Identification of novel HSP90α/β isoform selective inhibitors using structure-based drug design. demonstration of potential utility in treating CNS disorders such as huntington's disease

Article

pubs.acs.org/jmc

Identiﬁcation of Novel HSP90α/β Isoform Selective Inhibitors Using

Structure-Based Drug Design. Demonstration of Potential Utility in

Treating CNS Disorders such as Huntington’s Disease

Justin T. Ernst,*^,†Timothy Neubert,^†Michael Liu,^†Samuel Sperry,^†Harmon Zuccola,^‡Amy Turnbull,^†

Beth Fleck,^†William Kargo,^†Lisa Woody,^†Peggy Chiang,^†Dao Tran,^†Weichao Chen,^†Phillip Snyder,^‡

Timothy Alcacio,^†Azin Nezami,^‡James Reynolds,^†Khisal Alvi,^†Lance Goulet,^†and Dean Stamos^†

^†Vertex Pharmaceuticals, 11010 Torreyana Road, San Diego, California 92121, United States

^‡Vertex Pharmaceuticals, 130 Waverly Street, Cambridge, Massachusetts 02139, United States

S

* Supporting Information

ABSTRACT: A structure-based drug design strategy was used to

optimize a novel benzolactam series of HSP90α/β inhibitors to

achieve >1000-fold selectivity versus the HSP90 endoplasmic

reticulum and mitochondrial isoforms (GRP94 and TRAP1,

respectively). Selective HSP90α/β inhibitors were found to be

equipotent to pan-HSP90 inhibitors in promoting the clearance of

mutant huntingtin protein (mHtt) in vitro, however with less cellular

toxicity. Improved tolerability proﬁles may enable the use of

HSP90α/β selective inhibitors in treating chronic neurodegenerative

indications such as Huntington’s disease (HD). A potent, selective,

orally available HSP90α/β inhibitor was identiﬁed (compound 31)

that crosses the blood−brain barrier. Compound 31 demonstrated

proof of concept by successfully reducing brain Htt levels following

oral dosing in rats.

disease,⁷thus decreasing levels of this protein in the CNS of

patients carrying a copy of the mutant allele should reduce or

INTRODUCTION

■

HSP90 is a molecular chaperone that is responsible for the

eliminate disease progression. Selective siRNA knockdown of

proper folding of multiple client proteins.¹Numerous small

the cytosolic forms of HSP90 (α and β) is suﬃcient to promote

molecule HSP90 inhibitor programs have been disclosed in the

the degradation of mutant Huntingtin (mHtt).⁴On the basis of

literature and have been reviewed extensively.²The majority of

these data, we initiated a structure-based drug design strategy to

these programs have targeted the ATP-binding site of the N-

identify novel, brain penetrating HSP90α/β isoform selective

inhibitors for treating HD and potentially other neuro-

degenerative disorders.

In a recent publication, we described the correlation between

terminal domain, which regulates the turnover of folded client

proteins.³All of the HSP90 inhibitors that have entered clinical

trials bind to this site, and to date these studies have been

primarily focused on treating oncology indications.²The use of

chemotype-dependent binding conformations of HSP90α/β

HSP90 inhibitors outside of oncology has been limited, due

predominately to modest tolerability proﬁles. We have recently

disclosed a strategy to decrease mechanism-based toxicities

and isoform selectivity.⁴Several published inhibitors, such as 1

(SNX-0723),⁸possess a modest level of HSP90α/β isoform

associated with inhibiting HSP90.⁴This strategy involves

selectivity. The molecular basis for this selectivity is

hypothesized to be driven by the stabilization of an extended

selectively targeting the cytosolic isoforms of HSP90 (inducible

α-helical conformation in the residue 104−111 region of

α and constitutive β) versus the mitochondria (HSP75/

TRAP1) and endoplasmic reticulum (GRP94) isoforms. This

approach reduces the number of client proteins aﬀected and

retains the functions of GRP94 and TRAP1, which have been

HSP90α/β (Figure 1A).⁴This conformation is believed to be

less energetically favorable or not accessible in the case of

binding GRP94 and TRAP1. Compounds such as 2 (AUY-

922),⁹which have little to no isoform selectivity, do not

shown to be essential for ER and mitochondrial viability,

respectively.⁵

strongly interact with this region of the HSP90 isoforms

(Figure 1B). Thus, for chemical series such as the benzamide-

tetrahydroindolones (i.e., 1), a signiﬁcant level of isoform

Mutant and wild-type Huntingtin (Htt) have been reported

to be client proteins of HSP90, therefore, inhibition of HSP90

(via inhibiting N-terminal ATP-ase activity) results in

degradation of these proteins via the proteosome.⁶Mutant

Htt is the toxic species that is responsible for Huntington’s

Received: January 8, 2014

Published: March 27, 2014

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Figure 1. Isoform selectivity proﬁles and X-ray structures of compounds 1 ((A) PDB code 4NH8) and 2 ((B) PDB code 2VCI) bound to the N-

terminal domain of HSP90α. Residues 104−111 of HSP90α are highlighted in red.

selectivity is obtainable from taking advantage of conforma-

tional aspects of inhibitor binding modes versus residue

diﬀerences in the binding pockets of the four isoforms.

RESULTS AND DISCUSSION

■

The fact that compound 1 possesses some degree of selectivity

versus GRP94 and TRAP1 and was reported to be CNS

penetrating⁸made this compound an attractive starting point

for further optimization. Thus, the main objective of our

approach was to further improve isoform selectivity (K_ivalues

for GRP94 and TRAP1 > 10 μM) while optimizing cellular

potency (mHtt clearance) and CNS exposure proﬁles. An

additional goal was to identify novel chemical matter through

the optimization process. An analysis of the binding mode of 1

to HSP90α (Figure 2) indicates that the 3-methylpyrrole

portion of the tetrahydroindolone core is directed toward the

Figure 2. Binding mode of compound 1 (PDB code 4NH8). The key

helical residues 104−111. Given the impact of this region of the

binding residues of HSP90α are highlighted in blue.

protein on isoform selectivity properties, a set of analogues

utilizing novel tricyclic-tetrahydroindolone motifs was prepared

(analogues 4−10 in Table 1), thus expanding the part of the

scaﬀold directed toward the residue 104−111 helix. These

systems were chosen primarily in an attempt to maximize the ﬁt

into a hydrophobic cleft formed by residues I110, A111, and

V136. In some cases, polar groups were incorporated to probe

for additional interactions with backbone hydrogen bonding

groups or water molecules at the solvent interface.

The N-Me octahydro-β-carboline analogue 8 was identiﬁed

from this set as a suitable starting point for further

optimization, although overall the approach failed to yield a

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Table 1. SAR Studies for Benzamide-tetrahydroindolone Analogues

a

Values were determined using ﬂuorescence polarization-based binding assays.

scaﬀold with improved HSP90α/β binding aﬃnities versus the

parent tetrahydroindolone structure 3.¹⁰The basicity of 8 (pK_a

= 7.6), however, was deemed to be attractive because many

HSP90 inhibitors, including the benzamide-tetrahydroindolone

series (i.e., 3), suﬀer from poor aqueous solubility. The

inactivity of the 7-membered N-Me β-carboline analogue 9

along with reduced binding aﬃnity for the cyclohexene

derivative 7 suggested that the N-Me moiety in 8 is forming

a speciﬁc interaction in the active sites of HSP90α/β. Because

compound 8 has similar potency to tetrahydroindolone 3, this

interaction was thought to oﬀset energetic penalties due to the

incorporation of the additional ring system. An X-ray structure

was obtained for compound 8 bound to HSP90α. The structure

indicated that a hydrogen bond mediated by a water molecule is

present between the basic center of the octahydro-β-carboline

and N51 (Figure 3A). Also, the piperidine ring occupies the

hydrophobic cleft formed in part by helix 104−111 as designed

(Figure 3B). Comparing the X-ray structures for 1 and

compound 8 bound to HSP90α revealed that the binding

modes of the benzamide-tetrahydroindolone systems are highly

conserved (Figure 3C). As expected, incorporation of the

analogous benzamide functionality of 1 into 8 resulted in a 20-

fold increase in HSP90α/β binding aﬃnities (compound 11 in

Table 2). However, increases in GRP94 and TRAP1 binding

aﬃnities were also observed. Comparing the isoform selectivity

ratios of 1 and 11 indicated that the additional piperidine ring

was unsuccessful in improving this property despite forming

additional interactions with helix 104−111. Therefore, it was

deemed that a diﬀerent approach would ultimately have to be

employed to further increase the isoform selectivity of this

system.

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presumably would adopt conformations less ideal for optimal

hydrogen bonding in the active site.

To test this hypothesis, benzolactam 23 was prepared (Table

3). Lactam 23 has similar HSP90α/β binding aﬃnities

compared to primary amide 21 but has improved isoform

selectivity and cellular potency. The X-ray structure of 23 with

HSP90α conﬁrmed the predicted binding mode, with the

benzylic methylene of the lactam ﬁtting tightly with the side

chain of V186 (Figure 5A). Also, the orientation of the lactam

hydrogen bond donor/acceptor pair is identical to that

observed in the case of the primary amide system (Figure

5B). To better understand the ﬂexibility of the lactam system,

analogues 24−26 were prepared. The data for these analogues

clearly indicate the sensitive nature of the hydrogen bond

donor/acceptor pair, with both the 5- and 7-membered lactam

systems being inactive compared to 6-membered lactam 25.

Given the rigidiﬁcation of the hydrogen bond donor/

acceptor system in the lactam, replacement of the ortho aniline

side chain in 23 with a carbon linked side chain was examined.

In the primary amide-based systems (i.e., 14), the N−H of the

aniline side chain forms an intramolecular hydrogen bond to

the carbonyl of the amide. This intramolecular hydrogen bond

is needed to reduce the free rotation of the amide and accounts

for as much as a 20-fold increase in HSP90α/β binding aﬃnity

(comparing 14 with 27). However, in the case of the lactam

system, it was hypothesized that the requirement for the

intramolecular hydrogen bonding was no longer necessary.

Replacement of the aniline was viewed as advantageous because

it eliminates a potential genotoxic risk factor and further

improves the CNS lead-like properties of the system by

removing a hydrogen bond donor and reducing PSA.

Compound 28 was prepared and was found to have very

similar potency and isoform selectivity properties compared to

the analogous aniline analogue 23. Comparing the X-ray

structures of 23 and 28 bound to HSP90α revealed that the

binding modes of the two inhibitors are well conserved, with

the exception of the alkyl side chains which adopt diﬀerent

poses (Figure 6). Removal of the aniline moiety results in

increased conformational mobility of the alkyl side chain due to

loss of the intramolecular hydrogen bond, however, this

increased ﬂexibility has little impact on the HSP90α/β binding

aﬃnities and isoform selectivity.

Compound 28 was evaluated in human HD patient derived

ﬁbroblasts for its eﬀects on HSP90 client protein levels and

HSP70 induction (Figure 7). Dose dependent reductions were

observed for Htt (IC₅₀= 27 nM) as well as client proteins

CDK4 and AKT. Also, increases in HSP70 were clearly

observed at concentrations of 28 that produced client protein

clearance, consistent with the mechanism of inhibiting

HSP90α/β.

The pharmacokinetic properties of compound 28 were

evaluated in rats using subcutaneous and intravenous delivery

(Table 4). Compound 28 suﬀers from high plasma clearance

and limited brain exposure. Evaluation of 28 in a MDR1-

MDCK permeability assay¹¹indicated that it was a P-gp eﬄux

substrate because the ratio of basal-to-apical permeability to

apical-to-basal permeability (BA/AB) is 42. This property is

most likely the key driver of the observed poor brain to plasma

ratio. SAR studies indicated that the basic amine functionality

was a major contributor to the scaﬀold being an eﬄux substrate

(data not shown). In addition, in vitro metabolite identiﬁcation

studies using human liver microsomes indicated rapid turnover

due to oxidation of the piperidine ring system. These data

Figure 3. X-ray structure of 8 (blue) bound to HSP90α (gray ribbon),

PDB code 4O04: (A) hydrogen bonding highlighted from basic

nitrogen to N51 via a water molecule; (B) spaceﬁll model of 8

highlighting the ﬁt of the piperidine system into a hydrophobic pocket

formed by I110, A111, and V136; (C) overlap of the X-ray structures

for 8 and 1 (pink, HSP90α is yellow ribbon).

Compound 11 was tested in a cellular assay to determine the

eﬀects on mHtt clearance and was found to be moderately

potent. Removal of the ﬂuorine substituent (compound 12)

appeared to have little eﬀect on cellular potency and isoform

selectivity. A focused set of compounds was prepared at this

point to explore the eﬀects of aniline substitution on isoform

selectivity and cellular potency (13−22). Although a large,

diverse set of side chains was tolerated at this position, this

approach did not lead to the identiﬁcation of a system that was

both highly potent for mHtt clearance (IC₅₀mHtt < 50 nM)

and isoform selective (K_ivalues for GRP94 and TRAP1 > 10

μM).

An analysis of the X-ray structures of HSP90α and GRP94

bound to 1 revealed that there was a subtle amino acid

diﬀerence in the active sites adjacent to the benzamide moiety

that could oﬀer the opportunity to further modulate isoform

selectivity (Figure 4).⁴In the case of HSP90α and β, residue

186 is a valine. The analogous residue in GRP94 and TRAP1 is

an isoleucine, thus providing slightly increased steric bulk at this

position (Figure 4A). Analysis of the GRP94-bound structure

of 1 revealed that there is a tight ﬁt for this isoleucine and the

ﬂuorine substituent of the benzamide (Figure 4B). It was

hypothesized that optimization of benzylic substitution ortho to

the primary amide could maximize the ﬁt with V186 in the case

of HSP90α/β but sterically clash with the extra methyl of the

analogous isoleucine residue in GRP94 and TRAP1. To ensure

benzylic substitution would not adversely aﬀect the orientation

of the primary amide for optimal hydrogen bonding, cyclization

of this position to the amide was envisioned to rigidify the

system. This strategy oﬀered the advantages of removing an

unneeded hydrogen bond donor of the primary amide and

reducing the overall PSA of the scaﬀold, thus improving the

physicochemical properties of the system to favor CNS

penetration. In addition, it was deemed that the resulting

lactam structures further contributed to the novelty of the

chemical series. In fact, previously reported SAR studies

suggested that secondary amides were not tolerated at this

position,¹⁰although in these cases the amides were acyclic and

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Table 2. Aniline SAR Studies in the Octahydro-β-carboline Series

a

b

Values were determined using ﬂuorescence polarization-based binding assays. Values were derived from an in-cell western assay for mHtt in HEK

cells.

suggested that the piperidine ring was a liability for both eﬄux

and metabolic stability, and thus it was decided that removal of

the basic system was necessary. The disadvantages of this

strategy were that aqueous solubility and cellular potency

properties could be compromised. Previous SAR studies (i.e.,

comparison of 12 and 29 in Figure 8) indicated that the basic

systems generally had greater aqueous solubility and cellular

potency versus nonbasic systems with similar HSP90α/β

binding aﬃnities.

It was decided to revisit the 3-methyl tetrahydroindolone

system as a replacement for the octahydro-β-carboline moiety

in the benzolactam series (compound 30 in Table 5).

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GRP94 and TRAP1, has a >100-fold window between the IC₅₀

for mHtt clearance (6 nM) and the CC₅₀for cytotoxicity (>10

μM). Compound 1, which has modest selectivity, has a much

reduced CC₅₀/IC₅₀mHtt ratio (ratio = 4) compared to 31.

Finally, for 2, which has little to no isoform selectivity, the

CC₅₀/IC₅₀mHtt ratio is essentially one. Overall, these data

support the hypothesis that HSP90α/β isoform selective

inhibitors are equally eﬃcacious for Htt clearance and less

cytotoxic versus pan-HSP90 inhibitors.

Compound 31 was progressed into rat PK/PD studies to

assess its eﬀects on Htt concentrations in brain tissue. It has

been previously shown that the HSP90-mediated clearance of

Htt protein is nonselective for mutant forms of the protein

versus wild-type Htt.⁶Therefore, our initial proof of concept

studies were carried out in nontransgenic rats using wild-type

Htt as the biomarker for eﬃcacy. The rat PK properties of 31

are shown in Table 7. Compound 31 is orally available and

successfully crosses the blood−brain barrier. A 5 mg/kg po

dose given once a day for 1 week was well tolerated and

successfully reduced brain Htt levels by > 50% (Figure 10).

These data provide the ﬁrst in vivo proof of concept

demonstrating that selective HSP90α/β inhibition with a

small molecule can aﬀect brain Htt levels. Overall, these results

support the continued study of the HSP90α/β-mediated

reduction of Htt protein levels as a strategy to treat HD.

Figure 4. (A) Overlap of the X-ray structures of 1 (pink) bound to

HSP90α (yellow ribbon, V186 highlighted in yellow) and 1 (blue)

bound to GRP94 (green ribbon, isoleucine at analogous position to

V186 highlighted in green), PDB code 4NH9; (B) X-ray structure of 1

(blue spaceﬁll model, benzylic F in orange) bound to GRP94 (green

ribbon, key isoleucine highlighted as green spaceﬁll model).

Fortunately, in this case, the potency and selectivity proﬁles of

30 were found to be quite similar to 28, although the aqueous

solubility, as predicted, was much reduced (4 μM versus 122

μM for 28). The rat pharmacokinetic properties of 30 are

shown in Table 4. Overall, a 5-fold improvement in brain

exposure was observed versus compound 28. Evaluation of 30

in the MDR1-MDCK permeability system indicated that

removal of the basic ring successfully mitigated the eﬄux

liability (BA/AB = 1) and is most likely the reason for the

observed increase in the brain to plasma ratio. Also, as

predicted, removal of the piperidine ring system resulted in

reduced plasma clearance.

Given the attractive CNS exposure properties of 30, further

SAR studies were conducted within the lactam−tetrahydroin-

dolone system in an attempt to improve cellular potency while

maintaining good PK and isoform selectivity properties.

Substitution ortho to the amide system was once again revisited

due to the precedented ﬂexibility at this position. Also, an

analysis of the X-ray structure of 30 bound to HSP90α (Figure

9A) indicated that substitution at this position could be used to

“shield” the amide hydrogen bond donor/acceptor system from

solvent and lower the dielectric constant in this part of the

binding pocket.¹²In theory, this could result in increased

binding aﬃnity by strengthening the existing hydrogen bonds

to D93 and T184. A series of analogues was prepared with

carbon linked substitution ortho to the lactam. From these

studies, the cyclopentyl analogue 31 was identiﬁed as having

excellent HSP90α/β binding aﬃnities, cellular potency, and

isoform selectivity properties (Table 5).

An X-ray structure of 31 bound to HSP90α was obtained

(Figure 9B) and indicated that the cyclopentane side chain

completely blocks access of solvent to the hydrogen bond

donor/acceptor system of the lactam. Furthermore, ITC

measurements for the binding of 30 and 31 to HSP90α

indicated that the diﬀerence in binding aﬃnities was due to a

1.6 kcal/mol increase in binding enthalpy for 31. Both of these

results are consistent with the “shielding” hypothesis.

With compound 31 identiﬁed, a set of studies was conducted

to probe whether isoform selectivity versus GRP94 and TRAP1

translates into reduced cytotoxicity in vitro. Compounds 31, 1,

and 2 were tested in an HD-patient ﬁbroblast cell system for

mHtt reduction and cytotoxicity (Table 6). These three

compounds represent varying degrees of isoform selectivity.

Compound 31, which shows no measurable binding aﬃnity to

SYNTHETIC CHEMISTRY

■

The synthesis of compound 3 was previously reported.¹⁰

Analogues 4−10 were prepared using similar methodology.

The corresponding tetrahydroindolone systems 42 were treated

with NaH in DMF and then coupled with 4-ﬂuorocyanoben-

zene under high temperature. Hydrolysis of the nitrile

intermediates 43 with NaOH and H₂O₂in a mixture of

EtOH and DMSO yielded the desired benzamide products

(Scheme 1).

The aza-tetrahydrocarbazolone systems 50−52, needed for

the preparation of products 4−6, were synthesized from

condensation of the appropriately substituted amino-halopyr-

idines 44−46 with dimedone, followed by an intramolecular

Heck cyclization (Scheme 2).¹³

The octahydrocarbazolone intermediate 54, which was

required for the synthesis of 7, was synthesized using a

published modiﬁed Knorr procedure.¹⁴Keto-oxime 53 and

dimedone were heated with Zn in AcOH and H₂O to aﬀord the

desired tricyclic system (Scheme 3).

The syntheses of β-carboline systems 59−61, the building

blocks necessary for the preparation of analogues 8−10, are

shown in Scheme 4. Amino acetals 56−58 were combined with

formaldehyde to form the corresponding imines, which were

then reacted with tetrahydroindolone 55 regioselectively at the

2-position.¹⁵Acidic hydrolysis of the acetals resulted in

cyclization to the 3-position of the tetrahydroindolone system

and subsequent reduction of the oleﬁn intermediates using

catalytic hydrogenation yielded 59−61. In the case of 61, the

Bn protecting group was also removed under these reduction

conditions. The secondary amine of 61 was reprotected with a

BOC group to yield 62 (Scheme 5), which was then used in the

SNAr coupling reaction with 4-ﬂuorocyanobenzene. Removal

of the BOC group and hydrolysis of the nitrile aﬀorded

compound 10.

Octahydro-β-carboline analogue 11 was synthesized as

shown in Scheme 6. (S)-Tetrahydrofuran-3-amine was added

to 4-cyano-3,5-diﬂuorobromobenzene (63) to yield 64.

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Table 3. SAR Studies in the Octahydro-β-carboline Series

a

b

Values were determined using ﬂuorescence polarization-based binding assays. Values were derived from an in-cell western assay for mHtt in HEK

cells.

Buchwald coupling of 64 with 59 and subsequent nitrile

hydrolysis yielded compound 11. Analogues 12−22 were

prepared by coupling octahydro-β-carboline 59 with 3-ﬂuoro-5-

cyanobromobenzene to yield intermediate 65 (Scheme 7).

Various primary amines were incorporated via Buchwald aryl

amination reactions followed by nitrile hydrolyses to yield the

desired compounds 12−22.

Benzolactam 23 synthesis (Scheme 8) began with subjecting

ketone 66 to Schmidt reaction conditions to give a mixture of

lactam regioisomers which favored intermediate 67 (9:1 ratio).

Isopropyl amine was coupled with 67, with exclusive displace-

ment of the ﬂuorine ortho to the amide. Finally, an SNAr

reaction with 59 yielded 23. Lactam products 24−26 were

prepared in one step via coupling of the commercially available

ﬂuorobenzolactams (68) with 59 (Scheme 9).

Two diﬀerent synthetic protocols were developed to

incorporate carbon linked side chains ortho to the primary

amide (i.e., 27) or lactam systems (28, 30−41). The synthesis

of analogue 27 is shown in Scheme 10. Suzuki coupling of 3-

ﬂuoro-5-cyanobromobenzene with (E)-2-(2-cyclopropylvinyl)-

4,4,5,5-tetramethyl-1,3,2-dioxaborolane aﬀorded oleﬁn 69.

Coupling of 69 with 59, catalytic hydrogenation of the oleﬁn,

and ﬁnally hydrolysis of the nitrile gave the desired product 27.

Lactams 28 and 30−41 were prepared starting from

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Figure 5. (A) X-ray structure of 23 (tan spaceﬁll model) bound to

HSP90α (V186 highlighted as yellow spaceﬁll model), PDB code

4O05; (B) X-ray structure of 23 (tan) bound to HSP90α (gray

ribbon) overlaid with X-ray structure of 8 bound to HSP90α (yellow

ribbon).

Figure 8. Eﬀects of basic amine functionality on solubility and cellular

potency properties.

to the amide. These intermediates (70) were then coupled with

tetrahydroindolone 42 (where R₁= H, R₂= Me) under high

temperature conditions to yield the desired products 28 and

30−41.

CONCLUSION

■

In summary, a structure-based design approach was utilized that

took advantage of chemotype-dependent binding conforma-

tions, as well as a subtle amino acid diﬀerence in the active sites

of HSP90α and β, to identify a novel benzolactam series with

>1000-fold selectivity versus HSP90 isoforms GRP94 and

TRAP1. Additional optimization, which included a hydrogen

bond “shielding” strategy, resulted in the identiﬁcation of

compound 31, a potent, selective, orally available HSP90α/β

inhibitor with good CNS exposure. Compound 31 was found

to be equipotent to pan-HSP90 inhibitors in promoting the

clearance of mHtt protein in vitro, however with less cellular

toxicity. Ultimately, compound 31 was used to demonstrate in

vivo proof of concept by successfully reducing brain Htt levels

following oral dosing in rats. Compound 31 has been advanced

into further preclinical studies to probe the eﬀects of mHtt

reduction on delaying disease onset and reversing HD and

other neurodegenerative phenotypes in transgenic animal

models.

Figure 6. Overlay of X-ray structures of 23 (tan) bound to HSP90α

(gray ribbon) and 28 (orange) bound to HSP90α (yellow ribbon),

PDB code 4O07.

EXPERIMENTAL SECTION

■

Figure 7. Eﬀect of compound 28 (increasing concentrations in μM

versus DMSO control) on HSP90 client protein and HSP70 levels in

human HD patient derived ﬁbroblasts. Proteins were quantiﬁed by

Western blot and normalized to a GAPDH loading control.

General. All reagents and solvents were used as purchased from

commercial sources. Flash column chromatography was performed

with a Teledyne ISCO CombiFlash Rf system using normal-phase

silica columns (230−400 mesh). ¹H NMR spectra were recorded on a

Bruker Advance-400 spectrometer at 400 MHz or a Bruker Advance-

500 spectrometer at 500 MHz. ¹³C NMR spectra were recorded on a

Bruker Advance-400 spectrometer at 100 MHz or a Bruker Advance-

500 spectrometer at 125 MHz. Coupling constants (J) are expressed in

hertz (Hz). Chemical shifts (δ) of NMR are reported in parts per

diﬂuorobenzolactam 67 (Scheme 11). Various Grignard

reagents were added to 67, and analogous to SNAr addition

of amines, yielded selective displacement of the ﬂuorine ortho

Table 4. Pharmacokinetic Properties of Compounds 28 and 30 in Rat

dose

AUC plasma (μg·h/mL)

Cl (mL/min/kg)

57.7

V_d(L/kg)

T_1/2(h)

B/P (T = 2 h)

28 @ 1 mg/kg iv

28 @ 10 mg/kg sc

30 @ 1 mg/kg iv

30 @ 10 mg/kg sc

0.28

2.94

0.64

3.58

7.97

2.4

7.0

0.1

0.5

25.7

4.08

2.2

11.6

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Table 5. SAR Studies in the Benzolactam−Tetrahydroindolone Series

a

b

Values were determined using ﬂuorescence polarization-based binding assays. Values were derived from an in-cell western assay for mHtt in HEK

cells.

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Figure 10. In vivo eﬃcacy data (reduction of total brain Htt levels) for

compound 31; (A) rat vehicle control group (n = 5); (B) rats treated

with 5 mg/kg 31 po once a day for 1 week (n = 6).

Figure 9. X-ray structures of (A) 30 (PDB code 4O09) and (B) 31

(4O0B) bound to HSP90α. The degree of lactam carbonyl “shielding”

by the iBu and cyPent side chains of 30 and 31 is highlighted using

space ﬁll models. Water molecules at the edge of the binding cavity are

also highlighted (red plus symbols).

aqueous buﬀer. Calorimety experiments were performed at 25 °C,

stirring at a rate of 307 RPM, with a reference power of 5 μCal s⁻¹.

The HSP90 was injected consecutively (10−20 μL per injection) until

the ligand in the cell reached saturation. Data were analyzed with the

Origin 7.0 software package using a one-site model to estimate the

enthalpy of binding and association constant.

Table 6. Binding Aﬃnity, Cellular Potency, and Toxicity

Properties for Compounds 31, 1, and 2

Pharmacokinetic Studies. In vivowork for the pharmacokinetic

studies was performed at Vertex Pharmaceuticals (San Diego, CA).

Groups of male Sprague−Dawley rats (n = 3 per dose group) were

administered as single doses of 28, 30, or 31. Compounds were

formulated in 10% dimethylisosorbide, 15% ethanol, 35% polyethylene

glycol (PEG400), and 40% dextrose (5% in water, D5W) for

intravenous administration at a nominal dose level of 1 mg/kg.

Compounds were formulated as suspensions in 0.5% carboxymethyl-

cellulose and 0.5% sodium dodecylsulfate in water for oral gavage

administration at a nominal dose level of 5 mg/kg. Compounds were

formulated in 60% miglyol 810, 25% water, 10% dimethyl sulfoxide,

and 5% Tween 80 for subcutaneous administration at a nominal dose

level of 10 mg/kg. For intravenously dosed animals, blood samples

(approximately 0.25 mL each) were collected via a carotid artery

catheter at 0.017, 0.083, 0.17, 0.25, 0.50, 1, 2, 4, 8, 12, and 24 h post

dose. For orally dosed animals, blood samples (approximately 0.25 mL

each) were collected via a carotid artery catheter at 1, 2, 4, 8, 12, 24,

48, and 72 h postdose. For subcutaneously dosed animals, blood

samples (approximately 0.25 mL each) were collected at 0.25, 0.5, 1, 2,

4, 8, 12, and 24 h postdose. Each blood sample was collected into a

tube that was chilled and contained potassium EDTA as the

anticoagulant. Plasma was separated and stored at approximately

−80 °C until analysis. Animals dosed for brain distribution were

euthanized at 2 h post dose and, following perfusion with heparinized

saline, were decapitated and brains harvested. Brains were stored in

conical tubes at approximately −80 °C until analysis. Upon thawing,

whole rat brains were homogenized in three volume equivalents of

water. Following protein precipitation with acetonitrile containing an

internal standard, plasma and brain samples were analyzed using a

liquid chromatography/tandem mass spectrometry (LC/MS/MS)

method to determine the plasma and brain concentrations of 28, 30,

and 31. Plasma concentration versus time data were subjected to

noncompartmental pharmacokinetic analysis to determine AUC, Cl,

31

1

2

a

K_iHSP90α (μM)

0.005 0.002

>10.0

0.003 0.002

0.375 0.022

1.195 0.090

0.030 0.004

0.002 0.0001

0.004 0.003

0.016 0.012

0.007 0.0001

a

K_iGRP94 (μM)

a

K_iTRAP1 (μM)

>10.0

IC₅₀mHtt HD

ﬁbroblast (μM)

0.006 0.004

b

c

CC₅₀mHtt HD

ﬁbroblast (μM)

>10.0

0.140 0.028

0.008 0.001

a

Values were determined using ﬂuorescence polarization-based

binding assays. Values were derived from a Western blot assay for

mHtt in human HD ﬁbroblasts. Viability values determined in human

b

c

HD ﬁbroblasts.

million (ppm) units relative to an internal control (TMS). Microwave

reactions were performed with a Biotage Initiator focused beam

microwave reactor (300 W). HPLC puriﬁcation was performed on a

Waters FractionLink system equipped with a Phenomenex Luna C18

column (5 μm, 75 mm × 30 mm). Analytical purity was assessed on a

Waters Acquity Ultra Performance UPLC system equipped with an

Acquity UPLC BEH C18 column (1.7 μm, 2.1 mm × 30 mm), and all

compounds tested were determined to be >95% pure using this

method. High resolution mass spectroscopy (HRMS) was performed

using an Exactive Plus benchtop Oribtrap mass spectrometer

connected to an UltiMate 3000 UHPLC system (Thermo Scientiﬁc).

The mass spectrometer was operated in positive ionization mode.

Acquisition was a full scan from m/z 150 to 1000 with a resolution of

70000.

Isothermal Calorimetry. HSP90 was extensively dialyzed against

50 mM HEPES, pH 7.5, and 100 mM NaCl, and the ﬁnal dialysate was

retained. Both protein and dialysis buﬀer were aliquoted and stored at

−70 °C. Compounds were stored as 10 mM stock solutions in 100%

DMSO. The DMSO stocks were diluted with the retained dialysate,

and DMSO was added to the protein solution to obtain equal volume

percentage of DMSO in the protein and ligand solution. All

calorimetry experiments were performed in a VP-ITC instrument

(GE/Microcal, Northampton, MA). For each experiment, the protein

solution (100 μM in buﬀer) was loaded into the syringe and

compound (10 μM in identical buﬀer) into the sample cell. This

method was necessary due to the low solubility of compounds in

V_d, and T_1/2

.

Measurement of Mutant Huntingtin Clearance in HEK Cells.

mHtt protein levels were measured using an in-cell Western blot assay

in HEK cell lines stably expressing the full-length mHtt Q73 gene.

Stable inducible cell lines were generated using the Flp-In TRex

system from Life Technologies (catalogue no. R780-07). In brief, full-

length mHtt Q73 protein overexpression was induced in HEK cells

with 50 nM doxycycline and treated with compounds or DMSO.

Table 7. Pharmacokinetic Properties of Compounds 31 in Rat

dose

AUC plasma (μg·h/mL)

Cl (mL/min/kg)

32.4

V_d(L/kg)

T_1/2(h)

B/P (T = 6 h)

% F

1 mg/kg iv

5 mg/kg po

0.53

0.87

3.9

1.9

3.1

0.6

34

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a

Scheme 1. General Methodology to Construct Benzamides 3−10

a

Reagents and conditions: (a) NaH, 4-ﬂuorocyanobenzene, DMF, 100−150 °C; (b) NaOH, H₂O₂, EtOH, DMSO, rt.

a

Scheme 2. Synthesis of aza-Tetrahydrocarbazolone Systems

a

Reagents and conditions: (a) dimedone, PTSA, benzene or toluene, reﬂux; (b) Pd(PPh₃)₄, Cs₂CO₃, DMF, 150 °C.

Following 24 h of incubation, cells were ﬁxed in 10% formalin, washed,

permeabilized, blocked (30 min), and stained with a Htt speciﬁc

antibody (mouse monoclonal mAB2166 Millipore 1:1000 dilution) for

2 h at room temperature. Following incubation, cells were washed and

incubated with a goat antimouse IRDye secondary antibody (LI-COR

Biosciences) and DRAQ-5 DNA dye (for cell viability) (Biostatus

Limited) diluted 1:1000 and 1:10000, respectively, in blocking buﬀer

for 1 h at room temperature. Plates were scanned on the LI-COR

Odyssey NIR Imaging system. Ratiometric analysis of Htt signal to

DRAQ-5 dye was used to generate dose−response curves and IC₅₀

values. IC₅₀values reported with standard deviation measurements are

means of duplicate experiments.

Scheme 3. Synthesis of Octahydrocarbazolone Intermediate

54

a

Reagents and conditions: (a) dimedone, Zn, AcOH, H₂O, 80 °C.

a

Scheme 4. Synthesis of β-Carboline Intermediates

Determination of HSP90α, HSP90β, Grp94, and Trap-1 K_i

Values. K_ivalues were determined using a ﬂuorescence polarization

assay system. The assay was used to identify compounds that inhibit

ﬂuorescein-conjugated geldanamycin (FITC-GA) binding to the

ATPase site of the HSP90 isoforms. In brief, FITC-GA was dispensed

into wells containing compound for a ﬁnal concentration of 5 nM

FITC-GA. HSP90α, HSP90β, GRP94, or TRAP1 recombinant protein

(Assay Designs, catalogue no. SPP-776, no. SPP-777, Prospec Protein

Specialists, catalogue no. HSP-091) in buﬀer (50 mM KCl, 5 mM

MgCl₂, 20 mM HEPES, pH 7.3−7.5, 0.1% CHAPS (Sigma C5070),

0.1% bovine gamma-globulin (Sigma G7516), and 2 mM dithiothreitol

(Sigma 646563)) was then added to the well at ﬁnal concentration of

10 nM. IC₅₀values were determined by measuring the amount of

ﬂuorescence polarization in the well after 2 h at varying concentrations

of test compound (Perkin-Elmer Envision, 485 nm excitation, 535 nm

emission, 505 nm dichroic). K_ivalues were calculated using the

method of Nikolovska.¹⁶K_i= [I]50/([L]50/K_d+ [P]0/K_d+1). [I]50

= concentration of free inhibitor at 50% inhibition, [L]50 =

concentration of free ﬂuorescence labeled ligand molecule at 50%

inhibition, [P]0 = concentration of free protein at 0% inhibition, K_d=

dissociation constant of the protein−ligand complex. All reported K_i

values are means of duplicate experiments.

a

Reagents and conditions: (a) formaldehyde, H₂O, AcOH, rt−75 °C;

(b) 8 M HCl, 60−70 °C; (c) 10% Pd on C, EtOH, 1 atm to 30 psi H₂,

rt−70 °C.

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a

Scheme 5. Synthesis of β-Carboline Analogue 10

a

Reagents and conditions: (a) (BOC)₂O, Et₃N, DCM, rt; (b) NaH, 4-ﬂuorocyanobenzene, DMF, 150 °C; (c) TFA, DCM, rt; (d) NaOH, H₂O₂,

EtOH, DMSO, rt.

a

Scheme 6. Synthesis of Octahydro-β-carboline Analogue 11

a

Reagents and conditions: (a) (S)-tetrahydrofuran-3-amine, DIEA, DMSO, rt; (b) CuI, 59, K₂CO₃, N,N′-dimethylethane-1,2-diamine, 1,4-dioxane,

100 °C; (c) NaOH, H₂O₂, EtOH, DMSO, rt.

a

Scheme 7. Synthesis of Octahydro-β-carboline Analogues 12−22

a

Reagents and conditions: (a) NaH, 3-ﬂuoro-5-cyanobromobenzene, DMF, 150 °C; (b) R₁NH₂, Pd(OAc)₂, dppf, NaOtBu, toluene, 110 °C; (c)

NaOH, H₂O₂, EtOH, DMSO, rt.

a

Scheme 8. Synthesis of Benzolactam Analogue 23

Scheme 9. Synthesis of Benzolactam Analogues 24−26

a

Reagents and conditions: (a) NaH, 59, DMF, 150 °C.

a

Reagents and conditions: (a) methanesulfonic acid, NaN₃, DCM, 0

°C to rt; (b) isopropylamine, Et₃N, DMF, 180 °C; (c) NaH, 59, DMF,

180 °C.

loaded onto a 4−12% Bis-tris gel, transferred onto a PVDF membrane.

Following transfer, blots were washed with PBS containing 0.1%

Tween 20, blocked in 5% nonfat milk PBST for 1 h, and probed with

primary antibody directed against Htt protein and GAPDH as a

loading control (mAb2166 Millipore 1:3000, Santa Cruz Biotechnol-

ogy 25−778 1:5000). Following overnight incubation at 4 °C, blots

were washed and probed with 2° HRP antibody. The blots were then

developed using CL-Xposure clear blue X-ray ﬁlm. GAPDH expression

level is used as a loading control. For the determination of Htt IC₅₀

values, the density of the Htt band at a speciﬁc compound

Determination of Huntingtin IC₅₀and CC₅₀Values in HD

Patient Derived Fibroblasts. Compounds were tested in primary

human ﬁbroblasts for the ability to reduce the levels of Htt protein.

HD patient derived ﬁbroblasts were plated at 7.5 × 10⁴cells per well

and dosed with varying concentrations of compound or DMSO for 48

h. Following incubation, cells were washed with PBS and lysed in

Laemeli sample buﬀer with β-mercaptoethanol. Cell lysates were

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were washed and probed with 2° HRP antibody. The blot was then

developed using CL-Xposure clear blue X-ray ﬁlm. GAPDH expression

level was used as a loading control.

Scheme 10. Synthesis of Octahydro-β-carboline Analogue

27

a

Determination of Htt Protein Levels in Rat Brains. Male

Sprague−Dawley rats weighing approximately 200 g were adminis-

tered vehicle (0.5% methylcellulose, 0.5% sodium lauryl sulfate) or 31

(5 mg/kg) at a 5 mL/kg dose volume by oral gavage. At the

appropriate time point following dosing, animals were brieﬂy

anesthetized in an isoﬂurane chamber (30 s) and then decapitated.

Brains were dissected, and a horizontal corticostriatal slice

(approximately 1 mm thick in diameter) was removed, ﬂash frozen

in liquid nitrogen, placed into Covaris S-series glass tubes, and stored

at −80 °C. Cortical-striatal slices were homogenized in 1 mL of lysate

buﬀer (1× PBS with 0.1% Tween 20, 0.1% SDS, protease inhibitors)

using a Covaris E210 ultrasonicator (Covaris incorporated) to

generate a brain lysate. Brain lysates were loaded onto a 4−12% Bis-

tris gel, transferred onto a PVDF membrane. Following transfer, blots

were washed with PBS containing 0.1% Tween 20, blocked in 5%

nonfat milk PBST for 1 h, and probed with primary antibody directed

against Htt protein and GAPDH as a loading control (mAb2166

Millipore 1:3000, Santa Cruz Biotechnology 25-778 1:5000).

Following overnight incubation at 4 °C, blots were washed and

probed with 2° HRP antibody. The blot was then developed using CL-

Xposure clear blue X-ray ﬁlm. GAPDH expression level was used as a

loading control. The intensity of the Htt protein band in the

compound treated animals was normalized to the corresponding

GAPDH protein band. The normalized density of the Htt protein

band in compound treated rats was then calculated as a percent of the

normalized density of the Htt protein band in the vehicle controls.

General Procedure for the Preparation of 4-(Substituted-

6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-1-yl)-

benzamides (3−8). To a stirring solution of substituted-6,6-

dimethyl-6,7-dihydro-1H-indol-4(5H)-one (0.15 mmol) in DMF

(250 μL), under N₂, was added NaH (0.15 mmol, 60% in mineral

oil). The solution was stirred at ambient temperature for 10 min,

followed by the addition of 4-ﬂuorobenzonitrile (0.46 mmol). The

solution was heated at 150 °C for 5 min and then quenched with

MeOH (100 μL) and DMSO (500 μL). The solution was ﬁltered and

puriﬁed by HPLC (10−90% ACN in water with HCl modiﬁer) to

obtain the nitrile intermediate 43. The nitrile intermediate 43 was

dissolved in EtOH (500 μL) and DMSO (50 μL). To this solution was

added NaOH (1.0 M in water, 50 μL) and H₂O₂(30% in water, 30

μL). The resulting mixture was stirred at rt for 1 h. The reaction was

then evaporated to dryness under reduced pressure, diluted with

DMSO (500 μL), ﬁltered, and puriﬁed by preparative HPLC (10−

90% ACN in water with HCl modiﬁer) to obtain the desired product.

4-(3,6,6-Trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-1-yl)-

benzamide (3). Yield 62%. ¹H NMR (500 MHz, DMSO-d₆) δ 8.11 (s,

1H), 8.00 (d, J = 8.4 Hz, 2H), 7.52−7.49 (m, 3H), 6.87 (s, 1H), 2.70

(s, 2H), 2.26 (s, 2H), 2.21 (s, 3H), 0.98 (s, 6H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 194.47, 167.61, 142.26, 140.91, 133.23, 129.35, 124.44,

121.42, 119.13, 118.92, 52.40, 36.98, 35.62, 28.41, 11.79. HRMS

(ESI): [M + H]⁺calcd for C₁₈H₂₀N₂O₂, 297.1598; found, 297.1599.

4-(7,7-Dimethyl-9-oxo-6,7,8,9-tetrahydro-5H-pyrido[3,2-b]indol-

a

Reagents and conditions: (a) 2-[(E)-2-cyclopropylvinyl]-4,4,5,5-

tetramethyl-1,3,2-dioxaborolane, PdCl₂dppf, tBuNH₂, iPrOH, H₂O,

90 °C; (b) NaH, 59, DMF, 170 °C; (c) 10% Pd on C, EtOAc, AcOH,

1 atm H₂, rt; (d) NaOH, H₂O₂, EtOH, DMSO, rt.

Scheme 11. Synthesis of Tetrahydroindolone−Benzolactam

Derivatives 28 and 30−41

a

Reagents and conditions: (a) R₁MgCl, THF, 110 °C; (b) NaH, 59 or

3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-indol-4-one, DMF, 180 °C.

concentration was compared to a DMSO treated control cell sample

present on each exposure. IC₅₀values are deﬁned as the concentration

of compound in which the density of the Htt protein band is 50% of

the DMSO control. For determination of CC₅₀values, HD patient

derived ﬁbroblasts were plated at 7.5 × 10⁴cells per well and dosed

with varying concentrations of compound or DMSO for 48 h.

Following incubation, cell viability was determined using the Cell Titer

Glo kit (Promega G7571) according to manufacturer’s instructions.

The signal in compound treated wells was normalized to solvent

control wells and CC₅₀values were calculated. All reported IC₅₀and

CC₅₀values are means of duplicate experiments.

1

5-yl)benzamide Hydrochloride (4). Yield 55%. H NMR (400 MHz,

DMSO-d₆) δ 8.61 (d, J = 5.4 Hz, 1H), 8.17 (m, 4H), 7.73 (d, J = 8.3

Hz, 2H), 7.59 (m, 2H), 2.93 (s, 2H), 2.53 (s, 2H), 1.10 (s, 6H).

HRMS (ESI): [M + H]⁺calcd for C₂₀H₁₉N₃O₂, 334.1550; found,

334.1551.

Characterization of HSP90 Client Protein and HSP70 Levels

in HD Patient Derived Fibroblasts. Compounds were tested in

primary human ﬁbroblasts for the ability to reduce the levels of HSP90

client proteins and increase the levels of HSP70. Human ﬁbroblasts

were plated at 7.5 × 10⁴cells per well and dosed with varying

concentrations of compound or DMSO for 48 h. Following

incubation, cells were washed with PBS and lysed in Laemeli sample

buﬀer with β-mercaptoethanol. Cell lysates are loaded onto a 4−12%

Bis-tris gel, transferred onto a PVDF membrane. Following transfer,

blots are washed with PBS containing 0.1% Tween 20, blocked in 5%

nonfat milk PBST for 1 h, and probed with primary antibody directed

against HSP70, CDK4, AKT, and GAPDH as a loading control (no.

4876 Cell Signal 1:1000, SC-260 Santa Cruz Biotechnology 1:2000,

no. 4619 Cell Signaling 1:1000, Santa Cruz Biotechnology 25-778

1:5000, respectively). Following overnight incubation at 4 °C, blots

4-(7,7-dimethyl-5-oxo-7,8-dihydro-5H-pyrido[3,4-b]indol-9(6H)-

1

yl)benzamide Hydrochloride (5). Yield 70%. H NMR (400 MHz,

DMSO-d₆) δ 9.61 (s, 1H), 8.82 (d, J = 8.0 Hz, 1H), 8.41 (d, J = 6.7

Hz, 1H), 8.30−8.10 (m, 3H), 7.98 (d, J = 8.7 Hz, 2H), 7.68 (s, 1H),

3.16 (s, 2H), 2.56 (s, 2H), 1.14 (s, 6H). HRMS (ESI): [M + H]⁺calcd

for C₂₀H₁₉N₃O₂, 334.1550; found, 334.1552.

4-(7,7-Dimethyl-5-oxo-7,8-dihydro-5H-pyrido[2,3-b]indol-9(6H)-

1

yl)benzamide Hydrochloride (6). Yield 74%. H NMR (400 MHz,

DMSO-d₆) δ 8.38 (dd, J = 7.7, 1.5 Hz, 1H), 8.27 (dd, J = 4.7, 1.5 Hz,

1H), 8.14 (s, 1H), 8.09 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H),

7.54 (s, 1H), 7.34 (dd, J = 7.8, 4.8 Hz, 1H), 2.86 (s, 2H), 2.45 (s, 2H),

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1.08 (s, 6H). ¹³C NMR (125 MHz, DMSO-d₆) δ 193.56, 167.78,

152.22, 149.36, 144.27, 137.42, 134.60, 129.04, 127.95, 119.73, 117.11,

110.36, 51.92, 36.54, 35.57, 28.41. HRMS (ESI): [M + H]⁺calcd for

C₂₀H₁₉N₃O₂, 334.1550; found, 334.1552.

(30% in water, 3 mL). The solution was stirred at rt for 1 h. The

reaction was acidiﬁed to pH 4 using AcOH and puriﬁed via silica gel

chromatography using 10% MeOH in DCM to obtain the desired

1

compound as a white solid (3.5g, 10.4 mmol, yield 75%). H NMR

4-(2,2-Dimethyl-4-oxo-3,4,5,6,7,8-hexahydro-1H-carbazol-9(2H)-

yl)benzamide (7). Yield 25%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.08

(s, 1H), 8.01 (d, J = 8.5 Hz, 2H), 7.48 (s, 1H), 7.45 (d, J = 8.5 Hz,

2H), 2.72−2.67 (m, 2H), 2.53−2.49 (m, 2H), 2.36−2.30 (m, 2H),

2.22 (s, 2H), 1.73−1.69 (m, 4H), 0.99 (s, 6H). HRMS (ESI): [M +

H]⁺calcd for C₂₁H₂₄N₂O₂, 337.1911; found, 337.1919.

(400 MHz, D₂O) δ 7.92 (d, J = 8.6 Hz, 2H), 7.41 (d, J = 8.6 Hz, 2H),

4.05 (s, 2H), 3.45 (t, J = 6.1 Hz, 2H), 3.04 (s, 2H), 2.56 (s, 2H), 2.37

(s, 2H), 1.98 (s, 1H), 0.96 (s, 6H). HRMS (ESI): [M + H]⁺calcd for

C₂₀H₂₃N₃O₂, 338.1863; found, 338.1868.

2-Fluoro-6-(((S)-tetrahydrofuran-3yl)amino)-4-(2,7,7-tri-

methyl-5-oxo-decahydro-1H-pyrido[3,4-b] indol-9(9aH)-yl)-

benzamide (11). To a solution of (S)-4-bromo-2-ﬂuoro-6-((tetrahy-

drofuran-3-yl) amino)benzonitrile 64 (625 mg, 2.19 mmol) and 2,7,7-

trimethyl-1,3,4,6,8,9-hexahydropyrido[3,4-b]indol-5-one 59 (509 mg,

2.19 mmol) in 1,4-dioxane (7.5 mL), under N₂in a microwave vessel,

was added K₂CO₃(1.5 g, 11.0 mmol). CuI (626 mg, 3.28 mmol) and

N,N′-dimethylethane-1,2-diamine (289 mg, 359 μL, 3.28 mmol) were

added. The microwave vessel was sealed with a septa cap and heated at

100 °C in an oil bath for 19 h. The mixture was ﬁltered through a bed

of Celite followed by EtOAc (300 mL). The ﬁltrate was evaporated to

dryness under reduced pressure. The residue was puriﬁed by silica gel

chromatography using 10% MeOH in DCM to obtain the nitrile

intermediate. The nitrile intermediate was dissolved in EtOH (1.5 mL)

and DMSO (300 μL). To this solution was added NaOH (1.0 M in

water, 300 μL) and H₂O₂(30% in water, 300 μL). The solution was

stirred at rt for 1 h. The reaction was acidiﬁed to pH 4 using AcOH

and puriﬁed by silica gel chromatography using 10% MeOH in DCM

to obtain the desired compound as a white solid (50 mg, 0.11 mmol,

yield 5%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.79 (s, 2H), 7.31 (d, J =

6.9 Hz, 1H), 6.55 (dd, J = 11.2, 1.8 Hz, 1H), 6.44 (s, 1H), 4.15 (s,

1H), 3.93−3.77 (m, 2H), 3.75−3.71 (m, 1H), 3.56−3.53 (m, 1H),

3.23 (s, 2H), 2.70 (s, 2H), 2.64−2.55 (m, 4H), 2.30 (s, 3H), 2.23 (s,

3H), 1.79−1.66 (m, 1H), 1.00 (s, 6H). HRMS (ESI): [M + H]⁺calcd

for C₂₅H₃₁FN₄O₃, 455.2453; found, 455.2456.

General Procedure for the Preparation of 2-(Aminoalkyl)-4-

(2,7,7-trimethyl-5-oxo-3,4,5,6,7,8-hexahydro-1H-pyrido[3,4-b]-

indol-9(2H)-yl)benzamides (12−22). A microwave vessel was

loaded with 2-bromo-4-(2,7,7-trimethyl-5-oxo-3,4,6,8-tetrahydro-1H-

pyrido[3,4-b]indol-9-yl)benzonitrile 65 (0.73 mmol), amine (0.73

mmol), sodium tert-butoxide (1.45 mmol), Pd(OAc)₂(0.072 mmol),

and dppf (0.072 mmol). The vessel was purged with N₂, and toluene

(6 mL) was added via syringe. The mixture was heated at 110 °C for

20 min via microwave irradiation. The mixture was ﬁltered through a

bed of silica gel (1 g) using 10% MeOH in DCM (50 mL). The ﬁltrate

was then evaporated to dryness under reduced pressure to obtain the

desired nitrile intermediate. The nitrile intermediate was dissolved in

EtOH (1.5 mL) and DMSO (300 μL). To this solution was added

NaOH (1.0 M in water, 300 μL) and H₂O₂(30% in water, 300 μL).

The solution was stirred at rt for 1 h. The reaction was acidiﬁed to pH

4 using AcOH and puriﬁed via silica gel chromatography using 10%

MeOH in DCM or puriﬁed by preparative HPLC (10−90% ACN in

water with HCl modiﬁer) to obtain the desired compound.

4-(2,7,7-Trimethyl-5-oxo-3,4,5,6,7,8 hexahydro-1H-pyrido[3,4-b]-

1

indol-9(2H)-yl)benzamide Hydrochloride (8). Yield 38%. H NMR

(400 MHz, CD₃OD) δ 8.10 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 8.6 Hz,

2H), 4.29−4.22 (m, 2H), 3.6−3.724 (m, 1H), 3.57−3.39 (m, 1H),

3.25−3.10 (m, 3H), 2.99 (s, 3H), 2.83−2.13 (m, 5H), 1.10 (s, 3H),

1.03 (s, 3H). HRMS (ESI): [M + H]⁺calcd for C₂₁H₂₅N₃O₂,

352.2020; found, 352.2021.

4-(2,8,8-Trimethyl-6-oxo-2,3,4,5,6,7,8,9-octahydroazepino-

[3,4-b]indol-10(1H)-yl)benzamide Hydrochloride (9). A solution

of 2-(1,3-dioxolan-2-yl)-N-methyl-ethanamine 57 (530 mg, 4.0 mmol)

and formaldehyde (300 μL of 37% w/w in water, 4.0 mmol) in AcOH

was added slowly to 6,6-dimethyl-5,7-dihydro-1H-indol-4-one 55 (660

mg, 4.0 mmol) in AcOH (5 mL) at rt. The reaction was stirred for 5 h

at 75 °C, diluted with water, and then extracted with Et₂O. The

aqueous layer was basiﬁed with NaHCO₃to pH 9 and extracted with

DCM. The DCM layer was dried (MgSO₄), ﬁltered, and concentrated

under reduced pressure to yield the intermediate acetal (900 mg). The

acetal was stirred in aq HCl (10 mL of 8 M, 80.0 mmol) for 25 min at

70 °C, diluted with water, and then basiﬁed with 1.0 M aq NaOH

solution to pH 10. The mixture was extracted with DCM, and the

combined organics were dried (MgSO₄), ﬁltered, and concentrated

under reduced pressure to yield the oleﬁn intermediate (50 mg). The

oleﬁn intermediate was dissolved in EtOH (2 mL), and to this solution

was added 10% Pd on C (50 mg, 0.46 mmol). The resulting mixture

was stirred for 10 min under 1 atm of H₂. The mixture was ﬁltered

through a bed of Celite to give 2,8,8-trimethyl-2,3,4,5,7,8,9,10-

octahydroazepino[3,4-b]indol-6(1H)-one 60 (49 mg, yield 3%) as a

white solid. To a stirring solution of 2,8,8-trimethyl-2,3,4,5,7,8,9,10-

octahydroazepino[3,4-b]indol-6(1H)-one 60 (37.0 mg, 0.15 mmol) in

DMF (250 μL), under N₂, was added NaH (6.0 mg, 0.15 mmol, 60%

in mineral oil). The solution was stirred at ambient temperature for 10

min, followed by the addition of 4-ﬂuorobenzonitrile (56 mg, 0.46

mmol). The solution was heated at 100 °C for 8 h and then quenched

with MeOH (100 μL) and DMSO (500 μL). The solution was ﬁltered

and puriﬁed by HPLC (10−90% ACN in water with HCl modiﬁer) to

obtain the nitrile intermediate. The nitrile intermediate was dissolved

in EtOH (500 μL) and DMSO (50 μL). To this solution was added

NaOH (1.0 M in water, 50 μL) and H₂O₂(30% in water, 30 μL). The

mixture was stirred at rt for 1 h. The reaction was evaporated to

dryness under reduced pressure, diluted with DMSO (500 μL),

ﬁltered, and puriﬁed by preparative HPLC (10−90% ACN in water

with HCl modiﬁer) to obtain the desired product as a white solid (11

mg, 0.031 mmol, yield 21%). ¹H NMR (400 MHz, CD₃OD) δ 8.11 (d,

J = 7.6 Hz, 2H), 7.49−7.38 (m, 2H), 4.36 (d, J = 15.2 Hz, 1H), 4.11

(d, J = 15.2 Hz, 1H), 3.70 (s, 1H), 3.64−3.33 (m, 4H), 3.23−3.02 (m,

1H), 2.79 (s, 3H), 2.47 (d, J = 2.9 Hz, 2H), 2.37 (s, 2H), 2.25−2.17

(m, 1H), 2.10−1.95 (m, 1H), 1.04 (s, 6H). HRMS (ESI): [M + H]⁺

calcd for C₂₂H₂₇N₃O₂, 366.2176; found, 366.2172.

(S)-2-((Tetrahydrofuran-3-yl)amino)-4-(2,7,7-trimethyl-5-oxo-

3,4,5,6,7,8-hexahydro-1H-pyrido[3,4-b]indol-9(2H)-yl)benzamide

1

(12). Yield 68%. H NMR (400 MHz, DMSO-d₆) δ 8.49 (d, J = 6.8

Hz, 1H), 7.96 (s, 1H), 7.74 (d, J = 8.3 Hz, 1H), 7.31 (s, 1H), 6.57 (d, J

= 1.9 Hz, 1H), 6.52 (dd, J = 8.2, 1.9 Hz, 1H), 4.22−4.01 (m, 1H),

3.93−3.68 (m, 3H), 3.54 (dd, J = 8.9, 2.9 Hz, 1H), 3.20 (s,1H), 3.17

(s, 2H), 2.79−2.68 (m, 2H), 2.63−2.54 (m, 3H), 2.29 (s, 2H), 2.26−

2.21 (m, 2H), 1.91 (s, 1H), 1.77−1.68 (m, 1H), 1.00 (s, 6H). HRMS

(ESI): [M + H]⁺calcd for C₂₅H₃₂N₄O₃, 437.2547; found, 437.2547.

2-(Cyclopentylamino)-4-(2,7,7-trimethyl-5-oxo-3,4,5,6,7,8-hexa-

hydro-1H-pyrido[3,4-b]indol-9(2H)-yl)benzamide Hydrochloride

4-(7,7-Dimethyl-5-oxo-3,4,5,6,7,8-hexahydro-1H-pyrido[3,4-

b]indol-9(2H)-yl)benzamide (10). A mixture of tert-butyl 7,7-

dimethyl-5-oxo-1,3,4,6,8,9-hexahydropyrido[3,4-b]indole-2-carboxylate

62 (4.4 g, 13.9 mmol), 4-ﬂuorobenzonitrile (2.1 g, 18.0 mmol), NaH

(556 mg, 13.9 mmol, 60% in mineral oil), and DMF (44 mL) was

heated at 150 °C for 5 min. The solution was directly puriﬁed via silica

gel chromatography using 30% EtOAc in hexanes to obtain the nitrile

intermediate. The nitrile intermediate (5 g, 11.9 mmol), TFA (27.2 g,

18.4 mL, 238 mmol), and DCM (15 mL) were stirred under N₂at rt

for 30 min. The solution was concentrated to dryness under reduced

pressure and then dissolved in EtOH (15 mL) and DMSO (1 mL). To

this solution was added NaOH (5.0 M in water, 3 mL) and H₂O₂

1

(13). Yield 73%. H NMR (400 MHz, DMSO-d₆) δ 11.21 (s, 1H),

8.01 (s, 1H), 7.80 (d, J = 8.3 Hz, 1H), 7.31 (s, 1H), 6.55 (d, J = 1.9

Hz, 1H), 6.48 (dd, J = 8.3, 2.0 Hz, 1H), 4.20−4.18 (m, 2H), 3.93−

3.76 (m, 1H), 3.60−3.54 (m, 1H), 3.35−3.29 (m, 1H), 3.10−3.00 (m,

2H), 2.83 (d, J = 4.7 Hz, 3H), 2.76 (d, J = 16.5 Hz, 1H), 2.35 (d, J =

15.9 Hz, 1H), 2.21 (d, J = 15.9 Hz, 1H), 2.09−1.96 (m, 2H), 1.77−

1.51 (m, 4H), 1.51−1.35 (m, 2H), 1.51 (s, 3H), 0.97 (s, 3H). HRMS

(ESI): [M + H]⁺calcd for C₂₆H₃₄N₄O₂, 435.2755; found, 435.2756.

3395

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Article

2-((Cyclopropylmethyl)amino)-4-(2,7,7-trimethyl-5-oxo-

3,4,5,6,7,8-hexahydro-1H-pyrido[3,4-b]indol-9(2H)-yl)benzamide

(14). Yield 77%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.35 (t, J = 5.1 Hz,

1H), 7.93 (s, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.27 (s, 1H), 6.50 (d, J =

2.0 Hz, 1H), 6.46 (dd, J = 8.2, 2.0 Hz, 1H), 3.18 (d, J = 5.5 Hz, 3H),

3.00 (dd, J = 6.5, 5.4 Hz, 2H), 2.73−2.68 (m, 2H), 2.59−2.51 (m,

3H), 2.28 (s, 3H), 2.22 (s, 2H), 1.13−1.02 (m, 1H), 0.99 (s, 6H),

0.57−0.42 (m, 2H), 0.27−0.14 (m, 2H). HRMS (ESI): [M + H]⁺

calcd for C₂₅H₃₂N₄O₂, 421.2598; found, 421.2599.

(m, 2H), 2.84 (d, J = 4.7 Hz, 3H), 2.76 (d, J = 16.5 Hz, 1H), 2.39−

2.16 (m, 2H), 1.28−1.12 (m, 6H), 1.05 (s, 3H), 0.97 (s, 3H). HRMS

(ESI): [M + H]⁺calcd for C₂₄H₃₂N₄O₂, 409.2598; found, 409.2600.

2-(Neopentylamino)-4-(2,7,7-trimethyl-5-oxo-3,4,5,6,7,8-hexahy-

dro-1H-pyrido[3,4-b]indol-9(2H)-yl)benzamide Hydrochloride (22).

1

Yield 89%. H NMR (400 MHz, DMSO-d₆) δ 11.00 (s, 1H), 8.01 (s,

1H), 7.81 (d, J = 8.3 Hz, 1H), 7.31 (s, 1H), 6.58 (d, J = 2.0 Hz, 1H),

6.44 (dd, J = 8.3, 2.0 Hz, 1H), 4.17 (d, J = 4.6 Hz, 2H), 3.62−3.58 (m,

1H), 3.41−3.18 (m, 1H), 3.06−3.01 (m, 2H), 2.93 (d, J = 5.7 Hz,

2H), 2.83 (d, J = 4.7 Hz, 3H), 2.73 (d, J = 16.5 Hz, 1H), 2.42 (d, J =

5.9 Hz, 2H), 2.22 (d, J = 5.9 Hz, 2H), 1.05 (s, 3H), 0.99−0.96 (m,

12H). HRMS (ESI): [M + H]⁺calcd for C₂₆H₃₆N₄O₂, 437.2911;

found, 437.2912.

2-((2-Methoxyethyl)amino)-4-(2,7,7-trimethyl-5-oxo-3,4,5,6,7,8-

hexahydro-1H-pyrido[3,4-b]indol-9(2H)-yl)benzamide Hydrochlor-

1

ide (15). Yield 62%. H NMR (400 MHz, DMSO-d₆) δ 11.12 (s,

1H), 7.98 (s, 1H), 7.79 (d, J = 8.3 Hz, 1H), 7.29 (s, 1H), 6.58 (d, J =

2.0 Hz, 1H), 6.49 (dd, J = 8.2, 2.0 Hz, 1H), 4.20−4.15 (m, 2H), 3.60−

3.53 (m, 3H), 3.38−3.22 (m, 6H), 3.09−3.01 (m, 2H), 2.83 (d, J = 4.7

Hz, 3H), 2.76 (d, J = 16.6 Hz, 1H), 2.35 (d, J = 15.9 Hz, 2H), 2.20 (d,

J = 15.9 Hz, 2H), 1.05 (s, 3H), 0.97 (s, 3H). HRMS (ESI): [M + H]⁺

calcd for C₂₄H₃₂N₄O₃, 425.2547; found, 425.2548.

2-((Tetrahydro-2H-thiopyran-4-yl)amino)-4-(2,7,7-trimethyl-5-

oxo-3,4,5,6,7,8-hexahydro-1H-pyrido[3,4-b]indol-9(2H)-yl)-

benzamide Hydrochloride (16). Yield 72%. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.40 (s, 1H), 7.99 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H),

7.35 (s, 1H), 6.61 (d, J = 1.9 Hz, 1H), 6.47 (dd, J = 8.3, 1.9 Hz, 1H),

3.55−3.40 (m, 2H), 3.29−3.25 (m, 1H), 3.05−3.02 (m, 2H), 2.89−

2.85 (m, 3H), 2.83−2.61 (m, 6H), 2.25−2.09 (m, 6H), 1.60−1.48 (m,

2H), 1.05 (s, 3H), 0.96 (s, 3H). HRMS (ESI): [M + H]⁺calcd for

C₂₆H₃₄N₄O₂S, 467.2476; found, 467.2478.

9-(8-(Isopropylamino)-1-oxo-1,2,3,4-tetrahydroisoquinolin-

6-yl)-2,7,7-trimethyl-3,4,6,7,8,9-hexahydro-1H-pyrido[3,4-b]-

indol-5(2H)-one (23). A microwave vessel was loaded with 6,8-

diﬂuoro-3,4-dihydro-2H-isoquinolin-1-one 67 (250 mg, 1.37 mmol),

Et₃N (414 mg, 570 μL, 4.10 mmol), and DMF (250 μL). To this

solution was added propan-2-amine (403 mg, 586 μL, 6.83 mmol).

The mixture was heated at 180 °C for 10 min via microwave

irradiation. The residue was puriﬁed via silica gel chromatography

using 30% EtOAc in hexanes to obtain the intermediate aniline (239

mg). The aniline was added to a preformed solution of 2,7,7-trimethyl-

1,3,4,6,8,9-hexahydropyrido[3,4-b]indol-5-one 59 (250 mg, 1.08

mmol), NaH (86 mg, 2.15 mmol, 60% in mineral oil), and DMF

(1.2 mL). The solution was heated via microwave irradiation at 180 °C

for 10 min. The reaction was quenched with MeOH (300 μL),

neutralized with AcOH, and puriﬁed via silica gel chromatography

using 3% MeOH in DCM to obtain a white solid (152 mg, 0.35 mmol,

2-(Propylamino)-4-(2,7,7-trimethyl-5-oxo-3,4,5,6,7,8-hexahydro-

1H-pyrido[3,4-b]indol-9(2H)-yl)benzamide Hydrochloride (17).

1

Yield 81%. H NMR (400 MHz, DMSO-d₆) δ 11.04 (s, 1H), 8.05

yield 26%). H NMR (400 MHz, DMSO-d₆) δ 8.80 (d, J = 7.6 Hz,

(d, J = 39.9 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.32 (s, 1H), 6.53 (s,

1H), 6.48 (d, J = 8.2 Hz, 1H), 4.22−4.18 (m, 2H), 3.61−3.56 (m 1H),

3.33−3.29 (m,1H), 3.55−3.00 (m, 2H), 3.04 (s, 2H), 2.84 (d, J = 4.4

Hz, 3H), 2.76 (d, J = 16.5 Hz, 1H), 2.35 (d, J = 16.0 Hz, 1H), 2.23 (d,

J = 16.0 Hz, 1H), 1.60−1.50 (m, 2H), 1.17−0.89 (m, 9H). HRMS

(ESI): [M + H]⁺calcd for C₂₄H₃₂N₄O₂, 409.2598; found, 409.2597.

2-(Cyclopropylamino)-4-(2,7,7-trimethyl-5-oxo-3,4,5,6,7,8-hexa-

hydro-1H-pyrido[3,4-b]indol-9(2H)-yl)benzamide Hydrochloride

1H), 7.83 (s, 1H), 6.40 (d, J = 1.7 Hz, 1H), 6.33 (d, J = 1.6 Hz, 1H),

3.71−3.64 (m, 1H), 3.21 (s, 2H), 3.17 (s, 2H), 2.86−2.80 (m, 2H),

2.73−2.65 (m, 2H), 2.63−2.57 (m, 4H), 2.29 (s, 3H), 2.23 (s, 2H),

1.16 (d, J = 6.2 Hz, 6H), 1.00 (s, 6H). HRMS (ESI): [M + H]⁺calcd

for C₂₆H₃₄N₄O₂, 435.2755; found, 435.2755.

General Procedure for the Preparation of 4-(2,7,7-Trimeth-

yl-5-oxo-3,4,5,6,7,8-hexahydro-1H-pyrido[3,4-b]indol-9(2H)-

yl)benzolactams (24−26). A microwave vessel was loaded with

2,7,7-trimethyl-1,3,4,6,8,9-hexahydropyrido[3,4-b]indol-5-one 59

(0.22 mmol) and DMF (1 mL). NaH (0.32 mmol, 60% in mineral

oil) was added under N₂, and the solution was stirred for 10 min. The

ﬂuorobenzolactam 68 (0.32 mmol) was added, and the solution was

heated via microwave irradiation at 150 °C for 10 min. The reaction

was diluted with DMSO (500 μL), ﬁltered, and puriﬁed by HPLC

(10−90% ACN in water with HCl modiﬁer) to obtain the desired

product.

1

(18). Yield 71%. H NMR (400 MHz, DMSO-d₆) δ 11.15 (s, 1H),

8.02 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.35 (s, 1H), 6.88 (d, J = 2.1

Hz, 1H), 6.57 (dd, J = 8.3, 2.1 Hz, 1H), 4.24−4.20 (m, 3H), 3.62−

3.55 (m, 1H), 3.39−3.24 (m, 1H), 3.17 (s, 1H), 3.09−3.05 (m, 2H),

2.87−2.75 (m, 4H), 2.49−2.42 (m, 1H), 2.38 (dd, J = 16.0 Hz, 1H),

2.28 (dd, J = 16.0 Hz, 1H), 1.05 (s, 3H), 0.98 (s, 3H), 0.91−0.70 (m,

2H), 0.60−0.30 (m, 2H). HRMS (ESI): [M + H]⁺calcd for

C₂₄H₃₀N₄O₂, 407.2442; found, 407.2442.

2-((2,2-Diﬂuoroethyl)amino)-4-(2,7,7-trimethyl-5-oxo-3,4,5,6,7,8-

2,7,7-Trimethyl-9-(1-oxo-isoindolin-5-yl)-3,4,6,7,8,9-hexahydro-

1

hexahydro-1H-pyrido[3,4-b]indol-9(2H)-yl)benzamide Hydrochlor-

1H-pyrido[3,4-b]indol-5(2H)-one Hydrochloride (24). Yield 62%. H

1

ide (19). Yield 58%. H NMR (400 MHz, DMSO-d₆) δ 10.83 (s,

NMR (400 MHz, DMSO-d₆) δ 10.81 (s, 1H), 8.78 (s, 1H), 7.87 (d, J

= 8.0 Hz, 1H), 7.64 (s, 1H), 7.47 (dd, J = 8.0, 1.7 Hz, 1H), 4.48 (s,

2H), 4.20−4.15 (m, 2H), 3.65−3.58 (m, 1H), 3.32−3.28 (m, 1H),

3.07−3.03 (m, 2H), 2.85−2.81 (m, 3H), 2.72 (d, J = 16.6 Hz, 1H),

2.35 (d, J = 16.0 Hz, 1H), 2.25 (d, J = 16.0 Hz, 1H), 1.05 (s, 3H), 0.98

(s, 3H). HRMS (ESI): [M + H]⁺calcd for C₂₂H₂₅N₃O₂, 364.2020;

found, 364.2021.

2,7,7-Trimethyl-9-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)-

3,4,6,7,8,9-hexahydro-1H-pyrido[3,4-b]indol-5(2H)-one Hydrochlor-

ide (25). Yield 82%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.30 (s, 1H),

8.11 (s, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.40−7.29 (m, 2H), 4.21 (s,

2H), 3.69−3.62 (m, 1H), 3.35−3.22 (m, 4H), 3.08−2.98 (m, 4H),

2.89−2.86 (m, 3H), 2.76−2.68 (m, 1H), 2.35 (d, J = 15.6 Hz, 1H),

2.25 (d, J = 15.6 Hz, 1H), 1.06 (s, 3H), 0.98 (s, 3H). ¹³C NMR (100

MHz, DMSO-d₆) δ 193.07, 163.45, 143.42, 141.41, 137.54, 129.41,

128.75, 125.03, 124.47, 121.57, 116.44, 112.70, 51.73, 50.68, 48.52,

41.60, 35.63, 35.18, 28.50, 27.54, 27.41, 19.57. HRMS (ESI): [M +

H]⁺calcd for C₂₃H₂₇N₃O₂, 378.2176; found, 378.2178.

1H), 8.65 (s, 1H), 8.06 (s, 1H), 7.82 (d, J = 8.3 Hz, 1H), 7.43 (s, 1H),

6.77 (s, 1H), 6.69−6.52 (m, 1H), 6.48−5.96 (m, 1H), 3.73 (t, J = 15.8

Hz, 2H), 3.62−3.57 (m, 1H), 3.35−3.18 (m, 1H), 3.09−3.01 (m, 2H),

2.89−2.84 (m, 3H), 2.77 (d, J = 16.5 Hz, 1H), 2.35 (d, J = 15.9 Hz,

2H), 2.23 (d, J = 15.9 Hz, 2H), 1.05 (s 3H), 0.97 (s, 3H). HRMS

(ESI): [M + H]⁺calcd for C₂₃H₂₈F₂N₄O₂, 431.2253; found, 431.2252.

2-(((3-Methyloxetan-3-yl)methyl)amino)-4-(2,7,7-trimethyl-5-

oxo-3,4,5,6,7,8-hexahydro-1H pyrido[3,4-b]indol-9(2H)-yl)-

benzamide Hydrochloride (20). Yield 77%. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.79 (s, 1H), 8.06 (d, J = 36.2 Hz, 1H), 7.80 (d, J = 8.3

Hz, 1H), 7.33 (s, 1H), 6.66 (d, J = 1.9 Hz, 1H), 6.46 (dd, J = 8.3, 1.9

Hz, 1H), 4.22−4.15 (m, 2H), 3.62−3.57 (m, 3H), 3.45−3.22 (m, 3H),

3.15−3.09 (m, 2H), 3.06−3.01 (m, 2H), 2.85 (d, J = 4.7 Hz, 3H), 2.74

(d, J = 16.5 Hz, 1H), 2.35 (d, J = 15.9 Hz, 1H), 2.22 (d, J = 15.9 Hz,

1H), 1.05 (s, 3H), 1.01−0.97 (m, 6H). HRMS (ESI): [M + H]⁺calcd

for C₂₆H₃₄N₄O₃, 451.2704; found, 451.2687.

2-(Isopropylamino)-4-(2,7,7-trimethyl-5-oxo-3,4,5,6,7,8-hexahy-

dro-1H-pyrido[3,4-b]indol-9(2H)-yl)benzamide Hydrochloride (21).

2,7,7-Trimethyl-9-(1-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepin-

1

Yield 64%. H NMR (400 MHz, DMSO-d₆) δ 10.93 (s, 1H), 7.98 (s,

7-yl)-3,4,6,7,8,9-hexahydro-1H-pyrido[3,4-b]indol-5(2H)-one Hydro-

1

1H), 7.79 (d, J = 8.3 Hz, 1H), 7.32 (s, 1H), 6.54 (d, J = 1.9 Hz, 1H),

6.46 (dd, J = 8.3, 2.0 Hz, 1H), 4.19 (d, J = 4.9 Hz, 2H), 3.81−3.64 (m,

1H), 3.62−3.58 (m 1H), 3.39−3.21 (m, 1H), 3.17 (s, 1H), 3.09−3.01

chloride (26). Yield 67%. H NMR (400 MHz, DMSO-d₆) δ 9.99 (s,

1H), 8.22 (t, J = 5.9 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.1

Hz, 1H), 4.25−4.20 (m, 2H), 3.64−3.60 (m, 1H), 3.10−2.93 (m, 4H),

3396

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Journal of Medicinal Chemistry

Article

2.89−2.87 (m, 3H), 2.85−2.81 (m, 2H), 2.73 (d, J = 16.5 Hz, 1H),

2.46−2.42 (m, 1H), 2.35 (d, J = 15.9 Hz, 1H), 2.25 (d, J = 15.9 Hz,

1H), 2.04−1.77 (m, 2H), 1.05 (s, 3H), 0.98 (s, 3H). HRMS (ESI): [M

+ H]⁺calcd for C₂₄H₂₉N₃O₂, 392.2333; found, 392.2334.

(m, 1H), 3.90−3.70 (m, 3H), 3.54 (dd, J = 8.9, 3.0 Hz, 1H), 2.66 (s,

2H), 2.56−2.53 (m, 2H), 2.36 (s, 2H), 2.31−2.13 (m, 3H), 1.88−1.60

(m, 5H), 1.00 (s, 6H). HRMS (ESI): [M + H]⁺calcd for C₂₅H₃₁N₃O₃,

422.2438; found, 422.2440.

General Procedure for the Preparation of 8-Substituted-6-

(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-1-yl)-3,4-di-

hydroisoquinolin-1(2H)-one (30−41). A microwave vessel was

loaded with 6,8-diﬂuoro-3,4-dihydro-2H-isoquinolin-1-one 67 (0.44

mmol). To this was added a solution of the Grignard reagent (1.75

mmol). The mixture was heated at 110 °C via microwave irradiation

for 5 min. The reaction was quenched with 10% MeOH in DCM (1

mL) and evaporated to dryness under reduced pressure. The residue

was passed through a short plug of silica gel (1 g) using 10% MeOH in

DCM (8 mL). The ﬁltrate was evaporated to dryness under reduced

pressure, and the residue was added to a preformed solution of 3,6,6-

trimethyl-6,7-dihydro-1H-indol-4(5H)-one (0.22 mmol), NaH (0.44

mmol, 60% in mineral oil), and DMF (800 μL) that had been stirred

at rt for 15 min. The reaction was then heated via microwave

irradiation at 180 °C for 15 min. The reaction was diluted with DMSO

(500 μL), ﬁltered, and puriﬁed by preparative HPLC (10−99% ACN

in water with HCl modiﬁer) to obtain the desired product.

8-Isobutyl-6-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-

1-yl)-3,4-dihydroisoquinolin-1(2H)-one (30). Yield 52%. ¹H NMR

(500 MHz, DMSO-d₆) δ 7.93 (s, 1H), 7.23 (s, 1H), 7.10 (s, 1H), 6.84

(s, 1H), 3.28 (s, 2H), 3.02 (d, J = 6.8 Hz, 2H), 2.90 (t, J = 5.9 Hz,

2H), 2.71 (s, 2H), 2.25 (s, 2H), 2.21 (s, 3H), 1.86 (dt, J = 13.2, 6.4 Hz,

1H), 0.98 (s, 6H), 0.85 (d, J = 6.6 Hz, 6H). HRMS (ESI): [M + H]⁺

calcd for C₂₄H₃₀N₂O₂, 379.2380; found, 379.2382.

8-Cyclopentyl-6-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-

indol-1-yl)-3,4-dihydroisoquinolin-1(2H)-one (31). Yield 48%. ¹H

NMR (400 MHz, DMSO-d₆) δ δ 8.03 (s, 1H), 7.26 (d, J = 2.0 Hz,

1H), 7.21 (d, J = 2.0 Hz, 1H), 6.88 (s, 1H), 4.46−4.17 (m, 1H), 3.3−

3.22 (m, 2H), 2.92−2.88 (m, 2H), 2.76−2.71 (m, 2H), 2.26 (s, 2H),

2.22 (s, 3H), 2.04−1.99 (m, 2H), 1.82−1.76 (m, 2H), 1.65−1.50 (m,

4H), 0.99 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 193.60, 164.55,

149.95, 142.13, 141.47, 139.62, 126.81, 121.18, 120.81, 120.30, 118.66,

118.37, 51.95, 36.61, 35.07, 34.55, 30.05, 27.93, 25.32, 11.25. HRMS

(ESI): [M + H]⁺calcd for C₂₅H₃₀N₂O₂, 391.2380; found, 391.2376.

8-Cyclohexyl-6-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-

indol-1-yl)-3,4-dihydroisoquinolin-1(2H)-one (32). Yield 67%. ¹H

NMR (400 MHz, DMSO-d₆) δ 8.02−7.98 (m, 1H), 7.25 (s, 1H), 7.22

(s, 1H), 6.87 (s, 1H), 4.08−3.95 (m, 1H), 3.31−3.24 (m, 2H), 2.93−

2.88 (m, 2H), 2.77−2.70 (m, 2H), 2.26 (s, 2H), 2.22 (s, 3H), 1.83−

1.67 (m, 5H), 1.55−1.11 (m, 5H), 0.99 (s, 6H). HRMS (ESI): [M +

H]⁺calcd for C₂₆H₃₂N₂O₂, 405.2537; found, 405.2540.

2-(2-Cyclopropylethyl)-4-(2,7,7-trimethyl-5-oxo-3,4,5,6,7,8-

hexahydro-1H-pyrido[3,4-b]indol-9(2H)-yl)benzamide Hydro-

chloride (27). A mixture of (E)-2-(2-cyclopropylvinyl)-4-ﬂuoroben-

zonitrile 69 (63 mg, 0.34 mmol) and 2,7,7-trimethyl-1,3,4,6,8,9-

hexahydropyrido[3,4-b]indol-5-one 59 (78 mg, 0.34 mmol), DMF

(1.5 mL), and NaH (12 mg, 0.50 mmol, 60% in mineral oil) was

heated at 170 °C via microwave irradiation for 1 h. The mixture was

partitioned between EtOAc and water. The organic phase was dried,

ﬁltered, and concentrated under reduced pressure. The residue was

puriﬁed by silica gel chromatography using 0−30% MeOH in DCM to

obtain the intermediate oleﬁn. The oleﬁn product was added to EtOAc

(945 μL), AcOH (315 μL), and 10% Pd on C (35 mg, 0.33 mmol)

and stirred under 1 atm of H₂for 30 min. The mixture was ﬁltered,

and the ﬁltrate was concentrated under reduced pressure. The residue

was puriﬁed by silica gel chromatography using 0−30% MeOH in

DCM to obtain the intermediate nitrile. The nitrile intermediate was

dissolved in EtOH (378 μL) and DMSO (38 μL). To this solution was

added NaOH (1.0 M in water, 37 μL) and H₂O₂(30% in water, 10

μL). The solution was stirred at rt for 1 h. The reaction was acidiﬁed

to pH 4 using AcOH and puriﬁed by preparative HPLC (10−90%

ACN in water with HCl modiﬁer) to obtain a white solid (12 mg, 0.03

mmol, yield 9%). ¹H NMR (400 MHz, CDCl₃) δ 7.48 (d, J = 8.0 Hz,

1H), 7.06 (d, J = 2.0 Hz, 1H), 7.01 (dd, J = 8.0, 2.1 Hz, 1H), 5.81−

5.70 (bs, 2H), 3.19 (s, 2H), 2.97−2.85 (m, 4H), 2.65−2.60 (m, 2H),

2.37 (s, 2H), 2.34 (s, 3H), 2.26 (s, 2H), 1.50 (dd, J = 15.1, 7.2 Hz,

2H), 0.99 (s, 6H), 0.70−0.62 (m, 1H), 0.45−0.27 (m, 2H), 0.06−0.13

(m, 2H). HRMS (ESI): [M + H]⁺calcd for C₂₆H₃₃N₃O₂, 420.2646;

found, 420.2648.

9-(8-Isobutyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)-

2,7,7-trimethyl-3,4,6,7,8,9-hexahydro-1H-pyrido[3,4-b]indol-

5(2H)-one (28). A microwave vessel was loaded with 2,7,7-trimethyl-

1,3,4,6,8,9-hexahydropyrido[3,4-b]indol-5-one 59 (250 mg, 1.076

mmol) and DMF (1.3 mL). NaH (86 mg, 2.15 mmol, 60% in

mineral oil) was added under N₂, and the solution was stirred for 15

min. To this solution was added 6-ﬂuoro-8-isobutyl-3,4-dihydro-2H-

isoquinolin-1-one (238 mg, 1.08 mmol), and the solution was heated

via microwave at 180 °C for 10 min. The reaction was quenched with

MeOH (300 μL), neutralized with AcOH, and puriﬁed via silica gel

chromatography using 3% MeOH in DCM to obtain a white solid

(156 mg, 33.6 mmol, yield 33%). ¹H NMR (400 MHz, DMSO-d₆) δ δ

7.97 (s, 1H), 7.20 (d, J = 2.0 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 3.19

(bs, 1H), 3.17 (s, 3H), 3.02 (d, J = 6.9 Hz, 2H), 2.95−2.89 (m, 2H),

2.74−2.68 (m, 2H), 2.62−2.55 (m, 3H), 2.29−2.56 (m, 2H), 2.23 (bs,

1H), 1.96−1.82 (m, 2H), 0.99 (s, 6H), 0.85 (d, J = 6.6 Hz, 6H).

HRMS (ESI): [M + H]⁺calcd for C₂₇H₃₅N₃O₂, 434.2802; found,

434.2804.

8-(Tetrahydro-2H-pyran-4-yl)-6-(3,6,6-trimethyl-4-oxo-4,5,6,7-

tetrahydro-1H-indol-1-yl)-3,4-dihydroisoquinolin-1(2H)-one (33).

1

Yield 42%. H NMR (400 MHz, DMSO-d₆) δ 8.05 (s, 1H), 7.29−

7.26 (m, 2H), 6.91 (s, 1H), 4.30−4.20 (m, 1H), 3.94 (d, J = 10.7 Hz,

2H), 3.46−3.37 (m, 2H), 3.32−3.27 (m, 2H), 2.95−2.86 (m, 2H),

2.75−2.73 (m, 2H), 2.26 (s, 2H), 2.22 (s, 3H), 1.75−1.54 (m, 4H),

1.00 (s, 6H). HRMS (ESI): [M + H]⁺calcd for C₂₅H₃₀N₂O₃,

407.2329; found, 407.2331.

(S)-4-(2,2-Dimethyl-4-oxo-3,4,5,6,7,8-hexahydro-1H-carba-

zol-9(2H)-yl)-2-((tetrahydrofuran-3 yl)amino)benzamide Hy-

drochloride (29). A microwave vessel was loaded with 2-bromo-4-

(7,7-dimethyl-5-oxo-1,2,3,4,6,8-hexahydrocarbazol-9-yl)benzonitrile

(30 mg, 0.08 mmol), (S)-tetrahydrofuran-3-amine (9.3 mg, 0.08

mmol), Pd(OAc)₂(1.7 mg, 0.008 mmol), DPPF (4.1 mg, 0.008

mmol), sodium tert-butoxide (14.5 mg, 0.15 mmol), and toluene (600

μL). The vessel was purged with N₂, and the mixture was heated via a

preheated oil bath at 150 °C for 20 min. The mixture was directly

passed through a short plug of silica gel (1 g) using EtOAc (5 mL).

The ﬁltrate was concentrated under reduced pressure to obtain the

nitrile intermediate. The nitrile intermediate was dissolved in EtOH

(400 μL) and DMSO (100 μL). To this solution was added NaOH

(1.0 M in water, 100 μL) and H₂O₂(30% in water, 30 μL). The

solution was stirred at rt for 1 h. The reaction was acidiﬁed to pH 4

using AcOH, diluted with DMSO (500 μL), ﬁltered, and puriﬁed by

HPLC (10−90% ACN in water with HCl modiﬁer) to obtain a white

8-Cyclobutyl-6-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-

indol-1-yl)-3,4-dihydroisoquinolin-1(2H)-one (34). Yield 62%. ¹H

NMR (400 MHz, DMSO-d₆) δ 7.98 (s, 1H), 7.26 (s, 1H), 7.24 (s,

1H), 6.91 (s, 1H), 4.60−4.49 (m, 1H), 3.32−3.23 (m, 2H), 2.89 (t, J =

6.2 Hz, 2H), 2.74 (s, 2H), 2.35−2.26 (m, 2H), 2.26 (s 2H), 2.23 (s,

3H), 2.10−1.97 (m, 2H), 1.96−1.86 (m, 1H), 1.79−1.70 (m, 1H),

1.00 (s, 6H). HRMS (ESI): [M + H]⁺calcd for C₂₄H₂₈N₂O₂,

377.2224; found, 377.2228.

8-Butyl-6-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-1-

yl)-3,4-dihydroisoquinolin-1(2H)-one (35). Yield 43%. ¹H NMR (500

MHz, DMSO-d₆) δ 7.94 (s, 1H), 7.22 (s, 1H), 7.16 (s, 1H), 6.86 (s,

1H), 3.13−3.07 (m, 2H), 2.90 (t, J = 6.0 Hz, 2H), 2.72 (s, 2H), 2.55

(s, 2H), 2.26 (s, 2H), 2.21 (s, 3H), 1.59−1.47 (m, 2H), 1.37−1.27 (m,

2H), 0.99 (s, 6H), 0.88 (t, J = 7.3 Hz, 3H). HRMS (ESI): [M + H]⁺

calcd for C₂₄H₃₀N₂O₂, 379.2380; found, 379.2378.

1

solid (15 mg, 0.036 mmol, yield 45%). H NMR (400 MHz, DMSO-

8-(tert-Butyl)-6-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-

indol-1-yl)-3,4-dihydroisoquinolin-1(2H)-one (36). Yield 72%. ¹H

NMR (400 MHz, DMSO-d₆) δ 8.12 (s, 1H), 7.37 (s, 1H), 7.26 (s,1H),

d₆) δ 8.49 (d, J = 6.7 Hz, 1H), 7.95 (bs, 1H), 7.74 (d, J = 8.3 Hz, 1H),

7.29 (bs, 1H), 6.57 (s, 1H), 6.51 (dd, J = 8.3, 1.9 Hz, 1H), 4.22−4.06

3397

dx.doi.org/10.1021/jm500042s | J. Med. Chem. 2014, 57, 3382−3400

Journal of Medicinal Chemistry

Article

6.87 (s,1H), 3.24 (dd, J = 11.2, 5.4 Hz, 2H), 2.87 (t, J = 5.4 Hz, 2H),

2.73 (s, 2H), 2.27 (s, 2H), 2.23 (s, 3H), 1.49 (s, 9H), 1.01 (s, 6H).

HRMS (ESI): [M + H]⁺calcd for C₂₄H₃₀N₂O₂, 379.2380; found,

379.2381.

8-Benzyl-6-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-1-

yl)-3,4-dihydroisoquinolin-1(2H)-one (37). Yield 45%. ¹H NMR (400

MHz, DMSO-d₆) δ 7.88 (s, 1H), 7.35−7.28 (m, 5H), 7.26−7.17 (m,

1H), 7.06 (s, 1H), 6.47 (s, 1H), 4.01 (s, 2H), 3.41−3.28 (m, 2H),

2.94−2.88 (m, 2H), 2.25 (s, 2H), 2.19−2.10 (m, 5H), 0.96 (s, 3H),

0.93 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 193.15, 161.93,

145.57, 143.41, 142.04, 140.07, 137.40, 128.83, 128.57, 127.93, 127.50,

126.19, 123.72, 121.74, 117.08, 116.41, 52.22, 35.56, 34.95, 29.28,

28.25, 28.01, 11.34. HRMS (ESI): [M + H]⁺calcd for C₂₇H₂₈N₂O₂,

413.2224; found, 413.2229.

8-Phenyl-6-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-1-

yl)-3,4-dihydroisoquinolin-1(2H)-one (38). Yield 64%. ¹H NMR (500

MHz, DMSO-d₆) δ 7.94 (s, 1H), 7.41 (s, 1H), 7.39−7.23 (m, 5H),

7.14 (d, J = 1.7 Hz, 1H), 6.93 (s, 1H), 2.98 (t, J = 5.9 Hz, 2H), 2.75 (s,

2H), 2.55 (s, 2H), 2.25 (s, 2H), 2.20 (s, 3H), 0.99 (s, 6H). HRMS

(ESI): [M + H]⁺calcd for C₂₆H₂₆N₂O₂, 399.2067; found, 399.2067.

8-(4-Fluorophenyl)-6-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-

1H-indol-1-yl)-3,4-dihydroisoquinolin-1(2H)-one (39). Yield 73%. ¹H

NMR (500 MHz, DMSO-d₆) δ 7.95 (d, J = 14.1 Hz, 1H), 7.41 (d, J =

1.7 Hz, 1H), 7.33 (dd, J = 8.6, 5.5 Hz, 2H), 7.26−7.07 (m, 3H), 6.92

(s, 1H), 3.42−3.38 (m, 2H), 2.97 (t, J = 6.0 Hz, 2H), 2.74 (s, 2H),

2.24 (s, 2H), 2.20 (s, 3H), 0.98 (s, 6H). HRMS (ESI): [M + H]⁺calcd

for C₂₆H₂₅FN₂O₂, 417.1973; found, 417.1975.

8-(3-Fluorophenyl)-6-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-

1H-indol-1-yl)-3,4-dihydroisoquinolin-1(2H)-one (40). Yield 59%. ¹H

NMR (500 MHz, DMSO-d₆) δ 7.98 (s, 1H), 7.44 (s, 1H), 7.44−7.39

(m, 1H), 7.23−7.10 (m, 4H), 6.94 (s, 1H), 2.98 (t, J = 6.1 Hz, 2H),

2.76 (s, 2H), 2.55 (s, 2H), 2.25 (s, 2H), 2.20 (s, 3H), 0.99 (s, 6H).

HRMS (ESI): [M + H]⁺calcd for C₂₆H₂₅FN₂O₂, 417.1973; found,

417.1976.

yellow solid (7.0 g, 32.7 mmol, yield 41%). LCMS (C18 column

eluting with 1−99% ACN/water gradient over 3 min with TFA

modiﬁer) M + 1: 295.5 (1.20 min). ¹H NMR (400 MHz, DMSO-d₆) δ

8.48−8.25 (m, 2H), 8.10 (dd, J = 7.9, 1.6 Hz, 1H), 7.06 (dd, J = 7.9,

4.7 Hz, 1H), 6.21 (s, 1H), 2.52 (s, 2H), 2.10 (s, 2H), 1.03 (s, 6H).

7,7-Dimethyl-7,8-dihydro-5H-pyrido[3,2-b]indol-9(6H)-one

(50). A stirring solution of 2-chloropyridin-3-amine 44 (2.0 g, 15.6

mmol), 5,5-dimethylcyclohexane-1,3-dione (2.8 g, 20.2 mmol), p-

TsOH-H₂O (20 mg, 0.11 mmol), and toluene (50 mL) was heated at

reﬂux for 24 h. The reaction was cooled and added to 50 mL of aq

NaHCO₃solution. The mixture was extracted with DCM (3 × 50

mL). The organic fractions were combined, dried (MgSO4), ﬁltered,

and concentrated under reduced pressure to yield 3-((2-chloropyridin-

3-yl)amino)-5,5-dimethylcyclohex-2-enone 47 (3.9 g, 15.6 mmol, yield

100%) as a white solid. A portion of the obtained 3-((2-chloropyridin-

3-yl)amino)-5,5-dimethylcyclohex-2-enone 47 (700 mg, 2.79 mmol)

was combined with Cs₂CO₃(2.73 g, 8.38 mmol), Pd(PPh₃)₄(322 mg,

0.28 mmol), and DMF (5 mL) and heated at 150 °C for 2 h. The

reaction mixture was cooled and diluted with DCM. The mixture was

passed through a short plug of silica gel (3 g) using DCM, and the

eluent was concentrated under reduced pressure to give a white solid

1

(455 mg, 2.13 mmol, yield 76%). H NMR (400 MHz, DMSO-d₆) δ

13.60 (s, 1H), 8.62 (d, J = 7.8 Hz, 1H), 8.53 (d, J = 5.7 Hz, 1H), 7.73

(dd, J = 8.1, 6.0 Hz, 1H), 3.09 (s, 2H), 2.53 (s, 2H), 1.12 (d, J = 12.5

Hz, 6H).

7,7-Dimethyl-6,7,8,9-tetrahydro-5H-pyrido[3,4-b]indol-5-

one (51). A microwave vessel was loaded with 3-[(4-chloro-3-

pyridyl)amino]-5,5-dimethyl-cyclohex-2-en-1-one 48 (400 mg, 1.60

mmol), Pd(PPh₃)₄(369 mg, 0.32 mmol), and Cs₂CO₃(1.60 g, 4.8

mmol). The vessel was purged with N₂and heated at 150 °C for 6 min

via microwave irradiation. The mixture was diluted with MeOH (5

mL) and DCM (100 mL) and ﬁltered. The ﬁltrate was evaporated to

dryness under reduced pressure. The residue was puriﬁed via silica gel

chromatography using 10% MeOH in DCM to obtain a light-yellow

1

8-(4-Methoxyphenyl)-6-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahy-

solid (300 mg, 1.40 mmol, yield 88%). H NMR (400 MHz, DMSO-

dro-1H-indol-1-yl)-3,4-dihydroisoquinolin-1(2H)-one (41). Yield

d₆) δ 12.29 (s, 1H), 8.74 (s, 1H), 8.25 (d, J = 5.3 Hz, 1H), 7.82 (d, J =

5.3 Hz, 1H), 2.91 (s, 2H), 2.37 (s, 2H), 1.09 (s, 6H).

1

61%. H NMR (400 MHz, DMSO-d₆) δ 7.93 (s, 1H), 7.38 (d, J =

2.1 Hz, 1H), 7.33−7.20 (m, 2H), 7.14 (d, J = 2.2 Hz, 1H), 7.04−6.85

(m, 3H), 3.79 (s, 3H), 3.48−3.40 (m, 2H), 2.97 (t, J = 6.0 Hz, 2H),

2.76 (s, 2H), 2.26 (s, 2H), 2.22 (s, 3H), 1.01 (s, 6H). ¹³C NMR (100

MHz, DMSO-d₆) δ 193.55, 163.38, 158.20, 143.84, 143.07, 141.63,

139.21, 133.46, 129.72, 126.75, 125.00, 121.48, 120.98, 118.73, 118.37,

112.99, 55.02, 52.01, 36.50, 35.17, 29.86, 27.95, 11.28. HRMS (ESI):

[M + H]⁺calcd for C₂₇H₂₈N₂O₃, 429.1729; found, 429.2173.

3-((4-Chloropyridin-3-yl)amino)-5,5-dimethylcyclohex-2-

enone (48). A stirring mixture of 4-chloropyridin-3-amine 45 (1.0 g,

7.8 mmol), 5,5-dimethylcyclohexane-1,3-dione (3.3 g, 23.3 mmol), and

p-TsOH-H₂O (150 mg, 0.78 mmol) in toluene (100 mL) was heated

at reﬂux for 3 h using a Dean−Stark apparatus to remove water. The

cooled solution was partitioned between DCM (500 mL) and

saturated aq NaHCO₃solution (300 mL). The organic portion was

evaporated to dryness under reduced pressure. The residue was

puriﬁed via silica gel chromatography using 5% MeOH in DCM to

obtain the desired product (530 mg, 2.12 mmol, yield 27%). ¹H NMR

(400 MHz, CDCl₃) δ 8.62 (s, 1H), 8.35 (d, J = 5.2 Hz, 1H), 7.40 (d, J

= 5.2 Hz, 1H), 6.08 (s, 1H), 5.39 (s, 1H), 2.61 (s, 2H), 2.26 (s, 2H),

1.09 (s, 6H).

3-((3-Bromopyridin-2-yl)amino)-5,5-dimethylcyclohex-2-

enone (49). A stirring solution of 2-amino-3-bromopyridine 46 (10 g,

57.8 mmol), 5,5-dimethyl-1,3-cyclohexanedione (24.3 g, 173.4 mmol),

p-TsOH-H₂O (1.1 g, 5.78 mmol), and toluene (400 mL) was heated

at reﬂux under N₂for 20 h using a Dean−Stark apparatus to remove

water. The reaction mixture was concentrated under reduced pressure.

The residue was dissolved in DCM (1 L) and washed with saturated

aq NaHCO₃solution (500 mL). The organic portion was removed

and washed with saturated aq NaHCO₃solution (3 × 500 mL), water

(500 mL), and saturated aq NaCl solution (500 mL). The organic

portion was dried over Na₂SO₄and ﬁltered. The ﬁltrate was

concentrated under reduced pressure, and the residue was puriﬁed

by silica gel chromatography using 5−40% EtOAc in hexanes to give a

7,7-Dimethyl-6,7,8,9-tetrahydro-5H-pyrido[2,3-b]indol-5-

one (52). A stirring mixture of 3-(3-bromopyridin-2-ylamino)-5,5-

dimethylcyclohex-2-enone 49 (5.0 g, 16.9 mmol), Pd(PPh₃)₄(980 mg,

0.85 mmol), Cs₂CO₃(8.3 g, 25.4 mmol), and DMF (20 mL) was

heated at 150 °C for 3 h. The reaction mixture was ﬁltered through a

pad of Celite, and the ﬁlter cake was washed with DCM (50 mL). The

ﬁltrate was washed with saturated aq NaCl solution (150 mL), dried

over Na₂SO₄(250 g), and concentrated under reduced pressure. The

residue was a puriﬁed by silica gel chromatography using 5% MeOH in

1

DCM to give a tan solid (1.3 g, yield 36%). H NMR (400 MHz,

DMSO-d₆) δ 12.36 (s, 1H), 8.22 (ddd, J = 9.4, 6.3, 1.6 Hz, 2H), 7.19

(dd, J = 7.7, 4.8 Hz, 1H), 2.88 (s, 2H), 2.36 (s, 2H), 1.10 (s, 6H).

2,2-Dimethyl-2,3,5,6,7,8-hexahydro-1H-carbazol-4(9H)-one

(54). To a stirring solution of 2E)-2-hydroxyiminocyclohexanone 53

(50 mg, 0.39 mmol), 5,5-dimethylcyclohexane-1,3-dione (55 mg, 0.39

mmol), AcOH (2.8 mL), and H₂O (1.2 mL) was added Zn powder

(51 mg, 0.79 mmol). The mixture was heated at 80 °C for 19 h. The

mixture was added to H₂O (50 mL) and neutralized to pH 7 with

saturated aq NaHCO₃. The mixture was extracted with EtOAc (2 × 20

mL), and the organic fractions were combined, dried (MgSO₄),

ﬁltered, and concentrated under reduced pressure. The residual oil was

puriﬁed via silica gel chromatography using 10−70% EtOAc in hexanes

to yield the desired product as a white solid (25 mg, 0.12 mmol, yield

1

29%). H NMR (400 MHz, MeOD) δ 2.64 (d, J = 2.8 Hz, 4H), 2.52

(s, 2H), 2.27 (s, 2H), 1.79 (dd, J = 17.4, 11.6 Hz, 4H), 1.11 (s, 6H).

2,7,7-Trimethyl-3,4,6,7,8,9-hexahydro-1H-pyrido[3,4-b]-

indol-5(2H)-one (59). To a stirring solution of 2,2-dimethoxy-N-

methyl-ethanamine 56 (18.1 g, 19.5 mL, 152.1 mmol) and AcOH (60

mL), under N₂, at rt was added 37% formaldehyde in water (12.3 g,

11.3 mL, 152.1 mmol). The solution was stirred at rt for 10 min. This

solution was then added dropwise to a stirring solution of 6,6-

dimethyl-5,7-dihydro-1H-indol-4-one 55 (25 g, 152.1 mmol) and

AcOH (250 mL) over a period of 30 min. The solution was then

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Article

stirred at rt for 24 h. The solution was partitioned between water (700

mL) and Et₂O (50 mL). The aqueous portion was carefully neutralized

with 30% aq NH₄OH solution and extracted with DCM (4 × 200

mL). The organic portions were combined, dried (Na₂SO₄), and

evaporated to dryness. The brown oil was puriﬁed by silica gel

chromatography using 5% MeOH in DCM to obtain the acetal

intermediate as a yellow oil (37.0 g). The acetal intermediate was

added to aq HCl (170 mL of 8 M, 1.4 mol) and was heated at 70 °C

for 5 min. The solution was quickly cooled to 25 °C and injected via

syringe into a N₂purged vessel containing Pd on C (36.2 g, 33.9

mmol) and EtOH (200.0 mL). The mixture was shaken at 25 °C for

24 h under 1 atm of H₂at 30 psi using a Parr apparatus. The solution

was ﬁltered through a bed of Celite, and the ﬁltrate was concentrated

under reduced pressure. The residual oil was dissolved in water (100

mL) and basiﬁed to pH 10 using 30% aq NH₄OH. The precipitate was

ﬁltered, washed with water (20 mL), and vacuum-dried to obtain the

between Et₂O (250 mL) and water (350 mL). The organic layer was

evaporated to dryness under reduced pressure, and the residue was

puriﬁed via silica gel chromatography using 30% EtOAc in hexanes to

give a white solid (7.9 g, 27.7 mmol, yield 82%). ¹H NMR (400 MHz,

DMSO) δ 7.00−6.85 (m, 2H), 6.78 (d, J = 6.2 Hz, 1H), 4.32−4.15

(m, 1H), 3.92−3.83 (m 2H), 3.76−3.68 (m, 1H), 3.69−3.62 (m, 1H),

2.33−2.09 (m, 1H), 2.04−1.77 (m, 1H).

2-Bromo-4-(2,7,7-trimethyl-5-oxo-3,4,5,6,7,8-hexahydro-1H-

pyrido[3,4-b]indol-9(2H)yl)benzonitrile (65). 2,7,7-trimethyl-

1,3,4,6,8,9-hexahydropyrido[3,4-b]indol-5-one 59 (750 mg, 3.23

mmol) was dissolved in DMF (7.5 mL), and to it was added NaH

(258 mg, 6.46 mmol, 60% in mineral oil). The mixture was allowed to

stir for 5 min. To this mixture was added 2-bromo-4-ﬂuoro-

benzonitrile (1.6 g, 8.1 mmol), and the mixture was stirred at 150

°C for 10 min. The reaction was cooled and puriﬁed by silica gel

chromatography using 20% MeOH in DCM to aﬀord a white solid

1

(970 mg, 2.35 mmol, yield 73%). H NMR (400 MHz, DMSO-d₆) δ

desired compound as a white solid (5.1 g, 21.9 mmol, yield 14%). H

8.13 (d, J = 8.3 Hz, 1H), 8.04 (d, J = 2.0 Hz, 1H), 7.65 (dd, J = 8.3, 2.0

Hz, 1H), 3.21 (s, 2H), 2.76−2 0.71 (m, 2H), 2.61−2.57 (m, 4H), 2.29

(s, 3H), 2.24 (s, 2H), 0.99 (s, 6H).

NMR (400 MHz, DMSO-d₆) δ 10.93 (s, 1H), 2.66−2.52 (m, 5H),

2.50 (dd, J = 3.6, 1.8 Hz, 3H), 2.34 (s, 3H), 2.14 (s, 2H), 1.03 (d, J =

9.9 Hz, 6H).

6,8-Diﬂuoro-3,4-dihydroisoquinolin-1(2H)-one (67). To a

stirring solution of 5,7-diﬂuoroindan-1-one 66 (10 g, 59.5 mmol) in

MeSO₃H (60 mL) at 0 °C was added NaN₃(4.1 g, 62.4 mmol) in

portions over 10 min (exothermic). The mixture was stirred at 0 °C

for 30 min, then allowed to warm to rt and stirred for 2 h. The mixture

was poured onto ice (150 g), and to this solution was added 1.0N

NaOH aq solution until pH 10 was reached. The organic layer was

separated and concentrated to a volume of 15 mL. Et₂O (50 mL) was

added, and the mixture was ﬁltered and vacuum-dried to give a white

7,7-Dimethyl-3,4,6,7,8,9-hexahydro-1H-pyrido[3,4-b]indol-

5(2H)-one (61). To a stirring solution of N-benzyl-2,2-dimethoxy-

ethanamine 58 (29.7 g, 152.1 mmol) and AcOH (60 mL), under N₂,

at rt was added formaldehyde (12.3 g, 11.3 mL, 152.1 mmol, 37%

solution in water). The solution was stirred at rt for 10 min. This

solution was then added dropwise to a stirring solution of 6,6-

dimethyl-5,7-dihydro-1H-indol-4-one 55 (25.0 g, 152.1 mmol) and

AcOH (250 mL) over a period of 30 min. The solution was then

stirred at rt for 19 h. The solution was concentrated under reduced

pressure. The residue was carefully neutralized with saturated aq

NaHCO₃solution and extracted with DCM (3 × 200 mL). The

organic portions were combined, dried (Na₂SO₄), and evaporated

under reduced pressure to give a yellow oil. The oil was puriﬁed by

silica gel chromatography using 50% EtOAc in hexanes to obtain a

semipure oil. The obtained oil was dissolved in EtOAc (300 mL), and

to this solution was added Et₂O (200 mL). The formed precipitate was

ﬁltered and vacuum-dried to obtain the intermediate acetal as a white

solid (44 g). The acetal intermediate was dissolved in aq HCl (455 mL

of 8 M, 3.64 mmol) and heated at 70 °C for 15 min. The solution was

quickly cooled to 25 °C and injected via syringe into a N₂purged

vessel containing 10% Pd on C (43.1 g, 40.5 mmol) and EtOH (450

mL). The mixture was shaken at 25 °C for 20 h under H₂at 16 psi

using a Parr apparatus. The solution was ﬁltered through a bed of

Celite, and the ﬁltrate was concentrated under reduced pressure. The

residual oil was dissolved in water (100 mL) and basiﬁed to pH 10

using 30% aq NH₄OH. The solution was extracted with DCM (8 ×

100 mL). The organic portions were combined, dried (Na₂SO₄), and

concentrated under reduced pressure. The residual solid was triturated

with Et₂O (100 mL), and the resulting white solid was dried under

1

solid (5.2 g, 28.4 mmol, yield 48%). H NMR (400 MHz, CDCl₃) δ

7.28 (d, J = 4.5 Hz, 1H), 6.81 (d, J = 2.4 Hz, 1H), 6.80−6.78 (m, 1H),

6.77 (s, 1H), 3.56−3.48 (m, 2H), 2.97 (dd, J = 12.3, 5.8 Hz, 2H).

(E)-2-(2-Cyclopropylvinyl)-4-ﬂuorobenzonitrile (69). A mix-

ture of 2-bromo-4-ﬂuoro-benzonitrile (100 mg, 0.50 mmol), 2-[(E)-2-

cyclopropylvinyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (97 mg, 104

μL, 0.50 mmol), tert-butylamine (110 mg, 157 μL, 1.50 mmol), and

Pd(dppf)Cl₂(37 mg, 0.05 mmol), was taken up in 2-propanol (3.3

mL) and water (1.6 mL). This mixture was stirred at 90 °C under N₂

for 19 h. The mixture was ﬁltered, and the ﬁltrate was concentrated

under reduced pressure. The residue was partitioned between EtOAc

and water. The aqueous phase was extracted twice with EtOAc. The

organic phases were combined, dried (MgSO₄), ﬁltered, and

concentrated under reduced pressure. The residue was puriﬁed by

silica gel chromatography with 0−100% EtOAc in hexanes to obtain a

1

white solid (67 mg, 0.36 mmol, yield 72%). H NMR (400 MHz,

CDCl₃) δ 7.58 (dd, J = 8.6, 5.6 Hz, 1H), 7.24−7.18 (m, 1H), 6.94

(ddd, J = 8.5, 7.9, 2.5 Hz, 1H), 6.80 (dd, J = 15.6, 1.5 Hz, 1H), 5.92

(dd, J = 15.6, 9.3 Hz, 1H), 1.79−1.61 (m, 1H), 1.01−0.91 (m, 2H),

0.68−0.52 (m, 2H).

1

vacuum (8.8 g, 40.3 mmol, yield 26%). H NMR (400 MHz, DMSO-

d₆) δ 11.40 (s, 1H), 8.75 (bs, 1H), 4.07 (s, 2H), 3.27−3.22 (m, 2H),

ASSOCIATED CONTENT

2.87−2.79 (m, 2H), 2.62 (s, 2H), 2.19 (s, 2H), 1.02 (s, 6H).

■

tert-Butyl 7,7-Dimethyl-9-oxo-3,4,6,7,8,9-hexahydro-1H-

pyrido[4,3-b]indole-2(5H)-carboxylate (62). To a stirring suspen-

sion of 7,7-dimethyl-2,3,4,6,8,9-hexahydro-1H-pyrido[3,4-b]indol-5-

one 61 (1.0 g, 4.581 mmol) and DCM (20.00 mL), under N₂, at rt

was added Et₃N (556 mg, 766 μL, 5.50 mmol) and di-tert-butyl

dicarbonate (1.2 g, 1.3 mL, 5.50 mmol). The mixture was stirred at rt

for 30 min. The mixture was diluted with MeOH (5 mL) and directly

puriﬁed via silica gel chromatography using 5% MeOH in DCM to

S

* Supporting Information

Analytical data for all compounds and X-ray crystallography

information. This material is available free of charge via the

Internet at http://pubs.acs.org.

Accession Codes

New protein/ligand coordinates have been deposited in the

PDB with codes 4O04, 4O05, 4O07, 4O09, and 4O0B,

respectively.

1

obtain a white solid (1.2g, 3.77 mmol, yield 82%). H NMR (400

MHz, DMSO-d₆) δ 11.06 (s, 1H), 4.31 (s, 2H), 3.50 (t, J = 5.7 Hz,

2H), 3.32 (s, 2H), 2.58 (s, 2H), 2.15 (s, 2H), 1.41 (s, 9H), 1.02 (s,

6H).

(S)-4-Bromo-2-ﬂuoro-6-((tetrahydrofuran-3-yl)amino)-

benzamide (64). To a stirring solution of 4-bromo-2,6-diﬂuoro-

benzonitrile 63 (7.4 g, 33.7 mmol) in DMSO (74 mL) was added

DIEA (10.5 g, 14.1 mL, 80.9 mmol), followed by the addition of (S)-

tetrahydrofuran-3-amine hydrochloride (5.0 g, 40.5 mmol) under N₂.

The mixture was stirred at rt for 19 h. The solution was partitioned

AUTHOR INFORMATION

■

Corresponding Author

*Phone: 858-404-8409. E-mail: Justin_ernst@vrtx.com.

Notes

The authors declare no competing ﬁnancial interest.

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dx.doi.org/10.1021/jm500042s | J. Med. Chem. 2014, 57, 3382−3400

Journal of Medicinal Chemistry

Article

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ACKNOWLEDGMENTS

■

The Berkeley Center for Structural Biology is supported in part

by the National Institutes of Health, National Institute of

General Medical Sciences, and the Howard Hughes Medical

Institute. The Advanced Light Source is supported by the

Director, Oﬃce of Science, Oﬃce of Basic Energy Sciences, of

the U.S. Department of Energy under contract no. DE-AC02-

05CH11231.

ABBREVIATIONS USED

■

HSP90, heat shock protein 90; HD, Huntington’s disease; Htt,

Huntingtin; mHtt, mutant Huntingtin; CNS, central nervous

system; SAR, structure−activity relationship; HOAc, acetic

acid; DMF, dimethylformamide; EtOH, ethanol; DMSO,

dimethyl sulfoxide; dppf or DPPF, 1,10 bis-

(diphenylphosphino)ferrocene; THF, tetrahydrofuran; TFA,

triﬂuoroacetic acid; MsOH, methanesulfonic acid; PBS,

phosphate-buﬀered saline; LCMS or LC-MS, liquid chroma-

tography−mass spectrometry; HPLC, high pressure liquid

chromatography; HRMS, high-resolution mass spectroscopy

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Silinski, M. A.; Hu, M.; Partridge, J. M.; Scott, A.; DuBois, L. G.;

Freed, T.; Steed, P. M.; Ommen, A. J.; Smith, E. D.; Hughes, P. F.;

Woodward, A. R.; Hanson, G. J.; McCall, W. S.; Markworth, C. J.;

Hinkley, L.; Jenks, M.; Geng, L.; Lewis, M.; Otto, J.; Pronk, B.;

Verleysen, K.; Hall, S. E. Discovery of novel 2-aminobenzamide

inhibitors of heat shock protein 90 as potent, selective and orally active

antitumor agents. J. Med. Chem. 2009, 52, 4288−4305.

(11) Polli, J. W.; Wring, S. A.; Humphreys, J. E.; Huang, L.; Morgan,

J. B.; Webster, L. O.; Serabjit-Singh, C. S. Rational use of in vitro P-

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Identification of novel HSP90α/β isoform selective inhibitors using structure-based drug design. demonstration of potential utility in treating CNS disorders such as huntington's disease

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