Discovery of 4-((3′R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-1′-ethyl-2-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3-indoline]-5′-carboxamido)bicyclo[2.2.2]octane-1-carboxylic Acid (AA-115/APG-115): A Potent and Orally Active Murine Double Minute 2 (MDM2) Inhibitor in Clinical Development

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Discovery of 4‑((3′R,4′S,5′R)‑6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-

1′-ethyl-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-

5′-carboxamido)bicyclo[2.2.2]octane-1-carboxylic Acid (AA-115/APG-

115): A Potent and Orally Active Murine Double Minute 2 (MDM2)

Inhibitor in Clinical Development

Angelo Aguilar,^†Jianfeng Lu,^†Liu Liu,^†Ding Du,^†Denzil Bernard,^†Donna McEachern,^†

Sally Przybranowski,^†Xiaoqin Li,^‡Ruijuan Luo,^‡Bo Wen,^‡Duxin Sun,^‡Hengbang Wang,^§,#

Jianfeng Wen,^§,#Guangfeng Wang,^§,#Yifan Zhai,^§,#Ming Guo,^§,#Dajun Yang,^§,#,

and Shaomeng Wang*^,†

^†University of Michigan Comprehensive Cancer Center, and Departments of Internal Medicine, Pharmacology, and Medicinal

Chemistry, University of Michigan, 1600 Huron Parkway, Ann Arbor, Michigan 48109, United States

^‡Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States

^§Jiangsu Ascentage Biomed Development Inc., China Medical City, Taizhou, Jiangsu 225300, China

^#Suzhou Ascentage Pharma Inc., Suzhou, Jiangsu 215123, China

Department of Experimental Research, Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China,

Collaborative Innovation Center for Cancer Medicine, 651 Dongfeng Road East, Guangzhou, China

S

* Supporting Information

ABSTRACT: We previously reported the design of spirooxindoles

with two identical substituents at the carbon-2 of the pyrrolidine

core as potent MDM2 inhibitors. In this paper we describe an

extensive structure−activity relationship study of this class of

MDM2 inhibitors, which led to the discovery of 60 (AA-115/APG-

115). Compound 60 has a very high aﬃnity to MDM2 (K_i< 1 nM),

potent cellular activity, and an excellent oral pharmacokinetic

proﬁle. Compound 60 is capable of achieving complete and long-

lasting tumor regression in vivo and is currently in phase I clinical

trials for cancer treatment.

Our laboratory has designed and optimized spirooxindoles as

a new class of MDM2 inhibitors and has advanced compound 2

(MI-77301) into clinical development.¹⁸One shortcoming of 2

and its earlier analogues that we have found is that they slowly

isomerize in solution.^21,22To overcome this stability issue, we

recently reported the design of new spirooxindoles containing

two identical substituents at C-2 of the pyrrolidine ring, which

can undergo a rapid and irreversible conversion to a single

diastereoisomer.^21,22In the present study, we report an

extensive structure−activity relationship (SAR) study for this

class of MDM2 inhibitors. This study has led to the discovery

of 60 (AA-115/APG-115), a highly potent, chemically stable

and eﬃcacious MDM2 inhibitor, which has entered clinical

development for cancer treatment (ClinicalTrials.gov identiﬁer

for 60: NCT02935907).

INTRODUCTION

■

The tumor suppressor function of p53 is compromised in

essentially all human cancers. In about half of human cancers,

TP53, the gene encoding p53 protein, is mutated or deleted,

and this inactivates the tumor suppressor function of p53.¹In

the remaining 50%, p53 retains its wild-type status but its

function is eﬀectively inhibited by the murine double minute 2

(MDM2) protein through a direct protein−protein inter-

action.²⁻⁸Through direct binding, the MDM2 protein blocks

the transactivation domain of p53, transports p53 from the

nucleus to the cytoplasm, and ubiquitinates p53 for

proteasomal degradation.⁶Small-molecule inhibitors designed

to block the MDM2−p53 interaction (hereafter called MDM2

inhibitors) can liberate the tumor suppressor function of

p53⁹⁻¹³and may have a promising therapeutic potential for

cancer treatment. Intense research eﬀorts have resulted in

advancement of several potent, selective, non-peptide MDM2

inhibitors (1−5),¹⁴⁻²⁰shown in Figure 1, into clinical

development.

Received: November 17, 2016

A

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Figure 1. Chemical structures of representative MDM2 inhibitors in clinical trials.

Figure 2. (A) Chemical structure of compound 6 and ﬁve regions (A−E) where chemical modiﬁcations were made. (B) Cocrystal structure of 2

(light blue spheres, PDB code 5TRF) and model of the binding mode of 6 (green sticks) in complex with MDM2.

We began by replacing the 4-hydroxycyclohexyl group at site

D in compound 6 with a methyl group, giving compound 7.

Since compounds 6 and 7 have similar binding aﬃnities to

MDM2, we have used either compound 6 or 7 as the template

for modiﬁcations of site A (Table 1). Replacement of the

cyclohexyl group at site A with 4-piperidinyl group led to

compound 8, which has no appreciable binding up to 2 μM to

MDM2. Methylation or acetylation of the amine group in 8

resulted in compound 9 or 10, respectively, and these also have

very weak aﬃnities for MDM2. Replacement of the cyclohexyl

group at site A in 6 with 4-tetrahydro-2H-pyran generated

compound 11, which has a moderate binding aﬃnity to MDM2

(K_i= 75.9 nM). Introduction of two ﬂuorine substituents at the

4-position of the cyclohexyl group in compound 6 resulted in

compound 12, which has a good aﬃnity to MDM2 (K_i= 9.8

nM) but is 4 times less potent than 6. However, introduction of

two methyl groups at the same 4-position in 6 yielded

compound 15, which has a K_ivalue of <1 nM to MDM2 and is

several times more potent than 6. Replacement of the

RESULTS AND DISCUSSION

■

Our exploration started with lead molecule 6²¹(Figure 2A) that

binds to MDM2 with K_i= 2.9 nM, inhibits the growth of the

SJSA-1 cancer cell line with IC₅₀= 190 nM, and achieves strong

tumor growth inhibition but not complete tumor regression in

an SJSA-1 osteosarcoma xenograft model in mice.²¹In a search

for superior compounds, we have performed extensive

modiﬁcations of 6.

To guide our chemical modiﬁcations, we modeled the

structure of compound 6 in a complex with MDM2 based upon

the cocrystal structure of compound 2 complexed with human

MDM2 (Figure 2B).¹⁸Our model shows that the oxindole-

phenyl, 3-chloro-2-ﬂuorophenyl, and cyclohexyl groups of 6

project into the Trp23, Leu26, and Phe19 p53 binding pockets

of the MDM2 protein and the 4-hydroxyl of the N-

cyclohexylcarboxamide group forms a hydrogen bond with

Lys94 of MDM2 (Figure 2B). Guided by this model, we

performed extensive modiﬁcations on ﬁve regions of the

molecule (labeled as sites A−E, Figure 2A).

B

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c

Table 1. Structure−Activity Relationship of Pyrrolidine-2-spirocyclohexyl Substituent

a

b

c

Value of one experiment. Reference 21. Values are average of at least two tests.

a

Table 2. SAR of the B and C Aryl Rings

a

Values are average of at least two tests.

cyclohexyl group with a cyclobutyl group generated compound

13,²¹which is 6 times less potent than 6. Installation of methyl

groups at the 3-position of the cyclobutyl ring led to compound

14, which is equally potent as compound 6 and 7 times more

potent than compound 13. Hence, our SAR data for site A

clearly show that a hydrophobic group is highly desirable for

achieving a strong binding aﬃnity to MDM2, and cyclohexyl,

4,4′-dimethylcyclohexyl, and 3,3′-cyclobutyl groups are the

most preferred.

C in Figure 2A, respectively). To improve solubility, we

inserted one or more nitrogen atoms into the aryl rings,

producing compounds 16, 17, 18, and 19 (Table 2). Insertion

of a nitrogen into the 3-chloro-2-ﬂuorophenyl substituent (ring

B) gave 16, which has a K_iof 77 nM, 26 times weaker than 6.

Interestingly, although the Trp23 pocket is hydrophobic in

nature, compounds 17, 18, and 19 containing a pyridinyl or a

pyrimidinyl oxindole group (ring C) are all quite potent with K_i

= 13−14 nM. Our data, however, are consistent with a previous

study, which showed that a compound containing a

chloropyridyl oxindole group is a potent MDM2 inhibitor.²³

Next, we explored modiﬁcations of the 3-chloro-2-

ﬂuorophenyl and the oxindolephenyl substituents (sites B and

C

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c

Table 3. Carboxamide (E) Bioisostere Replacement

a

b

c

Value of only one experimnent. NT = not tested. Values are average of at least two experiments.

This previous study also showed that replacing the oxindole

with a thienopyrrolone led to potent MDM2 inhibitors.²³We

therefore synthesized compound 20 containing a thienopyrro-

lone, which has a K_iof 6.5 nM and is 2-fold less potent than 6.

We found that the thiophene group in 20 can be oxidized

during the oxidative removal of the chiral auxiliary, preventing

further investigation of this compound.

Our modeling of 6 and the cocrystal structure of 2 (Figure

2B) showed that the carbonyl of the amide (site E) forms a

hydrogen bond with His96 of the MDM2 protein but the

amino group does not interact with the MDM2 protein. We

evaluated a series of ﬁve-membered heteroaromatic rings as the

amide bioisoteres (Table 3). Among these replacements, we

found that heteroaromatic rings with carboxylic acid sub-

stituents, e.g., 21, 22, 26, and 27 have the best binding aﬃnities

with K_ivalues between 1 and 3 nM. Unfortunately, these

compounds show weak cell growth inhibitory activity (IC₅₀> 2

μM, Table 3) in the SJSA-1 cancer cells, probably because the

negatively charged acid group in these compounds decreases

their cell permeability. We therefore converted the acid group

in compound 21 to an amide but found that the resulting

compound 28 is 20 times less potent than 21.

substituent of 6 forms a hydrogen bond with Lys94 of the

MDM2 protein (Figure 2B). We synthesized a series of

compounds containing either a terminal carboxylic acid or a

sulfone to investigate the eﬀect on binding (compounds 29−

38) (Table 4). These modiﬁcations resulted in a series of

compounds with high aﬃnities. In particular, compounds 31,

32, and 33 bind to MDM2 with K_i< 1 nM and are more than 3

times more potent than 6.

We tested the cell growth inhibitory activity of compounds

29−38 in the SJSA-1 cell line, and the data are summarized in

Table 4. Among this series of compounds, 32, 33, and 38 have

the best antiproliferative activity with IC₅₀values of 0.22, 0.15,

and 0.24 μM, respectively.

We further evaluated compound 33 for its pharmacodynamic

(PD) eﬀect in the SJSA-1 xenograft tissue. Our PD data showed

that a single, oral administration of 33 at 100 mg/kg is very

eﬀective in inducing upregulation of MDM2, p53, and p21

proteins at both 3 and 6 h time-points in the SJSA-1 tumor

tissue, indicating strong activation of p53 (Figure 3A). Upon

the basis of its promising PD data, we evaluated compound 33

for its antitumor eﬃcacy in the SJSA-1 xenograft model (Figure

3B,C). While compound 33 can eﬀectively retard tumor

growth, it failed to achieve tumor regression. During the course

of our research, we also observed that compound 33 can

Our modeling of 6 and the cocrystal structure of 2 also

showed that the 4-hydroxyl on the cyclohexylcarboxamide

D

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d

Table 4. SAR of Carboxamide Substituent (D)

a

b

c

d

Reference 21. NT = not tested. Value of one time testing. Values are average of at least two tests.

Figure 3. (A) Pharmacodynamics (PD) eﬀect of 33 in SJSA-1 tumors in mice. Mice were given a single, oral dose of the vehicle, or 33 at 100 mg/kg

and sacriﬁced at indicated time points. West blotting was performed on harvested tumor tissue to probe MDM2, p53, p21, PAPR, cleaved PARP (cl-

PAPR) proteins. (B) Antitumor activity of 33 in the SJSA-1 osteosarcoma tumor xenograft model in mice. Compound 33 was administered via oral

gavage at 100 mg/kg daily for 14 days. (C) Body weight change of mice during administration of 33.

decompose in solution, particularly in cell culture media

(panels A and B in Scheme 1). To analyze the potential

structural features aﬀecting the stability of 33, we determined

the stability of compound 31 with a ﬂexible butanoic acid

group, compound 6 with a 4-hydroxylcyclohexyl group, and 33.

Compound 6 had negligible (<1%) decomposition (panel C in

Scheme 1). On the other hand, compound 31 showed 18%

decomposition after 2 days in cell culture media, which is

greater than that observed for compound 33 (panel D in

Scheme 1). On the basis of these results, we proposed that the

carboxylic group assists in the decomposition of 33.

We next performed further chemical modiﬁcations of 33 with

the goal to improve its chemical stability, cellular potency, and

in vivo eﬃcacy.

We investigated if replacement of the cyclohexyl group with

an even more rigid aryl group can prevent the decomposition.

A series of compounds containing an N-arylcarboxamide

substituent were synthesized and tested (Table 5). These

compounds all display very high binding aﬃnities to MDM2,

superior to that of 6 and equivalent to that of 33. The cell

growth inhibition of SJSA-1 cells by these compounds was

determined (Table 5). Compounds 39 (MI-1061)²¹and 44

displayed the best cellular activity among the compounds in this

series and are 3 times more potent than compound 33 in

inhibition of cell growth in the SJSA-1 cell line. Compound 39

was found in our previous study²¹to be very stable in solution.

Furthermore, 39 was capable of achieving partial tumor

regression in the SJSA-1 xenograft model as shown in our

previous report.²¹

We next combined all the favorable modiﬁcations on sites A

and D and designed and synthesized compound 54 (Scheme

2). Compound 54 has a very high binding aﬃnity to MDM2

E

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a

Scheme 1. Proposed Mechanism of Decomposition for 33

a

(A) Stability of compound 33 in various solutions. Compound 33 was incubated in CH₃CN/H₂O, MeOH/H₂O, or cell culture media, and the %

composition of 33 in the solutions was determined daily for 2 days by UPLC (ultraperformance liquid chromatography) and plotted. (B, C, D)

UPLC/MS spectrum of 33, 6, and 31, respectively, after incubation for 2 days in cell culture media. Peaks are labeled with retention time followed by

molecular weight.

(IC₅₀= 2.4 nM, K_i< 1 nM, Scheme 2) and potently inhibits cell

growth of SJSA-1 cells with IC₅₀= 13 nM (Scheme 2).

We assessed the antitumor activity of compound 54 in the

SJSA-1 xenograft model in mice, with compound 2 included as

the control (Figure 4). Compound 54 eﬀectively inhibits tumor

growth in a dose-dependent manner compared to the vehicle

control. While compound 54 at 50 mg/kg daily dosing

eﬀectively retards tumor growth, at 100 mg/kg it achieves a

maximum of 87% tumor regression during the treatment.

However, while compound 2 at 100 mg/kg achieves complete

tumor regression, compound 54 fails to do so.

Pharmacokinetic (PK) studies in rats (Table 6) showed that

with oral administration, compounds 39 and 54 achieve

considerably lower C_maxand AUC values than compound 2,

suggesting that further improvement of the oral PK for

compounds 39 and 54 is needed in order to achieve stronger in

vivo antitumor activity. Since 39 and 54 achieve comparable

tumor regression in the SJSA-1 xenograft model and 39 is less

hydrophobic than 54, we have selected compound 39 for

further optimization of its PK properties and in vivo eﬃcacy.

In our attempt to further improve the oral PK properties of

compound 39 while retaining its high binding aﬃnity to

MDM2 and cellular potency, we investigated the replacement

of the benzoic acid with nonclassical benzoic acid mimetics

such as a bicyclo[1.1.1]pentane-1-carboxylic acid²⁴or a

bicyclo[2.2.2]octane-1-carboxylic acid. These eﬀorts led to the

design and synthesis of compounds 55 and 56 (Scheme 3).

Compound 55 binds to MDM2 with a high aﬃnity (IC₅₀= 6.4

nM, K_i< 1 nM) but is 5 times less potent than compound 39 in

inhibition of cell growth in the SJSA-1 cell line. In comparison,

compound 56 has a high binding aﬃnity to MDM2 (IC₅₀= 3.7

nM, K_i< 1 nM) and is as potent as compound 39 in inhibition

of cell growth in the SJSA-1 cell line (Scheme 3 and Table 7).

A PK study in rats showed that 56 has a higher plasma

exposure than compound 2 based upon both the C_max(8234 vs

4547 μg/L) and AUC values (73603 vs 17230 h·μg/L).

However, our PD experiment showed that oral administration

of compound 56 at 100 mg/kg induces only modest p53

activation and no cleavage of PARP and caspase-3 in the SJSA-1

xenograft tumor tissue in mice (Figure 6A). Consistent with the

PD data, compound 56 at 100 mg/kg administered daily for 14

days demonstrates only a very moderate tumor growth

inhibition in the SJSA-1 xenograft model in mice (Figure

5A). Hence, the antitumor activity of 56 is much inferior to that

achieved by 2, 39, and 54. Because of its excellent in vitro

cellular potency and excellent plasma exposure, the modest p53

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d

Table 5. SAR of (Aryl acid)carboxamides

a

b

c

d

Value of one time testing. NT = not tested. Reference 21. Values are average of at least three tests.

Scheme 2. Design of Compound 54 by Combining the Optimal Substituents of Sites A and D

a

Reference 21.

activation and antitumor activity achieved by compound 56

suggests that this compound has a poor penetration into the

xenograft tumor tissue. Accordingly, we investigated strategies

with which to improve the tissue penetration while retaining

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Figure 4. (A) Antitumor activity of compound 54, in comparison to compound 2 in the SJSA-1 osteosarcoma tumor xenograft model in mice.

Compound 54 was administered via oral gavage at 50 or 100 mg/kg daily for 14 days and compound 2 was administered via oral gavage at 100 mg/

kg daily for 14 days. (B) Changes in body weight of mice during animal dosing.

Table 6. Pharmacokinetics Table for Comparison of 39, 54, 56, 59, 60, and 2 in Rats

compound

39

54

56

59

60

2

po dose (mg/kg)

po C_max(ng/mL)

po AUC (h·ng/mL)

po T_1/2(h)

25

1553

6799

4.0

968

8234

4391

35205

3.9

5453

39083

4.6

4547

17230

1.6

10188

4.5

73603

4.3

iv dose (mg/kg)

iv AUC (h·ng/mL)

10

5

8633

1.16

2.14

31.5

14767

0.690

2.45

27.6

82241

0.119

0.734

35.0

28825

0.352

1.10

48.6

38265

0.257

1.25

40.3

4630

1.03

2.98

74.9

iv CL (L h⁻¹kg⁻¹

iv V_ss(L/kg)

)

F (%)

antitumor eﬃcacy

86% regression

87% regression

no regression

100% regression

Scheme 3. Replacement of the Benzoic Acid in Compound 39 with Nonclassical Benzoic Acid Bioisosteres

high binding aﬃnity to MDM2, potent cellular activity, and

good oral bioavailability of compound 56.

Compound 57 binds to MDM2 with a high aﬃnity but is less

potent than 56 and is 2 times less potent than 56 in inhibition

of cell growth in the SJSA-1 cell line (Table 7). Compound 58

is 2 times less potent than 56 in binding to MDM2 and is 4

times less potent than 56 in inhibition of cell growth in the

SJSA-1 cell line (Table 7). The PD study showed that oral

administration of 57 or 58 at 100 mg/kg only has a modest

eﬀect on activation of p53 and induction of cleavage of PARP

and caspase-3 (Figure 6B).

In comparison, compounds 59 and 60 bind to MDM2 with a

high binding aﬃnity (K_i< 1 nM, Table 7). Both compounds

potently inhibit cell growth in the SJSA-1 cell line with IC₅₀

values of 70 and 60 nM, respectively, and are thus as potent as

compound 56 (Table 7). PD experiments showed that a single,

oral dose of 59 and 60 at 100 mg/kg achieves strong p53

We investigated three strategies to improve the tissue

penetration: (1) decreasing the lipophilicity of the molecule;

(2) reducing the acidity of carboxylic acids; and (3) increasing

the basicity of the nitrogen atom in the pyrrolidine core.²⁵

Fluorine substitution on aliphatic groups is known to

decrease lipophilicity.²⁶⁻²⁸We therefore synthesized com-

pound 57 with a 4,4-diﬂuorocyclohexyl group at carbon-2 of

the pyrrolidine core (Table 7). For the second strategy, we

replaced the carboxylic acid group with an acyl sulfonamide

bioisostere, which resulted in 58 (Table 7). For the third

strategy, we installed a methyl or ethyl group on the nitrogen of

the pyrrolidine core, which led to 59 and 60, respectively

(Table 7).

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c

Table 7. SAR of Benzoic Acid Nonclassical Bioisosteres and Alkylation of the Pyrrolidine Nitrogen

a

b

c

Value from one experiment. Reference 21. Values are average of at least two independent experiments.

Figure 5. (A) Antitumor activity of compounds 39 and 56 in the SJSA-1 osteosarcoma xenograft model in mice. (B) Body weight change of mice

during administration period.

Figure 6. Pharmacodynamic (PD) analysis of the eﬀect of MDM2 inhibitors in SJSA-1 tumors. Mice bearing SJSA-1 tumors were dosed with a single

oral dose of 56, 57, 58, 59, 60 and 2 at 100 mg/kg and the tumors were harvested at 6 and 24 hours for western blot analysis. (A, B) PD of 56, 57

and 58 show modest activation of p53 as seen with the low levels of p53, MDM2, and p21; (C) Compounds 59, 60 and 2 show robust activation of

p53 as seen with increased levels of p53, MDM2, ubiquitinated MDM2 (ub-MMD2) and p21, and induce strong apoptosis as seen with increased

levels of cleaved PARP (cl-PAPR) and cleaved caspase-3 (cl-Casp3).

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Figure 7. (A) Antitumor activity of compound 59, 60 in comparison to 56 in the SJSA-1 osteosarcoma xenograft model in mice. Compound 59 and

60 were administered at 30 mg/kg or 100 mg/kg daily for 14 days and 56 at 100 mg/kg daily for 14 days. (B) Animal weight changes of mice during

the experiment.

a

Table 8. Cell Growth Inhibition Activity of 56, 59, and 60 in Diﬀerent Human Cancer Cell Lines

cell growth inhibition (IC₅₀)

cell line

SJSA-1

tumor type

p53 status

56

59

60

osteosarcoma

leukemia

wild-type

null

89 33 (nM)

26.7 5.1 (μM)

62 26 (nM)

36 19 (nM)

12.3 2.5 (μM)

137 31 (nM)

14 2 (μM)

70 21(nM)

25 6 (μM)

56 18 (nM)

30 15 (nM)

24 5 (μM)

117 33 (nM)

18 8 (μM)

60 22 (nM)

22.7 4.7 (μM)

38 5 (nM)

Saos2

RS4;11

wild-type

null

LNCaP

prostate cancer

colon cancer

18 13 (nM)

22 7.2 (μM)

104 36 (nM)

PC3

HCT116

HCT116 p53−/−

wild-type

knockout

8

1 (μM)

a

IC₅₀values are average of at least three independent experiments.

Figure 8. (A) Antitumor activity of compound 60 in the RS4; 11 acute lymphoblastic leukemia xenograft model in mice. Compound 60 was

administered at 50 mg/kg, 75 mg/kg, or 100 mg/kg daily for 14 days and 200 mg/kg weekly for 3 weeks. (B) Animal body weight of mice during

administration.

activation, with the eﬀect persisting for 24 h in the SJSA-1

xenograft tumor tissue (Figure 6C). Compounds 59 and 60

also achieve strong apoptosis induction in the SJSA-1 xenograft

tumor, based upon cleavage of PARP and caspase-3 (Figure

6C).

We next tested 59 and 60 for the antitumor activity in the

SJSA-1 xenograft model (Figure 7). Compound 59 or 60 orally

administered at 30 mg/kg daily for 14 days eﬀectively inhibits

tumor growth. Compound 59 or 60 orally administered at 100

mg/kg daily for 14 days eﬀectively achieves complete and long-

lasting tumor regression. In comparison, compound 56 at 100

mg/kg inhibits tumor growth only moderately in the same

experiment (Figure 7A). Compounds 56, 59, and 60 cause no

or minimal weight loss during the entire experiment (Figure

7B).

Our PK studies in rats with oral administration showed that

59 and 60 achieve a C_maxvalue similar to that of 2 but have a 2

times higher AUC than compound 2 (Table 6).

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Figure 9. Stability comparison of compounds 2 and 60 in (A) 1:1 CH₃CN to H₂O; (B) 1:1 MeOH to H₂O; (C) cell culture media.

a

Scheme 4. Synthesis of Compounds 6−15

a

Reagents and conditions: (a) toluene, reﬂux, 2 h; (b) R₁NH₂, THF, reﬂux overnight; (c) CH₃CN/H₂O (1:1), CAN, 15 min; (d) CH₃CN/H₂O,

overnight.

a

Scheme 5. Synthesis of Compounds 16−20

a

Reagents and conditions: (a) MeOH, piperidine, reﬂux.

We evaluated compounds 56, 59, and 60 for their activity

and selectivity in a number of human cancer cell lines of

diﬀerent tumor types (Table 8). Consistent with their

mechanism of action, these three compounds potently inhibit

cell growth in cancer cell lines with wild-type p53 and display

outstanding selectivity in cancer cell lines with deleted p53.

Compound 60 is the most potent compound, achieving IC₅₀

values of 38, 18, and 104 nM in the RS4;11 acute leukemia,

LNCaP prostate cancer, and HCT116 colon cancer cell lines,

respectively.

We next evaluated compound 60 for its antitumor activity in

the RS4;11 acute leukemia xenograft model in mice.

Compound 60 eﬀectively inhibits tumor growth in a dose-

dependent manner and achieves partial tumor regression at 100

mg/kg, daily, oral administration for 14 days, or at 200 mg/kg

weekly dosing for 3 weeks (Figure 8). Compound 60 is also

well tolerated at all doses and schedules in this experiment.

We evaluated the chemical stability of compound 60 in three

diﬀerent solutions and found that this compound is very stable

in solutions for a period of 7 days (Figure 9). In comparison,

compound 2 slowly isomerizes in solutions and retains only

93−96% purity after 7 days (Figure 9).

CHEMISTRY

■

To explore achiral substituents in carbon-2 of the pyrrolidine

core, the compounds in Table 1 were synthesized using the

method²¹outlined in Scheme 4. The azomethine ylide

generated in situ by reaction of the chiral amine (62) with

the cyclic ketone (63) was allowed to react with 61 in a 1,3-

dipolar cycloaddition reaction, generating the chiral spiroox-

K

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a

Scheme 6. Synthesis of Compounds 21, 27, and 28

a

Reagents and conditions: (a) DCM/AcOH (1:1), paraformaldehyde, NaBH(OAc)₃; (b) THF/MeOH/H₂O, LiOH·H₂O; (c) DCE, CDI, DIEA, 50

°C 30 min, then NH₄OH; (d) DCM, Lawesson’s reagent, reﬂux overnight; (e) (1) THF, BrCH₂COR, Et₃N, reﬂux overnight, (2) DME, TFAA,

pyridine, −20 °C to rt, 30 min; (f) THF/MeOH/H₂O (1:1:1), NaOH, rt, prep-HPLC.

a

Scheme 7. Synthesis of Compounds 22−26

a

Reagents and conditions: (a) DCE, CDI, DIEA, DMAP, 50 °C for 30 min, then 71, 74, 76, 78, and reﬂux; (b) (1) THF, Deoxo-Fluor, −20 °C for

30 min, (2) BrCCl₃, DBU at rt overnight; (c) THF/H₂O (2:1), LiOH·H₂O, rt for 30 min, then preparative HPLC; (d) pyridine, 100 °C for 4 h; (e)

DCM, PPh₃, I₂, Et₃N, rt; (f) DCM, Lawesson’s reagent, reﬂux overnight.

indoles (6a−15a). Reaction of this morpholine (6a−15a) with

an aliphatic amine led to the open-ring compound (6b−15b).

Upon oxidation with ceric ammonium nitrate (CAN), 6c−15c

were formed as a mixture of two diastereoisomers. “Aging” in

acetonitrile/water resulted in formation of the single

diastereoisomers 6−15.

Scheme 5 shows the method by which the B and C aryl rings

were replaced. First the intermediates 16b−20b were

synthesized by condensation of the pyrrolidones (16a−20a)

with an arylaldehyde. Subsequently, the compounds (16−20)

in Table 2 were obtained by using the intermediates (16b−

20b), which are equivalent to 61, in the synthetic method

outlined in Scheme 4.

Compounds containing various ﬁve-membered heteroaro-

matic rings as bioisostere replacements for the carboxamide

group E were synthesized as outlined in Schemes 6 and 7. The

N-methylpyrrolidine (65) was generated by reductive amina-

tion of 64 with paraformaldehyde and NaBH(OAc)₃.

Hydrolysis of the methyl ester (65) was followed by conversion

of the acid (66) to the corresponding carboxamide (67).

Reﬂuxing of 67 with Lawesson’s reagent produced the

thioamide (68) which was reacted with an α-bromoketone in

a Hantzsch thiazole synthesis reaction, generating the thiazoles

69 and 70.²⁹Basic hydrolysis of the esters produced the acid

analogues 21 and 27. Compound 21 was further elaborated by

amide coupling, generating the carboxamide (28).

L

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a

Scheme 8. Synthesis of Compounds in Tables 4 and 5

a

Reagents and conditions: (a) DCM, HATU, DIEA, alkylamine; (b) THF/MeOH/H₂O (1:1:1), LiOH·H₂O for alkyl esters or NaOH for aryl

esters; (c) DCM, Ph₂POCl, DIEA 30 min, then arylamine and DMAP; (d) DCE, CDI, DIEA, DMAP, 50 °C for 30 min, then R₂NH₂and reﬂux.

a

Several ﬁve-membered heteroaromatic rings were formed

from the carboxylic acid (66). The carboxylic acid (66) was

activated with CDI (1,1′-carbonyldiimidazole), and this was

followed by addition of commercially available compounds 71,

74, 76, 78, resulting in the formation of intermediates 72, 75,

77, 79, respectively. Cyclization to the respective ﬁve-

membered heteroaromatic compounds (73,³⁰23,³¹24,³²25,

80) was accomplished under the conditions outlined in Scheme

7. The carboxylic acid compounds 22 and 26 were produced by

hydrolysis of the esters in compounds 73 and 80, respectively.

The compounds in Tables 4 and 5 were obtained by the

synthesis outlined in Scheme 8. Activation of the carboxylic

acid (29) by HATU (1-[Bis(dimethylamino)methylene]-1H-

1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexaﬂuorophosphate)

followed by addition of an alkylamine gave the N-alkylcarbox-

amide intermediates 30, 36, 38, 31−35, 37, 55, and 56. When

N-arylamines were used, HATU failed to accomplish the

coupling, but the coupling was successful when the acid was

activated with Ph₂POCl ¹⁷and produced the N-arylcarbox-

amides 39−48, 50, and 51. Basic hydrolysis of the esters

produced the carboxylic acid compounds listed in Tables 4 and

5. Further elaboration of the acids (56 to give 58; 39 to give 49

and 52; and 44 to give 53) was accomplished by CDI activation

of the carboxylic acid followed by reaction with the respective

amine.

Scheme 9. Synthesis of Compound 59 and 60

a

Reagents and conditions: (a) DCM/AcOH (1:1), paraformaldehyde

or acetaldehyde, NaBH(OAc)₃, rt overnight.

inhibitors. In particular, compound 60 has a very high binding

aﬃnity to MDM2 (K_i< 1 nM), potently activates wild-type

p53, inhibits cell growth with low nanomolar IC₅₀values in

human cancer cell lines carrying wild-type p53, and

demonstrates an outstanding cellular selectivity over human

cancer cell lines with deleted p53. Compound 60 is very stable

in solutions, has excellent oral pharmacokinetics, and eﬀectively

activates p53 in the SJSA-1 xenograft tumor tissue in mice

following a single oral administration. Signiﬁcantly, 60 achieves

complete and long-lasting regression of the SJSA-1 xenograft

tumors in mice and demonstrates strong antitumor activity in

the RS4;11 acute leukemia model at well tolerated dose

schedules. Compound 60 has been advanced into phase I

clinical development for the treatment of human cancer.

Reductive amination of 56 with paraformaldehyde or

acetaldehyde produced 59 and 60 as shown in Scheme 9.

EXPERIMENTAL SECTION

CONCLUSION

■

General Information. Unless otherwise stated, all commercial

reagents were used as supplied without further puriﬁcation and all

reactions were performed under a nitrogen atmosphere in dry solvents

under anhydrous conditions. NMR spectra were obtained on either a

In this study, we have performed an extensive SAR study on

spirooxindoles containing two substituents at the carbon-2 of

the pyrrolidine core as MDM2 inhibitors. Extensive mod-

iﬁcations in ﬁve diﬀerent regions of the molecule have led to

discovery of a number of potent and highly eﬃcacious MDM2

1

Bruker 300 UltraShield spectrometer at a H frequency of 300 MHz

and ¹³C frequency of 75 MHz or Bruker 400 Ascend spectrometer at a

M

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¹H frequency of 400 MHz and ¹³C frequency of 100 MHz. Chemical

triﬂuoroacetic acid and stirred. After 30 min the triﬂuoroacetic acid

was removed and the crude was puriﬁed by HPLC to give 8 (TFA

salt). H NMR (300 MHz, CD₃OD) δ ppm 8.24 (br s, 1H), 7.64 (t,

1H, J = 7.2 Hz), 7.49 (dd, 1H, J = 2.2, 8.1 Hz), 7.27 (t, 1H, J = 7.3

Hz), 7.13−7.03 (m, 2H), 6.68 (s, 1H), 4.79 (d, 1H, J = 9.6 Hz), 4.64

(d, 1H, J = 9.6 Hz), 3.70 (t, 1H, J = 13.1 Hz), 3.44−3.18 (m, 3H), 2.77

(d, 3H, J = 4.3 Hz), 2.39 (d, 1H, J = 14.5 Hz), 2.10−1.88 (m, 2H),

1.50−1.26 (m, 1H). ESI-MS m/z 477.17 (M + 1)⁺.

shifts (δ) are reported in parts per million (ppm) relative to an internal

standard. The ﬁnal products were puriﬁed on a preparative HPLC

(Waters 2545, quaternary gradient module) with a SunFire Prep C18

OBD 5 μm, 50 mm × 100 mm reverse phase column. The mobile

phase was a gradient of solvent A (H₂O with 0.1% TFA) and solvent B

(MeOH with 0.1% TFA or MeCN with 0.1% TFA) at a ﬂow rate of 40

mL/min and 1%/2 min increase of solvent B or a ﬂow rate of 60 mL/

min and 1%/min increase of solvent B. All ﬁnal compounds have

purity of ≥95% as determined by Waters ACQUITY UPLC using

reverse phase column (SunFire, C18-5 μm, 4.6 mm × 150 mm) and a

solvent gradient of A (H₂O with 0.1% of TFA) and solvent D (MeOH

with 0.1% of TFA) or A (H₂O with 0.1% of TFA) and solvent B

(CH₃CN with 0.1% of TFA). ESI mass spectrum analysis was

performed on a Thermo-Scientiﬁc LCQ Fleet mass spectrometer. No

PAINS liability was found in any of the compounds presented as

determined by analysis in the online ﬁlter SmartsFilter (http://pasilla.

health.unm.edu/tomcat/biocomp/smarts ﬁ lter).

1

(3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N,1″-di-

methyl-2-oxodispiro[indoline-3,3′-pyrrolidine-2′,4″-piperi-

dine]-5′-carboxamide (9). Starting with 1-methylpiperidin-4-one in

place of cyclohexanone, compound 9 was prepared according to the

1

synthetic method described for the preparation of 6. H NMR (300

MHz, CD₃OD) δ ppm 8.25 (br s, 1H), 7.62 (t, 1H, J = 7.3 Hz), 7.47

(dd, 1H, J = 2.1, 8.2 Hz), 7.25 (t, 1H, J = 7.5 Hz), 7.12−7.01 (m, 2H),

6.77 (d, 1H, J = 1.6 Hz), 4.76 (d, 1H, J = 9.5 Hz), 4.62 (d, 1H, J = 9.5

Hz), 3.75 (t, 1H, J = 12.5 Hz), 3.52−3.40 (m, 2H), 3.24−3.12 (m,

1H), 2.86 (s, 3H), 2.76 (d, 3H, J = 4.0 Hz), 2.40 (d, 1H, J = 14.4 Hz),

2.12−1.86 (m, 2H), 1.54−1.34 (m, 1H); ESI-MS m/z 491.08 (M +

1)⁺.

Compounds 6, 13, 29, 39 and intermediate 64 were reported

previously.²¹Compounds 16b−20b were synthesized as reported

previously.²³

(3R,4′S,5′R)-1″-Acetyl-6-chloro-4′-(3-chloro-2-ﬂuorophen-

yl)-N-methyl-2-oxodispiro[indoline-3,3′-pyrrolidine-2′,4″-pi-

peridine]-5′-carboxamide (10). Starting with 1-acetylpiperidin-4-

one in place of cyclohexanone, compound 10 was prepared according

Compounds 7−20 were synthesized according to our reported

method²¹which is described below for the synthesis of compound 6.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-

((1r,4R)-4-hydroxycyclohexyl)-2″-oxodispiro[cyclohexane-1,2′-

pyrrolidine-3′,3″-indoline]-5′-carboxamide (6).²¹In a round-

bottom ﬂask, (E)-6-chloro-3-(3-chloro-2-ﬂuorobenzylidene)indolin-2-

one (500 mg, 1.62 mmol), (5R,6S)-5,6-diphenyl-2-morpholinone (492

mg, 1.94 mmol), and cyclohexanone (4.86 mmol) were suspended in

toluene (10 mL) and heated at reﬂux for 2 h at 140 °C. Then the

reaction was allowed to cool to room temperature and the solvent was

removed by rotoevaporation. The crude product was puriﬁed by

column chromatography (the compound was eluted with 100% DCM)

to give 665 mg (64% yield) of 6a as a pale yellow solid. trans-4-

Aminocyclohexanol (475 mg, 4.13 mmol) was added to a solution of

6a (530 mg, 0.826 mmol) in THF (20 mL) and heated at reﬂux

overnight. Then the reaction was cooled to room temperature and the

solvent was removed by rotoevaporation. The crude 6b was puriﬁed by

column chromatography to produce 224 mg of 6b. Cerium

ammonium nitrate (324 mg, 0.592 mmol) was added to a solution

of the resulting 6b (224 mg, 0.296 mmol) in MeCN (6 mL) and

stirred for 5 min at room temperature, then H₂O (6 mL) was added.

After stirring the reaction for an additional 10 min, it was quenched

with saturated sodium bicarbonate, brine was added, and the solution

was extracted with EtOAc. The EtOAc solution was dried over sodium

sulfate, ﬁltered through Celite and the solvent was removed by

rotoevaporation to produce crude 6c as a mixture of diastereisomers.

The crude material was dissolved in a 3:1 mixture of MeOH/H₂O that

was acidiﬁed with TFA and aged. After 2 days this solution was

puriﬁed by preparative HPLC. The combined fractions of the pure

compound were concentrated and then redissolved in a minimum

amount of MeCN, H₂O was added, and the solution was frozen and

lyophilized to produce the TFA salt of 6 (116 mg, 70% yield) as a

white powder. Chemical data were reported previously.²¹

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-

methyl-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-in-

doline]-5′-carboxamide (7). 7 was obtained using the synthetic

method described for the preparation of 6. ¹H NMR (300 MHz,

CD₃OD) δ ppm 8.29 (br s, 1H), 7.63 (t, 1H, J = 7.2 Hz), 7.51 (dd,

1H, J = 2.4, 8.2 Hz), 7.39 (t, 1H, J = 7.6 Hz), 7.21−7.07 (m, 2H), 6.77

(d, 1H, J = 1.5 Hz), 5.13 (d, 1H, J = 10.9 Hz), 4.83 (d, 1H, J = 11.0

Hz), 2.83 (d, 1H, J = 8.3 Hz), 2.73 (s, 3H), 2.17 (d, 1H, J = 15.3 Hz),

2.04−1.68 (m, 5H), 1.52 (q, 1H, J = 14.6 Hz), 1.31−1.09 (m, 2H);

ESI-MS m/z 476.25 (M + 1)⁺.

(3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-meth-

yl-2-oxodispiro[indoline-3,3′-pyrrolidine-2′,4″-piperidine]-5′-

carboxamide (8). Starting with tert-butyl 4-oxopiperidine-1-carbox-

ylate in place of cyclohexanone, compound 8-Boc (compound 8 with

N-Boc piperidine) was prepared according to the synthetic method

described for the preparation of 6. Compound 8-Boc was dissolved in

1

to the synthetic method described for the preparation of 6. H NMR

(300 MHz, CD₃OD) δ ppm 7.63 (t, 1H, J = 7.8 Hz), 7.47 (dd, 1H, J =

2.6, 8.2 Hz), 7.31 (t, 1H, J = 8.3 Hz), 7.15−7.04 (m, 2H), 6.75 (d, 1H,

J = 1.7 Hz), 4.77 (d, 1H, J = 10.1 Hz), 4.61 (d, 0.5H, J = 16.4 Hz,

rotamer), 4.43 (d, 0.5H, J = 11.9 Hz, rotamer), 4.00 (d, 0.5H, J = 13.1

Hz, rotamer), 3.85 (d, 0.5H, J = 12.6 Hz, rotamer), 3.81−3.68 (m,

1H), 2.76 (s, 3H), 2.56−2.40 (m, 1H), 2.16−1.76 (m, 5H), 1.45−1.11

(m, 2H); ESI-MS m/z 519.17 (M + 1)⁺.

(3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-

((1r,4R)-4-hydroxycyclohexyl)-2-oxo-2″,3″,5″,6″-

tetrahydrodispiro[indoline-3,3′-pyrrolidine-2′,4″-pyran]-5′-

carboxamide (11). Starting with tetrahydro-4H-pyran-4-one in place

of cyclohexanone, compound 11 was prepared according to the

1

synthetic method described for the preparation of 6. H NMR (300

MHz, CD₃OD) δ ppm 8.19 (d, J = 7.9 Hz 1H), 7.63 (ddd, 1H, J = 1.5,

6.5, 7.9 Hz), 7.51 (dd, 1H, J = 2.3, 8.2 Hz), 7.37 (t, 1H, J = 8.3 Hz),

7.19−7.07 (m, 2H), 6.80 (d, 1H, J = 1.9 Hz), 5.02 (d, 1H, J = 10.8

Hz), 4.74 (d, 1H, J = 10.8 Hz), 4.11−3.93 (m, 2H), 3.87 (dd, 1H, J =

3.9, 12.4 Hz), 3.69−3.55 (m, 2H), 3.50−3.38 (m, 1H), 2.62 (d, 1H, J

= 13.2 Hz), 2.26−2.12 (m, 1H), 2.04−1.73 (m, 4H), 1.70−1.17 (m,

5H), 1.08 (ddd, 1H, J = 3.5, 12.7, 24.0 Hz); ESI-MS m/z 562.67 (M +

1)⁺.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-4,4-di-

ﬂuoro-N-((1r,4R)-4-hydroxycyclohexyl)-2″-oxodispiro-

[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-carboxamide

(12). Starting with 4,4-diﬂuorocyclohexan-1-one in place of cyclo-

hexanone, compound 12 was prepared according to the synthetic

method described for the preparation of 6. ¹H NMR (300 MHz,

CD₃OD) δ ppm 8.12 (d, 1H, J = 8.1 Hz), 7.62 (t, 1H, J = 7.2 Hz),

7.49 (dd, 1H, J = 2.3, 8.2 Hz), 7.33 (t, 1H, J = 8.3 Hz), 7.16−7.05 (m,

2H), 6.78 (d, 1H, J = 1.9 Hz), 4.77 (d, 1H, J = 10.3 Hz), 3.70−3.41

(m, 2H), 2.74−1.64 (m, 11H), 1.48−1.21 (m, 4H), 1.18−1.02 (m,

1H); ESI-MS m/z 596.75 (M + 1)⁺.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-

((1r,4R)-4-hydroxycyclohexyl)-3,3-dimethyl-2″-oxodispiro-

[cyclobutane-1,2′-pyrrolidine-3′,3″-indoline]-5′-carboxamide

(14). Starting with 3,3-dimethylcyclobutan-1-one in place of cyclo-

hexanone, compound 14 was prepared according to the synthetic

method described for the preparation of 6. ¹H NMR (400 MHz,

CD₃OD) δ ppm 7.61 (dd, 1H, J = 1.9, 8.2 Hz), 7.52 (ddd, 1H, J = 1.5,

6.4, 7.9 Hz), 7.39 (ddd, 1H, J = 1.5, 7.3, 8.6 Hz), 7.18−7−11 (m, 2H),

6.89 (d, 1H, J = 1.9 Hz), 4.92 (d, 1H, J = 10.9 Hz), 4.46 (d, 1H, J =

10.9 Hz), 3.68−3.58 (m, 1H), 3.50−3.39 (m, 1H), 2.78 (dd, 2H, J =

14.5, 39.1 Hz), 2.37 (d, 1H, J = 14.2 Hz), 1.95−1.76 (m, 3H), 1.69−

1.59 (m, 1H), 1.38−1.17 (m, 7H), 0.98 (ddd, 1H, J = 3.6, 12.9, J =

24.3 Hz), 0.54 (s, 3H); ESI-MS m/z 560.25 (M + H)⁺.

N

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(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-

((1r,4R)-4-hydroxycyclohexyl)-4,4-dimethyl-2″-oxodispiro-

[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-carboxamide

(15). Starting with 4,4-dimethylcyclohexan-1-one in place of cyclo-

hexanone, compound 15 was prepared according to the synthetic

method described for the preparation of 6. ¹H NMR (400 MHz,

CD₃OD) δ ppm 7.62 (t, 1H, J = 7.9 Hz), 7.48 (dd, 1H, J = 1.4, 7.8

Hz), 7.35−7.25 (m, 1H), 7.15−7.04 (m, 2H), 6.78 (d, 1H, J = 1.7 Hz),

4.73 (d, 1H, J = 9.9 Hz), 3.67−3.57 (m, 1H), 3.52−3.43 (m, 1H),

2.08−1.64 (m, 8H), 1.58−1.42 (m, 2H), 1.41−1.20 (m, 6H), 0.98 (s,

3H), 0.73 (s, 3H); ESI-MS m/z 588.25 (M + H)⁺.

1H), 4.58 (d, 1H, J = 10.3 Hz), 4.45 (d, 1H, J = 10.4 Hz), 3.68−3.57

(m, 1H), 3.53−3.43 (m, 1H), 2.16 (d, 1H, J = 11.4 Hz), 2.00−1.79

(m, 4H), 1.77−1.46 (m, 7H), 1.40−1.07 (m, 6H); ESI-MS m/z 566.25

(M + H)⁺.

2-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-1′-

methyl-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-in-

dolin]-5′-yl)thiazole-4-carboxylic Acid (21). Ethyl 3-bromo-2-

oxopropanoate (48 mg, 0.244 mmol) and triethylamine (0.033 mL,

0.244 mmol), were added to a solution of 68 (30 mg, 0.061 mmol) in

THF (3 mL) and reﬂuxed overnight. Then the reaction was cooled,

and the solvent was removed. The crude product was redissolved in

DME (3 mL), and to this solution, at −20 °C, was added triﬂuoracetic

anhydride (0.025 mL, 0.183 mmol) and pyridine (0.025 mL, 0.305

mmol), and then the reaction was allowed to warm to rt. After 30 min,

saturated sodium bicarbonate solution was added and the reaction was

extracted with EtOAc. The EtOAc solution was dried over sodium

sulfate, ﬁltered, and puriﬁed by column chromatography to produce 18

mg of compound 69. Lithium hydroxide monohydrate (50 mg, 1.19

mmol) was added to a solution of 69 (18 mg, 0.031 mmol) in THF/

MeOH/H₂O (1:1:1, 3 mL). After 2 h the reaction was quenched with

ammonium chloride, and brine was added and extracted with EtOAc.

The EtOAc solution was dried over sodium sulfate, ﬁltered,

concentrated and the crude was puriﬁed by preparative HPLC to

(3′R,4′R,5′R)-6″-Chloro-4′-(5-chloropyridin-3-yl)-N-((1r,4R)-

4-hydroxycyclohexyl)-2″-oxodispiro[cyclohexane-1,2′-pyrroli-

dine-3′,3″-indoline]-5′-carboxamide (16). Starting with (E)-6-

chloro-3-((5-chloropyridin-3-yl)methylene)indolin-2-one (16b) in

place of (E)-6-chloro-3-(3-chloro-2-ﬂuorobenzylidene)indolin-2-one

(61), compound 16 was prepared according to the synthetic method

1

described for the preparation of 6. H NMR (300 MHz, CD₃OD) δ

ppm 8.45 (d, 1H, J = 2.1 Hz), 8.24 (d, 1H, J = 1.7 Hz), 7.89 (s, 1H),

7.60 (d, 1H, J = 8.2 Hz), 7.14 (dd, 1H, J = 1.8, 8.2 Hz), 6.78 (d, 1H, J

= 1.8 Hz), 5.10 (d, 1H, J = 10.9 Hz), 4.47 (d, 1H, J = 10.9 Hz), 3.73−

3.57 (m, 1H), 3.50−3.36 (m, 1H), 2.83 (d, 1H, J = 12.5 Hz), 2.17 (d,

1H, J = 14.3 Hz), 2.03−1.70 (m, 8H), 1.70−1.13 (m, 7H), 1.08−0.88

(m, 1H); ESI-MS m/z 543.75 (M + H)⁺.

1

produce compound 21 (TFA salt). H NMR (300 MHz, CD₃OD) δ

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-

((1r,4R)-4-hydroxycyclohexyl)-2″-oxo-1″,2″-dihydrodispiro-

[cyclohexane-1,2′-pyrrolidine-3′,3″-pyrrolo[3,2-c]pyridine]-5′-

carboxamide (17). Starting with (E)-6-chloro-3-(3-chloro-2-ﬂuoro-

benzylidene)-1,3-dihydro-2H-pyrrolo[3,2-c]pyridin-2-one (17b) in

place of (E)-6-chloro-3-(3-chloro-2-ﬂuorobenzylidene)indolin-2-one

(61), compound 17 was prepared according to the synthetic method

ppm 8.34 (s, 1H), 7.70 (t, 1H, J = 6.8 Hz), 7.53 (d, 1H, J = 7.1 Hz),

7.27 (t, 1H, J = 7.5 Hz), 7.13−7.03 (m, 2H), 6.73 (d, 1H, J = 1.8 Hz),

5.78−5.59 (m, 1H), 4.81−4.68 (m, 1H), 3.10 (s, 3H), 2.51−2.38 (m,

1H), 2.25 (d, 1H, J = 15.1 Hz), 2.13−1.94 (m, 1H), 1.87−1.10 (m,

7H); ESI-MS m/z 560.33 (M + H)⁺.

General Procedure A: Amide Coupling to Compound 66.

CDI (3 equiv), DIEA (5 equiv), DMAP (1 equiv) were added to a

suspension of compound 66 (1 equiv) in 1,2-dichloroethane and

heated at 50 °C. After 30 min, the respective amine (5 equiv) was

added and the reaction was reﬂuxed overnight. Then the solvent was

removed and the crude product was puriﬁed to produce the

carboxamide.

1

described for the preparation of 6. H NMR (400 MHz, CD₃OD) δ

ppm 8.32 (s, 1H), 7.58 (t, 1H, J = 6.7 Hz), 7.36−7.27 (m, 1H), 7.11

(t, 1H, J = 8.6 Hz), 6.81 (s, 1H) 3.67−3.57 (m, 1H), 3.55−3.45 (m,

1H), 2.01−1.51 (m, 10H), 1.42−1.00 (m, 8H); ESI-MS m/z 561.25

(M + H)⁺.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-

((1r,4R)-4-hydroxycyclohexyl)-2″-oxo-1″,2″-dihydrodispiro-

[cyclohexane-1,2′-pyrrolidine-3′,3″-pyrrolo[2,3-b]pyridine]-5′-

carboxamide (18). Starting with (E)-6-chloro-3-(3-chloro-2-ﬂuoro-

benzylidene)-1,3-dihydro-2H-pyrrolo[2,3-b]pyridin-2-one (18b) in

place of (E)-6-chloro-3-(3-chloro-2-ﬂuorobenzylidene)indolin-2-one

(61), compound 18 was prepared according to the synthetic method

2-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-1′-

methyl-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-in-

dolin]-5′-yl)oxazole-4-carboxylic Acid (22). Compound 72 was

produced according to general procedure A. At −20 °C, Deoxo-Fluor

(50 μL) was added to a solution of 72 (51 mg) in THF (3 mL). After

30 min bromotrichloromethane (500 μL) and DBU (400 μL) were

added and the reaction was allowed to warm to room temperature.

After 5 h the reaction was quenched with saturated sodium

bicarbonate (stirred for 5 min) and then extracted with EtOAc. The

EtOAc solution was dried over sodium sulfate, ﬁltered, and puriﬁed by

column chromatography to produce 34 mg of compound 73. Lithium

hydroxide monohydrate (60 mg, 1.43 mmol) was added to a solution

of 73 (34 mg, 0.061 mmol) in THF/H₂O (1:1, 4 mL). After 2 h the

reaction was quenched with TFA, concentrated and the crude was

redissolved in 3:1 MeOH/H₂O and puriﬁed by HPLC to produce 22

1

described for the preparation of 6. H NMR (300 MHz, CD₃OD) δ

ppm 7.89 (dd, 1H, J = 2.14, 7.84 Hz), 7.61 (t, 1H, J = 7.43 Hz), 7.40

(t, 1H, J = 7.14 Hz), 7.17 (t, 1H, J = 8.40 Hz), 7.12 (d, 1H, J = 7.96

Hz), 5.07−4.94 (m, 1H), 4.78 (d, 1H, J = 10.91 Hz), 3.71−3.57 (m,

1H), 3.51−3.39 (m, 1H), 2.89−2.66 (m, 1H), 2.17−2.02 (m, 1H),

1.99−1.43 (m, 10H), 1.43−1.11 (m, 5H), 1.09−0.93 (m, 1H); ESI-

MS m/z 561.58 (M + H)⁺.

(3′R,4′S,5′R)-2″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-

((1r,4R)-4-hydroxycyclohexyl)-6″-oxo-6″,7″-dihydrodispiro-

[cyclohexane-1,2′-pyrrolidine-3′,5″-pyrrolo[2,3-d]pyrimidine]-

5′-carboxamide (19). Starting with (E)-2-chloro-5-(3-chloro-2-

ﬂuorobenzylidene)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one

(19b) in place of (E)-6-chloro-3-(3-chloro-2-ﬂuorobenzylidene)-

indolin-2-one (61), compound 19 was prepared according to the

1

(TFA salt) as a white powder. H NMR (400 MHz, CD₃OD) δ ppm

8.50 (s, 1H), 7.62−7.53 (m, 2H), 7.24 (t, 1H, J = 7.3 Hz), 7.07 (dd,

1H, J = 1.7, 8.2 Hz), 7.03 (t, 1H, J = 8.1 Hz), 6.73 (d, 1H, J = 1.8 Hz),

5.34 (d, 1H, J = 10.3 Hz), 5.02 (d, 1H, J = 11.6 Hz), 2.97 (s, 3H), 2.33

(d, 1H, J = 13.1 Hz), 2.15 (d, 1H, J = 14.1 Hz), 1.95−1.85 (m, 1H),

1.74−1.44 (m, 5H), 1.38−1.26 (m, 1H), 1.19−1.05 (m, 1H); ESI-MS

m/z 544.42 (M + H)⁺.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-1′-

methyl-5′-(3-methyl-1,2,4-oxadiazol-5-yl)dispiro[cyclohexane-

1,2′-pyrrolidine-3′,3″-indolin]-2″-one (23). Starting with 50 mg of

compound 66, compound 75 was obtained using general procedure A.

Crude 75 was dissolved in pyridine (5 mL) and heated to 100 °C.

After 5 h the solution was cooled and the solvent was removed by

rotoevaporation and then the crude product was puriﬁed by HPLC to

produce 23 (TFA salt) as a white powder. ¹H NMR (400 MHz,

CD₃OD) δ ppm 7.60 (ddd, 1H, J = 1.5, 6.4, 7.9 Hz), 7.50 (dd, 1H, J =

3.0, 8.2 Hz), 7.25 (ddd, 1H, J = 1.6, 7.2, J = 8.6 Hz), 7.07−7.01 (m,

2H), 6.72 (d, 1H, J = 1.9 Hz), 5.30 (d, 1H, J = 11.0 Hz), 4.98 (d, 1H, J

= 11.1 Hz), 2.99 (s, 3H), 2.32 (s, 3H), 2.24 (d, 1H, J = 13.2 Hz),

1

synthetic method described for the preparation of 6. H NMR (400

MHz, CD₃OD) δ ppm 8.55 (d, 1H, J = 2.2 Hz), 7.55 (t, 1H, J = 7.8

Hz), 7.39 (t, 1H, J = 7.0 Hz), 7.16 (t, 1H, J = 7.9 Hz), 3.67−3.56 (m,

1H), 3.53−3.43 (m, 1H), 1.99−1.48 (m, 10H), 1.42−1.04 (m, 8H);

ESI-MS m/z 562.33 (M + H)⁺.

(3′R,4′R,5′R)-2″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-

((1r,4R)-4-hydroxycyclohexyl)-5″-oxo-4″,5″-dihydrodispiro-

[cyclohexane-1,2′-pyrrolidine-3′,6″-thieno[3,2-b]pyrrole]-5′-

carboxamide (20). Starting with (Z)-2-chloro-6-(3-chloro-2-ﬂuoro-

benzylidene)-4,6-dihydro-5H-thieno[3,2-b]pyrrol-5-one (20b) in place

of (E)-6-chloro-3-(3-chloro-2-ﬂuorobenzylidene)indolin-2-one (61),

compound 20 was prepared according to the synthetic method

1

described for the preparation of 6. H NMR (400 MHz, CD₃OD) δ

ppm 7.40 (td, 2H, J = 6.8, 15.1 Hz), 7.15 (t, 1H, J = 8.0 Hz), 6.71 (s,

O

DOI: 10.1021/acs.jmedchem.6b01665

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

2.19−2.10 (m, 1H), 1.86−1.47 (m, 6H), 1.21 (ddd, 1H, J = 5.0, 12.6

Hz, 13.9 Hz), 1.15−1.02 (m, 1H); ESI-MS m/z 514.92 (M + H)⁺.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-1′-

methyl-5′-(5-methyl-1,3,4-oxadiazol-2-yl)dispiro[cyclohexane-

1,2′-pyrrolidine-3′,3″-indolin]-2″-one (24). Starting with 77 mg of

compound 66, 63 mg of 77 was obtained using general procedure A. A

solution of 77 (30 mg, 0.056 mmol) in DCM (3 mL) was slowly

added to a solution of PPh₃(30 mg, 0.112 mmol), Et₃N (31 μL), and

iodine (28 mg, 0.112 mmol) in DCM (1 mL). After 3 h, H₂O and

brine were added and extracted with EtOAc. The EtOAc solution was

dried over sodium sulfate, ﬁltered, concentrated and the crude product

was puriﬁed by HPLC to produce compound 24 (TFA salt) as a white

of acid compound 29 (1 equiv) in DCM. After 10 min, alkylamine (4

equiv) was added. After 12 h, the reaction was concentrated and

puriﬁed by column chromatography to produce alkyl amide

compounds.

General Procedure C: Hydrolysis of Alkyl Esters. LiOH·H₂O

(3 equiv) was added to a solution of ester (1 equiv) in THF/MeOH/

H₂O (1:1:1). After 2 h, TFA was added and the solution was

concentrated, redissolved in MeOH/H₂O (3:1), and puriﬁed by

reverse phase preparative HPLC. The pure fractions were combined

and lyophilized to give the carboxylic acid products (TFA salt) as a

white powder.

General Procedure D: Amide Coupling of Arylamines.

Ph₂POCl (3 equiv) and DIEA (5 equiv) were added to a suspension

of compound 29 (1 equiv) in DCM. After 30 min, arylamine (4 equiv)

and DMAP (1 equiv) were added. After 12 h, the reaction was

concentrated and puriﬁed by column chromatography to produce

arylamide compounds.

General Procedure E: Hydrolysis of Aryl Esters. NaOH (3

equiv) was added to a solution of ester (1 equiv) in THF/MeOH/

H₂O (1:1:1). After 2 h, TFA was added and the reaction was

concentrated, redissolved in MeOH/H₂O (3:1), and puriﬁed by

reverse phase preparative HPLC. The pure fractions were combined

and lyophilized to give the carboxylic acid products as the TFA salt as

a white powder.

1

powder. H NMR (400 MHz, CD₃OD) δ ppm 7.60 (t, 1H, J = 7.9

Hz), 7.51 (dd, 1H, J = 2.9, 8.2 Hz), 7.25 (t, 1H, J = 8.3 Hz), 7.08−7.00

(m, 2H), 6.72 (d, 1H, J = 1.9 Hz), 5.29 (d, 1H, J = 11.1 Hz), 4.95 (d,

1H, J = 10.8 Hz), 2.95 (s, 3H), 2.52 (s, 3H), 2.24 (d, 1H, J = 13.8 Hz),

2.15 (dd, 1H, J = 2.3, 14.1 Hz), 1.86−1.02 (m, 8H); ESI-MS m/z

515.25 (M + H)⁺.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-1′-

methyl-5′-(5-methyl-1,3,4-thiadiazol-2-yl)dispiro[cyclohexane-

1,2′-pyrrolidine-3′,3″-indolin]-2″-one (25). Lawesson’s reagent

(45 mg, 0.112 mmol) was added to a solution of 77 (30 mg, 0.056

mmol) in DCM and reﬂuxed. After standing overnight the solvent was

removed and the crude was puriﬁed by HPLC to produce compound

1

25 (TFA salt) as a white powder. H NMR (400 MHz, CD₃OD) δ

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-

(methylsulfonyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (30). Starting with compound 29

in place of 66, compound 30 was obtained according to general

ppm 7.69 (t, 1H, J = 7.9 Hz), 7.46 (dd, 1H, J = 2.8, 8.2 Hz), 7.25 (t,

1H, J = 8.4 Hz), 7.08 (dt, 1H, J = 1.1, 8.1 Hz), 7.03 (dd, 1H, J = 2.0,

8.2 Hz), 6.72 (d, 1H, J = 1.9 Hz), 5.50 (d, 1H, J = 10.2 Hz), 4.63 (d,

1H, J = 11.0 Hz), 3.00 (s, 3H), 2.71 (s, 3H), 2.27 (d, 1H, J = 13.0 Hz),

2.23−2.15 (m, 1H), 1.88 (t, 1H, J = 11.6 Hz), 1.83−1.60 (m, 4H),

1.60−1.49 (m, 1H), 1.31−1.07 (m, 2H); ESI-MS m/z 531.17 (M +

H)⁺.

1

procedure A. H NMR (300 MHz, CD₃OD) δ ppm 7.61 (t, 1H, J =

7.5 Hz), 7.53 (dd, 1H, J = 2.1, 8.2 Hz), 7.33 (t, 1H, J = 8.0 Hz), 7.17−

7.06 (m, 2H), 6.76 (d, 1H, J = 1.7 Hz), 4.98 (d, 1H, J = 10.4 Hz), 3.09

(s, 3H), 2.60 (d, 1H, J = 13.7 Hz), 2.09 (d, 1H, J = 16.1 Hz), 2.01−

1.43 (m, 6H), 1.34−1.06 (m, 2H); ESI-MS m/z 540.08 (M + 1)⁺.

4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-2″-

oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamido)butanoic Acid (31). 31 was obtained according to

2-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-1′-

methyl-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-in-

dolin]-5′-yl)-5-methyloxazole-4-carboxylic Acid (26). Starting

with 44 mg of compound 66, 25 mg of 79 was obtained using general

procedure A. Cyclization to generate 80 (13 mg) was accomplished

according to the same procedure used to obtain 24. Lithium hydroxide

monohydrate (30 mg, 0.715 mmol) was added to a solution of 80 (13

mg, 0.023 mmol) in THF/H₂O (1:1, 2 mL). After 2 h the reaction was

quenched with TFA, concentrated and the crude was redissolved in

3:1 MeOH/H₂O and puriﬁed by HPLC to produce 26 (TFA salt) as a

1

general procedure B followed by general procedure C. H NMR (300

MHz, CD₃OD) δ ppm 7.64 (t, 1H, J = 7.1 Hz), 7.51 (dd, 1H, J = 2.2,

8.3 Hz), 7.39 (t, 1H, J = 7.5 Hz), 7.17 (t, 1H, J = 8.1 Hz), 7.11 (dd,

1H, J = 1.6, 8.2 Hz), 6.78 (d, 1H, J = 1.6 Hz), 5.12 (d, 1H, J = 11.0

Hz), 4.80 (d, 1H, J = 11.1 Hz), 3.21−3.04 (m, 1H), 2.81 (d, 1H, J =

7.4 Hz), 2.17 (d, 1H, J = 12.0 Hz), 2.06 (t, 2H, J = 7.4 Hz), 2.01−1.84

(m, 3H), 1.84−1.40 (m, 5H), 1.32−1.12 (m, 2H); ESI-MS m/z 548.42

(M + 1)⁺.

(1R,3r)-3-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophen-

yl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-

5′-carboxamido)cyclobutane-1-carboxylic Acid (32). 32 was

obtained by general procedure B followed by general procedure C. ¹H

NMR (300 MHz, CD₃OD) δ ppm 7.65 (t, 1H, J = 7.0 Hz), 7.49 (dd,

1H, J = 1.7, 8.0 Hz), 7.40 (t, 1H, J = 7.3 Hz), 7.18 (t, 1H, J = 8.0 Hz),

7.11 (d, 1H, J = 8.3 Hz), 6.79 (s, 1H), 5.06 (d, J = 10.8, 1H), 4.79 (d,

1H, J = 10.8 Hz), 4.47 (p, 1H, J = 8.1 Hz), 2.96−2.71 (m, 2H), 2.63−

2.38 (m, 2H), 2.29−2.08 (m, 2H), 2.05−1.83 (m, 4H), 1.76 (d, 2H, J

= 16.2 Hz), 1.62−1.39 (m, 1H), 1.33−1.11 (m, 2H) ESI-MS m/z

560.50 (M + 1)⁺.

(1R,4r)-4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophen-

yl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-

5′-carboxamido)cyclohexane-1-carboxylic Acid (33). 33 was

obtained by general procedure B followed by general procedure C. ¹H

NMR (300 MHz, CD₃OD) δ ppm 7.64 (t, 1H, J = 7.1 Hz), 7.49 (d,

1H, J = 8.1 Hz), 7.39 (t, 1H, J = 7.5 Hz), 7.17 (t, 1H, J = 8.1 Hz), 7.11

(d, 1H, J = 8.1 Hz), 6.79 (s, 1H), 5.08 (d, 1H, J = 11.1 Hz), 4.79 (d,

1H, J = 11.1 Hz), 3.72−3.54 (m, 1H), 2.84 (d, 1H, J = 8.4 Hz), 2.25−

2.07 (m, 2H), 2.04−1.84 (m, 6H), 1.77 (d, 2H, J = 12.0 Hz), 1.63 (d,

1H, J = 13.0 Hz), 1.56−1.34 (m, 3H), 1.32−1.11 (m, 3H), 0.99−0.82

(m, 1H); ¹³C NMR (75 MHz, CD₃OD) δ ppm 179.01, 178.21,

167.66, 157.76 (d, J_C−F= 248.87 Hz), 145.27, 137.07, 132.35, 129.50,

128.91, 126.48 (d, J_C−F= 4.74 Hz), 123.39, 122.45, 122.31, 122.05,

121.87, 111.77, 73.24, 67.76, 62.25, 46.72, 46.68, 43.15, 32.33, 32.09,

32.07, 31.37, 28.79, 28.72, 25.35, 23.11, 21.70; ESI-MS m/z 588.33 (M

+ 1)⁺.

1

white powder. H NMR (400 MHz, CD₃OD) δ ppm 7.60 (t, 1H, J =

7.2 Hz), 7.52 (dd, 1H, J = 2.7, 8.1 Hz), 7.21 (t, 1H, J = 8.3 Hz), 7.07−

6.98 (m, 2H), 6.70 (d, 1H, J = 1.9 Hz), 5.07 (d, 1H, J = 10.8 Hz), 4.91

(d, 1H, J = 11.7 Hz), 2.83 (s, 3H), 2.59 (s, 3H), 2.31−2.23 (m, 1H),

2.12−2.04 (m, 1H), 1.85−1.02 (m, 8H); ESI-MS m/z 558.75 (M +

H)⁺.

2-(2-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-

1′-methyl-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-

indolin]-5′-yl)thiazol-4-yl)acetic Acid (27). Compound 27 was

produced using the same synthetic strategy used for 21. ¹H NMR (400

MHz, CD₃OD) δ ppm 7.65 (t, 1H, J = 6.7 Hz), 7.57 (d, 1H, J = 7.1

Hz), 7.42 (s, 1H), 7.27 (t, 1H, J = 8.2 Hz), 7.13−7.04 (m, 2H), 6.75

(d, 1H, J = 1.9 Hz), 5.92−5.70 (m, 1H), 3.78 (s, 2H), 3.20 (s, 3H),

2.58−2.44 (m, 1H), 2.26 (d, 1H, J = 13.8 Hz), 2.22−2.08 (m, 1H),

1.83−1.43 (m, 5H), 1.38−1.27 (m, 1H), 1.25−1.13 (m, 1H); ESI-MS

m/z 574.08 (M + H)⁺.

2-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-1′-

methyl-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-in-

dolin]-5′-yl)thiazole-4-carboxamide (28). Starting with acid 21 in

place of 66, compound 28 was produced using general procedure A.

1

Crude 28 was puriﬁed by HPLC to produce 28 as its TFA salt. H

NMR (400 MHz, CD₃OD) δ ppm 8.11 (s, 1H), 7.75 (t, 1H, J = 7.2

Hz), 7.42 (d, 1H, J = 8.1 Hz), 7.26 (t, 1H, J = 8.46 Hz), 7.09 (t, 1H, J

= 7.83), 7.01 (dd, 1H, J = 1.7, 8.1 Hz), 6.71 (d, 1H, J = 1.8 Hz), 3.00

(s, 3H), 2.36−2.16 (m, 2H), 2.09−2.00 (m, 1H), 1.91−1.52 (m, 5H),

1.38−1.24 (m, 1H), 1.21−1.06 (m, 1H); ESI-MS m/z 559.42 (M +

H)⁺.

General Procedure B: Amide Coupling of Alkylamines.

HATU (2 equiv) and DIEA (4 equiv) were added to a suspension

P

DOI: 10.1021/acs.jmedchem.6b01665

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Journal of Medicinal Chemistry

Article

(1S,4s)-4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophen-

yl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-

5′-carboxamido)cyclohexane-1-carboxylic Acid (34). 34 was

obtained according to general procedure B followed by general

(d, 1H, J = 15.8 Hz), 2.04−1.85 (m, 3H), 1.77 (d, 2H, J = 11.9 Hz),

1.52 (q, 1H, J = 13.7 Hz), 1.33−1.10 (m, 2H); ESI-MS m/z 596.33

(M + 1)⁺.

2-(4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-

2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamido)phenyl)acetic Acid (42). 42 was obtained according

1

procedure C. H NMR (300 MHz, CD3OD) δ ppm 7.67 (t, 1H, J =

6.61 Hz), 7.49 (dd, 1H, J = 2.0, 8.1 Hz), 7.37 (t, 1H, J = 7.5 Hz), 7.16

(t, 1H, J = 7.9 Hz), 7.10 (dd, 1H, J = 1.7, 8.3 Hz), 6.79 (d, 1H, J = 1.7

Hz), 5.20 (d, 1H, J = 11.3 Hz), 4.76 (d, 1H, J = 11.2 Hz), 3.89−3.75

(m, 1H), 2.84 (d, 1H, J = 9.1 Hz), 2.41−2.29 (m, 1H), 2.20 (d, 1H, J =

15.3 Hz), 2.03−1.12 (m, 16H); ESI-MS m/z 588.50 (M + H)⁺.

(1R,4r)-4-(((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuoro-

phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-in-

doline]-5′-carboxamido)methyl)cyclohexane-1-carboxylic

Acid (35). 35 was obtained according to general procedure B followed

by general procedure C. ¹H NMR (300 MHz, CD₃OD) δ ppm 8.36−

8.26 (m, 1H), 7.67 (t, 1H, J = 6.8 Hz), 7.52 (d, 1H, J = 8.2 Hz), 7.42

(t, 1H, J = 7.5 Hz), 7.20 (t, 1H, d J = 8.0 Hz), 7.12 (dd, 1H, J = 1.4, 8.2

Hz), 6.78 (d, 1H, J = 1.5 Hz), 5.14 (d, 1H, J = 11.1 Hz), 4.78 (d, 1H, J

= 11.3 Hz), 3.48−3.34 (m, 1H), 2.90−2.64 (m, 2H), 2.19 (d, 1H, J =

11.3 Hz), 2.09−1.70 (m, 8H), 1.61−1.11 (m, 8H), 0.79−0.59 (m,

2H); ESI-MS m/z 602.58 (M + 1)⁺.

1

to general procedure D followed by general procedure E. H NMR

(400 MHz, CD₃OD) δ ppm 7.70 (t, 1H, J = 7.8 Hz), 7.52 (dd, 1H, J =

2.5, 8.2 Hz), 7.47 (d, 2H, J = 8.6 Hz), 7.35 (t, 1H, J = 8.1 Hz), 7.24 (d,

2H, J = 8.6 Hz), 7.16 (t, 1H, J = 8.0 Hz), 7.10 (dd, 1H, J = 1.9, 8.2

Hz), 6.78 (d, 1H, J = 1.9 Hz), 5.20 (d, 1H, J = 9.8 Hz), 4.93 (d, 1H, J

= 10.8 Hz), 3.57 (s, 2H), 2.87−2.69 (m, 1H), 2.13 (d, 1H, J = 13.3

Hz), 1.99−1.70 (m, 5H), 1.64−1.48 (m, 1H), 1.27−1.12 (m, 2H);

ESI-MS m/z 596.42 (M + H)⁺.

4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-2″-

oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamido)-2-methoxybenzoic Acid (43). 43 was obtained

according to general procedure D followed by general procedure E. ¹H

NMR (300 MHz, CD₃OD) δ ppm 7.83 (d, 1H, J = 8.51 Hz), 7.70 (t,

1H, J = 6.92 Hz), 7.62 (s, 1H), 7.52 (dd, 1H, J = 2.17, 8.08 Hz), 7.35

(t, 1H, J = 7.08 Hz), 7.20−7.02 (m, 3H), 6.78 (d, 1H, J = 1.71 Hz),

5.24−5.09 (m, 1H), 4.94 (d, 1H, J = 10.49 Hz), 3.89 (s, 3H), 2.80−

2.58 (m, 1H), 2.09 (d, 1H, J = 14.12 Hz), 2.01−1.47 (m, 6H), 1.26−

1.09 (m, 2H); ESI-MS m/z 612.42 (M + H)⁺.

4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-2″-

oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamido)-3-methoxybenzoic Acid (44). 44 was obtained

according to general procedure D followed by general procedure E. ¹H

NMR (300 MHz, CD₃OD) δ ppm 8.26 (d, 1H, J = 8.6 Hz), 7.79 (t,

1H, J = 7.1 Hz), 7.66 (dd, 1H, J = 1.4, 8.4 Hz), 7.60 (s, 1H), 7.51 (dd,

1H, J = 2.2, 8.3 Hz), 7.40 (t, 1H, J = 8.2 Hz), 7.21 (t, 1H, J = 8.6 Hz),

7.10 (dd, 1H, J = 1.8, 8.1 Hz), 6.79 (d, 1H, J = 1.7 Hz), 5.49−5.23 (m,

1H), 3.80 (s, 3H), 2.68−2.47 (m, 1H), 2.21−1.52 (m, 7H), 1.35−1.07

(m, 2H); ESI-MS m/z 612.17 (M + 1)⁺.

4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-2″-

oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamido)-2-ﬂuorobenzoic Acid (45). 45 was obtained

according to general procedure D followed by general procedure E.

¹H NMR (300 MHz, CD₃OD) δ ppm 7.90 (t, 1H, J = 8.4 Hz), 7.74−

7.61 (m, 2H), 7.55 (dd, 1H, J = 2.5, 8.2 Hz), 7.38 (t, 1H, J = 7.2 Hz),

7.26 (dd, 1H, J = 1.7, 8.6 Hz), 7.19 (t, 1H, J = 8.2 Hz), 7.12 (dd, 1H, J

= 1.8, 8.2 Hz), 6.80 (d, 1H, J = 1.7 Hz), 5.31 (d, 1H, J = 10.8 Hz), 4.97

(d, 1H, J = 10.8 Hz), 2.94−2.84 (m, 1H), 2.20 (d, 1H, J = 15.7 Hz),

2.06−1.85 (m, 3H), 1.79 (d, 2H, J = 10.9 Hz), 1.65−1.43 (m, 1H),

1.34−1.11 (m, 2H); ESI-MS m/z 600.42 (M + H)⁺.

4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-2″-

oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamido)-3-ﬂuorobenzoic Acid (46). 46 was obtained

according to general procedure D followed by general procedure E.

¹H NMR (300 MHz, CD₃OD) δ ppm 8.21 (t, 1H, J = 8.0 Hz), 7.91−

7.79 (m, 1H), 7.79−7.67 (m, 2H), 7.52 (dd, 1H, J = 2.4, 8.2 Hz), 7.37

(t, 1H, J = 7.3 Hz), 7.25−7.06 (m, 2H), 6.79 (d, 1H, J = 1.7 Hz), 5.41

(d, 1H, J = 9.4 Hz), 2.81−2.67 (m, 1H), 2.14 (d, 1H, J = 14.7 Hz),

2.00−1.84 (m, 3H), 1.84−1.70 (m, 2H), 1.68−1.47 (m, 1H), 1.41−

1.09 (m, 2H); ESI-MS m/z 600.83 (M + H)⁺.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(1,1-

dioxidotetrahydro-2H-thiopyran-4-yl)-2″-oxodispiro-

[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-carboxamide

1

(36). 36 was obtained according to general procedure B. H NMR

(300 MHz, CD₃OD) δ ppm 7.64 (t, 1H, J = 7.1 Hz), 7.49 (dd, 1H, J =

2.1, 8.1 Hz), 7.41 (t, 1H, J = 7.5 Hz), 7.18 (t, 1H, J = 9.9 Hz), 7.11

(dd, 1H, J = 1.6, 8.2 Hz), 6.79 (d, 1H, J = 1.6 Hz), 5.10 (d, 1H, J =

11.1 Hz), 4.80 (d, 1H, J = 11.2 Hz), 4.10−3.94 (m, 1H), 3.27−2.91

(m, 3H), 2.88−2.74 (m, 2H), 2.35−1.64 (m, 10H), 1.52 (q, 1H, J =

13.9 Hz), 1.31−1.11 (m, 2H); ESI-MS m/z 594.50 (M + 1)⁺.

2-(4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-

2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamido)piperidin-1-yl)acetic Acid (37). 37 was obtained

according to general procedure B followed by general procedure C. ¹H

NMR (300 MHz, CD₃OD) δ ppm 7.66 (t, 1H, J = 7.1 Hz), 7.48 (dd,

1H, J = 2.1, 8.2 Hz), 7.39 (t, 1H, J = 7.3 Hz), 7.17 (t, 1H, J = 8.0 Hz),

7.10 (dd, 1H, J = 1.6, 8.1 Hz), 6.79 (d, 1H, J = 1.7 Hz), 5.12 (d, 1H, J

= 11.0 Hz), 4.80 (d, 1H, J = 10.9 Hz), 4.05−3.89 (m, 2H), 3.79−3.39

(m, 2H), 3.29−3.04 (m, 2H), 2.79 (d, 1H, J = 9.4 Hz), 2.17 (d, 2H, J =

9.4 Hz), 2.03−1.42 (m, 8H), 1.42−1.33 (m, 2H), 1.31−1.10 (m, 2H);

ESI-MS m/z 603.67 (M + 1)⁺.

(3′R,4′S,5′R)-N-(1-Acetylazetidin-3-yl)-6″-chloro-4′-(3-

chloro-2-ﬂuorophenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrro-

lidine-3′,3″-indoline]-5′-carboxamide (38). 38 was obtained

1

according to general procedure B. H NMR (300 MHz, CD₃OD) δ

ppm 7.64 (t, 1H, J = 7.2 Hz), 7.50 (dd, 1H, J = 1.8, 8.2 Hz), 7.40 (t,

1H, J = 7.7 Hz), 7.17 (t, 1H, J = 8.1 Hz), 7.11 (dd, 1H, J = 1.7, 8.2

Hz), 6.79 (s, 1H), 5.10 (d, 1H, J = 10.6 Hz), 4.64−4.36 (m, 2H),

4.29−4.11 (m, 1H), 4.09−3.56 (m, 2H), 2.79 (d, 1H, J = 9.8 Hz), 2.16

(d, 1H, J = 14.9 Hz), 2.01−1.68 (m, 8H), 1.64−1.37 (m, 1H), 1.34−

1.11 (m, 2H); ESI-MS m/z 560.08 (M + 1)⁺.

3-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-2″-

oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamido)benzoic Acid (40). 40 was obtained according to

general procedure D followed by general procedure E. ¹H NMR (300

MHz, CD3OD) δ ppm 8.14 (s, 1H), 7.84−7.67 (m, 3H), 7.55 (dd,

1H, J = 1.9, 8.2 Hz), 7.48−7.34 (m, 2H), 7.19 (t, 1H, J = 8.0 Hz), 7.12

(dd, 1H, J = 1.6, 8.2 Hz), 6.79 (d, 1H, J = 1.6 Hz), 5.29 (d, 1H, J =

11.0 Hz), 4.97 (d, 1H, J = 10.7 Hz), 2.96−2.84 (m, 1H), 2.18 (d, J =

14.0 Hz 1H), 2.08−1.85 (m, 3H), 1.78 (d, 2H, J = 12.2 Hz), 1.63−

1.42 (m, 1H), 1.35−1.13 (m, 2H); ESI-MS m/z 582.58 (M + H)⁺.

4-(((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-2″-

oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamido)methyl)benzoic Acid (41). 41 was obtained

according to general procedure B followed by general procedure E.

¹H NMR (300 MHz, CD₃OD) δ ppm 7.87 (d, 2H, J = 8.2 Hz), 7.66

5-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-2″-

oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamido)picolinic Acid (47). 47 was obtained according to

1

general procedure D followed by general procedure E. H NMR (300

MHz, CD₃OD) δ ppm 8.80 (s, 1H), 8.27 (d, 1H, J = 8.8 Hz), 8.15 (d,

1H, J = 8.7 Hz), 7.71 (t, 1H, J = 7.2 Hz), 7.55 (d, 1H, J = 8.7 Hz), 7.37

(t, 1H, J = 7.1 Hz), 7.18 (t, 1H, J = 7.9 Hz), 7.12 (d, 1H, J = 8.1 Hz),

6.80 (s, 1H), 5.30 (d, 1H, J = 11.1 Hz), 5.00 (d, 1H, J = 10.8 Hz),

2.90−2.75 (m, 1H), 2.16 (d, 1H, J = 16.9 Hz), 2.06−1.84 (m, 3H),

1.78 (d, 2H, J = 13.2 Hz), 1.66−1.42 (m, 1H), 1.32−1.11 (m, 2H);

ESI-MS m/z 583.96 (M + 1)⁺.

5-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-2″-

oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamido)furan-2-carboxylic Acid (48). 48 was obtained

according to general procedure D followed by general procedure E.

¹H NMR (300 MHz, CD₃OD) δ ppm 7.66 (t, 1H, J = 7.23 Hz), 7.49

(t, 1H, J = 7.0 Hz), 7.50 (dd, 1H, J = 2.3, 8.3 Hz), 7.40 (t, 1H, J = 7.6

Hz), 7.22−7.08 (m, 2H), 7.04 (d, 2H, J = 8.2 Hz), 6.78 (d, 1H, J = 1.6

Hz), 5.20 (d, 1H, J = 11.2 Hz), 4.80 (d, 1H, J = 11.1 Hz), 4.66 (d, 1H,

J = 15.3 Hz), 4.20 (d, 1H, J = 15.3 Hz), 2.83 (d, 1H, J = 10.0 Hz), 2.20

Q

DOI: 10.1021/acs.jmedchem.6b01665

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(dd, 1H, J = 2.46, 8.24 Hz), 7.31 (t, 1H, J = 7.44 Hz), 7.22 (d, 1H, J =

3.60 Hz), 7.16−7.04 (m, 2H), 6.76 (d, 1H, J = 1.68 Hz), 6.51 (d, 1H, J

= 3.53 Hz), 5.06−4.90 (m, 2H), 2.53−2.37 (m, 1H), 2.06−1.50 (m,

7H), 1.25−1.00 (m, 2H); ESI-MS m/z 572.42 (M + H)⁺.

68.33, 62.99, 46.84, 35.75, 34.45, 32.21, 30.00, 28.07, 27.96, 23.63;

ESI-MS m/z 610.25 (M + H)⁺.

3-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-2″-

oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamido)bicyclo[1.1.1]pentane-1-carboxylic Acid (55). 55

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-

((methylsulfonyl)carbamoyl)phenyl)-2″-oxodispiro-

[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-carboxamide

(49). Starting with compound 39 in place of 66, compound 49 was

obtained according to general procedure A. ¹H NMR (300 MHz,

CD₃OD) δ ppm 7.89 (d, 2H, J = 8.8 Hz), 7.78−7.66 (m, 3H), 7.52

(dd, 1H, J = 2.5, 8.4 Hz), 7.36 (t, 1H, J = 7.8 Hz), 7.17 (t, 1H, J = 7.6

Hz), 7.11 (dd, 1H, J = 1.7, 8.2 Hz), 6.79 (d, 1H, J = 1.7 Hz), 5.31−

5.15 (m, 1H), 3.35 (s, 3H), 2.89−2.70 (m, 1H), 2.13 (d, 1H, J = 13.6

Hz), 2.03−1.70 (m, 5H), 1.68−1.46 (m, 1H), 1.35−1.12 (m, 2H);

ESI-MS m/z 659.67 (M + H)⁺.

1

was obtained using the same synthetic strategy described for 56. H

NMR (300 MHz, CD₃OD) δ ppm 7.61 (t, 1H, J = 6.55 Hz), 7.49 (dd,

1H, J = 2.34, 8.20 Hz), 7.39 (t, 1H, J = 6.90 Hz), 7.15 (t, 1H, J = 8.53

Hz), 7.10 (dd, 1H, J = 1.94, 8.22 Hz), 6.78 (d, 1H, J = 1.88 Hz), 4.98

(d, 1H, J = 10.87 Hz), 4.78 (d, 1H, J = 10.92 Hz), 2.84−2.71 (m, 1H),

2.26 (s, 6H), 2.14 (d, 1H, J = 13.90 Hz), 2.02−1.67 (m, 5H), 1.60−

1.38 (m, 1H), 1.31−1.10 (m, 2H); ESI-MS m/z 572.25 (M + H)⁺.

4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-2″-

oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamido)bicyclo[2.2.2]octane-1-carboxylic Acid (56).

HATU (616 mg, 1.62 mmol) and DIEA (0.550 mL, 3.24 mmol)

were added to a suspension of acid 29 (500 mg, 1.08 mmol) in DCM

(15 mL) and stirred. After 10 min, methyl 4-aminobicyclo[2.2.2]-

octane-1-carboxylate (396 mg, 2.16 mmol) and DMAP (132 mg, 1.08

mmol) were added to the reaction. After overnight, the solvent was

removed in vacuo and the crude was puriﬁed by column

chromatography to give 549 mg of interemediate 56-ester. LiOH·

H₂O (110 mg, 2.62 mmol) and sodium hydroxide (105 mg, 2.62

mmol) were added to a solution of interemidiate 56-ester (549 mg,

0.873 mmol) dissolved in a mixture of THF (3 mL), H₂O (3 mL), and

MeOH (3 mL). After the hydrolysis was complete, as determined by

TLC, the reaction was quenched with TFA (3 mL) and stirred. After 5

min, the solution was concentrated in vacuo (not to dryness) and the

resulting oil was redissolved in CH₃CN and H₂O (1:1) and the

solution was puriﬁed by preparative HPLC. The puriﬁed fractions

were combined, concentrated in vacuo, redissolved in H₂O, frozen,

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-2″-oxo-

N-(4-sulfamoylphenyl)dispiro- [cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (50). 50 was obtained according

to general procedure D. ¹H NMR (300 MHz, CD₃OD) δ ppm 7.60−

7.50 (m, 3H), 7.39 (dd, 1H, J = 2.24, 8.22 Hz), 7.30 (t, 1H, J = 6.96

Hz), 7.12−7.02 (m, 2H), 6.73 (d, 1H, J = 1.59 Hz), 6.64 (d, 2H, J =

8.68 Hz), 4.80 (d, 1H, J = 10.19 Hz), 4.62 (d, 1H, J = 10.28 Hz), 2.24

(d, 1H, J = 13.09 Hz), 1.94 (d, 1H, J = 13.01 Hz), 1.88−1.41 (m, 6H),

1.20−0.93 (m, 2H); ESI-MS m/z 617.17 (M + H)⁺.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-

(methylsulfonyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyr-

rolidine-3′,3″-indoline]-5′-carboxamide (51). 51 was obtained

1

according to general procedure D. H NMR (300 MHz, CD₃OD) δ

ppm 7.91 (d, 2H, J = 8.80 Hz), 7.82 (d, 2H, J = 8.85 Hz), 7.70 (t, 1H, J

= 6.71 Hz), 7.52 (dd, 1H, J = 2.42, 8.17 Hz), 7.35 (t, 1H, J = 7.58 Hz),

7.16 (t, 1H, J = 8.05 Hz), 7.10 (dd, 1H, J = 1.84, 8.21 Hz), 6.78 (d,

1H, J = 1.80 Hz), 5.20 (d, 1H, J = 14.03 Hz), 4.94 (d, 1H, J = 10.48

Hz), 3.09 (s, 3H), 2.80−2.63 (m, 1H), 2.11 (d, 1H, J = 13.18 Hz),

2.01−1.46 (m, 6H), 1.31−1.08 (m, 2H); ESI-MS m/z 616.58 (M +

H)⁺.

1

and lyophilized to give 56 (TFA salt) as a white powder. H NMR

(300 MHz, CD₃OD) δ ppm 7.63 (t, 1H, J = 6.84 Hz), 7.45 (d, 1H, J =

6.76 Hz), 7.35 (t, 1H, J = 7.21 Hz), 7.18−7.04 (m, 2H), 6.77 (dd, 1H,

J = 1.26 Hz), 4.68 (d, 1H, J = 10.61 Hz), 2.73−2.48 (m, 1H), 2.16−

1.98 (m, 1H), 1.98−1.43 (m, 18H), 1.27−1.02 (m, 2H); ¹³C NMR

(3′R,4′S,5′R)-N-(4-Carbamoylphenyl)-6″-chloro-4′-(3-chloro-

2-ﬂuorophenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-

3′,3″-indoline]-5′-carboxamide (52). Starting with compound 39

in place of 66, compound 52 was obtained according to general

(100 MHz, CD₃OD) δ ppm 180.79, 178.22, 167.86, 157.78 (d, J_C−F

248.9 Hz), 145.23, 137.07, 132.32, 129.39, 128.99, 126.45 (d, J_C−F

=

4.9 Hz), 123.39, 122.50, 122.17, 122.15 (d, J_C−F= 19.3 Hz), 111.77,

73.22, 67.72, 62.58, 52.82, 46.96 (d, J_C−F= 3.5 Hz), 38.96, 32.17,

31.36, 30.41(3C), 29.45(3C), 25.35, 23.08, 21.74; ESI-MS m/z 614.92

(M + H)⁺.

1

procedure A. H NMR (400 MHz, CD₃OD) δ ppm 7.85 (d, 2H, J =

8.9,Hz), 7.70 (t, 1H, J = 7.9 Hz), 7.65 (d, 2H, J = 8.8 Hz), 7.51 (dd,

1H, J = 2.4, 8.2 Hz), 7.34 (t, 1H, J = 7.1 Hz), 7.15 (t, 1H, J = 8.0 Hz),

7.09 (dd, 1H, J = 1.9, 8.2 Hz), 6.78 (d, 1H, J = 1.9 Hz), 5.22−5.10 (m,

1H), 4.93 (d, 1H, J = 9.6 Hz), 2.81−2.58 (m, 1H), 2.09 (d, 1H, J =

12.4 Hz), 1.99−1.67 (m, 5H), 1.67−1.50 (m, 1H), 1.28−1.10 (m,

2H); ESI-MS m/z 581.33 (M + H)⁺.

4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-4,4-

diﬂuoro-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-in-

doline]-5′-carboxamido)bicyclo[2.2.2]octane-1-carboxylic

Acid (57). 57 was obtained using the same synthetic strategy

1

(3′R,4′S,5′R)-N-(4-Carbamoyl-2-methoxyphenyl)-6″-chloro-

4′-(3-chloro-2-ﬂuorophenyl)-2″-oxodispiro[cyclohexane-1,2′-

pyrrolidine-3′,3″-indoline]-5′-carboxamide (53). Starting with

compound 44 in place of 66, compound 53 was obtained according to

described for 56. H NMR (300 MHz, CD₃OD) δ ppm 7.71 (s, 1H),

7.63 (t, 1H, J = 6.61 Hz), 7.50 (dd, 1H, J = 2.08, 8.18 Hz), 7.36 (t, 1H,

J = 7.54 Hz), 7.18−7.05 (m, 2H), 6.79 (d, 1H, J = 1.83 Hz) 4.96 (d,

1H, J = 10.48 Hz), 4.71 (d, 1H, J = 10.51 Hz), 2.78 (d, 1H, J = 14.25

Hz), 2.59−1.91 (m, 6H), 1.91−1.70 (m, 12H), 1.53−1.33 (m, 1H);

ESI-MS m/z 650.92 (M + H)⁺.

1

general procedure A. H NMR (400 MHz, CD₃OD) δ ppm 8.24 (d,

1H, J = 8.4 Hz), 7.75 (t, 1H, J = 7.0 Hz), 7.55−7.45 (m, 3H), 7.35 (t,

1H, J = 7.0 Hz), 7.15 (t, 1H, J = 7.9 Hz), 7.06 (dd, 1H, J = 1.9, 8.2

Hz), 6.76 (d, 1H, J = 1.9 Hz), 5.34−5.10 (m, 1H), 3.85 (s, 3H), 2.57−

2.31 (m, 1H), 2.18−1.96 (m, 2H), 1.91−1.57 (m, 5H), 1.25−1.05 (m,

2H); ESI-MS m/z 611.25 (M + H)⁺.

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-N-(4-

((methylsulfonyl)carbamoyl)-bicyclo[2.2.2]octan-1-yl)-2″-

oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-

carboxamide (58). CDI (49 mg, 0.303 mmol), DIEA (88 μL, 0.505

mmol), and DMAP (cat.) were added to a solution of 56 (62 mg,

0.101 mmol) in 1,2-dichloroethane (10 mL), and the reaction was

heated to 40 °C. After 20 min, methanesulfonamide (96 mg, 1.01

mmol) was added and the reaction was reﬂuxed. After overnight, the

sovent was removed in vacuo and the crude was puriﬁed by prepartive

4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-4,4-

dimethyl-2″-oxodispiro[cyclo- hexane-1,2'-pyrrolidine-3′,3″-

indoline]-5'-carboxamido)benzoic Acid (54). 54 was obtained

according to general procedure D followed by general procedure E. ¹H

NMR (400 MHz, CD₃OD) δ ppm 7.98 (d, 2H, J = 8.7 Hz), 7.70 (t,

1H, J = 7.7 Hz), 7.64 (d, 2H, J = 8.7 Hz), 7.57 (dd, 1H, J = 2.4, 8.2

Hz), 7.38 (t, 1H, J = 8.2 Hz), 7.19 (t, 1H, J = 8.1 Hz), 7.13 (dd, 1H, J

= 1.9, 8.2 Hz), 6.81 (d, 1H, J = 1.9 Hz), 5.29 (d, 1H, J = 10.5 Hz), 4.97

(d, 1H, J = 10.8 Hz), 2.75 (d, 1H, J = 13.6 Hz), 2.13 (dt, 1H, J = 4.2,

14.0 Hz), 2.04 (d, 1H, J = 13.4 Hz), 1.87 (dt, 1H, J = 3.4, 14.2 Hz),

1.64 (d, 1H, J = 14.7 Hz), 1.53−1.34 (m, 3H), 1.02 (s, 3H), 0.77 (s,

3H); ¹³C NMR (100 MHz, CD₃OD) δ ppm 178.48, 169.09, 168.51,

157.71 (d, J_C−F= 249.3 Hz), 145.30, 142.77, 136.94, 132.18,

131.88(2C), 129.50, 128.91, 128.25, 126.32 (d, J_C−F= 4.6 Hz),

123.29, 122.77, 122.28 (d, J_C−F= 19.1 Hz), 120.51(2C), 111.75, 72.59,

1

HPLC to give 58 (TFA salt) as a white solid. H NMR (300 MHz,

CD₃OD) δ ppm 7.64 (t, 1H, J = 7.23 Hz), 7.45 (dd, 1H, J = 1.93, 8.22

Hz), 7.36 (t, 1H, J = 7.23 Hz), 7.18−7.04 (m, 2H), 6.77 (d, 1H, J =

1.66 Hz), 4.69 (d, 1H, J = 10.70 Hz), 3.19 (s, 3H), 2.75−2.52 (m,

1H), 2.21−1.99 (m, 1H), 1.99−1.44 (m, 17H), 1.41−1.27 (m, 1H),

1.27−1.03 (m, 2H); ESI-MS m/z 691.42 (M + H)⁺.

4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-1′-

methyl-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-in-

doline]-5′-carboxamido)bicyclo[2.2.2]octane-1-carboxylic

Acid (59). Paraformaldehyde (15 mg, 0.506 mmol) was added to a

R

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solution of compound 56 (20 mg, 0.028 mmol) dissolved in AcOH (1

mL). After 15 min sodium triacetoxyborohydride (59 mg, 0.28 mmol)

was added, and after reacting overnight, the reaction was quenched

with saturated ammonium chloride solution and extracted with EtOAc.

The EtOAc solvent was evaporated in vacuo, and the resulting oil was

redissolved in a solution of MeCN and H₂O (1:1 with 0.1% TFA) and

puriﬁed by preparative HPLC. The pure 59 fractions were combined,

concentrate in vacuo, redissolved in H₂O (with minimum amount of

MeCN), frozen, and lyophilized to give 59 as the TFA salt as a white

Fluorescence Polarization-Based (FP-Based) Protein Binding

Assay, Cell Growth Inhibition Assay, Pharmacodynamics (PD)

Study in SJSA-1 Xenograft Model, and in Vivo Eﬃcacy Study.

These assays were performed according to the previously reported

procedures.²¹All the in vivo studies were performed under an animal

protocol (PRO00005315) approved by the University Committee on

Use and Care of Animals of the University of Michigan, in accordance

with the recommendations in the Guide for the Care and Use of

Laboratory Animals of the National Institutes of Health.

Computational Methods. Docking studies were performed using

our previously reported structure of MDM2 in complex with 2 (PDB

code 5TRF)¹⁸with the GOLD^33,34program (version 5.2). For each

genetic algorithm (GA) run, a maximum number of 200 000

operations were performed on a population of ﬁve islands of 100

individuals. Operator weights for crossover, mutation, and migration

were set to 95, 95, and 10, respectively, and the Chemscore³⁵ﬁtness

function was used to evaluate the docked conformations. Docking was

terminated after 10 runs for each ligand, and the top 10 conformations

were saved for analysis of the predicted binding modes.

1

powder. H NMR (300 MHz, CD₃OD) δ ppm 7.94 (s, 1H), 7.61−

7.52 (m, 2H), 7.40 (t, 1H, J = 7.32 Hz), 7.19−7.08 (m, 2H), 6.78 (d,

1H, J = 1.56 Hz), 4.99 (d, 1H, J = 11.86 Hz), 4.63 (d, 1H, J = 12.06

Hz), 3.27 (s, 3H), 2.61−2.48 (m, 1H), 2.32−2.14 (m, 2H), 1.88−1.40

(m, 18H), 1.37−1.12 (m, 1H); ¹³C NMR (100 MHz, CD₃OD) δ ppm

180.74, 178.21, 166.55, 157.74 (d, J_C−F= 249.6 Hz), 144.90, 137.11,

132.39, 129.80, 129.09, 126.30 (d, J_C−F= 4.7 Hz), 124.00, 123.43,

122.31 (d, J_C−F= 19.2 Hz), 121.63 (d, J_C−F= 13.6 Hz), 111.77, 77.98,

73.06, 67.67, 52.98, 46.56, 40.34, 38.97, 34.28, 30.38(3C), 29.44(3C),

28.97, 24.95, 23.57, 22.07; ESI-MS m/z 628.83 (M + H)⁺.

4-((3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-1′-

ethyl-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indo-

line]-5′-carboxamido)bicyclo[2.2.2]octane-1-carboxylic Acid

(60). 60 was obtained using the same synthetic strategy described

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.6b01665.

■

S

1

for 59. H NMR (300 MHz, CD₃OD) δ ppm 7.63 (t, 1H, J = 7.04

Hz), 7.56−7.48 (m, 2H), 7.42 (t, 1H, J = 7.39 Hz), 7.18 (t, 1H, J =

7.96 Hz), 7.10 (d, 1H, J = 8.06 Hz), 6.79 (s, 1H), 5.08−4.96 (m, 1H),

4.57 (d, 1H, J = 11.85 Hz), 4.18−3.99 (m, 1H), 3.87−3.69 (m, 1H),

2.70−2.54 (m, 1H), 2.36−2.13 (m, 2H), 1.94−1.45 (m, 18H), 1.39 (t,

3H, J = 6.65 Hz), 1.32−1.14 (m, 1H); ¹³C NMR (100 MHz, CD₃OD)

δ ppm 180.95, 180.06, 177.01, 157.71 (d, J_C−F= 247.1 Hz), 144.91,

135.39, 130.37, 129.74 (d, J_C−F= 1.6 Hz), 129.34 (d, J_C−F= 2.5 Hz),

126.86 (d, J_C−F= 12.5 Hz), 126.47, 125.46 (d, J_C−F= 4.6 Hz), 122.27,

121.50 (d, J_C−F= 19.7 Hz), 110.69, 74.84, 72.58, 71.05, 51.51, 48.09,

47.36, 39.17, 37.56, 30.84(3C), 29.61(3C), 29.25, 26.37, 24.25, 23.14,

17.04; ESI-MS m/z 642.50 (M + H)⁺.

Molecular formula strings and some data (CSV)

Structure representation (PDB)

AUTHOR INFORMATION

Corresponding Author

*E-mail: Shaomeng@umich.edu.

ORCID

■

Ding Du: 0000-0002-4615-5433

Shaomeng Wang: 0000-0002-8782-6950

Notes

(3′R,4′S,5′R)-6″-Chloro-4′-(3-chloro-2-ﬂuorophenyl)-1′-

methyl-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-in-

doline]-5′-carbothioamide (68). Intermediate 64²¹(1.0 g, 2.09

mmol) was dissolved in a mixture of DCM (10 mL) and AcOH (10

mL). Paraformaldehyde (1.13 g, 37.62 mmol) was added, and after 10

min NaBH(OAc)₃(4.43 g, 20.9 mmol) was added in portions. After 4

h, as determined by TLC, the reaction was complete. Saturated NH₄Cl

(aq) was added to the reaction which was then extracted with EtOAc.

The EtOAc solution was washed with NaOH (1 M) and brine, then

dried over Na₂SO₄, ﬁltered, concentrated, and puriﬁed by column

chromatography to give 764 mg of intermediate 65. Sodium hydroxide

(320 mg, 8.0 mmol) was added to a solution of intermediate 65 (764

mg, 1.6 mmol) dissolved in a mixture of THF (3 mL), H₂O (3 mL),

and MeOH (3 mL), and the solution was heated to 45−50 °C. After

hydrolysis was complete, as determined by TLC, the reaction was

cooled, neutralized with 2 M HCl, and extracted with EtOAc. The

EtOAc solution was dried over Na₂SO₄, ﬁltered, and concentrated to

give the acid intermediate 66 as a solid that was used without further

puriﬁcation. CDI (315 mg, 1.94 mmol) and DIEA (0.450 mL, 2.59

mmol) were added to a solution of 66 (300 mg, 0.648 mmol) in THF

(15 mL) and heated to 50 °C. After 30 min ammonium hydroxide was

was added. After standing overnight, H₂O was added and the reaction

was extracted with EtOAc. The EtOAc solution was dried over sodium

sulfate, ﬁltered, concentrated, and puriﬁed by column chromatography

to give 240 mg of compound 67. Lawesson’s reagent (102 mg, 0.252

mmol) was added to a solution of compound 67 (60 mg, 0.126 mmol)

in DCM (3 mL) and reﬂuxed overnight. Then the reaction was cooled,

the solvent was removed, and the crude was column chromatographed

The authors declare the following competing ﬁnancial

interest(s): The University of Michigan has ﬁled a patent on

AA-115 and its analogues, and Shaomeng Wang, Angelo

Aguilar, Jianfeng Lu, Liu Liu, and Donna McEachern are co-

inventors of the patent. The patent application covering AA-

115 and its analogues has been licensed by Ascentage Pharma

Group for clinical development in which Shaomeng Wang is a

co-founder, owns stock, and serves as a paid consultant.

Shaomeng Wang and the University of Michigan also receive a

research grant from Ascentage Pharma Group.

ACKNOWLEDGMENTS

■

This study is supported in part by grants from the National

Institutes of Health (Grant R01CA121279 to S.W.), the

National Cancer Institute Cancer Center Core grant

(P30CA046592) to the University of Michigan Comprehensive

Cancer Center, a grant from Ascentage Pharma Group to the

University of Michigan (to S.W.), the Key New Drug Creation

project of the major science and technology program (Grant

2012ZX09401005, China), National “863” Grant

(2012AA020305, China), and Science & Technology Innova-

tion Team Grant of Guangdong Province (Grant 2013Y114) to

Jiangsu Ascentage Biomed Development Inc.

1

to produce 60 mg of 68. H NMR (400 MHz, CD₃OD) δ ppm 7.66

(ddd, 1H, J = 1.4, 6.4, 7.9 Hz), 7.53 (dd, 1H, J = 2.5, 8.3 Hz), 7.38 (t,

1H, J = 7.3 Hz), 7.15 (t, 1H, J = 8.0 Hz), 7.10 (dd, 1H, J = 1.9, 8.2

Hz), 6.77 (d, 1H, J = 1.9 Hz), 5.48 (br s, 1H), 4.71 (d, 1H, J = 11.2

Hz), 3.37 (s, 3H), 2.38 (d, 1H, J = 14.2 Hz), 2.19−2.02 (m, 1H),

1.99−1.85 (m, 1H), 1.83−1.61 (m, 4H), 1.50−1.36 (m, 1H), 1.35−

1.16 (m, 2H); ESI-MS m/z 492.67 (M + H)⁺.

ABBREVIATIONS USED

■

THF, tetrahydrofuran; CAN, ceric ammonium nitrate; DCM,

dichloromethane; MeOH, methanol; DCE, 1,2-dichloroethane;

CDI, 1,1′-carbonyldiimidazole; AcOH, acetic acid; DIEA, N,N′-

S

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J. Med. Chem. XXXX, XXX, XXX−XXX

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induces complete and durable tumor regression. Cancer Res. 2014, 74,

5855−5865.

diisopropylethylamine; DME, 1,2-dimethoxyethane; TFA,

triﬂuoroacetic acid; TFAA, triﬂuoracetic anhydride; HPLC,

high performance liquid chromatography; UPLC, ultraperform-

ance liquid chromatography; DMAP, 4-(dimethylamino)-

pyridine; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; rt, room

temperature; HATU, 1-[bis(dimethylamino)methylene]-1H-

1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexaﬂuorophosphate

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L.; Kayser, F.; Liu, J. J.; Lo, M. C.; Long, A. M.; Lucas, B.; McGee, L.

R.; McIntosh, J.; Mihalic, J.; Oliner, J. D.; Osgood, T.; Peterson, M. L.;

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. Discovery of AMG 232, a

potent, selective, and orally bioavailable MDM2-p53 inhibitor in

clinical development. J. Med. Chem. 2014, 57, 1454−1472.

(20) Holzer, P.; Masuya, K.; Furet, P.; Kallen, J.; Valat-Stachyra, T.;

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CGM097): a highly potent and selective MDM2 inhibitor undergoing

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