NMR (400 MHz, DMSO-d6) d
10.47 (s, 1H), 10.21 (s, 1H), 7.91 (d, J = 9.29 Hz, 1H), 7.87 (d,
J = 8.78 Hz, 3H), 7.62 (d, J = 8.78 Hz, 2H), 7.55–7.60 (m, 1H), 7.36–
7.41 (m, 1H), 7.25 (br s, 1H), 7.17 (t, J = 8.03 Hz, 1H), 6.80 (d,
J = 6.27 Hz, 1H), 4.72 (d, J = 8.53 Hz, 1H), 4.57 (d, J = 9.54 Hz, 1H),
3.92 (br s, 1H), 3.45–3.50 (m, 1H), 1.22–1.33 (m, 1H), 0.87 (s,
9H), 0.78 (d, J = 13.74 Hz, 1H) ppm; HRMS (ES+) m/z Calcd for
C30H28Cl2F2N4O3+H [(M+H)+]: 601.1580, found: 601.1581.
4.17. Racemic (20S,30R,40S,50R)-6-chloro-40-(3-chloro-2-fluoro-
phenyl)-20-(2,2-dimethyl-propyl)-7-fluoro-2-oxo-1,2-dihydro-
spiro[indole-3,30-pyrrolidine]-50-carboxylic acid (4-carbamoyl-
2-methoxy-phenyl)-amide (27)
630.1933, found: 630.1934. [
a]
20 = ꢂ28.5° (c 0.07, MeOH).
D
4.14. General procedures for preparation of compounds 25, 26,
and 27 from corresponding racemic 4-, 5-, or 7-fluoro
intermediate 17 (R3 = F, R1 = CH2C(CH3)3)
A light yellow solid (82 mg, 92%). 1H NMR (400 MHz, DMSO-d6)
d 11.10 (s, 1H), 10.68 (s, 1H), 8.33 (d, J = 8.28 Hz, 1H), 7.93 (br s,
1H), 7.56–7.64 (m, 3H), 7.50 (dd, J = 1.51, 8.28 Hz, 1H), 7.36–7.42
(m, 1H), 7.30 (br s, 1H), 7.14–7.22 (m, 2H), 4.65–4.71 (m, 1H),
4.51 (d, J = 9.29 Hz, 1H), 3.89 (s, 3H), 3.83–3.93 (m, 1H),
3.72–3.80 (m, 1H), 1.30 (dd, J = 9.91, 13.93 Hz, 1H), 0.91
(s, 9H), 0.77 (d, J = 13.93 Hz, 1H) ppm; HRMS (ES+) m/z Calcd for
Step (A): In a manner similar to the method described in general
procedures for compounds 20–22 and 4, the corresponding
racemic 4-, 5-, or 7-fluoro carboxylic acid intermediate 17 (R3 = F,
R1 = CH2C(CH3)3, preparation in SI section) (0.33 g, 0.55 mmol),
was reacted with diisopropylethylamine (0.29 g, 2.2 mmol),
diphenylphosphinic chloride (0.26 g, 1.1 mmol), then reacted with
4-amino-3-methoxy benzonitrile (0.11 g, 0.8 mmol) or 4-amino-
benzonitrile (Aldrich) (0.2 g, 1.7 mmol) to give racemic coupled
product as a white solid (Yield 0.1 g, 31%).
Step (B): To the solution of above racemic coupled product
(0.1 g, 0.17 mmol) from (A) in DMSO (5 mL) at 0 °C was added an
aqueous solution (30% Aldrich) of H2O2 (0.39 g, 3.4 mmol), then
aqueous solution (1 N) of NaOH (1.7 mL, 1.7 mmol) was added
C
31H30Cl2F2N4O4+H [(M+H)+]: 631.1685, found: 631.1686.
Acknowledgments
The authors would like to thank Theodore Lambros for SFC puri-
fication, Gino Sasso for detailed 1H and NOE NMR analysis of ste-
reochemical assignments, Samir Serrano for high resolution mass
spectrometry, Simon Yang, Roche China for optical rotation, and
Francisco Talamas for proof reading of the manuscript.

Z. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 4001–4009
4009
Roveto, P.; Saiki, A. Y.; Shaffer, P.; Toteva, M.; Wang, Y.; Wang, Y. C.; Wortman,
S.; Yakowec, P.; Yan, X.; Ye, Q.; Yu, D.; Yu, M.; Zhao, X.; Zhou, J.; Zhu, J.; Olson, S.
H.; Medina, J. C. J. Med. Chem. 2014, 57, 1454.
Supplementary data
Supplementary data (the data include syntheses, X-ray crystal
data of 5 and 20, and biological assays) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmc.2014.05.072.
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