Discovery of 4-Aryl-7-hydroxyindoline-based P2Y1 antagonists as novel antiplatelet agents

Article abstract of DOI:10.1021/jm5006226

Adenosine diphosphate (ADP)-mediated platelet aggregation is signaled through two distinct G protein-coupled receptors (GPCR) on the platelet surface: P2Y₁₂ and P2Y₁. Blocking P2Y₁₂ receptor is a clinically well-validated strategy for antithrombotic therapy. P2Y₁ antagonists have been shown to have the potential to provide equivalent antithrombotic efficacy as P2Y₁₂ inhibitors with reduced bleeding in preclinical animal models. We have previously reported the discovery of a potent and orally bioavailable P2Y₁ antagonist, 1. This paper describes further optimization of 1 by introducing 4-aryl groups at the hydroxylindoline in two series. In the neutral series, 10q was identified with excellent potency and desirable pharmacokinetic (PK) profile. It also demonstrated similar antithrombotic efficacy with less bleeding compared with the known P2Y ₁₂ antagonist prasugrel in rabbit efficacy/bleeding models. In the basic series, 20c (BMS-884775) was discovered with an improved PK and liability profile over 1. These results support P2Y₁ antagonism as a promising new antiplatelet target.

Full text of DOI:10.1021/jm5006226

Article
pubs.acs.org/jmc
Discovery of 4‑Aryl-7-Hydroxyindoline-Based P2Y1 Antagonists as
Novel Antiplatelet Agents
Wu Yang,*,† Yufeng Wang,† Amy Lai,† Jennifer X. Qiao,† Tammy C. Wang,† Ji Hua,‡ Laura A. Price,‡
Hong Shen,§ Xue-qing Chen,§ Pancras Wong,‡ Earl Crain,‡ Carol Watson,‡ Christine S. Huang,§
Dietmar A. Seiffert,‡ Robert Rehfuss,‡ Ruth R. Wexler,† and Patrick Y. S. Lam†
‡
§
†Discovery Chemistry, Discovery Biology, and Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Research,
Post Office Box 5400, Princeton, New Jersey 08643-5400, United States
ABSTRACT: Adenosine diphosphate (ADP)-mediated platelet aggregation is signaled through two distinct G protein-coupled
receptors (GPCR) on the platelet surface: P2Y12 and P2Y1. Blocking P2Y12 receptor is a clinically well-validated strategy for
antithrombotic therapy. P2Y1 antagonists have been shown to have the potential to provide equivalent antithrombotic efficacy
as P2Y12 inhibitors with reduced bleeding in preclinical animal models. We have previously reported the discovery of a potent
and orally bioavailable P2Y1 antagonist, 1. This paper describes further optimization of 1 by introducing 4-aryl groups at the
hydroxylindoline in two series. In the neutral series, 10q was identified with excellent potency and desirable pharmacokinetic
(PK) profile. It also demonstrated similar antithrombotic efficacy with less bleeding compared with the known P2Y12 antagonist
prasugrel in rabbit efficacy/bleeding models. In the basic series, 20c (BMS-884775) was discovered with an improved PK and
liability profile over 1. These results support P2Y1 antagonism as a promising new antiplatelet target.
apparent developmental abnormalities. They showed no
spontaneous bleeding tendency but were resistant to ADP- or
collagen-induced acute thromboembolism.4 The first direct
demonstration that inhibition of P2Y1 activity could lead to an
INTRODUCTION
■
Adenosine diphosphate (ADP)-mediated platelet aggregation
plays a key role in thrombus formation. ADP activation of
platelets is signaled through two distinct G-protein coupled
receptors (GPCR) on the surface of platelets: P2Y12, which is
coupled to G-protein i (Gi), and P2Y1, which couples to
G-protein q (Gq). ADP is the endogenous activator of the two
receptors. Binding of ADP to the P2Y1 receptor results in
transitory increases in intracellular free Ca2+ and acts as the trigger
by stimulating platelets to form small, reversible aggregates.1
Binding of ADP to the P2Y12 receptors results in reduction of
cAMP (cyclic adenosine monophosphate) levels and acts as an
amplifier of the reaction, driving the reversible aggregates to an
irreversible state.2 Blocking ADP-driven platelet aggregation via
P2Y12 receptors is a clinically well-validated strategy for
antithrombotic therapy. Several successful marketed drugs such
as clopidogrel, prasugrel, and ticagrelor are all P2Y12 antagonists
used to treat peripheral artery disease and acute coronary
syndrome (Figure 1).3 However, there is no clinical validation for
P2Y1 antagonists as antithrombotic agents, though preclinically
there is ample evidence that blocking the P2Y1 receptor can
inhibit platelet aggregation and, consequently, thrombus forma-
tion. P2Y1 knockout mice were found to be viable with no
antithrombotic effect in vivo was reported by Leo
́
n et al.5 in a
model of thromboplastin-induced thromboembolism using a
selective P2Y1 antagonist, MRS-2179. Subsequently, MRS-25006
and a number of adenine nucleotide-based P2Y1 antagonists had
been reported to inhibit ADP activated platelet aggregation in
vitro and in vivo. Our in house study with MRS-2500 and
the P2Y12 antagonist clopidogrel in a rabbit efficacy/bleeding
model demonstrated that there are differential effects of P2Y1
antagonism versus P2Y12 antagonism on bleeding, with the P2Y1
antagonist MRS-2500 showing the same efficacy but less bleeding
compared with the P2Y12 antagonist clopidogrel.7 On the basis
of the genetic and pharmacological data, P2Y1 antagonism may
offer a new opportunity for the development of novel anti-
platelet agents with an improved therapeutic index over P2Y12
antagonism.
Received: April 21, 2014
Published: June 14, 2014
© 2014 American Chemical Society
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Figure 1. Selected P2Y12 and adenine nucleotide-based P2Y1 antagonists.
Figure 2. Generation of neutral P2Y1 antagonists from lead compound 1.
metabolic stability of this neutral series, functional groups that
had higher oxidation states (alcohol, acid, ester) or a CF3 group
generally known to be metabolically stable were introduced at the
C3 position.
RESULTS AND DISCUSSION
■
Adenine nucleotide-based P2Y1 antagonists such as MRS-2500
(Figure 1) exhibited potent antagonist activity. However, they
are unsuitable as oral drug candidates due to their lack of oral
bioavailability. As a result, nonnucleotide P2Y1 antagonists have
been sought in an attempt to find another chemotype with
potency, oral bioavailability, and desirable pharmacokinetic (PK)
properties. A number of novel chemotypes have been disclosed
as P2Y1 antagonists from high-throughput screening (HTS) or
virtual screening efforts.8 We had previously reported a series of
structure−activity relationship (SAR) studies on the optimiza-
tion of a biaryl urea HTS hit resulting in the discovery of 1,9
a potent and orally available P2Y1 antagonist with an IC50 of
120 pM in the FLIPR (fluorescent imaging plate reader) assay
and 130 nM in the functional assay (hPA: human platelet-rich
plasma was used in the presence of 10 μM ADP). Compound 1
exhibited excellent liver microsomal stability, with greater than
90% remaining after 10 min incubation with human or rat liver
microsomes. However, 1 showed potent inhibition of human
The general synthesis of analogues in Tables 1, 2, and 4
(10p and 10r) from the neutral series is shown in Scheme 1.
Substituted 2-methoxyanilines 3 were converted to their cor-
responding hydrazines 4 by treatment with sodium nitrate,
followed by reduction with tin(II) chloride in the presence of
concentrated HCl. Indolines 5 were prepared via Fisher indoline
synthesis by reacting the hydrazines 4 with various 2-substituted
propionaldehydes, followed by reduction of the intermediate
imines with NaBH4 or NaBH3CN. N-Arylation of indolines 5
with 1-bromo-2-nitrobenzene under Buchwald conditions
afforded compounds 6, which were reduced with Zn/NH4Cl
to give anilines 7. Ureas 8 could be obtained either by treat-
ment of 7 directly with isocyanate or by first converting 7 to
p-nitrophenylcarbamate, followed by reacting with aminohetero-
cycles in the presence of 4-dimethylaminopyridine (DMAP) at
80 °C. Demethylation of compounds 8 (when X = Cl and R = H)
with AlCl3 under microwave irradiation or with BCl3/Bu4NI
or with BBr3·Me2S at 85 °C afforded final compounds 2a−e in
Table 1. Reduction of 2e by LiBH4 provided 2f. The 4-aryl-
substituted analogues 9 were prepared via Suzuki coupling of
compounds 8 (X = Br, Ar = 4-OCF3Ph) with various boronic
acids, followed by demethylation with AlCl3 under microwave
condition or with BCl3/ Bu4NI.
̀
ether-a-go-go-related gene (hERG) in the patch clamp assay
(96% inhibition at 1 μM), though it did not cause significant QTc
prolongation in a rabbit EP (electrophysiology) study, most
likely due to its high protein binding (rabbit, 99.3% bound).
To further expand the SAR on this series and to mitigate the
potential off-target liabilities such as hERG inhibition, two closely
related series, the neutral series where spiropiperidine was
removed and the basic series where the spiropiperidine was
retained, were pursued simultaneously. The SAR discoveries in the
neutral series were readily applied to the basic series. In the neutral
series, removal of the basic nitrogen of the spiropiperidine resulted
in analogues as exemplified by 2a and 2b (Figure 2). Though 2a
and 2b retained most of the potency of1, liver microsomal stability
decreased. Subsequently, metabolic identification studies were
performed on these compounds and the C3 gem-dimethyl groups
were identified as the metabolic soft spots. To improve the
Alternatively, 4-(4-fluorophenyl) analogues with different urea
substitutions in Table 3 and 5-fluoro-4-aryl analogues in Tables 4
(10o and 10q) and 5 can be synthesized by a modified sequence
from Scheme 1 as shown in Scheme 2. The 4-aryl groups were
introduced earlier in the synthesis via Suzuki coupling of bromide
6 with various arylboronic acids to give compounds 11, which
were reduced to compounds 12 by Zn/NH4Cl. The ureas 9
were prepared by either treatment with isocyanates or first
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a
Scheme 1. General Synthesis of Analogues from the Neutral Series
a
Reagents and conditions: (a) NaNO2, HCl; (b) SnCl2, 34−92% for 2 steps; (c) HCl in dioxane, or H2SO4 in EtOH; (d) NaBH4 or NaBH3CN in
MeOH, 15−86%; (e) 1-bromo-2-nitrobenzene, Pd2(dba)3, BINAP, Cs2CO3, toluene, 115 °C, 57−96%; (f) Zn, NH4Cl, EtOAc/MeOH, 58−100%;
(g) OCN-p-OCF3Ph, CH2Cl2, 41−98%; (h) ClCO2-p-NO2Ph, K2CO3, CH2Cl2, then NH2Ar, DMAP, 80 °C, 5 h, 23−50%; (i) AlCl3, CH2Cl2,
microwave irradiation, 100 °C, 10 min, 13−22%; (j) BCl3, Bu4NI, CH2Cl2, −78 °C to rt, 15−82%; (k) BBr3·Me2S, 85 °C, 6 h, 22−34%;
(l) ArB(OH)2, Pd(PPh3)4, Na2CO3, 100 °C, 55−100%; (m) LiBH4, THF/toluene, 100 °C, 6 h, 23%; (n) NaOH/THF/MeOH, 6 days, 65%.
a
Scheme 2. Synthesis of 4-(4-Fluorophenyl) and 5-Fluoro-4-aryl Analogues
a
Reagents and conditions: (a) arylboronic acids, Pd(PPh3)4, Na2CO3, 100 °C, 84−98%; (b) Zn, NH4Cl, EtOAc/MeOH, 83−96%; (c) BCl3, Bu4NI,
CH2Cl2, −78 °C to rt, 39−90%; (d) OCN-p-OCF3Ph, CH2Cl2, 99%; (e) ClCO2-p-NO2Ph, K2CO3, CH2Cl2, then NH2Ar, DMAP, microwave
100 °C, 10 min, 56−96%; (f) 2-methyl- (or 2-chloro-) thiazole-4-carboxylic acid, DPPA, NEt3, tol. 100 °C, 1 h, 90−94%; (g) BCl3, Bu4NI, CH2Cl2,
−78 °C to rt, 26−97%; (h) AlCl3, CH2Cl2, microwave irradiation, 100 °C, 10 min, 13−22%.
activation with p-nitrophenyl chloroformate followed by reac-
tion with aminoheterocycles in the presence of DMAP at 80 °C.
For the preparation of 2-chloro or 2-methylthiazole urea analo-
gues 10k and 10l, Curtis rearrangement of 2-chloro- (or 2-methyl-)
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a
Table 1. Structure−Activity Relationship of C3-Substituted 7-Hydroxylindolines
a
FLIPR was tested with 1 μM 2-methylthio-ADP, n ≥ 2. bPA was tested with 10 μM ADP, n = 1 unless shown with SD. See ref 9f and SI in ref 9f
c
for detailed assay conditions. Liver microsome stability: percentage of compound (0.5 μM) remaining after incubation for 10 min with 1 mg/mL
human, rat, or mouse liver microsomes. NT, not tested.
d
thiazole-4-carboxylic acid produced isocyanates in situ, which
reacted with aniline 12b to give the ureas 9k and 9l. Standard
demethylation of ureas 9 provided compounds 10. Alternatively,
12b was demethylated first with BCl3/Bu4NI to give 13b,
which was converted to compounds 10 following the same urea
formation procedures as used to convert from compounds 12 to
compounds 9.
Table 2. Structure−Activity Relationship of 4-Aryl-
Substituted 7-Hydroxylindolines
The SAR of the C-3 modifications is shown in Table 1.
Replacement of a methyl group with a CH2CF3 group resulted in
2c, which showed a 5-fold loss in FLIPR potency and a 3-fold loss
in potency in the PA assay while maintaining the same level of
liver microsomal stability as that found for 2b. Replacement of
methyl group with ethyl ester improved FLIPR potency by 4-fold
but was 3-fold weaker in PA assay (2d vs 2b). However, when the
C3 ethyl ester substitution was combined with 5-chlorothiazolo-
[5,4-b]pyridine urea, the resulting compound 2e showed over
2-fold improvement in both FLIPR and PA potency compared
with 2a. Converting the ethyl ester to more polar groups such
as alcohol (2f), acid (2g), or amide (2h) all resulted in much
reduced P2Y1 activity. Though the C3 ethyl ester replacement
showed improved potency, the liver microsomal stability
decreased compared with the C3 gem-dimethyl analogues. As a
result, we shifted our focus to installing bulkier aromatic sub-
stitutions at C4 position on the indoline to block the metabolism
at C3 methyl groups.
a
b
c
FLIPR IC50
(nM)
PA IC50
(μM)
LM h, r, m
compd
Ar1
(% remaining)
10a
10b
10c
10d
10e
10f
Ph
0.37 0.04
0.37 0.20
0.15 0.07
0.33 0.14
0.13 0.04
1.51 0.59
0.52 0.28
1.8 0.04
0.18
76, 67, 48
62, 54, 48
67, 59, 56
87, 89, 83
84, 83, 78
96, 94, 89
81, 72, 79
90, 81, 66
2-F-Ph
0.08
0.24
3-F-Ph
4-F-Ph
0.16 0.05
0.21 0.07
0.23
4-Cl-Ph
4-CF3-Ph
2,4-diF-Ph
3,4-diF-Ph
3,5-diF-Ph
10g
10h
10i
0.16
0.4
0.57 0.20
0.18
63, 59, 64
a
b
FLIPR was tested with 1 μM 2-methylthio-ADP, n ≥ 2. PA was
tested with 10 μM ADP, n = 1 unless shown with SD. See ref 9f for
detailed assay conditions. Liver microsome stability: percentage of
compound (0.5 μM) remaining after incubation for 10 min with 1 mg/mL
human, rat, or mouse liver microsomes.
c
The SAR of C4 aryl substitutions is shown in Table 2.
Introducing the phenyl group at the C4 position resulted in
a 4-fold improvement in potency compared with 4-chloro
substitution as measured in the human PA inhibition assay
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a
Table 3. Structure−Activity Relationship of Urea Substitutions
a
FLIPR was tested with 1 μM 2-methylthio-ADP, n ≥ 2. bPA was tested with 10 μM ADP, n = 1 unless shown with SD. See ref 9f for detailed assay
c
conditions. Liver microsome stability: percentage of compound (0.5 μM) remaining after incubation for 10 min with 1 mg/mL human, rat, or
mouse liver microsomes.
(10a vs 2b). However, the metabolic stability of 10a was only
slightly improved in human and rat liver microsomes. A fluorine
“walk” around the C4 phenyl ring was performed to improve the
metabolic stability. Of the 2-, 3-, and 4-positions of the phenyl
ring, fluoro substitution at the 2- and 4-positions (10b and 10d)
provided slightly more potent compounds than at the 3-position
(10c) in PA assay. Between the 2- and 4-fluorophenyl analogues
(10b and 10d), the 4-fluorophenyl analogue 10d was meta-
bolically more stable than 10b. While increasing the size of
substitutions at the 4-position of phenyl from fluorine (10d) to
chlorine (10e) resulted in analogues with essentially equivalent
potency, the CF3 substitution (10f) appeared to be slightly
too large, as its potency decreased in both FLIPR and PA assays.
The 2,4-, 3,4-, and 3,4-bis-substitutions (10g−10i) did not
provide any additional boost in potency. As a result, 4-fluoro- and
4-chlorophenyl at the C4 position were chosen as templates to
study the SAR of urea substitution.
Shown in Table 3 are some examples of aryl- and heteroaryl-
substituted ureas with 4-fluorophenyl attached at the 4-position of
the indoline. The 5-chlorothiazolo[5,4-b]pyridine urea analogue
(10j) had comparable potency as the trifluoromethoxyphenyl
urea 10d. From a large number of heteroaryl ureas surveyed, the
2-chlorothiazole urea 10k was identified with excellent potency
(PA IC50 90 pM). The 2-chloro substitution on the thiazole
plays a crucial role in increasing potency, as the 2-methylthiazole
analogue (10l) was found to be 5-fold less potent in the platelet
aggregation assay. In an effort to improve the solubility of the
neutral series, phenylureas substituted with basic groups such as
pyrrolidines were prepared (10m and 10n). Pyrrolidine-
substituted phenylureas demonstrated superior potency in both
the FLIPR and PA assays; however, their poor liver microsomal
stability led to poor PK profiles. In a rat cassette PK study, at
0.5 mg/kg dose, 10m exhibited high clearance (34 mL·min−1·kg−1)
and a large volume of distribution (9.8 L/kg) with low oral
bioavailability (4%).
Though the 7-hydroxy-4-arylindoline series of compounds
demonstrated excellent potency, there was concern that
they might be prone to oxidation due to the electron-rich
nature of the ring system. To reduce the electron density of the
7-hydroxylindoline ring system, various electron-withdrawing
groups were introduced at the 5- and 6- positions, with only
fluorine being tolerated at either position (Table 4). The
5-fluoro analogue (10o) showed comparable potency and
microsomal stability when compared to the unsubstituted
compound 10d. Between the 5-fluoro (10o and 10q) and
6-fluoro analogues (10p and 10r), the 5-fluoro analogues
were slightly more potent (10o vs 10p and 10q vs 10r) with
comparable microsomal stability. As a result, subsequent SAR
study focused on the 5-fluoro series.
In addition to phenyl and substituted phenyl groups at the
C4 position, 5- and 6-membered heteroaryls were also explored
(Table 5). A variety of heterocycles such as chlorothiophene
(10s) as well as pyridines (10t and 10u), pyrimidine (10w),
and pyridazine (10x) afforded good potency. In general, small
hydrophobic substituents such as halogens and the CF3 group
helped maintain the potency of these analogues.
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(1.3 mL·min−1·kg−1), small volume of distribution (0.4 L/kg),
and good t1/2 (12 h) with moderate oral bioavailability of 10%.
As a result, 10q was chosen to proceed to rabbit efficacy and
bleeding models (in rabbit, 10q showed PA IC50 = 0.087 μM).
Antithrombotic activity was evaluated by thrombus weight
reduction in a rabbit model of electrically induced carotid artery
thrombosis (ECAT).11 Bleeding time (BT) was measured for up
to 20 min following cuticle incision. Compound 10q was infused
intravenously (iv), starting 30 min before electrical stimula-
tion and maintained throughout the experiment. As shown in
Figure 3, 10q at doses of 0.003 + 0.01, 0.01 + 0.033, 0.03 +
0.1, 0.1 + 0.033, and 0.3 + 1 mg/kg + mg·kg−1·h−1 iv reduced
thrombus formation by 22% 3%, 46% 3%, 50% 4%, 72%
3%, and 95% 3%, respectively, versus 3% 2% for the vehicle-
treated group (n = 5−6 per group). It increased BT by
(1.1 0.1)-fold, (1.4 0.1)-fold, (2.7 0.2)-fold, (3.7 0.2)-
fold, and (4.7 0.1)-fold versus (1 0.05)-fold for the vehicle
(n = 5−8 per group).
In rabbits, 10q prevented arterial thrombosis as effectively as
the P2Y12 antagonists clopidogrel and prasugrel. However, at 90%
antithrombotic dose, 10q produced 4.7-fold increase in BT, which
was less than the 6-fold BT increases produced by clopidogrel and
prasugrel at 80% antithrombotic dose in the same model.
Despite the encouraging profile of 10q, it could not proceed
further into development due to its lower oral bioavailability and
exposure when dosed as a suspension, a direct result of its poor
aqueous solubility (less than 1 μg/mL, amorphous). However,
the issue of lack of aqueous solubility of the neutral series could
be solved in the basic series. We also postulated that since the
bulky 4-aryl group could block the metabolism at C3, it might
also be able to shield the basic nitrogen of the spiropiperidine
from potential off-target side effects.
Table 4. 4-Aryl-5 (or 6)-F Analogues
a
b
c
FLIPR IC50
(nM)
PA IC50
(μM)
LM h, r, m,
compd
Ar1
(% remaining)
10d
10o
10p
10q
10r
4-F-Ph
5-F 4-F-Ph
6-F 4-F-Ph
0.33 0.14
0.23 0.12
0.45(n = 1)
0.16 0.05
0.19 0.07
0.39
87, 88, 83
73, 84, 77
80, 71, 66
83, 90, 79
5-F 4-Cl-Ph 1.06 0.48
6-F 4-Cl-Ph NT
0.24 0.09
0.30
88, 100, 80
a
b
FLIPR was tested with 1 μM 2-methylthio-ADP, n ≥ 2. PA was
tested with 10 μM ADP, n = 1 unless shown with SD. See ref 9f
c
for detailed assay conditions. Liver microsome stability: percentage
of compound (0.5 μM) remaining after incubation for 10 min with
1 mg/mL human, rat, or mouse liver microsomes.
From the 4-aryl-7-hydroxylindoline neutral series, a large
number of analogues were identified with desired in vitro profiles
(excellent P2Y1 potency and good liver microsomal stability). To
differentiate these compounds, rat cassette PK studies10 (4 in 1)
were utilized, from which 10q was found to have the best overall
profile. As shown in Table 4, 10q was found to be a potent P2Y1
antagonist (FLIPR IC50 1.1 nM; PA IC50 0.24 μM) with good
liver microsomal stability (greater than 80% remaining for all
species). In a rat discrete PK study, upon dosing in solution
(0.5 mg/kg iv and 15 mg/kg oral) with dimethylacetamide
(DMAc)/cremophor/EtOH/water (10/10/10/70) as the
vehicle, 10q exhibited a desired profile of low clearance
a
Table 5. 4-Heteroaryl Substitutions
a
b
FLIPR was tested with 1 μM 2-methylthio-ADP, n ≥ 2. PA was tested with 10 μM ADP, n = 1 unless shown with SD. See ref 9f for detailed
assay conditions. cLiver microsome stability: percentage of compound (0.5 μM) remaining after incubation for 10 min with 1 mg/mL human, rat, or
mouse liver microsomes.
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Figure 3. (A) Antithrombotic effects of 10q in rabbit ECAT model. Vehicle: 10% DMAc/2.5% PVP-K12/1% Pluronic F-108/D5W. (B) Effects
of 10q on bleeding time in rabbit cuticle bleeding time model. For clarity, only the bolus doses are shown on the figures. Means SEM are shown;
n = 5−6 per group.
a
Scheme 3. Synthesis of 4-Aryl-7-hydroxylspiroindoline Analogues
a
Reagents and conditions: (a) aldehyde 14; (b) H2SO4, 40 °C; (c) NaBH4, MeOH, 37−53%; (d) 1-bromo-2-nitrobenzene, Pd2(dba)3, BINAP,
Cs2CO3, 110 °C, 86−95%; (e) 4-F(or Cl)-phenylboronic acid, Pd(PPh3)4, K2CO3, dioxane, 100 °C, 49−100%; (f) Zn, NH4Cl, 54−85%; (g) Red-Al,
CH2Cl2, 61−88%; (h) ClCO2-p-NO2Ph, K2CO3, CH2Cl2, then NH2Ar, DMAP, 75 °C, 80%; (i) OCN-p-OCF3Ph, CH2Cl2, 54−85%; (j) BCl3,
Bu4NI, CH2Cl2, −78 °C to rt, 18−42%.
Synthesis of the 4-aryl-7-hydroxylspiroindoline analogues pro-
ceeded in a similar fashion to the neutral analogues and is shown
in Scheme 3. Fisher indoline synthesis, by reacting aldehyde 14
with (5-bromo-4-fluoro-2-methoxyphenyl)hydrazine followed
by reduction with NaBH4, afforded 15. Buchwald N-arylation
of 15 followed by Suzuki coupling of various boronic acids
provided compounds 17, which were sequentially reduced first
with Zn/NH4Cl and then with Red-Al to give compounds 18.
Ureas 19, prepared in the same ways as described for the neutral
series, were demethylated with BCl3/Bu4NI to generate the final
compounds 20.
The optimal aryl substitutions identified at the C4 position
and on the urea in the neutral series were “mixed and matched”
in the 5-fluoro-7-hydroxyspiroindoline basic series (Table 6).
A number of potent and metabolically stable analogues were
generated. Similar to the neutral series, rat cassette PK was used
to differentiate these analogues. Of the analogues prepared, 20c
was identified to demonstrate the best overall potency/PK profile. It
exhibited equivalent in vitro potency and liver microsomal stability
compared with 1 but with a superior PK profile (lower clearance,
smaller volume of distribution, comparable bioavailability, and
higher exposure) in a rat PK study. In the in vitro hERG patch clamp
assay, 20c also showed improvement over 1 (57% vs 96% inhibition
at 1 μM). Compound 20c demonstrated good selectivity with IC50
values greater than 10 μM against P2Y12 and P2Y2 receptors and a
panel of GPCRs. Compared with neutral compound 10q, 20c
showed marginally improved solubility (amorphous, 3 μg/mL at
pH = 3 vs less than 1 μg/mL for 10q as amorphous solid). On the
basis of its overall profile, 20c was also advanced to the rabbit
efficacy/bleeding models (20c rabbit PA IC50 = 0.59 μM) and
demonstrated similar antithrombotic efficacy with less bleeding
compared with known P2Y12 antagonist prasugrel.12
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Table 6. Structure−Activity Relationship of 4-Aryl-5-Fluorospiroindoline Analogues
continuously. H and 13C NMR spectra were taken on either Bruker
(400 MHz) or JEOL (500 MHz) Fourier transform spectrometers.
Chemical shifts were reported in parts per million (ppm) downfield
from internal standard (tetramethylsilane, TMS). Purification of final
compounds and intermediates was carried out via normal- or reverse-
phase chromatography. Normal-phase chromatography was performed
on an ISCO CombiFlash system with either an ISCO RediSep or a
Biotage silica gel disposable column eluted with EtOAc/hexanes or
methanol/CH2Cl2. Reverse-phase chromatography was performed on a
Shimadzu preparative system on a Phenomenex Luna or Sunfire C18
preparative column with appropriate gradients of MeCN/H2O/0.1%
trifluoroacetic acid (TFA) or methanol/H2O/0.1% TFA as eluent at a
flow rate of 40 mL/min. Reactions were monitored either by analytical
liquid chromatography−mass spectrometry (LCMS) or by thin-layer
1
CONCLUSIONS
■
To expand the SAR of a previously disclosed 7-hydroxyindoline
P2Y1 antagonist, 1, two closely related series, neutral and basic,
were pursued simultaneously. Removal of the basic nitrogen
from 1 provided the neutral series with 7−10-fold loss of potency
in human platelet aggregation assay and reduced liver micro-
somal stability. Introducing aryl groups to the 4-position of the
7-hydroxyindoline neutral series not only increased liver
microsomal stability, by blocking the metabolism at C3 position,
but also improved P2Y1 potency. As a result, 10q was identified
with excellent potency and a desired PK profile (low clearance,
small volume of distribution). Compound 10q also demon-
strated similar antithrombotic efficacy with less bleeding when
compared to the known P2Y12 antagonists clopidogrel and
prasugrel in the rabbit model of electrically induced carotid artery
thrombosis and cuticle bleeding. Applying the SAR from the
neutral series to the basic series led to the discovery of 20c, with
an improved PK and liability profile over 1. The results from in
vivo studies of 10q and 20c support P2Y1 receptor antagonism as
a promising drug target for the development of a new and safer
class of antiplatelet agents.
chromatography (TLC) on 0.25 mm E. Merck silica gel plates (60 F254
)
and were visualized with UV light or by staining with various agents.
LCMS data were obtained on a Shimadzu LC-10AT equipped with
a SIL-10A injector, a SPD-10AV detector, running on Discovery VP
software, and coupled with a Waters ZQ mass spectrometer running
MassLynx version 3.5 software by the following methods.
Method A: Phenomenex Luna C18 column (4.6 × 50 mm) eluted at
4 mL/min with a 4 min linear gradient from 0% to 100% B and then
1 min at 100% B (where A = 10% methanol/90% H2O/0.1% TFA and
B = 90% methanol/10% H2O/0.1% TFA).
Method B: Phenomenex Luna C18 column (2.0 × 30 mm) eluted
with a 2 min linear gradient from 0% to 100% B and then 1 min at
100% B (where A = 10% methanol/90% H2O/0.1% TFA and B = 90%
methanol/10% H2O/0.1% TFA).
EXPERIMENTAL SECTION
■
General Methods. All reagents and solvents, including anhydrous
solvents, were obtained from commercial sources and used as received.
All reactions were carried out under an atmosphere of argon and stirred
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Method C: Phenomenex Luna C18 column (2.0 mm × 30 mm)
eluted with a 2 min linear gradient from 0% to 100% B and then 1 min
at 100% B (where A = 2% methanol/98% H2O/0.1% formic acid and
B = 100% methanol/0.1% formic acid).
Purity of final compounds was determined under the following HPLC
conditions.
HPLC Method A (orthogonal): Sunfire C18 column (3.5 μm, 4.6 ×
150 mm) eluted at 1.0 mL/min with gradient 10−100% B/A (where
solvent A = 95% H2O/5% MeCN/0.05% TFA and solvent B = : 5%
H2O/95% MeCN/0.05% TFA) over 10 min, and then 100% B over
5 min.
HPLC Method B (orthogonal): Xbridge Phenyl column (3.5 μm,
4.6 × 150 mm) eluted at 1.0 mL/min gradient 10−100% B/A
(solvent A = 95% H2O/5% MeCN/0.05% TFA; solvent B = 5%
H2O/95% MeCN/0.05% TFA) over 10 min and then 100% B
over 5 min.
Ethyl 4-Chloro-7-methoxy-3-methyl-1-[2-({[4-(trifluoromethoxy)-
phenyl]carbamoyl}amino)phenyl]-2,3-dihydro-1H-indole-3-
carboxylate (8a−d). A solution of 7a (52 mg, 0.14 mmol) and
1-isocyanato-4-(trifluoromethoxy)benzene (35 mg, 0.17 mmol) in di-
chloromethane (DCM; 2 mL) was stirred at room temperature for 16 h.
The reaction mixture was concentrated and purified by flash chromato-
graphy, eluted with EtOAc/hexanes to give 8a−d (78 mg, 96% yield) as
a pale yellow solid. LCMS (ESI) m/z 564.1 (M + H)+.
Ethyl 4-Chloro-7-hydroxy-3-m ethyl-1-[2-({[4-
(trifluoromethoxy)phenyl]carbamoyl}amino)phenyl]-2,3-dihy-
dro-1H-indole-3-carboxylate (2d). To a solution of 8a−d (78 mg,
0.14 mmol) in DCM (3 mL) was added tetrabutylammonium iodide
(257 mg, 0.696 mmol) at −78 °C under argon, and BCl3 (1 M solution
in DCM, 0.7 mL, 0.7 mmol) was added dropwise. The reaction mixture
was gradually warmed up to room temperature and stirred for 16 h. The
reaction mixture was concentrated and purified by flash chromato-
graphy, eluted with EtOAc/hexanes to give 2d as a white solid (37 mg,
49% yield). LCMS (ESI) m/z 550.2 (M + H)+; purity 100% (by HPLC
method C). 1H NMR (400 MHz, chloroform-d) (mixture of rotamers,
major isomer reported) δ 8.30−8.22 (m, 2H), 7.49−7.40 (m, 3H),
7.35−7.29 (m, 1H), 7.22 (dd, J = 7.8, 1.3 Hz, 1H), 7.14 (d, J = 8.6 Hz,
2H), 7.06 (td, J = 7.6, 1.3 Hz, 1H), 6.69−6.59 (m, 2H), 4.34−4.25 (m,
2H), 4.16 (d, J = 9.9 Hz, 1H), 3.49 (d, J = 10.1 Hz, 1H), 1.85 (s, 3H),
1.35 (t, J = 7.2 Hz, 3H).
HPLC Method C (analytical): ZorbaxSB C18 column (4.6 × 75 mm)
eluted at 2.5 mL/min with gradient 0−100% B/A (solvent A = 90%
H2O/10% MeCN/0.2% H3PO4; solvent B = 10% H2O/90% MeOH/
0.2% H3PO4) over 8 min and then 100% B over 3 min.
Ethyl 4-Chloro-7-methoxy-3-methyl-2,3-dihydro-1H-indole-
3-carboxylate (5a). A solution of (5-chloro-2-methoxyphenyl)-
hydrazine hydrochloride (1.70 g, 8.13 mmol) and ethyl 2-methyl-3-
oxopropanoate hydrochloride (1.05 mg, 8.07 mmol) in dichloro-
methane (15 mL) was stirred at room temperature for 16 h before HCl
(8.07 mL of 4 M in dioxane, 32.3 mmol) was added. After 2 h, the
reaction mixture was concentrated and EtOH (15 mL) was added. At
0 °C, NaBH4 (1.22 g, 32.3 mmol) was added portionwise. After 10 min,
the reaction mixture was quenched with water, adjusted pH with 1 M
HCl to pH ∼7, concentrated, and extracted with EtOAc (2×) The
organic layer was dried over MgSO4, concentrated, and purified by flash
chromatography, eluted with EtOAc/hexanes to give 5a as a pale yellow
oil (413 mg, 19% yield). LCMS (electrospray ionization, ESI) m/z 270.1
(M + H)+. 1H NMR (400 MHz, chloroform-d) δ 6.68−6.60 (m, 2H),
4.28−4.13 (m, 2H), 3.98−3.85 (m, 2H), 3.81 (s, 3H), 3.46 (dd, J = 9.1,
1.3 Hz, 1H), 1.60 (s, 3H), 1.30−1.21 (m, 3H).
Ethyl 4-Chloro-7-methoxy-3-methyl-1-(2-nitrophenyl)-2,3-
dihydro-1H-indole-3-carboxylate (6a). A mixture of 5a (292 mg,
1.08 mmol), 1-bromo-2-nitrobenzene (262 mg, 1.30 mmol), rac-2,2′-
bis(diphenylphosphino)-1,1′-binaphthyl (BINAP; 47 mg, 0.076 mmol),
tris(dibenzylideneacetone)dipalladium [Pd2(dba)3; 30 mg, 0.032
mmol], and Cs2CO3 (847 mg, 2.60 mmol) in toluene (10 mL) was
purged with argon three times, sealed, and heated at 85 °C for 8 h.
Another portion of 1-bromo-2-nitrobenzene (260 mg, 1.30 mmol), rac-
BINAP (47 mg, 0.076 mmol), and Pd2(dba)3 (30 mg, 0.032 mmol) was
added to the reaction mixture, which was purged with argon three times,
sealed, and heated at 110 °C for 16 h. After cooling to room tem-
perature, the reaction mixture was filtered, concentrated, and purified by
flash chromatography, eluted with EtOAc/hexanes to give 6a (0.28 g,
65% yield) as an orange solid. LCMS (ESI) m/z 391.2 (M + H)+.
1H NMR (400 MHz, chloroform-d) δ 8.03 (dd, J = 8.3, 1.5 Hz, 1H),
7.52−7.41 (m, 1H), 7.20 (dd, J = 8.3, 1.0 Hz, 1H), 7.16−7.04 (m, 1H),
6.81 (d, J = 8.8 Hz, 1H), 6.65 (d, J = 8.6 Hz, 1H), 4.50 (d, J = 9.3 Hz,
1H), 4.27−4.19 (m, 2H), 3.91 (d, J = 9.3 Hz, 1H), 3.51 (s, 3H), 1.69 (s,
3H), 1.26−1.21 (m, 3H).
Ethyl 1-(2-Aminophenyl)-4-chloro-7-methoxy-3-methyl-2,3-
dihydro-1H-indole-3-carboxylate (7a). To a solution of 6a
(275 mg, 0.704 mmol) in MeOH (5 mL) and EtOAc (5 mL) were
added zinc (920 mg, 14.1 mmol) and NH4Cl (753 mg, 14.1 mmol). The
reaction mixture was stirred at room temperature. After 1 h, the reaction
mixture was filtered, concentrated, dissolved in dichloromethane, and
filtered. The filtrate was concentrated and purified by flash chromato-
graphy, eluted with EtOAc/hexanes to give 7a (210 mg, 83% yield) as an
off-white solid. LCMS (ESI) m/z 361.1 (M + H)+. 1H NMR (400 MHz,
acetonitrile-d3) δ 7.05−6.93 (m, 1H), 6.89−6.72 (m, 4H), 6.65−6.54
(m, 1H), 4.40 (d, J = 10.6 Hz, 0.5H), 4.31 (br s, 1H), 4.24−4.13 (m,
3H), 3.89 (d, J = 9.6 Hz, 0.5H), 3.59 (d, J = 9.9 Hz, 0.5H), 3.55 (s, 1.5H),
3.49 (s, 1.5H), 3.31 (d, J = 10.6 Hz, 0.5H), 1.63 (s, 1.5H), 1.57 (s, 1.5H),
1.28−1.17 (m, 3H).
Ethyl 4-Chloro-1-(2-{[(5-chloro-[1,3]thiazolo[5,4-b]pyridin-2-
yl)carbamoyl]amino}phenyl)-7-methoxy-3-methyl-2,3-dihy-
dro-1H-indole-3-carboxylate (8a−e). To a solution of 7a (450 mg,
1.25 mmol) and 4-nitrophenyl chloroformate (277 mg, 1.37 mmol) in
DCM (3 mL) was added K2CO3(345 mg, 2.49 mmol). The reaction was
stirred at room temperature (rt) overnight. The solid was filtered off,
and 5-chlorothiazolo[5,4-b]pyridin-2-amine (232 mg, 1.25 mmol) and
DMAP (5 mg, 0.033 mmol) were added. The reaction was sealed and
microwaved at 100 °C for 15 min. The solid was filtered off, con-
centrated, and purified by flash chromatography, eluted with EtOAc/
hexanes to give 8a−e as a white solid (164 mg, 23% yield). LCMS (ESI)
1
m/z 572.2 (M + H)+. H NMR (400 MHz, methanol-d4, 3:2 mixture
of isomers) δ 8.18−8.06 (m, 1H), 7.79−7.64 (m, 1H), 7.36 (dd, J = 8.6,
2.8 Hz, 1H), 7.25−6.94 (m, 3H), 6.91−6.75 (m, 2H), 4.53 (d, J =
10.9 Hz, 1H), 4.26−4.09 (m, 2H), 3.91−3.74 (m, 1H), 3.53 (s, 1.3H),
3.43 (s, 1.7H), 1.68 (s, 1.7H), 1.63 (s, 1.3H), 1.25−1.13 (m, 3H).
Ethyl 4-Chloro-1-(2-{[(5-chloro-[1,3]thiazolo[5,4-b]pyridin-2-
yl)carbamoyl]amino}phenyl)-7-hydroxy-3-methyl-2,3-dihydro-
1H-indole-3-carboxylate (2e). Compound 2e was prepared from
8a−e according to the same procedure as described for 2d. LCMS (ESI)
m/z 558.1 (M + H)+; purity >95%, retention time = 7.97 min (by HPLC
method C). 1H NMR (400 MHz, chloroform-d) δ 8.08 (d, J = 6.1 Hz,
1H), 7.69 (d, J = 8.3 Hz, 1H), 7.28 (m, 1H), 7.21−6.94 (m, 3H), 6.79−
6.51 (m, 2H), 4.42 (d, J = 10.4 Hz, 1H), 4.31−4.11 (m, 2H), 3.42 (d, J =
9.9 Hz, 1H), 1.71 (s, 3H), 1.25 (t, J = 6.9 Hz, 3H).
1-{2-[4-Chloro-7-hydroxy-3-(hydroxymethyl)-3-methyl-2,3-
dihydro-1H-indol-1-yl]phenyl}-3-(5-chloro-[1,3]thiazolo[5,4-b]-
pyridin-2-yl)urea (2f). To a solution of 2e (16 mg, 0.029 mmol) in
tetrahydrofuran (THF; 1 mL) were added LiBH4 (80 μL, 0.16 mmol,
2 M in THF) and toluene (1 mL). The reaction was heated at 100 °C for
6 h before cooling down to rt. The reaction mixture was quenched with
1 N HCl (1 mL), refluxed for 10 min, and then cooled down to rt. The
pH was adjusted to 7 with concentrated NaHCO3 (aq). The mixture was
extracted with EtOAc, dried over Na2SO4, filtered, concentrated, and
purified by flash chromatography, eluted with EtOAc/hexanes to give 2f
as a white solid (3.4 mg, 23% yield). LCMS (ESI) m/z 558.1 (M + H)+;
1
purity 97.9%, retention time = 7.42 min (by HPLC method C). H
NMR (400 MHz, chloroform-d) δ 8.74 (br s, 1H), 8.07 (d, J = 7.6 Hz,
1H), 7.73 (d, J = 8.3 Hz, 1H), 7.32 (d, J = 8.3 Hz, 1H), 7.22 (d, J =
10.1 Hz, 1H), 7.16−7.01 (m, 3H), 6.45 (d, J = 7.8 Hz, 1H), 6.30
(br s, 1H), 4.32 (d, J = 11.4 Hz, 1H), 4.07 (d, J = 11.1 Hz, 1H), 3.59
(d, J = 10.6 Hz, 1H), 3.27 (d, J = 8.6 Hz, 1H), 1.46 (s, 3H).
Compounds 2a−c and 2h. Compounds 2a−c and 2h were
prepared according to the procedure described for 2d.
1-[2-(4-Chloro-7-hydroxy-3,3-dimethyl-2,3-dihydro-1H-indol-1-
yl)phenyl]-3-(5-chloro-[1,3]thiazolo[5,4-b]pyridin-2-yl)urea (2a).
LCMS (ESI) m/z 500.3 (M + H)+; orthogonal HPLC purity >95%,
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retention time = 11.69 min (by HPLC method A), >95%, retention time
= 10.05 min (by HPLC method B). 1H NMR (400 MHz, chloroform-d)
δ 7.91 (br s, 1H), 7.51 (br s, 1H), 7.26 (m, J = 8.8 Hz, 2H), 7.13 (br s,
1H), 7.09−6.99 (m, 2H), 6.68 (d, J = 8.8 Hz, 1H), 6.54 (d, J = 8.8 Hz,
1H), 3.72 (d, J = 9.8 Hz, 1H), 3.12 (d, J = 9.8 Hz, 1H), 1.38 (s, 3H), 1.34
(s, 3H).
3-[2-(4-Chloro-7-hydroxy-3,3-dimethyl-2,3-dihydro-1H-indol-1-
yl)phenyl]-1-[4-(trifluoromethoxy)phenyl]urea (2b). LCMS (ESI)
m/z 492.4 (M + H)+; orthogonal HPLC purity 98.5%, retention time =
10.19 min (by HPLC method A), 98.1%, retention time = 11.70 min
(by HPLC method B). 1H NMR (400 MHz, methanol-d4) δ 8.00 (d, J =
8.2 Hz, 1H), 7.55−7.48 (m, 2H), 7.18 (d, J = 8.2 Hz, 2H), 7.15−7.10 (m,
1H), 6.95 (d, J = 3.8Hz, 2H), 6.82−6.72(m, 2H), 3.82 (d, J = 9.9 Hz, 1H),
3.22 (d, J = 9.9 Hz, 1H), 1.52 (s, 3H), 1.50 (s, 3H).
3-{2-[4-Chloro-7-hydroxy-3-methyl-3-(2,2,2-trifluoroethyl)-2,3-di-
hydro-1H-indol-1-yl]phenyl}-1-[4-(trifluoromethoxy)phenyl]urea
(2c). LCMS (ESI) m/z 560.2 (M + H)+; purity 95.8%, retention time =
8.26 min (by HPLC method C). 1H NMR (400 MHz, chloroform-d, 2:1
diastereomer mixture, major one reported) δ 7.87 (d, J = 8.1 Hz, 1H),
7.64 (d, J = 4.8 Hz, 1H), 7.35−7.24 (m, 3H), 7.21−7.07 (m, 3H), 6.81−
6.74 (m, 1H), 6.67−6.61 (m, 1H), 4.01 (d, J = 10.4 Hz, 1H), 3.26 (d, J =
10.6 Hz, 1H), 2.85−2.58 (m, 2H), 1.63 (s, 3H).
4-Chloro-1-(2-{[(6-fluoro-1,3-benzothiazol-2-yl)carbamoyl]-
amino}phenyl)-7-hydroxy-N,3-dimethyl-2,3-dihydro-1H-indole-3-
carboxamide (2h). LCMS (ESI) m/z 526.0 (M + H)+; purity 98.3%,
retention time = 7.05 min (by HPLC method C). 1H NMR (400 MHz,
methanol-d4,1:1 mixture of isomers, one reported) δ 8.11 (dd, J = 8.1,
1.3 Hz, 1H), 7.62−7.53 (m, 1.5H), 7.31−7.01 (m, 4.5H), 6.73 (d, J =
8.3 Hz, 1H), 6.64 (d, J = 6.8 Hz, 1H), 3.82 (d, J = 9.9 Hz, 1H), 3.66 (d,
J = 10.1 Hz, 1H), 2.76 (s, 3H), 1.66 (s, 3H).
4-Chloro-1-(2-{[(5-chloro-[1,3]thiazolo[5,4-b]pyridin-2-yl)-
carbamoyl]amino}phenyl)-7-methoxy-3-methyl-2,3-dihydro-
1H-indole-3-carboxylic acid (8a−g). To a solution of 8a−e (60 mg,
0.105 mmol) in THF (1 mL) and MeOH (1 mL) at rt was added NaOH
(1 mL, 1 N solution). The reaction was stirred at rt for 6 days. The
reaction mixture was partitioned between water and EtOAc. The EtOAc
layer was washed with water 3 times. The aqueous layer was acidified
with 1 N HCl and extracted with EtOAc until no more product was
detected in the aqueous layer. The combined EtOAc layers were dried
with Na2SO4 and concentrated to yield 8a−g as an off-white solid
(37 mg, 65%). LCMS (ESI) m/z 544.0 (M + H)+. 1H NMR (400 MHz,
chloroform-d) δ 8.14−8.02 (m, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.33−7.28
(m, 1H), 7.23−7.11 (m, 1H), 7.04−6.95 (m, 1H), 6.91−6.83 (m, 1H),
6.69 (d, J = 8.8 Hz, 1H), 6.52 (d, J = 8.2 Hz, 1H), 4.44 (d, J = 10.4 Hz,
1H), 3.49−3.37 (m, 4H), 1.66 (s, 3H).
4-Bromo-7-methoxy-3,3-dimethylindoline (5b). A solution of
4b (1.60 g, 7.37 mmol) and isobutyraldehyde (0.673 mL, 7.37 mmol)
in ethanol (30 mL) was stirred at room temperature. Sulfuric acid
(1.97 mL, 36.9 mmol) was added to the reaction mixture at 0 °C for 0.5 h.
The reaction was stirred at roomtemperature for 2 h and then diluted with
EtOAc and washed with saturated NaHCO3. The combined organics
were dried over MgSO4, filtered, and evaporated. The crude product was
dissolved in MeOH and cooled to 0 °C. Sodium borohydride (0.558 g,
14.7 mmol) was added portionwise and the reaction was stirred for 15 min
and then warmed to room temperature for 30 min. The reaction was
quenched with water and extracted with EtOAc. The combined organics
were dried over MgSO4 and evaporated to give the crude product, which
was purified by flash chromatography, eluted with EtOAc/hexanes to give
5b (0.45 g, 24%). LCMS (ESI) m/z 256, 258 (M + H, M + 2 + H)+. 1H
NMR (300 MHz, chloroform-d) δ 1.46 (s, 6H), 3.36 (s, 2H), 3.83 (s, 3H),
6.52 (d, J = 8.78 Hz, 1H), 6.74−6.89 (m, 1H).
4-Bromo-7-methoxy-3,3-dimethyl-1-(2-nitrophenyl)-2,3-di-
hydro-1H-indole (6b). A solution of 5b (1.61 g, 6.29 mmol) in toluene
(20 mL) was purged with nitrogen for 3 min and added to a sealable flask
containing 1-bromo-2-nitrobenzene (1.52 g, 7.54 mmol), Pd2(dba)3
(0.288 g, 0.314 mmol), rac-BINAP (0.391 g, 0.629 mmol), and Cs2CO3
(6.14 g, 18.86 mmol). The reaction was sealed and heated to 110 °C for
16 h. The reaction was cooled, filtered through Celite, and concentrated.
The crude mixture was purified by flash chromatography, eluted with
EtOAc/hexanes to give 6b as a brown solid (2.18 g, 92%). LCMS (ESI)
m/z 377.0 (M + H)+. 1H NMR (400 MHz, chloroform-d) δ 8.01 (dd,
J = 8.2, 1.6 Hz, 1H), 7.48−7.40 (m, 1H), 7.14 (dd, J = 8.3, 1.0 Hz, 1H),
7.09−7.03 (m, 1H), 7.01−6.97 (m, 1H), 6.57 (d, J = 8.6 Hz, 1H), 3.87−
3.72 (m, 2H), 3.53 (s, 3H), 1.50 (s, 6H).
2-(4-Bromo-7-methoxy-3,3-dimethyl-2,3-dihydro-1H-indol-
1-yl)aniline (7b). To a solution of 6b (1.62 g, 4.41 mmol) in ethanol
(40 mL) were added zinc (2.95 g, 45.1 mmol) and ammonium chloride
(2.41 g, 45.1 mmol), and the reaction was stirred at room temperature,
filtered through Celite, and concentrated. The crude product was
purified by flash chromatography, eluted with EtOAc/hexanes to give 7b
as a white solid (1.195 g, 76%). LCMS (ESI) m/z 347, 349 (M + H,
M + 2 + H)+. 1H NMR (400 MHz, chloroform-d) δ 7.07−6.84 (m, 1H),
6.79−6.70 (m, 1H), 6.69−6.63 (m, 1H), 6.57 (d, J = 8.6 Hz, 1H), 3.92
(br s, 3H), 3.76 (d, J = 9.9 Hz, 1H), 3.48 (s, 3H), 3.23 (d, J = 9.9 Hz, 1H),
1.51 (s, 3H), 1.47 (s, 3H).
3-[2-(4-Bromo-7-methoxy-3,3-dimethyl-2,3-dihydro-1H-
indol-1-yl)phenyl]-1-[4-(trifluoromethoxy)phenyl]urea (8b).
Compound 8b was prepared according to the procedure described for
1
8a−d. LCMS (ESI) m/z 552.0 (M + 2 + H)+. H NMR (400 MHz,
methanol-d4) δ 7.91 (d, J = 8.2 Hz, 1H), 7.42−7.39 (m, 2H), 7.06 (d, J =
8.2 Hz, 2H), 7.04−6.97 (m, 1H), 6.88−6.81 (m, 3H), 6.57−6.53 (m,
1H), 3.71 (d, J = 9.9 Hz, 1H), 3.34 (s, 3H), 3.10 (d, J = 9.9 Hz, 1H), 1.40
(s, 3H), 1.38 (s, 3H).
4-Chloro-1-(2-{[(5-chloro-[1,3]thiazolo[5,4-b]pyridin-2-yl)-
carbamoyl]amino}phenyl)-7-hydroxy-3-methyl-2,3-dihydro-
1H-indole-3-carboxylic acid (2g). To a solution of 8a−g (18 mg,
0.033 mmol) in dichloroethane (DCE; 1 mL) was added BBr3·Me2S
(50 μL, 0.05 mmol, 1 M in DCM). The reaction was heated at 85 °C for
12 h. The reaction was concentrated, redissolved in MeOH, and purified
by reverse-phase preparative HPLC. After removal of solvents, 2g was
obtained as a gray solid (4.2 mg, 23% yield). m/z 529.9 (M + H)+; purity
3-[2-(7-Methoxy-3,3-dimethyl-4-phenyl-2,3-dihydro-1H-
indol-1-yl)phenyl]-1-[4-(trifluoromethoxy)phenyl]urea (9a).
Compound 8b (52 mg, 0.094 mmol), phenylboronic acid (14 mg,
0.115 mmol), Pd(PPh3)4 (11 mg, 9.5 μmol), and Na2CO3 (0.12 mL,
2 M, 0.24 mmol) in dimethyl ether (DME; 2 mL) was bubbled with Ar
for 3 min. The reaction was sealed and refluxed at 100 °C for 50 h. The
reaction mixture was cooled down to rt and the solid was filtered off,
rinsed with EtOAc, concentrated, and purified by flash chromatography,
eluted with EtOAc/hexanes to give 9a as an off-white solid (51 mg,
1
94.4%, retention time = 7.12 min (by HPLC method C). H NMR
(400 MHz, acetonitrile-d3) δ 8.80 (d, J = 18.7 Hz, 1H), 8.15−8.08 (m,
1H), 7.92−7.86 (m, 1H), 7.44−7.38 (m, 1H), 7.32−7.20 (m, 1H),
7.18−7.02 (m, 2H), 6.87−6.66 (m, 2H), 4.64−4.53 (d, J = 10.9 Hz, 1H),
3.32 (d, J = 10.9 Hz, 1H), 1.61 (s, 3H).
1
99% yield). LCMS (ESI) m/z 548.1 (M + H)+. H NMR (400 MHz,
(5-Bromo-2-methoxyphenyl)hydrazine (4b). A suspension of
5-bromo-2-methoxyaniline (1.74 g, 8.62 mmol) in 6 M HCl (30 mL)
was cooled to −10 °C, and a solution of sodium nitrite (0.714 g,
10.4 mmol) in water (3 mL) was added dropwise. After the addition,
the reaction was stirred for an additional 30 min at −10 °C. A solution of
tin(II) chloride (4.90 g, 25.9 mmol) in concentrated HCl (10 mL) was
added slowly, resulting in precipitation. After 30 min, the precipitate
was filtered, washed with 1 M HCl, suspended in 10% NaOH solution,
and extracted with ether (3×). The combined organic layers were
dried over MgSO4 and evaporated to give 4b, which was used directly
in the next step without further purification. LCMS (ESI) m/z 200,
202 (M − NH3 + H, M − NH3 + 2 + H)+.
chloroform-d) δ 7.65 (d, J = 7.8 Hz, 1H), 7.39−7.29 (m, 7H), 7.17−7.02
(m, 5H), 6.76−6.73 (m, 2H), 3.66 (d, J = 10.1 Hz, 1H), 3.57 (s, 3H),
3.25 (d, J = 10.1 Hz, 1H), 1.06 (s, 3H), 0.96 (s, 3H).
3-[2-(7-Hydroxy-3,3-dimethyl-4-phenyl-2,3-dihydro-1H-
indol-1-yl)phenyl]-1-[4-(trifluoromethoxy)phenyl]urea (10a).
To a solution of 9a (51 mg, 0.093 mmol) in DCE (2 mL) was added
(CH3)2S·BBr3 (0.4 mL, 0.4 mmol, 1 M in DCM). The reaction was
heated at 85 °C for 16 h before cooling down to rt. HCl (1 N, 1 mL) was
added, and the mixture was heated at 65 °C for 1 h before cooling down
to rt. The reaction mixture was partitioned between water and EtOAc.
The combined EtOAc layers were dried over Na2SO4, concentrated, and
purified by flash chromatography, eluted with EtOAc/hexanes to give
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48 mg of off-white solid, which was further purified by reverse-phase
preparative HPLC to yield 10a as a white solid (7 mg, 14% yield). LCMS
(ESI) m/z 534.0 (M + H)+; purity 99%, retention time = 8.39 min
(by HPLC method C). 1H NMR (400 MHz, methanol-d4) δ 7.90−7.84
(m, 1H), 7.54−7.50 (m, 2H), 7.38−7.26 (m, 5H), 7.17 (d, J = 8.3 Hz,
2H), 7.11−6.94 (m, 3H), 6.62 (d, J = 8.1 Hz, 1H), 6.53 (d, J = 8.1 Hz,
1H), 3.73 (d, J = 9.9 Hz, 1H), 3.11 (d, J = 9.9 Hz, 1H), 1.07 (s, 3H),
1.06 (s, 3H).
6.56−6.51 (m, 1H), 3.74 (d, J = 9.9 Hz, 1H), 3.14 (d, J = 9.9 Hz, 1H), 1.12
(d, J = 4.8 Hz, 6H).
1-{2-[6-Fluoro-4-(4-fluorophenyl)-7-hydroxy-3,3-dimethyl-2,3-di-
hydro-1H-indol-1-yl]phenyl}-3-[4-(trifluoromethoxy)phenyl]urea
(10p). LCMS (ESI) m/z 570.3 (M + H)+; orthogonal HPLC purity
>94.8%, retention time = 10.53 min (by HPLC method A), >98%,
retention time = 11.93 min (by HPLC method B). 1H NMR (400 MHz,
chloroform-d) δ 7.80 (d, J = 7.8 Hz, 1H), 7.45−7.29 (m, 3H), 7.25−7.01
(m, 10H), 6.44 (d, J = 10.9 Hz, 1H), 3.59 (d, J = 9.6 Hz, 1H), 3.23 (d, J =
9.6 Hz, 1H), 1.00 (s, 3H), 0.95 (s, 3H).
1-{2-[4-(4-Chlorophenyl)-6-fluoro-7-hydroxy-3,3-dimethyl-2,3-di-
hydro-1H-indol-1-yl]phenyl}-3-[4-(trifluoromethoxy)phenyl]urea
(10r). LCMS (ESI) m/z 586.0 (M + H)+; orthogonal HPLC purity
97.8%, retention time = 10.87 min (by HPLC method A), 97.9%,
retention time = 12.59 min (by HPLC method B). 1H NMR (400 MHz,
chloroform-d) δ 8.01 (br s, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.64 (s, 1H),
7.39−7.28 (m, 4H), 7.25−7.16 (m, 5H), 7.09 (d, J = 8.3 Hz, 2H), 6.46
(d, J = 10.9 Hz, 1H), 3.54 (d, J = 9.6 Hz, 1H), 3.29 (d, J = 9.6 Hz, 1H),
1.01 (s, 3H), 0.90 (s, 3H).
4-(4-Fluorophenyl)-7-methoxy-3,3-dimethyl-1-(2-nitrophen-
yl)-2,3-dihydro-1H-indole (11b). To a mixture of 6b (407 mg,
1.08 mmol), 4-fluorophenylboronic acid (181 mg, 1.30 mmol), and
Pd(PPh3)4 (125 mg, 0.11 mmol) in DME (8 mL) was added Na2CO3
(2.7 mL, 2.70 mmol, 1 M aq). The reaction was bubbled through Ar for
3 min, sealed, and heated at 100 °C for 50 h before gradually cooling
down to rt. The solid was filtered off, rinsed with EtOAc, concentrated,
and purified by flash chromatography, eluted with EtOAc/hexanes to
give 11b as a red-orange solid (415 mg, 98% yield). LCMS (ESI) m/z
393.0 (M + H)+. 1H NMR (400 MHz, chloroform-d) δ 8.05 (dd, J = 8.3,
1.5 Hz, 1H), 7.44 (ddd, J = 8.4, 7.0, 1.8 Hz, 1H), 7.31−7.27 (m, 2H),
7.15 (dd, J = 8.3, 1.3 Hz, 1H), 7.09−6.97 (m, 3H), 6.76−6.66 (m, 2H),
3.77 (d, J = 9.3 Hz, 1H), 3.64 (d, J = 9.6 Hz, 1H), 3.60 (s, 3H), 1.17 (s,
3H), 1.02 (s, 3H).
Compounds 10b−i, 10p, and 10r. Compounds 10b−i, 10p, and
10r were prepared according to the procedure described for 10a.
3-{2-[4-(2-Fluorophenyl)-7-hydroxy-3,3-dimethyl-2,3-dihydro-
1H-indol-1-yl]phenyl}-1-[4-(trifluoromethoxy)phenyl]urea (10b).
LCMS (ESI) m/z 552.3 (M + H)+; purity 95.6%, retention time =
1
8.32 min (by HPLC method C). H NMR (400 MHz, methanol-d4)
δ 7.89 (d, J = 8.2 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H), 7.43−6.92 (m, 9H),
6.65 (d, J = 8.2 Hz, 1H), 6.53 (d, J = 7.1 Hz, 1H), 3.87−3.65 (m, 1H),
3.11 (d, J = 9.9 Hz, 1H), 1.14−0.99 (m, 6H).
3-{2-[4-(3-Fluorophenyl)-7-hydroxy-3,3-dimethyl-2,3-dihydro-
1H-indol-1-yl]phenyl}-1-[4-(trifluoromethoxy)phenyl]urea (10c).
LCMS (ESI) m/z 552.0 (M + H)+; purity >95%, retention time =
8.42 min (by HPLC method C). 1H NMR (400 MHz, methanol-d4) δ
7.95−7.83 (m, 1H), 7.54−7.49 (m, 2H), 7.37 (td, J = 7.9, 6.2 Hz, 1H),
7.17 (d, J = 8.3 Hz, 2H), 7.13−6.95 (m, 6H), 6.63 (d, J = 8.1 Hz, 1H),
6.53 (d, J = 8.1 Hz, 1H), 3.74 (d, J = 9.9 Hz, 1H), 3.12 (d, J = 9.9 Hz,
1H), 1.09 (s, 6H).
3-{2-[4-(4-Fluorophenyl)-7-hydroxy-3,3-dimethyl-2,3-dihydro-
1H-indol-1-yl]phenyl}-1-[4-(trifluoromethoxy)phenyl]urea (10d).
LCMS (ESI) m/z 552.0 (M + H)+; purity 99.8%, retention time =
8.34 min (by HPLC method C). 1H NMR (400 MHz, methanol-d4) δ
7.93−7.82 (m, 1H), 7.55−7.47 (m, 2H), 7.32−7.25 (m, 2H), 7.17 (d, J =
8.3 Hz, 2H), 7.13−7.05 (m, 3H), 7.04−6.95 (m, 2H), 6.62 (d, J = 8.1 Hz,
1H), 6.52 (d, J = 8.1 Hz, 1H), 3.74 (d, J = 9.9 Hz, 1H), 3.11 (d, J = 9.9
Hz, 1H), 1.07 (s, 6H).
2-[4-(4-Fluorophenyl)-7-methoxy-3,3-dimethyl-2,3-dihydro-
1H-indol-1-yl]aniline (12b). To a solution of 11b (415 mg,
1.06 mmol) in MeOH (5 mL) and EtOAc (5 mL) were added Zn
(1.3 g, 19.88 mmol) and NH4Cl (1.1 g, 20.56 mmol). The reaction was
stirred at rt for 1 h. The solid was filtered off, rinsed with MeOH, and
concentrated to yield a crude product, to which were added EtOAc and
Na2CO3 (1 N aq) to adjust pH > 7. The EtOAc layer was dried over
MgSO4 and concentrated to yield 12b as a white solid (369 mg, 96%
yield). LCMS (ESI) m/z 363.1 (M + H)+. 1H NMR (400 MHz,
chloroform-d) δ 7.32−7.26 (m, 2H), 7.11−6.91 (m, 4H), 6.81−6.65 (m,
3H), 6.59 (d, J = 8.1 Hz, 1H), 3.96 (br s, 2H), 3.64 (d, J = 9.6 Hz, 1H),
3.55 (s, 3H), 3.17 (d, J = 9.6 Hz, 1H), 1.15 (s, 3H), 1.03 (s, 3H).
1-(2-Aminophenyl)-4-(4-fluorophenyl)-3,3-dimethyl-2,3-di-
hydro-1H-indol-7-ol (13b). To a solution of 12b (1.3536 g,
3.73 mmol) in DCM (18.67 mL) was added tetrabutylammonium
iodide (TBAI; 7.90 g, 21.40 mmol). The reaction was cooled to −78 °C,
and BCl3 in heptanes (9.64 mL, 9.64 mmol) was gradually added. The
reaction was allowed to warm to rt over 3 h before it was recooled to
0 °C. Ice was added and MeOH was gradually added. After being stirred
for 30 min, the reaction was partitioned between DCM and water with a
small amount of saturated NaHCO3. Aqueous was extracted 2× with
DCM. The combined organic layers were dried over MgSO4, filtered,
and concentrated. Purification by flash chromatography, eluted with
EtOAc/hexanes, gave 13b as a dark green oil (1.17 g, 90% yield). LCMS
(ESI) m/z 349.0 (M + H)+. 1H NMR (400 MHz, chloroform-d) δ 7.29
(br s, 2H), 7.18−7.01 (m, 4H), 6.89−6.76 (m, 2H), 6.70 (d, J = 8.1 Hz,
1H), 6.55 (d, J = 8.1 Hz, 1H), 4.70 (s, 1H), 3.58 (d, J = 9.1 Hz, 1H), 3.24
(d, J = 8.6 Hz, 1H), 1.19 (br s, 3H), 1.04 (br s, 3H).
3-{2-[4-(4-Chlorophenyl)-7-hydroxy-3,3-dimethyl-2,3-dihydro-
1H-indol-1-yl]phenyl}-1-[4-(trifluoromethoxy)phenyl]urea (10e).
LCMS (ESI) m/z 568.0 (M + H)+; purity 99.7%, retention time =
8.62 min (by HPLC method C). 1H NMR (400 MHz, methanol-d4) δ
7.91−7.84 (m, 1H), 7.56−7.49 (m, 2H), 7.40−7.34 (m, 2H), 7.31−7.24
(m, 2H), 7.21−7.14 (m, 2H), 7.12−7.05 (m, 1H), 7.03−6.93 (m, 2H),
6.63 (d, J = 8.1 Hz, 1H), 6.52 (d, J = 8.1 Hz, 1H), 3.74 (d, J = 9.9 Hz,
1H), 3.11 (d, J = 9.9 Hz, 1H), 1.08 (s, 6H).
3-(2-{7-Hydroxy-3,3-dimethyl-4-[4-(trifluoromethyl)phenyl]-2,3-
dihydro-1H-indol-1-yl}phenyl)-1-[4-(trifluoromethoxy)phenyl]urea
(10f). LCMS (ESI) m/z 602.1 (M + H)+; purity 95.8%, retention time =
8.54 min (by HPLC method C). 1H NMR (400 MHz, methanol-d4) δ
7.88 (dd, J = 8.2, 1.1 Hz, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.55−7.46 (m,
4H), 7.17 (d, J = 8.2 Hz, 2H), 7.13−7.06 (m, 1H), 7.04−6.96 (m, 2H),
6.66 (d, J = 8.2 Hz, 1H), 6.56−6.51 (m, 1H), 3.75 (d, J = 9.9 Hz, 1H),
3.12 (d, J = 9.9 Hz, 1H), 1.08 (s, 6H).
3-{2-[4-(2,4-Difluorophenyl)-7-hydroxy-3,3-dimethyl-2,3-dihydro-
1H-indol-1-yl]phenyl}-1-[4-(trifluoromethoxy)phenyl]urea (10g).
LCMS (ESI) m/z 570.0 (M + H)+; purity 99.2%, retention time =
8.32 min (by HPLC method C). 1H NMR (400 MHz, methanol-d4) δ
7.89 (d, J = 7.8 Hz, 1H), 7.55−7.49 (m, 2H), 7.30 (d, J = 16.4 Hz, 1H),
7.18 (d, J = 8.6 Hz, 2H), 7.13−6.95 (m, 5H), 6.65 (d, J = 7.8 Hz, 1H),
6.51 (br s, 1H), 3.85−3.66 (m, 1H), 3.12 (d, J = 9.6 Hz, 1H), 1.14−0.98
(m, 6H).
3-{2-[4-(3,4-Difluorophenyl)-7-hydroxy-3,3-dimethyl-2,3-dihydro-
1H-indol-1-yl]phenyl}-1-[4-(trifluoromethoxy)phenyl]urea (10h).
LCMS (ESI) m/z 570.0 (M + H)+; purity 100%, retention time =
1
8.44 min (HPLC method C). H NMR (400 MHz, methanol-d4) δ
7.90−7.84 (m, 1H), 7.55−7.48 (m, 2H), 7.31−7.14 (m, 4H), 7.09 (ddd,
J = 8.3, 6.0, 2.4 Hz, 2H), 7.03−6.94 (m, 2H), 6.63 (d, J = 8.1 Hz, 1H),
6.53 (d, J = 8.1 Hz, 1H), 3.74 (d, J = 10.1 Hz, 1H), 3.12 (d, J = 9.9 Hz,
1H), 1.10 (d, J = 2.8 Hz, 6H).
3-{2-[4-(3,5-Difluorophenyl)-7-hydroxy-3,3-dimethyl-2,3-dihydro-
1H-indol-1-yl]phenyl}-1-[4-(trifluoromethoxy)phenyl]urea (10i).
LCMS (ESI) m/z 570.0 (M + H)+; purity 100%, retention time =
8.46 min (by HPLC method C). 1H NMR (400 MHz, methanol-d4) δ
7.92−7.84 (m, 1H), 7.55−7.49 (m, 2H), 7.17 (d, J = 8.3 Hz, 2H), 7.10
(ddd, J = 8.3, 6.3, 2.4 Hz, 1H), 7.03−6.86 (m, 5H), 6.64 (d, J = 8.3 Hz, 1H),
3-(5-Chloro-[1,3]thiazolo[5,4-b]pyridin-2-yl)-1-{2-[4-(4-fluo-
rophenyl)-7-hydroxy-3,3-dimethyl-2,3-dihydro-1H-indol-1-yl]-
phenyl}urea (10j). To a solution of 13b (36 mg, 0.10 mmol) in DCM
(10 mL) were added 4-nitrophenyl chloroformate (24.6 mg, 0.12 mmol)
and Na2CO3 (20 mg, 0.61 mmol). The reaction was stirred at rt for 3 h.
The solid was filtered off and used in the next step without further
purification. LCMS (ESI) m/z 514.3 (M + H)+. The crude product from
the above reaction was dissolved in DCM (3 mL), and 5-chlorothiazolo-
[5,4-b]pyridin-2-amine (37.1 mg, 0.2 mmol) and DMAP (6 mg,
0.05 mmol) were added. The reaction was sealed and microwaved at
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100 °C for 15 min. The solid was filtered off, concentrated, dissolved
in MeOH, and purified by reverse-phase preparative HPLC to give 10j
as a gray solid (8.5 mg, 15% for two steps). LCMS (ESI) m/z 560.0
(M + H)+; HPLC purity 99.2%, retention time = 8.59 min (by HPLC
method C). 1H NMR (400 MHz, methanol-d4) δ 8.11−7.98 (m, 1H),
7.74 (d, J = 8.3 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.33−7.26 (m, 2H),
7.18−7.01 (m, 5H), 6.61 (d, J = 7.4 Hz, 1H), 6.52 (d, J = 7.4 Hz, 1H), 3.72
(d, J = 9.6 Hz, 1H), 3.14 (d, J = 9.6 Hz, 1H), 1.10 (s, 3H), 1.07 (s, 3H).
3-(2-Chloro-1,3-thiazol-4-yl)-1-{2-[4-(4-fluorophenyl)-7-me-
thoxy-3,3-dimethyl-2,3-dihydro-1H-indol-1-yl]phenyl}urea
(9k). A solution of 2-chloro-1,3-thiazole-4-carboxylic acid (25 mg,
0.15 mmol), diphenylphosphoryl azide (DPPA; 44 mg, 0.16 mmol), and
NEt3 (77 μL, 0.55 mmol) in toluene (2 mL) was heated to 100 °C, and
12b (50 mg, 0.14 mmol) in toluene (2 mL) was added. The reaction was
heated at 100 °C for 1 h before cooling down to rt. The reaction mixture
was concentrated and purified by flash chromatography, eluted with
EtOAc/hexanes to give 9k as a white solid (20 mg, 28% yield). LCMS
(ESI) m/z 523.0 (M + H)+.
3-(2-Chloro-1,3-thiazol-4-yl)-1-{2-[4-(4-fluorophenyl)-7-hy-
droxy-3,3-dimethyl-2,3-dihydro-1H-indol-1-yl]phenyl}urea
(10k). To a solution of 9k (20 mg, 0.04 mmol) in DCM (2 mL) was
added AlCl3 (51 mg, 0.04 mmol). The reaction was sealed and micro-
waved at 100 °C for 10 min. The reaction mixture was quenched with
MeOH and purified by reverse-phase preparative HPLC to give 10k as
an off-white solid (10.4 mg, 53% yield). LCMS (ESI) m/z 509.0 (M +
H)+; purity 100%, retention time = 8.23 min (by HPLC method C).
1H NMR (400 MHz, methanol-d4) δ 7.91 (d, J = 7.8 Hz, 1H), 7.29 (dd,
J = 8.6, 5.6 Hz, 2H), 7.18 (s, 1H), 7.13−7.05 (m, 3H), 7.02−6.97 (m,
2H), 6.61 (d, J = 8.1 Hz, 1H), 6.51 (d, J = 8.1 Hz, 1H), 3.75 (d, J = 9.9
Hz, 1H), 3.07 (d, J = 9.9 Hz, 1H), 1.07 (d, J = 3.8 Hz, 6H).
1-{2-[4-(4-Fluorophenyl)-7-hydroxy-3,3-dimethyl-2,3-dihy-
dro-1H-indol-1-yl]phenyl}-3-(2-methyl-1,3-thiazol-4-yl)urea
(10l). Compound 10l was prepared according to the same procedure
described for 10k. LCMS (ESI) m/z 489.0 (M + H)+; purity 100%,
retention time = 7.98 min (by HPLC method C). 1H NMR (400 MHz,
methanol-d4) δ 7.95−7.88 (m, 1H), 7.32−7.25 (m, 2H), 7.13−7.05 (m,
3H), 7.03−6.94 (m, 2H), 6.90 (br s, 1H), 6.61 (d, J = 8.1 Hz, 1H), 6.50
(d, J = 8.1 Hz, 1H), 3.73 (d, J = 9.9 Hz, 1H), 3.08 (d, J = 9.9 Hz, 1H),
2.51 (s, 3H), 1.06 (d, J = 8.8 Hz, 6H).
Compounds 10m−o, 10q, and 10s−x. 1-{2-[4-(4-Fluorophenyl)-
7-hydroxy-3,3-dimethyl-2,3-dihydro-1H-indol-1-yl]phenyl}-3-{4-[1-
(2-methylpropyl)pyrrolidin-2-yl]phenyl}urea (10m). LCMS (ESI)
m/z 593.2 (M + H)+; purity 99.7%, retention time = 6.82 min (by
HPLC method C). 1H NMR (400 MHz, methanol-d4) δ 7.88−7.84 (m,
1H), 7.63−7.58 (m, 2H), 7.48−7.43 (m, 2H), 7.32−7.25 (m, 2H),
7.14−7.06 (m, 3H), 7.03−6.98 (m, 2H), 6.63 (d, J = 8.1 Hz, 1H), 6.53
(d, J = 8.1 Hz, 1H), 4.40−4.32 (m, 1H), 3.98 (s, 2H), 3.88 (dt, J = 12.3,
6.3 Hz, 1H), 3.75 (d, J = 9.9 Hz, 1H), 3.11 (d, J = 9.9 Hz, 1H), 2.97 (dd,
J = 12.9, 5.6 Hz, 1H), 2.84 (dd, J = 12.9, 8.6 Hz, 1H), 2.55−2.45 (m,
1H), 2.37−2.22 (m, 3H), 2.03−1.90 (m, 1H), 1.07 (d, J = 2.8 Hz, 6H),
0.95−0.86 (m, 6H).
>99.9%, retention time = 12.35 min (by HPLC method A), 100%,
retention time = 10.59 min (by HPLC method B). 1H NMR (400 MHz,
DMSO-d6) δ 9.62 (br s, 1H), 9.56 (s, 1H), 8.29 (s, 1H), 8.05 (d, J = 7.8
Hz, 1H), 7.61−7.55 (m, 2H), 7.54−7.49 (m, 2H), 7.36 (br s, 2H), 7.29
(d, J = 8.3 Hz, 2H), 7.05 (ddd, J = 8.4, 5.9, 2.9 Hz, 1H), 6.94−6.89 (m,
2H), 6.54 (d, J = 10.9 Hz, 1H), 3.82 (d, J = 10.1 Hz, 1H), 2.97 (d, J = 10.1
Hz, 1H), 1.00 (d, J = 16.7 Hz, 6H). 19F NMR (376 MHz, DMSO-d6) δ
−57.57, −123.72. 13C NMR (151 MHz, DMSO-d6) δ 152.40, 144.85,
144.77, 142.43, 140.72, 140.70, 139.23, 138.61, 133.77, 132.54, 132.45,
131.75, 127.92, 124.04, 122.72, 121.03, 119.40, 119.34, 117.65, 115.90,
115.77, 101.89, 101,71, 70.86, 29.19, 26.54. Anal. CalcdforC30H24N3O3Cl
F4: C, 61.51; H, 4.14; F, 12.96; N, 7.17; Cl, 6.05. Found: C, 61.68; H, 4.16;
N, 7.14.
1-{2-[4-(5-Chlorothiophen-2-yl)-5-fluoro-7-hydroxy-3,3-dimethyl-
2,3-dihydro-1H-indol-1-yl]phenyl}-3-[4-(trifluoromethoxy)phenyl]-
urea (10s). LCMS (ESI) m/z 592.0 (M + H)+; orthogonal HPLC purity
93.3%, retention time = 10.32 min (by HPLC method A), 98.0%, reten-
1
tion time = 12.49 min (by HPLC method B). H NMR (500 MHz,
DMSO-d6) δ 9.89 (br s, 1H), 9.55 (s, 1H), 8.28 (s, 1H), 8.03 (dd, J = 8.4,
0.8 Hz, 1H), 7.56 (d, J = 9.1 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H), 7.15 (d,
J = 3.9 Hz, 1 H), 7.00−7.09 (m, 1H), 6.95 (d, J = 3.9 Hz, 1H), 6.86−6.90
(m, 2H), 6.54 (d, J = 11.0 Hz, 1H), 3.82 (d, J = 10.2 Hz, 1H), 2.99 (d, J =
10.2 Hz, 1H), 1.13 (s, 6 H). 13C NMR (126 MHz, DMSO-d6) δ 155.89
(d, J = 238.1 Hz), 152.40, 146.20 (d, J = 11.6 Hz), 142.46, 139.21,
138.37, 133.81, 132.29, 132.10, 129.35, 128.26, 126.79, 124.16, 121.80,
121.74, 120.19 (q, J = 255.5 Hz), 119.50, 118.98, 107.46 (d, J = 18.5 Hz),
101.92 (d, J = 27.7 Hz), 70.80, 44.06 (d, J = 2.3 Hz), 28.92, 26.23. 19F
NMR (376 MHz, DMSO-d6) δ −57.17 (s, 3F), −120.93 (s, 1F).
1-(2-{5-Fluoro-7-hydroxy-3,3-dimethyl-4-[5-(trifluoromethyl)-
pyridin-2-yl]-2,3-dihydro-1H-indol-1-yl}phenyl)-3-[4-
(trifluoromethoxy)phenyl]urea (10t). LCMS (ESI) m/z 620.9 (M +
H)+; orthogonal HPLC purity 96.0%, retention time = 10.37 min
(by HPLC method A), 98.3%, retention time = 12.59 min (by HPLC
method B). 1H NMR (500 MHz, DMSO-d6) δ 9.90 (br s, 1H), 9.57 (s,
1H), 9.09 (dd, J = 1.5, 0.7 Hz, 1H), 8.30 (s, 1H), 8.28 (dd, J = 8.0, 2.2 Hz,
1H), 8.05 (dd, J = 8.3, 1.1 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.58 (d, J =
9.1 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 7.06 (ddd, J = 8.3, 6.4, 2.3 Hz, 1H),
6.87−6.96 (m, 2H), 6.61 (d, J = 11.3 Hz, 1H), 3.83 (d, J = 10.2 Hz, 1H),
3.00 (d, J = 10.2 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H). 13C NMR (126
MHz, DMSO-d6) δ 157.52, 154.98 (d, J = 237.0 Hz), 152.42, 145.87 (d,
J = 11.6 Hz), 145.61 (q, J = 4.6 Hz), 142.46, 141.28 (d, J = 3.5 Hz),
139.22, 138.61, 133.62, 133.58 (q, J = 2.3 Hz), 132.12, 126.45, 124.02
(q, J = 32.4 Hz), 124.00, 123.75 (q, J = 272.8 Hz), 121.82, 121.71,
121.62, 120.19 (q, J = 254.3 Hz),119.62, 118.97, 115.49 (d, J = 18.5 Hz),
101.90 (d, J = 26.6 Hz), 71.01, 43.84 (d, J = 2.3 Hz), 28.70, 25.88.
19F NMR (376 MHz, DMSO-d6) δ −57.23 (s, 3F), −60.88 (s, 3F),
−125.00 (s, 1F).
1-(2-{5-Fluoro-7-hydroxy-3,3-dimethyl-4-[6-(trifluoromethyl)-
pyridin-3-yl]-2,3-dihydro-1H-indol-1-yl}phenyl)-3-[4-
(trifluoromethoxy)phenyl]urea (10u). LCMS (ESI) m/z 621.3 (M +
H)+; orthogonal HPLC purity 98.4%, retention time = 10.10 min
(by HPLC method A), >95%, retention time = 11.39 min (by HPLC
method B). 1H NMR (400 MHz, methanol-d4) δ 8.69 (br s, 1H), 8.05
(br s, 1H), 7.93 (d, J = 8.3 Hz, 2H), 7.58−7.52 (m, 2H), 7.21 (d, J =
8.3 Hz, 2H), 7.16−7.10 (m, 1H), 7.05−7.00 (m, 2H), 6.54 (d, J =
10.9 Hz, 1H), 3.82 (d, J = 9.9 Hz, 1H), 3.17 (d, J = 10.1 Hz, 1H), 1.09
(d, J = 1.0 Hz, 6H). 13C NMR (126 MHz, DMSO-d6) δ 154.89 (d, J =
236.5 Hz), 152.38, 151.26, 145.72 (q, J = 34.3 Hz), 145.66 (d, J =
11.4 Hz), 142.43 (q, J = 1.9 Hz), 140.95, 140.39, 139.19, 138.37, 133.81,
133.39, 132.19, 124.19, 121.80, 121.75, 121.66 (q, J = 273.7 Hz), 120.19,
120.17 (q, J = 255.6 Hz), 119.48, 118.96, 111.98 (d, J = 19.1 Hz), 101.95
(d, J = 26.7 Hz), 70.71, 43.78, 29.26, 26.78.
3-{4-[1-(2,2-Dimethylpropyl)pyrrolidin-2-yl]phenyl}-1-{2-[4-(4-flu-
orophenyl)-7-hydroxy-3,3-dimethyl-2,3-dihydro-1H-indol-1-yl]-
phenyl}urea (10n). LCMS (ESI) m/z 607.2 (M + H)+; purity 97.6%,
retention time = 7.08 min (by HPLC method C). 1H NMR (400 MHz,
methanol-d4) δ 7.86 (d, J = 7.6 Hz, 1H), 7.63 (d, J = 8.6 Hz, 2H), 7.48 (d,
J = 8.6 Hz, 2H), 7.29 (dd, J = 8.7, 5.4 Hz, 2H), 7.13−7.06 (m, 3H),
7.03−6.99 (m, 2H), 6.62 (d, J = 8.1 Hz, 1H), 6.55−6.50 (m, 1H), 4.36
(d, J = 10.9 Hz, 1H), 4.10−4.00 (m, 1H), 3.75 (d, J = 9.9 Hz, 1H), 3.46−
3.38 (m, 1H), 3.15−3.01 (m, 3H), 2.52−2.39 (m, 1H), 2.37−2.23 (m,
3H), 1.07 (d, J = 3.0 Hz, 6H), 0.89 (s, 9H).
1-{2-[5-Fluoro-4-(4-fluorophenyl)-7-hydroxy-3,3-dimethyl-2,3-di-
hydro-1H-indol-1-yl]phenyl}-3-[4-(trifluoromethoxy)phenyl]urea
(10o). LCMS (ESI) m/z 570.0 (M + H)+; purity 98.8%, retention time =
8.42 min (by HPLC method C). 1H NMR (400 MHz, methanol-d4) δ
7.88 (d, J = 8.1 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H), 7.30 (br s, 2H), 7.21−
7.04 (m, 5H), 6.99 (br s, 2H), 6.45 (d, J = 10.6 Hz, 1H), 3.77 (d, J = 10.1
Hz, 1H), 3.10 (d, J = 9.9 Hz, 1H), 1.04 (d, J = 9.3 Hz, 6H).
1-{2-[4-(2,6-Difluoropyridin-4-yl)-5-fluoro-7-hydroxy-3,3-dimeth-
yl-2,3-dihydro-1H-indol-1-yl]phenyl}-3-[4-(trifluoromethoxy)-
phenyl]urea (10v). LCMS (ESI) m/z 589.0 (M + H)+; orthogonal
HPLC purity 99.6%, retention time = 9.98 min (by HPLC method A),
1
>99%, retention time = 11.67 min (by HPLC method B). H NMR
(400 MHz, methanol-d4) δ 7.80 (d, J = 7.8 Hz, 1H), 7.46−7.40 (m, 2H),
7.08 (d, J = 8.6 Hz, 2H), 7.04−6.97 (m, 1H), 6.93−6.86 (m, 4H), 6.40
(d, J = 10.9 Hz, 1H), 3.69 (d, J = 10.1 Hz, 1H), 3.06 (d, J = 10.1 Hz, 1H),
1.03 (s, 6H). 13C NMR (151 MHz, DMSO-d6) δ 160.04 (dd, J = 244.1,
1-{2-[4-(4-Chlorophenyl)-5-fluoro-7-hydroxy-3,3-dimethyl-2,3-di-
hydro-1H-indol-1-yl]phenyl}-3-[4-(trifluoromethoxy)phenyl]urea
(10q). LCMS (ESI) m/z 585.9 (M + H)+; orthogonal HPLC purity
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15.3 Hz), 153.90 (d, J = 237.2 Hz), 153.12 (d, J = 16.6 Hz), 152.38,
145.83 (d, J = 11.1 Hz), 142.44, 139.99, 139.18, 138.36, 133.76, 132.23,
124.17, 121.78, 121.74, 121.69, 120.17 (q, J = 255.2 Hz), 119.56, 118.97,
112.42 (d, J = 19.4 Hz), 108.98 (dd, J = 33.3, 4.2 Hz), 102.05 (d, J =
26.4 Hz), 70.76, 43.78, 29.05, 26.52.
7.47 (ddd, J = 8.3, 7.1, 1.5 Hz, 1H), 7.24 (d, J = 5.6 Hz, 2H), 7.20−7.15
(m, 1H), 7.13−7.04 (m, 3H), 6.57 (d, J = 10.6 Hz, 1H), 4.22 (br s, 2H),
3.84 (d, J = 9.9 Hz, 1H), 3.59 (s, 3H), 2.72 (t, J = 12.9 Hz, 1H), 2.56 (t,
J = 12.8 Hz, 1H), 1.81 (d, J = 13.4 Hz, 1H), 1.61 (td, J = 13.4, 4.3 Hz,
3H), 1.47 (dd, J = 13.6, 2.0 Hz, 1H), 1.18−1.03 (m, 9H).
1-{2-[5-Fluoro-4-(5-fluoropyrimidin-2-yl)-7-hydroxy-3,3-dimethyl-
2,3-dihydro-1H-indol-1-yl]phenyl}-3-[4-(trifluoromethoxy)phenyl]-
urea (10w). LCMS (ESI) m/z 571.9 (M + H)+; orthogonal HPLC
purity 99.0%, retention time = 10.83 min (by HPLC method A), 92.3%,
retention time = 9.34 min (by HPLC method B). 1H NMR (400 MHz,
methanol-d4) δ 8.84−8.75 (s, 2H), 7.89−7.76 (m, 1H), 7.50−7.37 (m,
2H), 7.17−7.07 (m, 2H), 7.04−6.97 (m, 1H), 6.93−6.84 (m, 2H), 6.40
(d, J = 11.0 Hz, 1H), 3.70 (d, J = 10.4 Hz, 1H), 3.06−3.01 (m, 1H), 1.02
(s, 3H), 0.98−0.92 (m, 3H). 13C NMR (151 MHz, DMSO-d6) δ 158.70
(d, J = 5.5 Hz), 155.44 (d, J = 238.6 Hz), 154.03 (d, J = 482.7 Hz),
152.40, 145.78 (d, J = 11.1 Hz), 145.12 (d, J = 19.4 Hz), 142.43, 141.30
(d, J = 4.2 Hz), 139.20, 138.63, 133.50, 131.63, 123.94, 121.82, 121.74,
121.68, 120.17 (q, J = 255.2 Hz), 119.50, 118.95, 115.01 (d, J = 18.0 Hz),
101.85 (d, J = 26.4 Hz), 70.90, 43.68, 28.29, 25.50.
2-{1′-(2,2-Dimethylpropyl)-5-fluoro-4-(4-fluorophenyl)-7-
methoxy-1,2-dihydrospiro[indole-3,4′-piperidine]-1-yl}aniline
(18b). To a solution of 17b (2.29 g, 4.28 mmol) in EtOAc (21.38 mL)
and MeOH (21.38 mL) were added ammonium chloride (4.57 g,
86 mmol) and zinc (5.59 g, 86 mmol). The reaction was stirred at rt for 1 h.
The solid was filtered off, rinsed with EtOAc, concentrated, and purified by
flash chromatography, eluted with EtOAc/hexanes to give aniline as a white
solid (1.44 g, 67% yield). LCMS (ESI) m/z 506.3 (M + H)+. To a solution
of the above aniline (1.44 g, 2.85 mmol) in DCM (28.5 mL) was added
RED-Al (4.34 mL, 14.24 mmol) dropwise for 20 min. The reaction turned
cloudy and was stirred at rt overnight. The reaction was quenched by
adding drops of aqueous NaHCO3 into the reaction mixture. It was then
diluted with DCM and washed with aqueous NaHCO3. The aqueous layer
was extracted again with DCM. The combined organic layers were dried
over MgSO4, concentrated, and purified by flash chromatography, eluted
with EtOAc/hexanes to give 18b as a white foam (858 mg, 61% yield).
LCMS (ESI) m/z 492.4 (M + H)+.
1-{2-[4-(5-Chloropyrazin-2-yl)-5-fluoro-7-hydroxy-3,3-dimethyl-
2,3-dihydro-1H-indol-1-yl]phenyl}-3-[4-(trifluoromethoxy)phenyl]-
1
urea (10x). LCMS (ESI) m/z 588.2 (M + H)+. H NMR (400 MHz,
methanol-d4) δ 8.79 (d, J = 1.8 Hz, 1H), 8.52 (t, J = 1.5 Hz, 1H), 7.91
(d, J = 7.5 Hz, 1H), 7.56−7.48 (m, 2H), 7.18 (d, J = 8.8 Hz, 2H), 7.10
(ddd, J = 8.3, 5.7, 3.1 Hz, 1H), 7.01−6.96 (m, 2H), 6.52 (d, J = 11.0 Hz,
1H), 3.78 (d, J = 9.7 Hz, 1H), 3.14 (d, J = 10.1 Hz, 1H), 1.14 (s, 3H),
1.10 (s, 3H).
3-(5-Chloro-[1,3]thiazolo[5,4-b]pyridin-2-yl)-1-(2-{1′-(2,2-di-
methylpropyl)-5-fluoro-4-(4-fluorophenyl)-7-methoxy-1,2-
dihydrospiro[indole-3,4′-piperidine]-1-yl}phenyl)urea (19c). To
a solution of18b (858 mg, 1.745 mmol) and K2CO3 (482 mg, 3.49 mmol)
in DCE (15 mL) was added 4-nitrophenyl chloroformate (387 mg,
1.920 mmol) portionwise, and the solution was stirred for 1 h at rt.
5-Chlorothiazolo[5,4-b]pyridin-2-amine (486 mg, 2.62 mmol) and
DMAP (213 mg, 1.745 mmol) were added, and the mixture was stirred
at 75 °C for 4.5 h. The reaction was diluted with DCM and washed with
1 N NaOH (30 mL × 2) and brine consecutively. The organic layer was
dried over MgSO4, concentrated, and purified by flash chromatography,
eluted with EtOAc/hexanes to give 19c as a pale yellow solid (981 mg,
1-(4-Bromo-5-fluoro-7-methoxy-1,2-dihydrospiro[indole-
3,4′-piperidine]-1′-yl)-2,2-dimethylpropan-1-one (15). A solu-
tion of (5-bromo-4-fluoro-2-methoxyphenyl)hydrazine (964 mg,
4.1 mmol) and aldehyde 14 (874 mg, 4.43 mmol) in DCM (15 mL)
was stirred at rt for 2 h before it was cooled down to 0 °C. HCl (4 mL,
4 M in 1,6-dioxane) was added and the reaction was gradually warmed
up to rt and stirred at rt overnight. The reaction mixture was
concentrated to yield a solid mixture, which was dissolved in MeOH
(20 mL) and cooled down to rt, and NaBH4 (621 mg, 16.4 mmol) was
added portionwise. After 10 min, the reaction mixture was quenched
with 1 N HCl, adjusted with concentrated NaHCO3 (aq) to neutral pH,
extracted with EtOAc (50 mL × 2), dried over Na2SO4, concentrated,
and purified by flash chromatography, eluted with EtOAc/hexanes to
give 15 as a beige solid (610 mg, 37% yield). LCMS (ESI) m/z 401.2
1
80% yield). LCMS (ESI) m/z 703.5 (M + H)+. H NMR (400 MHz,
chloroform-d) δ 8.04 (br s, 1H), 7.44 (br s, 1H), 7.35−7.27 (m, 1H),
7.22 (d, J = 8.6 Hz, 2H), 7.16−7.03 (m, 5H), 6.58 (d, J = 10.9 Hz, 1H),
3.81 (d, J = 10.4 Hz, 1H), 3.56 (s, 3H), 3.26 (d, J = 10.1 Hz, 1H), 2.36 (t,
J = 13.0 Hz, 2H), 2.10−1.93 (m, 2H), 1.82−1.70 (m, 2H), 1.65−1.58 (m,
1H), 1.39−1.11 (m, 3H), 0.63 (s, 9H).
3-(5-Chloro-[1,3]thiazolo[5,4-b]pyridin-2-yl)-1-(2-{1′-(2,2-di-
methylpropyl)-5-fluoro-4-(4-fluorophenyl)-7-hydroxy-1,2-
dihydrospiro[indole-3,4′-piperidine]-1-yl}phenyl)urea (20c). To
a solution of 19c (1.27 g, 1.806 mmol) in DCM (36.1 mL) was added
tetrabutylammonium iodide (4.00 g, 10.84 mmol). The reaction was
cooled down to −78 °C and degassed several times with vacuum/argon.
Trichloroborane (9.03 mL, 9.03 mmol) was added dropwise. The reac-
tion was slowly warmed to rt overnight. MeOH and H2O were added,
and the mixture was stirred for 30 min. The reaction was concentrated
and diluted with DCM; washed with saturated NaHCO3, NH4Cl, and
brine; dried over MgSO4; filtered; and concentrated. The crude mixture
was purified by flash chromatography, eluted with EtOAc/hexanes to
give 19c as a brownish solid, which was further purified by reverse-phase
preparative HPLC to give 20c as a brownish solid (430 mg, 30% yield).
Compound 20c could be converted to the methanesulfonate salt by
dissolving in EtOAc/EtOH, followed by addition of methanesulfonic
acid (MSA; 1.1 equiv). The salt precipitate was filtered and washed with
EtOH. LCMS (ESI) m/z 689.3 (M + H)+; orthogonal HPLC purity
100%, retention time = 7.71 min (by HPLC method A), 100%, retention
time = 9.29 min (by HPLC method B). 1H NMR (500 MHz, DMSO-d6)
δ 11.75 (s, 1H), 11.81 (s, 1H), 9.66 (s, 1H), 9.13 (br s, 1H), 8.75 (br s,
1H), 8.15 (dd, J = 8.20, 1.26 Hz, 1H), 8.04 (d, J = 8.51 Hz, 1H), 7.50 (J =
8.51 Hz, 1H), 7.50−7.24 (m, 4H), 7.20 (J = 8.20 Hz, 1H), 7.17 (dd, J =
7.88, 1.58 Hz, 1H), 7.06 (m, 1H), 6.56 (J = 10.7, 1H), 4.06 (d, J = 10.09
Hz, 1H), 3.27 (d, J = 10.40 Hz, 1H), 2.93−3.22 (m, 4H), 2.39 (dd, J =
13.24, 3.78 Hz, 1H), 2.21 (dd, J = 13.08, 3.63 Hz, 1H), 1.61−2.11 (m,
4H), 0.90 (s, 9H). 13C NMR (151 MHz, DMSO-d6) δ 162.75, 160.80,
159.16, 155.54, 154.63, 153.68, 151.32, 144.32, 144.12, 141.91, 138.03,
136.18, 134.40, 133.87, 133.30, 132.83, 129.58, 129.34, 125.65, 124.22,
123.19, 121.89, 119.04, 116.07, 115.48, 102.74, 65.70, 56.03, 49.80,
1
(M + H)+. H NMR (400 MHz, methanol-d4) δ 6.72 (dd, J = 10.5,
1.1 Hz, 1H), 4.46 (d, J = 13.1 Hz, 2H), 3.81 (s, 3H), 3.61 (s, 2H), 2.98 (t,
J = 12.5 Hz, 2H), 2.59 (td, J = 13.3, 4.2 Hz, 2H), 1.65 (d, J = 13.1 Hz,
2H), 1.31 (s, 9H).
1-{4-Bromo-5-fluoro-7-methoxy-1-(2-nitrophenyl)-1,2-
dihydrospiro[indole-3,4′-piperidine]-1′-yl}-2,2-dimethylpro-
pan-1-one (16). A mixture of 15 (610 mg, 1.53 mmol), 1-bromo-2-
nitrobenzene (370 mg, 1.83 mmol), Cs2CO3(1.5 g, 4.58 mmol), BINAP
(95 mg, 0.15 mmol), and Pd2(dba)3 (70 mg, 0.08 mmol) in toluene
(20 mL) was degassed with Ar, sealed, and heated at 110 °C for 16 h
before cooling down to rt. The solid was filtered off, rinsed with EtOAc,
concentrated, and purified by flash chromatography, eluted with EtOAc/
hexanes to give 16 as a brown solid (685 mg, 86% yield). LCMS (ESI)
m/z 522.3 (M + H)+. 1H NMR (400 MHz, chloroform-d) δ 7.92 (dd,
J = 8.3, 1.5 Hz, 1H), 7.44−7.38 (m, 1H), 7.13−7.02 (m, 2H), 6.51 (d,
J = 10.1 Hz, 1H), 4.53−4.33 (m, 2H), 4.11 (d, J = 9.9 Hz, 1H), 3.89 (d,
J = 9.9 Hz, 1H), 3.46 (s, 3H), 2.89−2.75 (m, 2H), 2.72−2.61 (m, 1H),
2.54 (td, J = 13.1, 4.5 Hz, 1H), 1.75 (d, J = 11.6 Hz, 1H), 1.54 (br s, 1H),
1.24 (s, 9H).
1-{5-Fluoro-4-(4-fluorophenyl)-7-methoxy-1-(2-nitrophenyl)-
1,2-dihydrospiro[indole-3,4′-piperidine]-1′-yl}-2,2-dimethyl-
propan-1-one (17b). A mixture of 16 (101 mg, 0.194 mmol),
4-fluorophenylboronic acid (33 mg, 0.233 mmol), Pd(PPh3)4 (24 mg,
0.019 mmol), and Na2CO3 (0.5 mL, 1 M aq) in DME (4 mL) was
degassed with Ar, sealed, and heated at 100 °C for 48 h before cooling
down to rt. The reaction mixture was partitioned between water and
ether, washed with brine, dried over MgSO4, concentrated, and purified
by flash chromatography, eluted with EtOAc/hexanes to give 17b as
an orange solid (88 mg, 85% yield). LCMS (ESI) m/z 536.4 (M + H)+.
1H NMR (400 MHz, chloroform-d) δ 8.05 (dd, J = 8.1, 1.5 Hz, 1H),
6162
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44.24, 30.97, 26.48. Anal. Calcd for C36H35ClF2N6O2S·0.93CH4SO3:
C, 55.37; H, 5.49; N, 10.09. Found: C, 55.28; H, 5.23; N, 9.98.
Compounds 20a and 20b. Compounds 20a and 20b were
prepared according to the procedure described for 20c.
1-(2-{4-(4-Chlorophenyl)-1′-(2,2-dimethylpropyl)-5-fluoro-7-hy-
droxy-1,2-dihydrospiro[indole-3,4′-piperidine]-1-yl}phenyl)-3-[4-
(trifluoromethoxy)phenyl]urea (20a). LCMS (ESI) m/z 697.5 (M +
H)+; purity 94.3%, retention time = 6.87 min (by HPLC method C).
1H NMR (400 MHz, methanol-d4, mixture of two rotamers) δ 7.96−
7.84 (m, 1H), 7.57−7.44 (m, 5H), 7.38−7.31 (m, 1H), 7.27−7.10 (m,
3H), 7.06−6.97 (m, 2H), 6.58−6.48 (m, 1H), 4.11−4.01 (m, 1H),
3.55−3.46 (m, 1H), 3.18−3.01 (m, 3H), 2.50−2.33 (m, 2H), 2.28−1.94
(m, 3H), 1.90−1.71 (m, 2H), 1.03−0.96 (s, 9H).
1-(2-{1′-(2,2-Dimethylpropyl)-5-fluoro-4-(4-fluorophenyl)-7-hy-
droxy-1,2-dihydrospiro[indole-3,4′-piperidine]-1-yl}phenyl)-3-[4-
(trifluoromethoxy)phenyl]urea (20b). LCMS (ESI) m/z 681.2 (M +
H)+; purity 95.9%, retention time = 6.66 min (by HPLC method C).
1H NMR (400 MHz, methanol-d4, mixture of two rotamers) δ 7.97−
7.86 (m, 1H), 7.58−7.51 (m, 2H), 7.51−7.32 (m, 2H), 7.31−7.22 (m,
2H), 7.19 (d, J = 8.8 Hz, 2H), 7.13 (ddd, J = 8.2, 6.1, 2.7 Hz, 1H), 7.06−
7.00 (m, 2H), 6.57−6.48 (m, 1H), 4.11−4.03 (m, 1H), 3.53−3.46 (m,
1H), 3.20−2.91 (m, 4H), 2.50−2.33 (m, 2H), 2.28−1.69 (m, 4H),
1.05−0.92 (s, 9H).
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AUTHOR INFORMATION
Corresponding Author
*Phone 609-818-6493; fax 609-818-3460; e-mail wu.yang@bms.
com.
■
(6) Hechler, B.; Nonne, C.; Roh, E. J.; Cattaneo, M.; Cazenave, J. P.;
Lanza, F.; Jacobson, K. A.; Gachet, C. MRS2500 [2-iodo-N6-methyl-
(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate], a potent,
selective, and stable antagonist of the platelet P2Y1 receptor with strong
antithrombotic activity in mice. J. Pharmacol. Exp. Ther. 2006, 316, 556−
563.
Notes
The authors declare no competing financial interest.
(7) (a) Bird, J. E.; Wang, X.; Smith, P. L.; Barbera, F.; Huang, C.;
Schumacher, W. A. A platelet target for venous thrombosis? P2Y1
deletion or antagonism protects mice from vena cava thrombosis. J.
Thromb. Thrombolysis 2012, 34, 199−207. (b) Wong, P. C.; Crain, E. J.;
Jiang X.; Bostwick, J. S.; Thibeault, C.; Ruel, R.; Rehfuss, R.;
Schumacher, W. A.; Ogletree, M. L. Differential effects of P2Y12 versus
P2Y1 antagonism on thrombosis, bleeding and ex vivo platelet
aggregation in rabbits. Circulation 2006, 114, II_248, abstract 1310.
(8) For recent publications on small-molecule nonnucleotide P2Y1
antagonists, see (a) Pfefferkorn, J. A.; Choi, C.; Winters, T.; Kennedy,
R.; Chi, L.; Perrin, L. A.; Lu, G.; Ping, Y.-W.; McClanahan, T.;
Schroeder, R.; Leininger, M. T.; Geyer, A.; Schefzick, S.; Atherton, J.
P2Y1 receptor antagonists as novel antithrombotic agents. Bioorg. Med.
Chem. Lett. 2008, 18, 3338−3343. (b) Morales-Ramos, A. I.; Mecom, J.
S.; Kiesow, T. J.; Graybill, T. L.; Brown, G. D.; Aiyar, N. V.; Davenport,
E. A.; Kallal, L. A.; Knapp-Reed, B. A.; Li, P.; Londregan, A. T.; Morrow,
D. M.; Senadhi, S.; Thalji, R. K.; Zhao, S.; Burns-Kurtis, C. L.; Marino, J.
P. Tetrahydro-4-quinolinamines identified as novel P2Y1 receptor
antagonists. Bioorg. Med. Chem. Lett. 2008, 18, 6222−6226. (c) Houston,
D.; Costanzi, S.; Jacobson, K. A.; Harden, T. K. Development of
selective high affinity antagonists, agonists, and radioligands for the P2Y1
receptor. Comb. Chem. High Throughput Screening 2008, 11, 410−419.
(d) Thalji, R. K.; Aiyar, N.; Davenport, E. A.; Erhardt, J. A.; Kallal, L. A.;
Morrow, D. M.; Senadhi, S.; Burns-Kurtis, C. L.; Marino, J. P., Jr.
Benzofuran-substituted urea derivatives as novel P2Y1 receptor
antagonists. Bioorg. Med. Chem. Lett. 2010, 20, 4104−4107. (e) Costanzi,
S.; Kumar, T. S.; Balasubramanian, R.; Harden, T. K.; Jacobson, K. A.
Virtual screening leads to the discovery of novel non-nucleotide P2Y1
receptor antagonists. Bioorg. Med. Chem. 2012, 20, 5254−5261. (f) Baqi,
Y.; Mueller, C. E. Convergent synthesis of the potent P2Y receptor
antagonist MG 50-3-1 based on a regioselective Ullmann coupling
reaction. Molecules 2012, 17, 2599−2615.
ACKNOWLEDGMENTS
■
We thank Dr. William R. Ewing for his help during preparation
of the manuscript and BBRC Chemical Synthesis Group and
BBRC Medicinal Chemistry Group for large-scale synthesis of
intermediate 6 and compound 20c.
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