Discovery of spirofused piperazine and diazepane amides as selective histamine-3 antagonists with in vivo efficacy in a mouse model of cognition

Article

pubs.acs.org/jmc

Discovery of Spirofused Piperazine and Diazepane Amides as

Selective Histamine‑3 Antagonists with in Vivo Eﬃcacy in a Mouse

Model of Cognition

Dean G. Brown,*^,†,∥Peter R. Bernstein,^†Andrew Griﬃn,^‡Steve Wesolowski,^†Denis Labrecque,^‡

Maxime C. Tremblay,^‡Mark Sylvester,^†,∥Russell Mauger,^†Phillip D. Edwards,^†Scott R. Throner,^†,∥

James J. Folmer,^†Joseph Cacciola,^†Clay Scott,^†,∥Lois A. Lazor,^†Mehrnaz Pourashraf,^†

Vijayaratnam Santhakumar,^†William M. Potts,^†Simon Sydserﬀ,^†Pascall Giguere,^§Carine Levesque,^§

̀

́

Mohammed Dasser,^§and Thierry Groblewski^‡,⊥

^†AstraZeneca CNS Discovery Research, 1800 Concord Pike, Wilmington, Delaware 19850, United States

^‡AstraZeneca Montrea

l, 7171 Frederick-Banting, Montreal, Quebec H4S 1Z9, Canada

^§OmegaChem, 480 Rue Perreault, Saint-Romuald, Queb

ec G6W 7 V6, Canada

́

S

* Supporting Information

ABSTRACT: A new series of potent and selective histamine-3

receptor (H₃R) antagonists was identiﬁed on the basis of an

azaspiro[2.5]octane carboxamide scaﬀold. Many scaﬀold mod-

iﬁcations were largely tolerated, resulting in nanomolar-potent

compounds in the H₃R functional assay. Exemplar compound 6s

demonstrated a selective proﬁle against a panel of 144 secondary

pharmacological receptors, with activity at only σ2 (62% at 10

μM). Compound 6s demonstrated free-plasma exposures above

the IC₅₀(∼50×) with a brain-to-plasma ratio of ∼3 following

intravenous dosing in mice. At three doses tested in the mouse novel object recognition model (1, 3, and 10 mg/kg s.c.), 6s

demonstrated a statistically signiﬁcant response compared with the control group. This series represents a new scaﬀold of H₃

receptor antagonists that demonstrates in vivo exposure and eﬃcacy in an animal model of cognition.

may reduce oﬀ-target issues.¹⁵The challenges of safety of H₃

INTRODUCTION

■

antagonists have been recently discussed in a review by

Plancher,¹¹in particular, hERG, phospholipidosis (PLD), and

mutagenicity, which can be mitigated in certain structural

classes but still remain an issue with this type of

pharmacophore. Because hERG tends to favor large hydro-

phobic groups often satisﬁed by hydrophobic aromatic

elements,¹⁶this series, which exempliﬁed the exclusion or

reduction of aromatic rings, oﬀered the opportunity to test our

hypothesis. Our investigation began with 6-azaspiro[2.5]octane

carboxamides (spirocyclopropyl core) and then expanded to

core modiﬁcations such as 7-azaspiro[3.5]nonane carboxamides

(spirocyclobutyl core) as well as ﬁve- and six-membered ring

spirofused analogues. A number of molecules made in this

eﬀort possessed two basic groups, which is a feature present in

the pharmacophore of other H₃antagonists such as the natural

products Aplysame-1¹⁷and Conessine^18a−c(Figure 1) as well

as the two synthetic examples from JNJ, namely, JNJ-5207852

and Bavisant (JNJ-31001074).^13e,f,19,20Both the piperazine and

diazepane scaﬀolds have been useful pharmacophoric elements

in the ﬁeld, with a wide amount of structural diversity of

The histamine-3 receptor (H₃R) is an autoreceptor that

operates presynaptically in histaminergic neurons.¹Evidence

suggests that selective blockade of H₃R results in increased

levels not only of histamine but also of noradrenaline,

acetylcholine, and dopamine.²As such, H₃R antagonists have

been the focus of many recent drug discovery eﬀorts, with

interests spanning sleep disorders, cognition, attention deﬁcit

hyperactivity disorder (ADHD), osteoarthritis, Tourette

syndrome, neuropathic pain, and metabolic disorders.³⁻¹¹

Over the years, selective antagonists at this receptor evolved

from analogues of the endogenous ligand histamine into more

advanced ligands that arguably are structurally distinct from

histamine because they lack the imidazole chemotype.¹²

Publications continue to emerge with a diversity of core

scaﬀolds capable of inhibiting H₃receptors.¹³Cardiovascular

safety has proven to be an area of recurring concern, potentially

because of oﬀ-target eﬀects such as those arising from human

ether-a-

̀

go-go-related gene (hERG) potassium channel binding.¹⁴

In the course of this project, we discovered a new class of

selective H₃R antagonists. Of interest to us in this class of

molecules was the lack of an aromatic ring that is present in

most structures within the published H₃literature. We

hypothesized that the lack of aromaticity in the pharmacophore

Received: September 24, 2013

A

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Figure 1. Representative H₃scaﬀolds with dibasic, piperazine, and diazepane scaﬀolds.

attachments to these cores. Three examples of advanced

compounds in the ﬁeld with these elements are shown in

Figure 1 (NNC-38-1049,²¹GSK 334429, and JNJ

39220675^22,23). We report herein the structure−activity

relationships (SAR), in vitro drug metabolism pharmacoki-

netics (DMPK), and in vivo assessment of example molecules

from this eﬀort.

protected piperidin-4-one 1. The spirocycle was then

introduced by reaction of CuCN and ethyldiazoacetate to

give ester 3. Deprotection of the Cbz group with catalytic

hydrogenation gave free amine 4, which was subsequently

alkylated under reductive amination conditions to give

intermediate ester 5. To ﬁnish the sequence, the ester was

cleaved, and the carboxamide was introduced by coupling to

the appropriate piperazine to give ﬁnal products 6a−e and 6g−

i.²⁴

An alternative method was employed to add versatility for

incorporating diversity elements to the route in Scheme 1,

which is illustrated in Scheme 2. Intermediate 3 from Scheme 1

was hydrolyzed to give intermediate acid 7, which was then

coupled with Boc-protected piperazine to provide 8. The Cbz

group on the piperidine nitrogen was then removed under

catalytic hydrogenation to provide 9 and subsequently reacted

with a ketone under reducing conditions to provide 10. At this

point, the piperazine was deprotected to provide free amine 11,

which was subjected to a second reductive amination to give

ﬁnal compound 6f.

CHEMISTRY

■

The synthesis of the basic core 6-azaspiro[2.5]octane

carboxamide scaﬀold is shown in Scheme 1. Intermediate

exocyclic oleﬁn 2 was generated by Wittig oleﬁnation of Cbz-

a

Scheme 1

Entry into analogues wherein the two N-alkyl groups were

reversed is outlined in Scheme 3. Intermediate 7 was coupled

with 1-(tetrahydro-2H-pyran-4-yl)piperazine to provide 12,

which in turn underwent reductive amination, providing 6j.

A series of spiropiperidine analogues were made in a similar

fashion where the piperazine ring was held constant with a

cyclohexyl substituent. The synthesis of these analogues is

provided in Scheme 4. Intermediate 7 was acylated with 1-

cyclohexylpiperazine to provide 13 and deprotected to give 6k.

This intermediate was acylated, alkylated, or aminated under

Buchwald−Hartwig conditions²⁵to provide title compounds

6l−q.

a

+

Reagents and conditions: (a) Ph₃PCH₃Br⁻, n-BuLi, THF, 54%; (b)

CuCN, ethyldiazoacetate, CH₂Cl₂, 45%; (c) EtOH, Pd/C, H₂, 44%;

(d) 1,2-DCE, dihydro-2H-pyran-4-(3H)-one, Na(OAc)₃BH, rt, 64%;

(e) LiOH, MeOH, quan.; (f) HBTU, DMF, DIPEA, amine, 34−80%.

Optically pure material was accessed through the route

illustrated in Scheme 5. Racemic Cbz-protected ester 3 was

separated by chiral supercritical ﬂuid chromatography (SFC) to

B

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a

provide pure stereoisomers 3a and 3b. The Cbz groups were

removed by hydrogenolysis to provide intermediate amines 4a

and 4b, which were assigned to their absolute relative

conﬁgurations using computed and observed vibrational

circular dichroism (VCD) spectra (Figure 5).²⁶

Scheme 2

Cbz-protected intermediates 3a and 3b were hydrolyzed with

aqueous sodium hydroxide to provide (R) and (S) stereo-

isomers 7a and 7b, respectively. The optically pure acids were

coupled to 1-isopropylpiperazine to provide Cbz-protected

piperazines 14a and 14b. Deprotection and reductive amination

were conducted in the same reaction to provide ﬁnal

compounds 6r and 6s.

The synthesis of an analogue in which the spiropiperidine

basic nitrogen was removed and replaced with a spirocyclohex-

ane is outlined in Scheme 6. Starting with ketone 15, Wittig

oleﬁnation was performed to provide ester 16 with 72% yield.

Cyclopropanation was then achieved using dimethylsulfonium

ylide under basic conditions to provide 17. Subsequent

hydrolysis and amide bond formation with cyclobutyl

piperidine provided ﬁnal compound 6t.

The construction of spirocyclobutyl analogues is detailed in

Scheme 7. The synthesis began with tert-butyl 4-methylenepi-

peridine-1-carboxylate 18, which was converted to dichlor-

oketone 19 by reaction with Cl₃COCl. The chlorine atoms

were subsequently removed with Zn to provide ketone 20. The

ketone was then reduced with NaBH₄and converted to the

mesylate, providing intermediate mesylate 21. The mesylate

was then displaced with cyanide and hydrolyzed to give Boc-

protected ester 23.

a

Reagents and conditions: (a) LiOH, THF, 94%; (b) HBTU, DIPEA,

tert-butyl piperazine-1-carboxylate, 69%; (c) EtOH, Pd/C, H₂, AcOH

(cat.), 99%; (d) EtOH, NaBH₃CN, AcOH (cat.), dihydro-2H-pyran-4-

(3H)-one; (e) CH₂Cl₂, TFA, 99%; (f) CH₂Cl₂, cyclohexanone, PS-

CNBH₄, 66%.

a

Scheme 3

Preparation of ﬁnal analogues on the cyclobutyl scaﬀold is

shown in bottom synthetic pathway of Scheme 7. Boc-

protected ester 23 was deprotected and subjected to reductive

amination with dihydro-2H-pyran-4-(3H)-one under catalytic

hydrogenation to give intermediate ester 28. Hydrolysis and

amide bond coupling with varying piperazines provided ﬁnal

compounds 29a−f.

An alternative synthesis illustrated in Scheme 8 was

employed to provide intermediates for analogues on the

cyclobutyl scaﬀold. Cbz-protected oleﬁn 2 (previously

described in Scheme 1) was reacted with dichloroketene

precursor to give intermediate dichloroketone 24. This was

reduced to ketone 25 with Zn and then converted to aldehyde

26 by Wittig oleﬁnation. The ﬁnal intermediate was then

obtained by 2,2,6,6-tetramethylpiperidine oxide (TEMPO)

oxidation to provide acid 27.

a

Reagents and conditions: (a) HBTU, 1-(tetrahydro-2H-pyran-4-

yl)piperazine dihydrochloride, 11%; (b) Pd/C, H₂, MeOH, cyclo-

butanone, 46%.

a

Scheme 4

Intermediate 27 was utilized to explore substitution

projecting from the piperidine nitrogen atom. The route

employed for these compounds is shown in the bottom

synthetic pathway of Scheme 8. Intermediate 27²⁷was reacted

with 1,4-cyclobutyl piperazine and 1,4-cyclobutyl diazepane to

form intermediate Cbz-protected piperazines 30g and 30j. The

Cbz group was removed, and various coupling strategies were

employed to derivatize the N atom, such as sulfonamide

formation (29g), Buchwald−Hartwig amination (29h),²⁵and

acylation (29i and 29j).

An azetidine analogue was prepared according to Scheme 9.

Spirofused azetidine 32 was acylated with p-nitrophenyl

carbonochloridate and then reacted with isopropyl piperidine

to provide intermediate 33. This intermediate was converted to

ﬁnal compound 34 under the reductive amination conditions

previously described in Scheme 8.

a

Reagents and conditions: (a) DMF, HBTU, DIPEA, 1-cyclo-

hexylpiperazine, 70%; (b) MeOH, Pd/C, H₂, rt, 95%; (c) 4-Cl-

pyridine, K₂CO₃, DMSO, 26%; (d) PhMe, Li(HMDS), PhBr,

PdCl₂[P(o-tol)₃]₂, 12%; (e) 1,2-DCE, R₂-CHO, Na(OAc)₃BH, rt,

30−60%.

Spirocyclic fused pyrrolidines attached to the 3 and 4

positions of the piperidine ring are illustrated in Schemes 10

C

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a

Scheme 5

a

Reagents and conditions: (a) Chiral SFC, quan.; (b) Pd/C, H₂, EtOH, rt, 59% for 4a and 82% for 4b; (c) NaOH, MeOH, H₂O, quan; (d) HBTU,

1-isopropylpiperazine, quan.; (e) Pd/C, H₂, MeOH, dihydro-2H-pyran-4-(3H)-one, 30 psi, 45% for 6r and 68% for 6s.

a

intermediate urea by reaction with p-nitrophenyl carbon-

ochloridate and 1,4-isopropyl piperazine. Deprotection of the

Boc group with HCl followed by reductive amination provided

41a. In an analogous fashion, Scheme 11 illustrates the

synthesis of cyclobutyl piperidines and diazpeines for the 3-

and 4-substituted piperidines. In this set, several N substituents

were explored, such as N-acyl and N-sulfonyl, following the

chemistry outlined in Scheme 11.

Scheme 6

Scheme 12 illustrates the synthetic protocol used to access

the piperidine replacement of the right-hand-side piperazine.

Boc-protected spirofused intermediate 42 was acylated with

benzoyl chloride to provide 43, which then underwent coupling

with 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid to

yield intermediate 44. This intermediate was subjected to

reductive amination with cyclobutanone to give ﬁnal compound

41g.

The ﬁnal synthetic protocol is shown in Scheme 13, which

illustrates the route employed to gain access to the spirofused

bis-piperidine scaﬀold. The scheme was analogous to that used

in Schemes 10 and 11, whereby starting spirofused amine 45

was converted to urea 46 to provide ﬁnal compound 41h using

protocols outlined in Scheme 13.

a

Reagents and conditions: (a) THF, NaH, (MeO)₂PCH₂CO₂Me,

cyclohexanone, 72%; (b) DMF, NaH, Me₃S⁺I⁻, 49%; (c) LiOH, THF,

quan.; (d) DMF, HBTU, DIPEA, cyclobutylpiperazine, 69%.

RESULTS AND DISCUSSION

■

and 11. For N-tetrahydropyran, isopropyl piperidine, and

diazepane analogues, the synthesis began with spirofused

intermediate 35 or 39 (Scheme 10). The free nitrogen of 35

was acylated with carbonyldiimidazole (CDI), and the

intermediate was methylated with methyl iodide and then

reacted with 1-isopropylpiperazine to give 36b. This was

converted into the ﬁnal test compounds through reductive

amination to provide 37b. Access to the 4-piperidine spirofused

cyclopentanes began with 39. This was converted to the

Initial investigations in this program began with the 6-

azaspiro[2.5]octane (spirocyclopropyl) core reported in Table

1. Functional data was determined using an agonist-stimulated

[³⁵S]GTPγS (GTPγS) binding assay with membranes from

CHO-K1 cells stably transfected with the long form of the

human H₃R (445 amino acids).²⁸To complement the

functional assay, a binding assay was also utilized that measured

the ability of test compounds to displace [³H]-N-α-methylhist-

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a

Scheme 7

a

Reagents and conditions: (a) Zn−Cu, Et₂O, Cl₃COCl, 40%; (b) NH₄Cl, MeOH, Zn, 97%; (c) NaBH₄, MeOH, 99%; (d) Et₃N, MsCl, CH₂Cl₂,

97%; (e) NaCN, TBAB, DMF, 57%; (f) EtOH, H₂SO₄; Boc₂O, 41%; (g) TFA, CH₂Cl₂, rt, 83%; (h) MeOH, dihydro-2H-pyran-4-(3H)-one, Pd/C,

H₂, rt, 58%; (i) MeOH, 1 N NaOH, reﬂux, quan.; (j) DMF, HBTU, amine, DIPEA, 76%; (k) CO₂Cl₂, CH₂Cl₂; DIPEA, R¹-amine, CH₂Cl₂, 32−65%.

a

Scheme 8

a

Reagents and conditions: (a) Et₂O, Zn−Cu, Cl₃COCl, DME, 40%; (b) MeOH, NH₄Cl, Zn, 74%; (c) THF, KOt-Bu, BuOH, Ph₃PCH₂OMe⁺Cl⁻,

48%; (d) CH₂Cl₂/NaHCO₃, NaOCl, TEMPO, NaBr, 72%; (e) DMF, DIEA, amine, HBTU, 78% or SO₂Cl₂, DIEA, CH₂Cl₂, quan.; (f) EtOH, Pd/C,

H₂50 psi, 88−96%; (g) CH₂Cl₂, TEA, MeSO₂Cl, 39%; (h) PhMe, Ph-Br, Pd(OAc)₂, Cs₂CO₃, BINAP, 110 °C, 18h, 46%; (i) CH₂Cl₂, DIPEA,

PhCOCl, 33−64%.

amine binding to human H₃R-K1 membranes.²⁹Because of the

a

Scheme 9

good correlation of the two assays and the translational

importance of the functional assay, the functional GTPγS assay

became the only SAR-driving assay utilized for the project, with

an occasional conﬁrmatory test using the binding assay. This

series of H₃antagonists was not routinely assessed for inverse-

agonist activity. However, 6d reduced basal GTPγS binding to

membranes expressing recombinant human H₃R, which is

consistent with inverse-agonist activity and has been reported

for other H₃antagonists.¹⁴Because the pharmacophore

possesses a lipophilic basic amine and prior H₃compounds

were known to have a history of hERG activity, the hERG data

(electrophysiological measurements in CHO cells) is also

reported even though only a few compounds in the series ever

demonstrated even a modest signal.

a

Reagents and conditions: (a) THF, 4-NO₂PhOCOCl, DIPEA, 1-

isopropylpiperazine, reﬂux, 72%; (b) HCl, MeOH, rt, quan.; (c)

MeOH, TEA, dihydro-2H-pyran-4-(3H)-one, Pd/C, H₂, rt, 28%.

Table 1 highlights the importance of the SAR of the N-alkyl

piperazine substituent. N-Me derivative 6a has relatively weak

E

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a

Scheme 10

Scheme 12

a

Reagents and conditions: (a) 2-MeTHF, PhCO₂H, DIPEA, 4-(4,6-

dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium, 61%; (b)

HCl, MeOH, quan.; (c) 2-MeTHF, DIPEA, 4-(4,6-dimethoxy-1,3,5-

triazin-2-yl)-4-methylmorpholin-4-ium, 1-(tert-butoxycarbonyl)-

piperidine-4-carboxylic acid, 22%; (d) dioxane, HCl, 64%; (e) DMF,

NaCNBH₄, cyclobutanone, 37%.

a

Reagents and conditions: (a) THF, CDI, Et₃N; (b) ACN, MeI; (c)

CH₂Cl₂, R¹-amine, Et₃N, 40% steps a−c; (d) HCl, iPrOH, Et₂O,

quan.; (e) NaBH(OAc)₃, 1,2-DCE, dihydro-2H-pyran-4-(3H)-one,

AcOH, Et₃N, 40−44%; (f) THF, 4-NO₂PhOCOCl, −78 °C, 1-

isopropylpiperazine, reﬂux, DIPEA, 28%; (g) HCl, MeOH, 75%; (h)

MeOH, TEA, dihydro-2H-pyran-4-(3H)-one, Pd/C, H₂, rt, 62%.

a

Scheme 13

activity in both the functional and binding assays (1100 and

1300 nM, respectively). The addition of isopropyl (6b),

cyclopropyl (6c), and cyclobutyl (6d) led to functional activity

in the double- and single-digit nanomolar region, with no

discernible hERG activity (GTPγS IC₅₀= 3.2, 19, and 3.6 nM,

respectively). The isopropyl and cyclobutyl appeared optimal,

as moving to larger groups such as cyclohexyl (6f) and

cycloheptyl (6g) demonstrated a subtle but gradual loss of

functional activity (GTPγS IC₅₀= 12 and 74 nM, respectively).

Polar modiﬁcations at the terminal N-alkyl piperazine, such as

N-4-pyridyl (6i), also led to decreased activity relative to the

small alkyl derivatives. Compound 6j was an attempt to

understand the promiscuity of the pharmacophore by swapping

a

Reagents and conditions: (a) THF, CDI, Et₃N, quan.; (b) ACN, MeI,

quan.; (c) CH₂Cl₂, Et₃N, 1-cyclobutyl-1,4-diazepane, 80%; (d) HCl,

iPrOH, quan.; (e) benzoyl chloride, CH₂Cl₂, Et₃N, 75%.

a

Scheme 11

a

Reagents and conditions: (a) THF, CDI, Et₃N; (b) ACN, MeI; (c) CH₂Cl₂, R¹-amine, Et₃N, 40% steps a−c; (d) HCl, iPrOH, Et₂O, quan.; (e)

NaBH(OAc)₃, 1,2-DCE, dihydro-2H-pyran-4-(3H)-one, AcOH, Et₃N, 36%; (f) CH₂Cl₂, PhCOCl, TEA, 48%; (g) PhSO₂Cl, TEA, 50%; (h) THF,

CDI, Et₃N, quan.; (i) ACN, MeI, quan.; (j) CH₂Cl₂, R¹-amine, Et₃N, 65−67%; (k) HCl, iPrOH, quan.; (l) CH₂Cl₂, PhCOCl, TEA, 85−88%; (m)

R₂SO₂Cl, TEA, 77−85%.

F

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Table 1. Spirocyclopropyl Analogues

a

Compounds were tested for activity at inhibiting agonist-stimulated [³⁵S]GTPγS binding to membranes from CHO-K1 cells stably transfected with

b

the long form of the H₃receptor (445 amino acids). Results are an average of n > 2 with standard error <20%. SPA binding assay utilized hH₃

c

transfected CHO membranes and [³H]-N-α-methylhistamine. Results are an average of n > 2 with standard error <50%. Values determined in

d

CHO-K1-hERG cells using electrophysiological measurements. logD values between octanol and water, as determined by shake-ﬂask HPLC

methods. nm, not measured.

the N-alkyl side pieces relative to 6d. A loss of ∼18-fold

functional activity relative to 6d was observed, suggesting that a

proper orientation of these two pieces signiﬁcantly contributes

to the binding and functional activity. A series of analogues was

explored where the N-alkyl piperazine derivative was ﬁxed as a

cyclohexyl group (6k−q), and the piperidine N-alkyl group was

subsequently explored. The absence of an N-alkyl group (6k)

led to a 30-fold drop in activity relative to 6f. Replacement of

the tetrahydro-2H-pyran in 6f with a cyclohexyl (6l) gave an

equipotent compound; however, hERG activity began to appear

in the series for the ﬁrst time (20.8 μM). This observation led

us to conclude that having two hydrophobic groups, one oﬀ of

the piperidine N atom and one oﬀ the piperazine N atom, led

to deterioration of the clean oﬀ-target proﬁle in compounds

such as 6f and hence having a polar group oﬀ the piperidine N

atom was important for avoiding these issues. Smaller

hydrophobic groups such as isopropyl 6m were also tolerated.

Addition of an aromatic ring at varying distances was also

explored. Direct attachment of a phenyl ring (6n) led to a

compound with poor binding activity (1670 nM), and as a

result, the functional activity was not measured. This

compound is expected to be a monobasic compound, given

the pK_aof the aniline nitrogen makes it unlikely to be

protonated at physiological pH. However, examples shown later

in Table 2 illustrate that certain monobasic compounds can

have good potency. The reason for the lack of activity for the

phenyl (6n) is unclear, but we speculate it has more to do with

a steric interaction than with a lack of protonatable amine at the

left-hand-side piperidine. Benzyl-linked compound 6o demon-

strated more potent H₃binding than the phenyl analogue (103

nM), although it was still not as potent as polar tetrahydro-2H-

pyran analogue 6f. Extension to the phenethyl analogue 6p

G

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Table 2. Azetidine and Pyrrolidne Analogues with N-Tetrahydropyran-2H-pyran

a

Compounds were tested for activity at inhibiting agonist-stimulated [³⁵S]GTPγS binding to membranes from CHO-K1 cells stably transfected with

b

the long form of the H₃receptor (445 amino acids). Results are an average of n > 2 with standard error <20%. Values determined in CHO-K1-

hERG cells using electrophysiological measurements. logD values between octanol and water, as determined by shake-ﬂask HPLC methods. nm, not

c

measured.

gave activity more comparable to 6f. Replacement of the

tetrahydro-2H-pyran with a 4-pyridyl (6q) led to a compound

with only a slight change from initial starting point 6f as well,

further reinforcing the promiscuity of this pocket with respect

to binding and functional activity. This promiscuity thus

provided opportunities to identify the best substituent from a

pharmacokinetic and oﬀ-target perspective while maintaining

intrinsic activity at the H₃receptor.

The separation and assignment of enantiomers of 6b was

conducted by separation on chiral SFC chromatography of

intermediates 3a and 3b and assignment based on calculated

and measured VCD spectra.²⁶(S) stereoisomer 6s demon-

strated a more favorable functional and binding proﬁle

compared with (R) stereoisomer 6r (GTPγS IC₅₀= 8.3 and

87 nM and binding IC₅₀= 0.8 and 16 nM, respectively).

Compound 6s was thus selected as a tool compound to

H

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Table 3. Spirofused Pyrrolidine and Piperidines with N Substituents

a

Compounds were tested for activity at inhibiting agonist-stimulated [³⁵S]GTPγS binding to membranes from CHO-K1 cells stably transfected with

b

the long form of the H₃receptor (445 amino acids). Results are an average of n > 2 with standard error <20%. Values determined in CHO-K1-

hERG cells using electrophysiological measurements. logD values between octanol and water, as determined by shake-ﬂask HPLC methods. nm, not

c

measured.

benchmark the pharmacokinetic and in vivo eﬃcacy of this

scaﬀold. Finally, a piperidine replacement, 4-cyclohexyl 6t, was

synthesized and tested and demonstrated a complete loss of

activity (>10 uM), thus reinforcing the importance of a

heteroatom in the left-hand-side piperidine position. Further-

more, the impact of solubility on this change was dramatic, as

might be expected, with 6t having a measured solubility of ∼1

μM (data not shown), whereas most other piperidine analogues

were >500 μM.

The SAR was next investigated on a 7-azaspiro[3.5]nonan-2-

yl core (spirocyclobutyl) and is reported in Tables 2 and 3. In

the spirocyclobutyl series, piperazine modiﬁcations on right-

hand-side 29a, 29b, and 29c provided very potent functional

antagonists (GTPγS IC₅₀= 0.8, 1.9, and 1.6 nM, respectively).

In comparison to the spirocyclopropyl matched pairs 6h, 6d,

and 6f, all three spirocyclobutyl compounds were more potent

(cf. GTPγS IC₅₀= 17, 3.6, 12 nM, respectively, for

spirocyclopropyl vs spirocyclobutyl matched pairs). One

possible explanation could be that the spirocyclopropyl

analogues are all racemic (whereas the achiral spirocyclobutyl

is not), so potentially pure separated spirocyclopropyl stereo-

isomers could be more potent (but only theoretically up to a

factor of 2). Because the spirocyclobutyl series is achiral, it

proved advantageous over spirocyclopropyl compounds with

regards to time spent on chiral separation and characterization.

Bicyclic variant 29d demonstrated comparable potency to the

acyclic analogues, suggesting that there was room for further

expansion at the α-position of the piperazine ring. Matched

pairs 29e and 29f were designed to understand the impact of

piperazine compared to 1,4-diazepane, where the isopropyl

group was held constant. The results indicated a negative

impact on the larger core (1,4-diazepane) and was consistent

with the trends observed in the spirocyclopropyl series (6d vs

6h). These results are inconsistent with the equipotent

matched pairs of 29a and 29b mentioned earlier, which

suggests that there is a unique trajectory and binding mode

utilized by the spirocyclobutyl core with an N-cyclobutyl right-

hand-side chain. Azetidine analogue 34 is an N-matched-pair

variant of 29f that demonstrated a signiﬁcant loss in potency

compared with 29f (GTPγS IC₅₀= 900 vs 1.7 nM,

respectively). This trend was not observed with pyrrolidine

analogues 37a, 37b, and 37c (Table 3). N-isopropyl derivative

I

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37b is ∼5−6-fold more potent than comparator azetidine

analogue 34. A more dramatic increase in potency is observed

on switching to 1,4-diazepane N-cyclobutyl 37a (GTPγS IC₅₀

=

4.4 nM). Exploration of the spirocyclic 4-pyrrolidine core

modiﬁcation led to 41a (GTPγS IC₅₀= 32 nM), which was ∼4-

fold more potent than spirocyclic 3-pyrrolidine matched pair

37b.

Figure 2. (left) Molecular overlays of Conesine (gray, small-molecule

X-ray structure) and 6s (purple, low-lying conformation). (right)

Molecular overlays of 29f (red), 34 (green), and Bavisant (light blue).

For 29g−I (Table 3), the N-cyclobutyl piperazine was held

constant, and series of N-sulfonyl (29g), N-phenyl (29h), and

N-benzoyl (29i) derivatives were made. All of these derivatives

were less potent than N-tetrahydro-2H-pyran comparator 29b

by ∼10−30-fold, as measured in the GTPγS functional assay.

The N-isopropyl 1,4-diazepane with the N-benzoyl attachment

to the piperidine ring was prepared (29j) and demonstrated

equal potency to N-tetrahydro-2H-pyran comparator 29e

(GTPγS IC₅₀= 12 vs 14 nM). N-linked spirocyclic

modiﬁcations such as azetidine, 3-pyrrolidine, 4-pyrrolidine,

and 4-piperidine were next explored. Replacement of the N-

tetrahydro-2H-pyran in 37a with N-benzoyl 37c gave a

compound with equal potency (GTPγS IC₅₀= 4.0 nM).

Replacement of the N-benzoyl with N-phenylsulfonyl (37d)

compared to 29f (K_ibinding = 0.3 nM, hH₃(GTPγS) IC₅₀

=

1.7 nM) and Bavisant (example of a potent, conformationally

constrained clinical candidate with reported in vitro K_ibinding

= 5.4 nM).^13fThe three structural overlays of these molecules

can be found that show very little distinction. It is possible that

either unanticipated electronic changes happen between the

cyclobutyl and azetinde linkers or that further subtleties of the

substantially large set of low-lying conformers exist, and simple

overlays based only on access to low-lying conformations are

not an accurate predictor of the bioactive conformation or

potential excluded volume for this linker. Furthermore, the

model would be most useful in predicting binding energetics

and not functional activity because the protein conformational

changes that must occur for a binding ligand to become a

functional ligand are unknown. From a binding perspective, it

may not be possible to distinguish potent binders (e.g., 29f K_i=

0.3 nM and 34 K_i= 97 nM) with a simple conformational

overlay approach. Rank ordering with this approach was not

possible, but it did serve to help understand the general

pharmacophore features within a loose-ﬁt model.

A subset of compounds was examined by in vitro DMPK

proﬁling, which is summarized in Table 4. All of the

compounds tested had high solubility (>500 μM) and

signiﬁcant free-drug concentrations, as determined in both rat

and human plasma protein binding assays (∼50−90% free in

most cases). Most compounds demonstrated high permeability,

as measured in the Madin-Darby canine kidney (MDCK)

permeability assay, except for an occasional molecule, such as

6e, which showed modest eﬄux and low permeability relative

to other compounds. In general, most compounds were not

signiﬁcantly metabolized by rat or human microsomes, with

many compounds measuring <4 μL/min/mg. Again, com-

pound 6e was an outlier, demonstrating relatively higher rat

clearance (43 μL/min/mg). It is not clear why the pyrrolidine

ring on the right-hand-side piperazine results in these modest

changes on these properties. None of the compounds tested

demonstrated any signiﬁcant inhibition of cytochrome P450

enzymes in a representative panel (CYP2D6, CYP3A4, and

CYP2C9). There was no observed diﬀerence between

separated chiral isomers and racemic compounds in this

panel of in vitro DMPK assays; thus, racemic compounds

were tested for expediency. Measured logD values are reported

in Tables 1−3. In general, the logD varied as expected. For

example, the tetrahydropyran substituent contributed to low

and sometimes negative logD values (e.g., 6s, logD = −0.26 and

6i, logD = −1.09), and hydrophobic substituents gave higher

logD values (e.g., 29h N-phenyl, logD = 3.25 and 29i N-

benzoyl, logD = 2.09). However, it did not appear that the

increasing logD contributed substantially to any increased

hERG liability or in vitro DMPK liability because most of the

compounds, regardless of logD value, maintained low hERG

and in vitro DMPK liabilities.

also led to a compound with similar potency (GTPγS IC₅₀

=

6.5 nM), suggesting a wide tolerance of functional groups with

the 3-pyrrolidine-1,4-diazepane-N-cyclobutyl motif. Extension

of the SAR with N-benzoyl, N-phenyl sulfonyl, and N-

isopropylsulfonyl (41b, 41c, and 41d) led to compounds

with single-digit nanomolar potency comparable to spirocyclic

3-pyrroldine matched pairs 37c and 37d. (N-isopropylsulfonyl

was not made in the 3-pyrrolidine series.) Analogues 41e and

41f were made to explore the comparison of N-1,4-cyclobutyl

piperazine to N-1,4-cyclobutyl diazepane with the N-benzoyl

and N-phenylsulfonyl groups attached on the piperidine. The

N-1,4-cyclobutyl piperazine was not as potent as the analogues

with the diazepane ring (41e, GTPγS IC₅₀= 13 nM and 41f,

GTPγS IC₅₀= 21 nM compared with 3.6 and 2.6 nM for

matched pairs with the diazepane ring). Compound 41g was

prepared to examine replacement of the piperazine ring with a

piperidine ring. A signiﬁcant drop in activity was observed

(GTPγS IC₅₀= 340 nM). The 4-piperidine right-hand side is

expected to have less of a planar geometry than the

corresponding piperazine ring, perhaps suggesting the

importance of this as a spatial requirement. Finally, 4-

spirocyclic piperidine ring core modiﬁcation 41h was prepared

and demonstrated ∼6-fold drop in activity compared to 4-

spirocyclic pyrrolidine analogue 41b.

Various molecular overlays were derived from conformations

generated with molecular mechanics methods (see Computa-

tional Spectral Simulations) and were used to help to try to

understand the topological features that may be essential for

activity. Conessine was chosen as a useful benchmark (hH₃K_i

binding = 5.4 nM, hH₃(GTPγS) IC₅₀= 11 nM)^18bgiven its

structural rigidity and availability of an X-ray structure.^18c

Compound 6s (hH₃K_ibinding = 0.8 nM, hH₃IC₅₀(GTPγS) =

8.3 nM) was found to have low-lying conformations with

appropriately placed diamines that mapped well to the structure

of Conessine (Figure 2a). Three other compounds were

modeled (29f, 34, and Bavisant; Figure 2b), and energetically

reasonable conformers were found in each case to superimpose

on one another. This set of structures highlights that even

though a gross approximation of pharmacophore match can be

gained from these types of analyses, the granularity of activity

may not be easily discerned. For example, 34 is weakly active

(hH₃K_ibinding = 97 nM, hH₃(GTPγS) IC₅₀= 900 nM)

J

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Table 4. In Vitro DMPK

a

b

c

d

e

f

g

h

entry

sol

hPPB

rPPB

MDCK perm

eﬄux

hClint

rClint

CYP 2D6 (IC₅₀)

CYP 3A4 (IC₅₀)

CYP 2C9 (IC₅₀)

6b

>500

87

69

5.4

8.4

3.9

<2.2

<1.7

13

1.8

1.5

3.3

>5.5

>2.0

0.7

6.2

3.3

1.5

6.0

4.5

<4

29

7.2

14

<4

8.3

20

18

24

20

<4

43

31

7

>20

nm

>20

nm

6c

nm

91

nm

80

nm

6d

nm

>20

nm

6e

50

65

nm

6l

81

62

>20

6s

nm

51

87

>20

nm

29b

29f

29g

41d

41e

nm

5.1

3.6

19

<4

16

34

25

nm

67

>20

nm

7.8

8.8

nm

>20

a

b

c

Equilibrium solubility (pH 7.4). Human plasma protein binding expressed as the percent free. Rat plasma protein binding expressed as the

d

e

percent free. Permeability measured in MDCK cell lines (A to B P_app1 × 10⁻⁶cm/s). Eﬄux as measured in MDCK cell lines (ratio of A-to-B P_app

over B-to-A P_app). Human liver microsomal clearance expressed as μL/min/mg. Rat liver microsomal clearance expressed as μL/min/mg. In vitro

f

g

h

inhibition of cytochrome P450 CYP2D6, CYP3A4, and CYP2C9 isoforms (IC₅₀values).

Compounds were proﬁled in a small panel of oﬀ-target

receptors as well as against closely related histamine receptors.

A sampling of some of these receptors is shown in Table 5.

>300 μM).³⁰Phospholipidosis (PLD) is a concern among the

lipophilic basic amine scaﬀolds prominent in the H₃ﬁeld³¹and

has been extensively studied by the J&J group. As an example,

compounds such as JNJ-5207852 can be predicted on the basis

of their in vitro PLD values to predict in vivo PLD ﬁndings.³²

The logD was measured for 6s and found to be −0.26, which

was consistent with many compounds in the dibasic series.

Representative logD values are recorded in Tables 1−3. It was

speculated that the low logD in the dibasic motif gave rise to

the lack of activity in the PLD assay; however, compounds of

higher logD were not tested in the PLD assay, which would

have provided a stronger basis for this hypothesis. On the basis

of the positive in vitro DMPK and oﬀ-target proﬁle,

compounds 29b and 6s were followed up with in vivo studies.

Pharmacokinetics. Table 6 summarizes the in vivo

pharmacokinetic (PK) data on six representative compounds.

All of the dibasic compounds (including 6s mentioned earlier)

had very high volumes of distribution and long half-lives. For

example, 29b and 29f had calculated volumes of distribution of

74 and 64 L/kg, respectively, with half-lives >10 h. For those

compounds with this proﬁle, the long half-life did not allow for

precise characterization of the volume of distribution.

Compound 6s demonstrated the lowest rate of clearance of

the three dibasic compounds by a factor of ∼4. The monobasic

compounds, such as 29g, 41d, and 41e, all demonstrated much

lower half-lives and volumes of distribution (e.g., 29g V_ss1.44

L/kg and t_1/2= 0.40) but had higher rates of clearance than 6s.

The high volume of distribution and long half-life for dibasic

compounds has been reported in the H₃area; for example, JNJ

5207852 is reported to have similar characteristics.¹⁹

Table 5. Representative Oﬀ-Target Activity

a

target

29b (activity at 10 μM)

6s (activity at 10 μM)

H₁

>10

nt

>10

H₂

H₄

>10

A2a

5HT1A

NAchE

NET

M₂

D₂

GABA

PDE₄

a

Representative binding values at 10 μM concentration against a

sampling of targets (Ricerca testing laboratories).

Neither 29b nor 6s demonstrated activity against the other

three histamine receptors (H₁, H₂, and H₄) or other biogenic

amine receptors sometimes seen with basic and hydrophobic

motifs. Compound 6s was further tested in expanded panel of

144 in vitro assays to assess any oﬀ-target liabilities (see

Supporting Information for panel list). The only hit from these

screens was modest σ2 receptor activity for 6s (62% at 10 μM),

with the remainder below the deﬁned criteria for activity for

that particular assay at 10 μM single-concentration testing.

Compound 6s was also found to be inactive in an Ames

mutagenicity screen as well as in a phospholipidosis assay (EC₅₀

A more comprehensive evaluation of pharmacokinetics and

central nervous system (CNS) penetration of test compound

Table 6. In Vivo DMPK and PK Parameters

a

b

c

d

e

f

Cpd

V_ss(L/kg)

t_1/2(h)

Clint (mL/min/kg)

n

dose (μmol/kg)

charge

6s

34.6 1.5

74.0 9.1

64

25.3 0.6

14.2 3.9

11.7

16.4 0.8

81.2 17.6

78

4

3.0

2.4

2.1

3.5

1.8

2.3

dibasic

29b

29f

29g

41d

41e

3

1

7

2

4

1.4 0.1

2.1 0.1

3.7 0.2

0.4 0.1

0.6 0.1

1.1 0.1

69.8 5.6

53 0.9

91 3.7

monobasic

a

b

c

Mean volume of distribution measured after IV dosing in male fasted rats. Mean half-life as measured from IV dosing in rats. Mean clearance as

d

e

f

measured from IV dosing in rats. Number of rats used in the experiment. Dose administered. Number of predicted basic groups.

K

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6s was completed in male Sprague−Dawley rats (Figure 3).

The plasma clearance calculated on the basis of area under the

would equate to a random (no preference) interaction. The

compound (6s) signiﬁcantly increased novel object exploration

relative to vehicle treatment in mice dosed with 1, 3, or 10 mg/

kg (s.c.) 60 min prior to the acquisition phase (p < 0.01, 0.05,

and 0.05, respectively, Figure 4). Even though the compound

Figure 3. Plasma concentrations and arterial pharmacokinetic

parameters of 6s following a 3 μmol/kg IV (fasted rats) dose and 3

μmol/kg PO (fed rats) dose in male Sprague−Dawley rats using a

crossover study design with a PO formulation of 10% DMA and 18%

SBECD, pH 4.73.

Figure 4. Compound 6s signiﬁcantly increased novel object

exploration 30 min after acquisition relative to vehicle treatment in

mice dosed with 1, 3, and 10 mg/kg 60 min prior to acquisition (p <

0.01, 0.05, and 0.05 respectively). All data are mean

SD ratio of

novel/familiar object interaction. Analysis was performed on manual

assessment of interaction scores using ANOVA followed by New-

man−Keuls posthoc analysis.

curve (AUC) from 0−24 h was high at 53 mL/min/kg. This

represents an overestimate of the true clearance, which is likely

to be in the moderate range but would require longer sampling

times to characterize fully. The volume of distribution was very

high and the apparent half-life was long at 15 h, but these

parameters could not be precisely characterized with conﬁdence

using a 24 h sampling protocol. The oral bioavailability,

estimated on the basis of comparison of the AUC (0−10 h)

following IV and oral administration, was high at 99%.

Approximately 14% of the dose was recovered in the urine

during the ﬁrst 24 h following intravenous bolus administration.

Compound 6s distributed into the CNS as demonstrated by a

brain/plasma ratio of 3.0 and cerebrospinal ﬂuid (CSF)/plasma

ratio of 0.19 (Table 7).

In Vivo Eﬃcacy. Compound 6s was tested for activity in a

mouse model of learning and recognition memory.³³Brieﬂy,

male CF-1 mice were placed in a test environment that

contained two identical objects and were allowed to explore the

environment (acquisition phase). The mice were removed from

the environment, and one of the objects was replaced with a

novel object. Fifteen minutes later, the mice were returned to

the test environment and monitored for time spent exploring

the novel object versus the familiar object. A ratio of novel to

familiar interaction was produced where a score of 50% (1:1)

had excellent oral bioavailability, the dosing for this eﬃcacy test

was routinely run with subcutaneous administration (s.c.) to

stay consistent with the protocol established for other

compounds.

CONCLUSIONS

■

We have identiﬁed a potent and selective series of H₃

antagonists originating from the azaspiro[2.5]octane carbox-

amide scaﬀold. Modiﬁcations of the spirocyclic core to

spirocyclobutyl, 3- and 4-pyrrolidine, and 4-piperidine were

largely tolerated. These molecules possess favorable oﬀ-target

proﬁles for both secondary pharmacology as well as favorable in

vitro DMPK properties. It has been our experience that basic

lipophilic amines tend to have multiparameter undesirable SAR

(oﬀ-target selectivity, microsomal clearance, phospholipidosis,

etc.) that often tracks with the primary and desired

pharmacology. To optimize to the primary pharmacology,

signiﬁcant chemistry resources are often employed to try to

separate the desired functional activity from the undesired

activity. We have found that this scaﬀold, as exempliﬁed by 6s,

lacked many of these undesirable features. This may be due in

part to the lack of aromatic rings or perhaps the low logD (e.g.,

Table 7. In Vivo DMPK Brain and Plasma Concentrations

a

b

c

d

f

g

Cpd

6s, IV, n = 4

plasma (nM)

180 40

Brain (nmol/kg)

540 120

CSF (nM)

34

Br/Pl

3.0 0.7

Cpd

e

C_max(nM)

t_max(h)

%F

99 18

6

6s, PO , n = 3

71 9.7

6.0

a

b

Sample taken after 1 h, IV dose (3 μmol/kg). Average plasma concentrations after 1 h with standard devation. Average brain concentration

reported with standard deviation. Brain concentrations are not corrected for vascular contamination of ∼2−3%. Nonspeciﬁc binding during CSF

c

d

e

sampling was not evaluated. Brain/plasma ratio. Oral route of administration with n = number of animals, details described in Figure 3. 3 μmol/

f

g

kg, 1:9 ratio of 10% DMA to 90% SBECD, pH 4.73, as vehicle. Maximal concentration of oral dose. Percent oral bioavailability.

L

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Figure 5. VCD Spectra of the Boltzmann-weighted average of the computed VCD spectra along with the experimental spectra for 4b (S) and 4a (R).

Conformations of compounds 6s, 29f, 34, and Bavisant were

6s = −0.26, measured logD). An exemplar compound from the

generated via Monte Carlo searching with the OPLS95 force ﬁeld in

spirocyclic cyclopropanes, 6s, demonstrated high brain

a continuum water model as implemented in MacroModel.³⁵

Molecular overlays comparing these conformational sets to the

exposures, high oral bioavailability, and eﬃcacy in a mouse

novel object recognition study. The high volumes of

small-molecule X-ray structure of Conessine were produced using

distribution and long half-lives of the dibasic compounds

shape and chemical feature matching with ROCS.³⁶Default settings

provide a signiﬁcant challenge in both interpretation of PK data

and prediction of pharmacodynamic (PD) response. Because

and parameters were used throughout the calculations.

Benzyl 4-Methylenepiperidine-1-carboxylate (2). A solution

the H₃receptor is hypothesized to be involved in wakefulness,

this may present a liability as the compound also possesses high

CNS exposure with an exceptionally long half-life. The

monobasic compounds provide a very diﬀerent PK proﬁle

with shorter half-lives and lower volumes of distribution that

potentially may have better alignment with the intended

indication. Compounds from both of these subtypes should

provide useful starting points and tools to investigate further

the importance of selective H₃antagonists for drug therapy in a

variety of potential disease states.

of methyltriphenylphosphonium bromide (88.2 mmol, 31.5 g) was

cooled in 600 mL of THF to 0 °C. To this solution, n-BuLi (2.5M, in

hexanes, 35.3 mL, 1.2 equiv) was slowly added whereupon a

precipitate gradually formed, and the reaction turned reddish-orange

in color. The reaction was stirred for 1 h at 0 °C, at which point benzyl

4-oxopiperidine-1-carboxylate 1 was added (73.5 mmol, 17.1 g, in 30

mL THF), and the reaction was stirred for an additional 1 h at 0 °C.

The volatiles were removed under reduced pressure, and the reaction

mixture was poured onto a pad of SiO₂and ﬂushed with a 4:1 mixture

of hexanes/EtOAc. The product was puriﬁed via column chromatog-

1

raphy (SiO₂, hexanes/EtOAc) as a clear oil (9.11 g, 54%). H NMR

(300 MHz, CDCl₃) δ 2.23 (t, J = 5.7 Hz, 4H), 3.53 (t, J = 5.9 Hz, 4H),

4.78 (s, 2H), 5.17 (s, 2H), 7.30−7.39 (m, 5H). MS m/z (ES+) [M +

H]⁺= 232.1.

EXPERIMENTAL SECTION

■

General Experimental. Proton magnetic resonance (¹H NMR)

spectra were recorded on a Bruker Avance DPX 300 or 500 MHz

spectrometer, and the chemical shifts are reported in parts per million

(δ) from a tetramethylsilane (TMS) internal standard. The MS

detection was performed with a Micromass Platform ZMD or LCZ

spectrometers using the indicated ionization method. High-resolution

mass spectra were recorded on an Agilent Technologies 6210 time-of-

ﬂight LC−MS spectrometer. Chemical purities of ﬁnal compounds

were determined to be >95% as determined by HPLC-MS analysis at

UV 220 nm. Unless otherwise indicated, all starting materials were

obtained from commercial suppliers. Preparatory HPLC procedure A:

Preparatory HPLC was conducted using a 21 × 150 mm, 5 μm Gemini

C18 column at pH 10 [0.125 M ammonium bicarbonate] with ACN/

H₂O as the mobile phase (solvent ﬂow was 50 mL/min and a gradient

from 10% acetonitrile to 93% acetonitrile over 33 min). Preparatory

HPLC procedure B: Preparative HPLC was conducted using a long,

high pH, shallow gradient method. The mobile phase was as follows:

30−50% B; A: H₂O with 15 mM ammonium bicarbonate and 0.375%

NH₄OH v/v, B: CH₃CN. The run was 25 min on XBridge Prep C18

OBD, 30 × 150 mm, Waters reverse-phase column. Preparatory HPLC

procedure C: Preparative HPLC was conducted using normal-phase

HPLC (mobile phase: CH₂Cl₂/MeOH/NH₄OH, ﬂow: 16 mL/min,

40 g column, gradient running from 100:0:1 to 80:20:1 over 1 h).

Analytical HPLC procedure D: Analytical HPLC used a high pH

gradient method (mobile phase: 5−95% B; A: H₂O with 10 mM

NH₄CO₃and 0.375% NH₄OH v/v, B: MeOH, 2.25 min run) on X-

Bridge C18, 2.1 × 30 mm, 5 μm particle size. Compounds 6a−g, 6j−

m, 6o−r, and 6s have been described in a previous patent application

by our group, and experimental details are included below.³⁴All ﬁnal

compounds were isolated in >95% purity as judged by LC−MS.

6-Benzyl 1-Ethyl 6-Azaspiro[2.5]octane-1,6-dicarboxylate

(3). Benzyl 4-methylenepiperidine-1-carboxylate 2 (39.6 mmol, 9.17

g) was dissolved in 1300 mL of anhydrous CH₂Cl₂followed by the

addition of CuCN (39.6 mmol, 3.54 g), and the reaction was stirred at

room temperature. Ethyl diazoacetate (87.2 mmol, 9.17 mL) was

dissolved in 10 mL of CH₂Cl₂and slowly added (0.2 mL/h, over

roughly 100 h). The solution was ﬁltered through SiO₂, and the

volatiles were removed. The residue was puriﬁed via column

chromatography (100% hexanes to 4:1 hexanes/EtOAc) to give the

title compound as a clear oil (5.68 g, 45% yield). ¹H NMR (300 MHz,

CDCl₃) δ 0.95 (dd, J = 8.0, 4.6 Hz, 1H), 1.20 (t, J = 5.0 Hz, 1H), 1.28

(t, J = 7.1 Hz, 3H), 1.42−1.48 (m, 2H), 1.56−1.61 (m, 1H), 1.69−

1.76 (m, 2H), 3.33−3.41 (m, 1H), 3.49−3.65 (m, 3H), 4.16 (q, J = 8.4

Hz, 2H), 5.16 (s, 2H), 7.35−7.38 (m, 5H). MS m/z (ES+) [M + H]⁺

= 318.2.

(R)-6-Benzyl 1-Ethyl 6-Azaspiro[2.5]octane-1,6-dicarboxy-

late (3a). The material was puriﬁed by chiral supercritical ﬂuid

chromatography (SFC, AD column with MeOH and 0.1% dimethyl

ethlyl amine, isocratic at 35%, 10 mL/min, main eluent CO₂, column

temperature 35 °C) to give the title compound with quantitative

recovery (t_R= 2.67 min). ¹H NMR (400 MHz, CDCl₃-d) δ 0.94 (dd, J

= 7.8, 4.7 Hz, 1H), 1.18 (t, J = 4.8 Hz, 1H), 1.27 (t, J = 7.2 Hz, 3H),

1.44 (br s, 2H), 1.57 (dd, J = 7.8, 5.4 Hz, 1H), 1.73 (br s, 2H), 3.30−

3.41 (m, 1H), 3.47−3.66 (m, 3H), 4.14 (q, J = 7.16 Hz, 2H), 5.14 (s,

2H), 7.29−7.35 (m, 1H), 7.35−7.40 (m, 4H). HRMS (TOF) m/z

20

calcd for C₁₈H₂₃NO₄(M + H)⁺, 318.1699; found, 318.1695. [α]_D

−70.4 (c 2.490, MeOH).

=

(S)-6-Benzyl 1-Ethyl 6-Azaspiro[2.5]octane-1,6-dicarboxy-

late (3b). The material was puriﬁed by chiral SFC chromatography

M

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as in 3a to give the title compound with quantitative recovery (t_R=

2.43 min). ¹H NMR (400 MHz, CDCl₃-d) δ 0.94 (dd, J = 8.2, 4.7 Hz,

1H), 1.18 (t, J = 4.88 Hz, 1H), 1.27 (t, J = 7.2 Hz, 3H), 1.44 (br s,

2H), 1.57 (dd, J = 8.2, 5.5 Hz, 1H), 1.72 (br s, 2H), 3.30−3.42 (m,

1H), 3.47−3.66 (m, 3H), 4.14 (q, J = 7.3 Hz, 2H), 5.14 (s, 2 H),

7.30−7.35 (m, 1H), 7.35−7.41 (m, 4H). HRMS (TOF) m/z calcd for

the starting material to give the title compound in 45% yield. The

material was converted to the citrate salt. ¹H NMR (300 MHz,

CDCl₃) δ 0.71−0.75 (m, 1H), 1.26 (t, J = 4.7 Hz, 1H), 1.32−1.51 (m,

2H), 1.55−1.82 (m, 8H), 2.28 (s, 3H), 2.38−2.66 (m, 6H), 2.67−2.78

(m, 1H), 3.39 (td, J = 11.7, 1.8 Hz, 2H), 3.46−3.60 (m, 2H), 3.66 (t, J

= 4.7 Hz, 2H), 3.72−3.84 (m, 1H), 4.04 (dd, J = 11.1, 4.0 Hz, 2H).

HRMS (TOF) m/z calcd for C₁₈H₃₂N₃O₂(M + H)⁺, 322.2489; found,

322.2499.

20

C₁₈H₂₃NO₄(M + H)⁺, 318.1699; found, 318.1699. [α]_D= +71.7 (c

2.306, MeOH).

Ethyl 6-Azaspiro[2.5]octane-1-carboxylate (4). To 6-benzyl 1-

ethyl 6-azaspiro[2.5]octane-1,6-dicarboxylate 3 (6.45 g, 20.3 mmol) in

ethanol (100 mL) was added 5% Pd/C (0.65 g, 0.30 mmol), and a

balloon ﬁlled with hydrogen was placed on top of the reaction. The

reaction was left to stir at room temperature overnight. The mixture

was ﬁltered through Celite, and the solvent was removed under

reduced pressure. The resulting oil was placed under high vacuum.

The crude material was chromatographed (SiO₂100 g, 5−10%

gradient of 7 N NH₃/MeOH in CH₂Cl₂). The solvent was removed

from the combined fractions under reduced pressure to give title

(4-Isopropylpiperazin-1-yl)(6-(tetrahydro-2H-pyran-4-yl)-6-

azaspiro[2.5]octan-1-yl)methanone (6b). This example was

prepared according to the procedure for compound 6d using

commercially available 1-isopropylpiperazine. ¹H NMR (500 MHz,

CDCl₃) δ 0.71 (dd, J = 7.8, 4.4 Hz, 1H), 1.04 (d, J = 6.5 Hz, 6H), 1.24

(t, J = 4.8 Hz, 1H), 1.36−1.47 (m, 2H), 1.58−1.67 (m, 5H), 1.69−

1.76 (m, 2H), 2.39−2.59 (m, 8H), 2.68−2.73 (m, 2H), 3.37 (t, J =

11.6 Hz, 2H), 3.51 (m, 1H), 3.63 (s, 2H), 3.76 (m, 1H), 4.02 (dd, J =

11.3, 4.3 Hz, 2H). HRMS (TOF) m/z calcd for C₂₀H₃₅N₃O₂(M +

H)⁺, 350.2802; found, 350.2799.

(4-Cyclopropylpiperazin-1-yl)(6-(tetrahydro-2H-pyran-4-yl)-

6-azaspiro[2.5]octan-1-yl)methanone (6c). This example was

prepared according to the procedure for compound 6d using

commercially available 1-cyclopropylpiperiazine. ¹H NMR (500

MHz, CDCl₃) δ 0.41−0.45 (m, 2H), 0.46−0.50 (m, 2H), 0.69−0.73

(m, 1H), 1.24 (t, J = 4.8 Hz, 1H), 1.35−1.46 (m, 2H), 1.57−1.66 (m,

5H), 1.69−1.76 (m, 3H), 2.43−2.72 (m, 9H), 3.37 (t, J = 11.2 Hz,

2H), 3.49 (m, 1H), 3.59 (s, 2H), 3.69 (m, 1H), 4.02 (dd, J = 11.1, 4.1

Hz, 2H). HRMS (TOF) m/z calcd for C₂₀H₃₃N₃O₂(M + H)⁺,

348.2646; found, 348.2654.

1

compound 4 (1.83g, 44%) as an amber oil. H NMR (300 MHz,

CDCl₃) δ 0.87 (dd, J = 8.0, 4.4 Hz, 1H), 1.13 (t, J = 4.9 Hz, 1H), 1.27

(t, J = 7.1 Hz, 3H), 1.41 (q, J = 5.2 Hz, 2H), 1.48−1.52 (m, 2H),

1.66−1.71 (m, 2H), 2.70−2.78 (m, 1H), 2.81−2.85 (m, 1H), 2.89 (t, J

= 5.4 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H). MS m/z (ES+) [M + H]⁺=

184.

(R)-Ethyl 6-Azaspiro[2.5]octane-1-carboxylate (4a). The

procedure was identical to 4b except (R)-6-benzyl 1-ethyl 6-

azaspiro[2.5]octane-1,6-dicarboxylate 3a was used to give the title

1

compound in 59% yield. H NMR (400 MHz, CDCl₃) δ 0.88 (dd,

(4-Cyclobutylpiperazin-1-yl)(6-(tetrahydro-2H-pyran-4-yl)-6-

azaspiro[2.5]octan-1-yl)methanone (6d). To ethyl 6-(tetrahydro-

2H-pyran-4-yl)-6-azaspiro[2.5]octane-1-carboxylate 5 (1.82 g, 6.81

mmol) were added MeOH (25 mL), 1 N LiOH (7.20 mL, 7.20

mmol), and water (1 mL). The cloudy reaction was left to stir at room

temperature. After 5 h, 1 N NaOH (7.2 mL, 7.2 mmol) and 20 mL

MeOH were added, and the reaction was stirred at room temperature.

After 2 h, solid NaOH (290 mg, 7.3 mmol) was added followed by 5

mL of THF, and the cloudy reaction was stirred overnight. The

reaction was then heated to reﬂux for 2 h, and the solvent was

removed under reduced pressure. The gummy residue was dissolved in

15 mL of water, and the pH was adjusted to ∼6 by adding 6 N HCl

(3.8 mL). The water was removed under reduced pressure, and

residual water was removed from the resulting material by azeotropic

distillation with MeOH. The addition and subsequent removal of

methanol under reduced pressure was performed several times, and

the resulting pale yellow solid was put under high vacuum. To this was

added 15 mL of MeOH, and the solution was decanted from the

insoluble material. The solvent was removed under reduced pressure

to give 2.45 g of crude 6-(tetrahydro-2H-pyran-4-yl)-6-azaspiro[2.5]-

octane-1-carboxylic acid, which was used as is without further

puriﬁcation (∼quan. recovery). MS m/z (ES+) [M + H]⁺= 240.

To the intermediate acid (150 mg, 0.63 mmol) were added HBTU

(238 mg, 0.63 mmol), DMF (2.0 mL), and DIPEA (0.328 mL, 1.88

mmol), and the reaction was stirred for 5 min. In a separate vial, 1-

cyclobutylpiperazine to (160 mg, 0.75 mmol), DMF (1.0 mL), and

DIPEA (0.263 mL, 1.50 mmol) were combined. This solution was

added to the ﬁrst reaction, and the yellow solution was stirred at room

temperature. After 1.5 h, the reaction was diluted with EtOAc (5 mL),

and the organic layer was separated. The aqueous layer was washed

three times with CH₂Cl₂and combined with the ﬁrst organic layer.

The organics were washed three times with saturated NaHCO₃and

twice with saturated NaCl, dried over Na₂SO₄, and concentrated under

reduced pressure, and the residue was put under high vacuum. The

crude material was chromatographed on 20 g silica with 10% (7 N

NH₃/MeOH)/CH₂Cl₂, and the combined puriﬁed fractions removed

of solvent under reduced pressure. The solid was triturated with Et₂O,

and the resulting precipitate was collected by ﬁltration to give the title

compound (77 mg, 34%) as a white solid. ¹H NMR (500 MHz,

CDCl₃) δ 0.71 (dd, J = 7.8, 4.4 Hz, 1H), 1.24 (t, J = 4.8 Hz, 1H),

1.35−1.45 (m, 2H), 1.56−1.75 (m, 9H), 1.84−1.92 (m, 2H), 2.01−

2.07 (m, 2H), 2.18−2.62 (m, 8H), 2.67−2.76 (m, 2H), 3.37 (t, J =

1H), 1.09−1.16 (m, 1H), 1.27 (t, J = 7.0 Hz, 3H), 1.34−1.47 (m, 2H),

1.50 (dd, J = 7.8, 5.4 Hz, 1H), 1.58 (br s, 1H), 1.61−1.76 (m, 2H),

2.68−2.79 (m, 1H), 2.80−2.87 (m, 1H), 2.89 (t, J = 5.8 Hz, 2H), 4.13

20

(q, J = 7.1 Hz, 2H). [α]_D= −115.1 (c 1.285, MeOH).

Stereochemistry assigned as (R) based on calculated and observed

VCD spectra (Figure 5).

(S)-Ethyl 6-Azaspiro[2.5]octane-1-carboxylate (4b). A mixture

of (S)-6-benzyl 1-ethyl 6-azaspiro[2.5]octane-1,6-dicarboxylate 3b

(0.50 g, 1.58 mmol), Pd/C (5%, 8.38 mg, 0.08 mmol), and ethanol

(31.5 mL) was shaken overnight in a Parr apparatus under a 20 psi

atmosphere of hydrogen. The mixture was ﬁltered over a Celite pad,

and the solvent was concentrated. The residue was loaded over a small

SiO₂pad using CH₂Cl₂and was puriﬁed by gravity ﬁltration using

CH₂Cl₂ﬁrst to elute the yellow colored impurity and then using 5%

MeOH, 10% acetone in CH₂Cl₂to give the title compound as a

1

colorless liquid (0.23 g, 82%). H NMR (400 MHz, CDCl₃) δ 0.89

(dd, J = 7.8, 4.3 Hz, 1H), 1.13 (t, J = 4.8 Hz, 1H), 1.27 (t, J = 6.8 Hz,

3H), 1.44 (dd, J = 9.3,4.7 Hz, 2H), 1.51 (dd, J = 7.8, 5.4 Hz, 1H),

1.64−1.79 (m, 2H), 2.34 (br s, 1H), 2.70−2.80 (m, 1H), 2.83−2.89

20

(m, 1H), 2.91 (t, J = 5.2 Hz, 2H), 4.14 (q, J = 6.7 Hz, 2H). [α]_D

=

+106.8 (c 1.367, MeOH). Stereochemistry was assigned as (S) based

on calculated and observed VCD spectra (Figure 5).

Ethyl 6-(Tetrahydro-2H-pyran-4-yl)-6-azaspiro[2.5]octane-1-

carboxylate (5). To ethyl 6-azaspiro[2.5]octane-1-carboxylate 4

(1.85 g, 10.0 mmol) in 1,2-dichloroethane (25 mL) was added

dihydro-2H-pyran-4(3H)-one (1.00 mL, 11.0 mmol), and the solution

was stirred 25 min. To this was added sodium triacetoxyborohydride

(2.50 g, 12.1 mmol), and the reaction was left to stir at room

temperature. After 5 h, the reaction was partitioned between EtOAc

and NaHCO₃(aq. sat.). The organic layer was removed, and the

solvent was evaporated to give a pale oil. The material was

chromatographed on SiO₂(50 g, 7 N NH₃/MeOH in CH₂Cl₂), and

the combined puriﬁed fractions were removed of solvent to give the

1

title compound (1.82 g, 64%) as a pale oil. H NMR (300 MHz,

CDCl₃) δ 0.87 (dd, J = 8.0, 4.5 Hz, 1H), 1.12 (t, J = 4.9 Hz, 1H), 1.26

(t, J = 7.1 Hz, 3H), 1.47−1.52 (m, 3H), 1.56−1.65 (m, 2H), 1.72−

1.78 (m, 4H), 2.39−2.61 (m, 5H), 3.37 (td, J = 11.7, 2.1 Hz, 2H), 4.02

(dd, J = 11.5, 4.5 Hz, 2H), 4.13 (q, J = 7.0 Hz, 2H). m/z (ES+) [M +

H]⁺= 268.

(4-Methylpiperazin-1-yl)(6-(tetrahydro-2H-pyran-4-yl)-6-

azaspiro[2.5]octan-1-yl)methanone (6a). The material was made

in an analogous fashion to compound 6d using 1-methylpiperiazine as

N

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11.2 Hz, 2H), 3.52 (m, 1H), 3.64 (t, J = 4.7 Hz, 2H), 3.75 (m, 1H),

4.02 (dd, J = 11.2, 4.1 Hz, 2H). HRMS (TOF) m/z calcd for

C₂₁H₃₅N₃O₂[M + H]⁺, 362.2802; found, 362.2808.

sieves, and cyclobutanone (0.04 mL, 0.45 mmol), and a balloon ﬁlled

with H₂(excess) was ﬁxed atop the reaction. This was stirred

overnight. The reaction was ﬁltered, the solvent was removed, and the

clear gum was put under high vacuum. The material was puriﬁed by

reverse-phase HPLC using procedure A. The fractions were collected,

and the solvent was removed, and the residue was dried under high

(4-Cyclopentylpiperazin-1-yl)(6-(tetrahydro-2H-pyran-4-yl)-

6-azaspiro[2.5]octan-1-yl)methanone (6e). This example was

prepared in an analogous fashion to compound 6d using 1-

cyclopentylpiperazine dihydrochloride.²⁴¹H NMR (500 MHz,

CDCl₃) δ 0.71 (dd, J = 7.8, 4.4 Hz, 1H), 1.24 (t, J = 4.8 Hz, 1H),

1.37−1.48 (m, 3H), 1.55−1.76 (m, 12H), 1.83−1.88 (m, 2H), 2.36−

2.62 (m, 9H), 2.70−2.74 (m, 1H), 3.37 (t, J = 11.0 Hz, 2H), 3.52 (m,

1H), 3.65 (t, J = 4.8 Hz, 2H), 3.76 (m, 1H), 4.03 (dd, J = 11.3, 4.1 Hz,

2H). HRMS (TOF) m/z calcd for C₂₂H₃₇N₃O₂(M + H)⁺, 376.2959;

found, 376.2957.

1

vacuum to give the title compound (0.08 g, 46%). H NMR (500

MHz, CDCl₃) δ 0.71 (m, 1H), 0.88 (M, 1H), 1.24 (t, J = 4.5 Hz, 1H),

1.39−1.49 (m, 2H), 1.60−1.75 (m, 7H), 1.86−1.90 (m, 2H), 2.01−

2.09 (m, 2H), 2.13−2.29 (m, 2H), 2.58−2.62 (m, 5H), 2.63−2.67 (m,

2H), 2.69−2.72 (m, 2H), 2.73−2.86 (m, 1H), 3.35 (t, J = 11.5 Hz,

2H), 3.60−3.63 (m, 2H), 3.77−3.79 (m, 1H), 4.00 (d, J = 11.5 Hz,

2H). HRMS (TOF) m/z calcd for C₂₁H₃₆N₃O₂(M + H)⁺, 362.2802;

found, 362.2801.

(4-Cyclohexylpiperazin-1-yl)(6-(tetrahydro-2H-pyran-4-yl)-

6-azaspiro[2.5]octan-1-yl)methanone (6f). Piperazin-1-yl(6-(tet-

rahydro-2H-pyran-4-yl)-6-azaspiro[2.5]octan-1-yl)methanone 11

(0.36 mmol, 0.19 g), cyclohexanone (1.08 mmol, 0.11 mL), 3 drops

of glacial acetic acid, and polystyrylmethyl trimethylammonium

cyanoborohydride (0.70 mmol, 0.19 g, 4.1 mmol/g of resin) were

slurried in 20 mL of CH₂Cl₂and stirred overnight at room

temperature. The solution was ﬁltered through a nylon ﬁlter, and

the volatiles were removed. The residue was puriﬁed via column

chromatography, and the product was eluted with CH₂Cl₂/CH₂Cl₂

containing 10% (2 M NH₃/MeOH) on SiO₂over 30 min. The

(4-Cyclohexylpiperazin-1-yl)(6-azaspiro[2.5]octan-1-yl)-

methanone (6k). To benzyl 1-(4-cyclohexylpiperazine-1-carbonyl)-6-

azaspiro[2.5]octane-6-carboxylate (3.21 g, 7.30 mmol) in MeOH (25

mL) was added 5% Pd/C (0.31 g, 0.15 mmol), and a balloon of

hydrogen was ﬁxed atop the reaction. The reaction was stirred at room

temperature. After 2 h, the reaction was ﬁltered through Celite and

concentrated. The ﬁltrate was removed of solvent, and the residual

white solid was put under high vacuum to give the title compound

1

(2.12 g, 95%). The material was used without further puriﬁcation. H

NMR (500 MHz, CDCl₃) δ 0.71−0.75 (m, 1H), 1.10−1.40 (m, 8H),

1.56−1.58 (m, 5H), 1.76−1.85 (m, 4H), 2.26−2.29 (m, 1H), 2.43−

2.46 (m, 1H), 2.52−2.57 (m, 2H). 2.64−2.66 (m, 1H), 2.74−2.79 (m,

2H), 2.80−2.94 (m, 2H), 3.46−3.49 (m, 1H), 3.60−3.65 (m, 2H),

3.75−3.78 (m, 1H). HRMS (TOF) m/z calcd for C₁₈H₃₁N₃O (M +

H)⁺, 306.2539; found, 306.2542.

1

product was isolated as a white solid (92 mg, 66% yield). H NMR

(300 MHz, DMSO-d₆) δ 0.61−0.65 (m, 1H), 0.95−0.98 (m, 1H),

1.07−1.22 (m, 6H), 1.30−1.55 (m, 6H), 1.55−1.82 (m, 6H), 2.25−

2.58 (m, 11H), 3.22−3.29 (m, 2H), 3.58−3.64 (m, 3H), 3.84−3.89

(m, 2H). HRMS (TOF) m/z calcd for C₂₃H₄₀N₃O₂(M + H)⁺,

390.3115; found, 390.3115.

(6-Cyclohexyl-6-azaspiro[2.5]octan-1-yl)(4-cyclohexylpiper-

azin-1-yl)methanone (6l). The title compound was prepared in

(4-Cycloheptylpiperazin-1-yl)(6-(tetrahydro-2H-pyran-4-yl)-

6-azaspiro[2.5]octan-1-yl)methanone (6g). This example was

prepared according to the procedure for compound 6d using

1

analogous fashion to compound 6b (0.03 g, 25%). H NMR (300

MHz, CDCl₃) δ 0.68−0.69 (m, 1H), 1.23−1.30 (m, 10H), 1.39−1.44

(m, 2H), 1.57−1.60 (m, 6H), 1.81−1.84 (m, 8H), 2.43−2.48 (m, 2H),

2.55−2.59 (m, 6H), 2.64−2.66 (m, 2H), 3.44−3.47 (m, 1H), 3.60−

3.63 (m, 2H), 3.73−3.75 (m, 1H). HRMS (TOF) m/z calcd for

C₂₄H₄₁N₃O (M + H)⁺, 388.3322; found, 388.3328.

1

commercially available 1-cycloheptylpiperazine. H NMR (500 MHz,

CDCl₃) δ 0.70 (dd, J = 7.8, 4.4 Hz, 1H), 1.24 (t, J = 4.8 Hz, 1H),

1.37−1.82 (m, 21H), 2.39−2.60 (m, 9H), 2.67−2.71 (m, 1H), 3.37 (t,

J = 11.7 Hz, 2H), 3.46 (m, 1H), 3.65 (s, 2H), 3.76 (m, 1H), 4.02 (dd, J

= 11.2, 4.1 Hz, 2H). HRMS (TOF) m/z calcd for C₂₄H₄₁N₃O₂(M +

H)⁺, 404.3272; found, 404.3278.

(4-Cyclohexylpiperazin-1-yl)(6-isopropyl-6-azaspiro[2.5]-

octan-1-yl)methanone (6m). The title compound was prepared in

1

analogous fashion to compound 6b (0.03 g, 26%). H NMR (300

(4-Cyclobutyl-1,4-diazepan-1-yl)(6-(tetrahydro-2H-pyran-4-

yl)-6-azaspiro[2.5]octan-1-yl)methanone (6h). To a solution of 6-

(tetrahydro-2H-pyran-4-yl)-6-azaspiro[2.5]octane-1-carboxylic acid

(0.10 g, 0.42 mmol) in DMF (2 mL) were added HOBT (0.096 g,

0.63 mmol), 1-cyclobutyl-1,4-diazepane dihydrochloride (0.11 g, 0.50

mmol), DIPEA (0.15 mL, 0.84 mmol), and EDCI (0.12 g, 0.63 mmol)

followed by DMF (2.0 mL). The reaction was then stirred overnight at

room temperature. The solvent was removed, and residual material

was puriﬁed with preparatory HPLC procedure A to provide the title

compound as a white solid (0.15, 46%). ¹H NMR (500 MHz, CDCl₃)

δ 0.70 (dd, J = 10.0, 5.0 Hz, 1H), 1.25−1.27 (m, 2H), 1.47−1.48 (m,

1H), 1.58−1.63 (m, 9H), 1.74−1.92 (m, 6H), 2.00−2.03 (m, 2H),

2.27−2.31 (m, 2H), 2.46−2.49 (m, 2H), 2.61−2.63 (m, 2H), 2.72−

2.74 (m, 1H), 2.84 (quin, J = 10.0 Hz, 1H), 3.37 (t, J = 10.0 Hz, 2H),

3.75−3.57 (m, 4H), 4.02 (dd, J = 10.0 Hz, 5.0 Hz, 2H). HRMS (TOF)

m/z calcd for C₂₂H₃₇N₃O₂(M + H)⁺, 376.2958; found, 376.2963.

(4-(Pyridin-4-yl)piperazin-1-yl)(6-(tetrahydro-2H-pyran-4-

yl)-6-azaspiro[2.5]octan-1-yl)methanone (6i). The material was

prepared in a similar fashion to compound 6d beginning with 1-

MHz, CDCl₃) δ 0.75−0.72 (m, 1H), 1.02−1.07 (m, 6H), 1.22−1.26

(m, 6H), 1.42−1.45 (m, 2H), 1.60−1.63 (m, 2H), 1.78−1.84 (m, 6H),

2.24−2.27 (m, 1H), 2.36−2.39 (m, 1H), 2.48−2.52 (m, 5H), 2.69−

2.71 (m, 2H), 2.78 (quin, J = 10.5 Hz, 1H), 3.47−3.49 (m, 1H), 3.58−

3.63 (m, 2H), 3.76−3.79 (m, 1H). HRMS (TOF) m/z calcd for

C₂₁H₃₇N₃O (M + H)⁺, 348.3009; found, 348.3010.

(4-Cyclohexylpiperazin-1-yl)(6-phenyl-6-azaspiro[2.5]octan-

1-yl)methanone (6n). To (4-cyclohexylpiperazin-1-yl)(6-

azaspiro[2.5]octan-1-yl)methanone 6k (0.10 g, 0.33 mmol) in toluene

(1.5 mL) were added bromobenzene (0.05 mL, 0.43 mmol), trans-

dichlorobis(tri-o-tolyl-phosphine)palladium(II) (0.03 g, 0.03 mmol),

and LiHMDS (1 M in toluene, 0.39 mL, 0.39 mmol). The reaction was

heated at 100 °C for 1 h. The reaction was quenched with 2 mL of

EtOAc, and the precipitate was ﬁltered oﬀ and put under high vacuum

overnight. The sample was chromatographed (SiO₂, CH₂Cl₂to 5% (7

N NH₃)/MeOH). The fractions were collected and dried to yield a

crude solid. The HCl salt was made by dissolving in 1 N HCl/MeOH,

and the solvent was removed. The solids were triturated with Et₂O,

ﬁltered, and put under high vacuum. The compound was further

puriﬁed by dissolving in MeOH and passing through 4 g Isolute NH₂

column with MeOH. The product was removed of solvent, dissolved

in ACN, and ﬁltered through a 0.45 μM frit. After sitting at room

temperature for a while, the material crystallized out, ﬁltered, and dried

under high vacuum at 50 °C for 5 h to give the title compound as a

white solid (0.015 g, 12%). ¹H NMR (500 MHz, CDCl₃) δ 0.71 (dd, J

= 7.8, 4.4 Hz, 1H), 1.10−1.24 (m, 5H), 1.32 (t, J = 4.5 Hz, 1H), 1.50−

1.65 (m, 3H), 1.74−1.82 (m, 6H), 2.76 (app t, J = 11 Hz, 1H), 2.46−

2.48 (m, 2H), 2.56−2.61 (m, 2H), 2.56−2.61 (m, 2H), 3.07−3.10 (m,

1H), 3.17−3.27 (m, 1H), 3.29−3.30 (m, 1H), 3.48−3.50 (m, 1H),

3.62−3.65 (m, 2H), 3.73−3.76 (m, 1H), 6.84 (t, J = 7 Hz, 1H), 6.95

1

(pyridin-4-yl)piperazine (0.09 g, 21%). H NMR (400 MHz, CDCl₃)

δ 0.79 (dd, J = 8.0, 4.8 Hz, 1H), 1.29 (t, J = 4.7 Hz, 1H), 1.32−1.42

(m, 1H), 1.42−1.53 (m, 1H), 1.53−1.74 (m, 4H), 1.74−1.95 (m, 3H),

2.37−2.60 (m, 3H), 2.60−2.69 (m, 1H), 2.69−2.81 (m, 1H), 3.26−

3.51 (m, 6H), 3.67−3.91 (m, 4H), 4.03 (dd, J = 11.1, 4.1 Hz, 2H),

6.67 (d, J = 6.2 Hz, 2H), 8.33 (d, J = 5.1 Hz, 2H). HRMS (TOF) m/z

calcd for C₂₂H₃₂N₄O₂(M + H)⁺, 385.2598; found, 385.2595.

(6-Cyclobutyl-6-azaspiro[2.5]octan-1-yl)(4-(tetrahydro-2H-

pyran-4-yl)piperazin-1-yl)methanone (6j). To benzyl 1-(4-

(tetrahydro-2H-pyran-4-yl)piperazine-1-carbonyl)-6-azaspiro[2.5]-

octane-6-carboxylate 12 (0.10 g, 0.23 mmol) were added MeOH (1.0

mL), Pd/C (4.82 mg, 4.53 μmol), oven-dried 3 Å powdered molecular

O

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(d, J = 8 Hz, 2H), 7.23 (m, 2H). HRMS (TOF) m/z calcd for

C₂₄H₃₅N₃O (M + H)⁺, 382.2852; found, 382.2850.

overnight in a Parr apparatus under 30 psi of a H₂atmosphere. The

reaction mixture was ﬁltered over a Celite bed, and the solvent was

concentrated. The product was puriﬁed by chromatography (SiO₂,

using 5% of (7 N NH₃in MeOH) in CH₂Cl₂as the eluent) to provide

(6-Benzyl-6-azaspiro[2.5]octan-1-yl)(4-cyclohexylpiperazin-

1-yl)methanone (6o). The title compound was prepared in

1

analogous fashion to compound 6b (0.07 g, 54%). H NMR (300

the title compound as white solid (1.68 g, 68.6%). H NMR (400

MHz, CDCl₃) δ 0.83−0.84 (m, 1H), 1.26−1.32 (m, 6H), 1.48−1.50

(m, 4H), 1.58−1.61 (m, 3H), 1.75−1.86 (m, 4H), 2.24−2.31 (m, 2H),

2.52−2.64 (m, 6H), 3.48−3.50 (m, 1H), 3.52 (s, 2H), 3.60−3.61 (m,

2H), 3.70−3.74 (m, 1H), 7.22−7.30 (m, 5H). HRMS (TOF) m/z

calcd for C₂₅H₃₇N₃O (M + H)⁺, 396.3009; found, 396.3004.

MHz, CD₃OD) δ 0.79 (dd, 1H), 1.09 (d, J = 6.2 Hz, 6H), 1.14 (t, J =

4.8 Hz, 1H), 1.40−1.51 (m, 1H), 1.51−1.74 (m, 5H), 1.78−1.89 (m,

3H), 2.42−2.50 (m, 1H), 2.50−2.62 (m, 5H), 2.63−2.79 (m, 4H),

3.40 (t, J = 11.9 Hz, 2H), 3.50 (ddd, J = 13, 7.6, 3.1 Hz, 1H), 3.70

(ddd, J = 6.4, 3.5, 3.3 Hz, 1H), 3.74 (t, J = 5.0 Hz, 2H), 3.99 (dd, J =

11.5, 4.4 Hz, 2H). HRMS (TOF) m/z calcd for C₂₀H₃₅N₃O₂(M +

(4-Cyclohexylpiperazin-1-yl)(6-phenethyl-6-azaspiro[2.5]-

octan-1-yl)methanone (6p). To (4-cyclohexylpiperazin-1-yl)(6-

azaspiro[2.5]octan-1-yl)methanone 6k (0.10 g, 0.33 mmol) was

added 1,2-dichloroethane (2 mL) and 2-phenylacetaldehyde (37 μL,

0.33 mmol). The reaction was stirred 30 min, and sodium

triacetoxyborohydride (0.76 g, 0.36 mmol) was added. The reaction

was stirred at room temperature overnight. To the reaction mixture

was added 1.5 mL of 1 N NaOH, and the mixture was stirred for 20

min. The aqueous layer was removed, to this were added Et₂O and

saturated NaCl, and the mixture was stirred for 5 min. The aqueous

layer was removed, the organic layers were dried over Na₂SO₄, the

solvent was removed, and the material was put under high vacuum at

50 °C overnight. The material was puriﬁed on 12 g silica gel eluting

with CH₂Cl₂to 10% (7 N NH₃/MeOH)/CH₂Cl₂. The product was

removed of solvent and put under high vacuum. The material was

dissolved in hot hexanes and cooled in a freezer to give a white solid

20

H)⁺, 350.2802; found, 350.2796. [α]_D= −31.1 (c 1.766, MeOH).

(4-Cyclobutylpiperazin-1-yl)(6-cyclohexylspiro[2.5]octan-1-

yl)methanone (6t). 6-Cyclohexylspiro[2.5]octane-1-carboxylic acid

was prepared by hydrolysis from 17 via a route analogous to

preparation of compound 7. To 6-cyclohexylspiro[2.5]octane-1-

carboxylic acid (0.20 g, 0.85 mmol) and O-benzotriazol-1-yl-

tetramethyluronium hexaﬂuorophosphate (0.32 g, 0.85 mmol) were

added DMF (10 mL) and DIPEA (0.74 mL, 4.23 mmol). The mixture

was stirred for 5 min, and solid 1-cyclobutylpiperazine·2HCl (0.27 g,

1.27 mmol) was added. The reaction was stirred for 15 min, and the

solvent was removed. The material was puriﬁed by preparatory HPLC

procedure A to give the title compound (0.30 g, 69%). ¹H NMR (500

MHz, DMSO-d₆) δ 0.60−0.62 (m, 1H), 0.82−0.85 (m, 1H), 1.09−

1.19 (m, 10H), 1.43−1.49 (m, 3H), 1.67−1.72 (m, 10H), 1.80−1.82

(m, 2H), 1.94−1.96 (m, 2H), 2.20−2.24 (m, 1H), 2.26−2.30 (m, 1H),

2.30−2.45 (m, 2H), 2.68−2.70 (m, 1H), 3.42−3.47 (m, 1H), 3.50−

3.55 (m, 1H), 3.65−3.68 (m, 1H), 3.72−3.75 (m, 1H). HRMS (TOF)

m/z calcd for C₂₃H₃₉N₂O (M + H)⁺, 359.3057; found, 359.3072.

6-(Benzyloxycarbonyl)-6-azaspiro[2.5]octane-1-carboxylic

acid (7). 6-Benzyl 1-ethyl 6-azaspiro[2.5]octane-1,6-dicarboxylate 3

(4.06 mmol, 1.29 g) was dissolved in MeOH (8 mL), THF (8 mL),

and H₂O (8 mL). To this was added LiOH (8.13 mmol, 0.33 g), and

the reaction was stirred overnight at room temperature. After the

reaction was complete, it was acidiﬁed to pH ∼1 with 1 N HCl and

extracted with EtOAc. The organics were dried over MgSO₄and

ﬁltered, and the residual material was dried under vacuum to give the

1

that was collected to give the ﬁnal compound. H NMR (500 MHz,

CDCl₃) δ 0.82−0.85 (m, 1H), 1.25−1.31 (m, 6H), 1.57−1.58 (m,

2H), 1.63−1.67 (m, 4H), 1.79−1.85 (m, 4H), 2.24−2.27 (m, 1H),

2.58−2.69 (m, 10H), 2.80−2.84 (m, 2H), 3.51−3.57 (m, 1H), 3.62−

3.64 (m, 2H), 3.79−3.81 (m, 1H), 7.18−7.21 (m, 3H), 7.27−7.30 (m,

2H), HRMS (TOF) m/z calcd for C₂₆H₃₉N₃O (M + H)⁺, 410.3165;

found, 410.3169.

(4-Cyclohexylpiperazin-1-yl)(6-(pyridin-4-yl)-6-azaspiro[2.5]-

octan-1-yl)methanone (6q). To (4-cyclohexylpiperazin-1-yl)(6-

azaspiro[2.5]octan-1-yl)methanone 6k (0.10 g, 0.33 mmol) were

added 4-chloropyridine hydrochloride (0.05 g, 0.33 mmol), K₂CO₃

(0.11 mg, 0.82 mmol), and DMSO (2 mL). The reaction was sealed

and placed in a 90 °C bath overnight. The reaction was diluted with

EtOAc/NaCl (sat.) and washed with NaCl (sat., 2×), dried over

Na₂SO₄, and stripped to give an orange oil. The material was puriﬁed

by chromatography (SiO₂, CH₂Cl₂to 10% (7 N NH₃/MeOH)/

CH₂Cl₂). The product was removed of solvent and put under high

vacuum at 50 °C overnight to give the title compound as a white solid

1

title compound as a white solid (1.10 g, 94% yield). H NMR (300

MHz, CDCl₃) δ 0.83−0.94 (m, 1H), 1.09−1.15 (m, 1H), 1.30−1.40

(m, 2H), 1.46−1.52 (m, 1H), 1.61−1.70 (m, 2H), 3.23−3.54 (m, 4H),

5.06 (s, 2H), 7.20−7.28 (m, 5H). MS m/z (ES+) [M + H]⁺= 290.1.

(R)-6-Benzyl 1-Ethyl 6-Azaspiro[2.5]octane-1,6-dicarboxy-

late (7a). The procedure was identical to 7b except (R)-6-benzyl 1-

ethyl 6-azaspiro[2.5]octane-1,6-dicarboxylate 3a was used to give the

title compound as a colorless oil (2.77 g, 100%). ¹H NMR (400 MHz,

CDCl₃) δ 1.03 (dd, 1H), 1.18−1.26 (m, 1H), 1.46 (br s, 2H), 1.59

(dd, J = 7.81, 5.47 Hz, 1H), 1.77 (br s, 2H), 3.41−3.50 (m, 1H),

3.50−3.61 (m, 3H), 5.14 (s, 2H), 7.30−7.34 (m, 1H), 7.35−7.40 (m,

4 H).

1

(0.02 g, 26%). H NMR (500 MHz, CDCl₃) δ 0.71 (dd, J = 7.8, 4.4

Hz, 1H), 1.46−1.30 (m, 6H), 1.46−1.48 (m, 1H), 1.50−1.55 (m, 1H),

1.54−1.58 (m, 2H), 1.63−1.69 (m, 2H), 1.74−1.81 (m, 4H), 2.24−

2.27 (m, 1H), 2.47−2.57 (m, 2H), 2.57−2.70 (m, 2H), 3.20−3.25 (m,

1H), 3.28−3.32 (m, 1H), 3.33−3.43 (m, 1H), 3.50−3.55 (m, 2H),

3.55−3.60 (m, 2H), 3.65−3.72 (m, 1H), 6.60 (d, J = 5.5 Hz, 2H), 8.26

(d, J = 5.5 Hz, 2H). HRMS (TOF) m/z calcd for C₂₃H₃₄N₄O (M +

H)⁺, 383.2805; found, 383.2805.

(S)-6-(Benzyloxycarbonyl)-6-azaspiro[2.5]octane-1-carbox-

ylic Acid (7b). NaOH (9.58 mL, 19.16 mmol) was added to a

solution of (S)-6-benzyl 1-ethyl 6-azaspiro[2.5]octane-1,6-dicarbox-

ylate (3.04 g, 9.58 mmol) in methanol (30 mL) and water (10 mL).

The reaction mixture was heated and stirred until all of the starting

material was consumed, as indicated by TLC. The solvent was

concentrated, and water was added. The solution was acidiﬁed to pH

∼2 using a 2 M HCl solution. The product was extracted with EtOAc

(3 × 50 mL). The organics were combined, dried over anhydrous

Na₂SO₄, and concentrated to provide the title compound (2.80 g,

(R)-(4-Isopropylpiperazin-1-yl)(6-(tetrahydro-2H-pyran-4-

yl)-6-azaspiro[2.5]octan-1-yl)methanone (6r). The procedure was

the same as 6s except (R)-benzyl 1-(4-isopropylpiperazine-1-carbon-

yl)-6-azaspiro[2.5]octane-6-carboxylate was used to give the title

compound (2.46 g, 45%) as a white solid. ¹H NMR (400 MHz,

CD₃OD) δ 0.79 (dd, 1H), 1.09 (d, J = 6.6 Hz, 6H), 1.14 (t, J = 4.8 Hz,

1H), 1.40−1.51 (m, 1H), 1.51−1.74 (m,5H), 1.77−1.89 (m, 3H), 2.46

(ddd, J = 11.3, 7.8, 3.1 Hz, 1H), 2.50−2.62 (m, 5H), 2.62−2.81 (m,

4H), 3.40 (t, J = 11.9 Hz, 2H), 3.50 (ddd, J = 13, 7.7, 3.3 Hz, 1H),

3.70 (dt, J = 6.6, 3.3 Hz, 1H), 3.74 (t, J = 5.0 Hz, 2H), 3.99 (dd, J =

11.0, 4.5 Hz, 2H). HRMS (TOF) m/z calcd for C₂₀H₃₅N₃O₂(M +

1

101%) as a colorless oil. H NMR (400 MHz, CDCl₃) δ 1.01 (dd,

1H), 1.17−1.25 (m, 1H), 1.45 (br s, 2H), 1.58 (dd, J = 7.81, 5.47 Hz,

1 H), 1.76 (br s, 2H), 3.38−3.48 (m, 1 H), 3.48−3.61 (m, 3H), 5.14

(s, 2H), 7.29−7.34 (m, 1H), 7.34−7.40 (m, 4H). MS (ES+) m/z [M +

20

H)⁺, 350.2802; found, 350.2796. [α]_D= +32.4 (c 1.766, MeOH).

H]⁺= 290.1. [α]_D= +69.2 (c 2.349, MeOH).

(S)-(4-Isopropylpiperazin-1-yl)(6-(tetrahydro-2H-pyran-4-

yl)-6-azaspiro[2.5]octan-1-yl)methanone (6s). Pd/C (5%, 0.015

g, 0.14 mmol) was added to a solution of (S)-benzyl 1-(4-

isopropylpiperazine-1-carbonyl)-6-azaspiro[2.5]octane-6-carboxylate

(2.80 g, 7.01 mmol) and dihydro-2H-pyran-4(3H)-one (1.05 g, 10.51

mmol) in methanol (28 mL). The reaction mixture was shaken

Benzyl 1-(4-(tert-Butoxycarbonyl)piperazine-1-carbonyl)-6-

azaspiro[2.5]octane-6-carboxylate (8). 6-(Benzyloxycarbonyl)-6-

azaspiro[2.5]octane-1-carboxylic acid 7 (1.90 mmol, 0.55 g), O-(7-

azabenzotriazole-1-yl)-N, N,N′,N′-tetramethyluronium hexaﬂuoro-

phosphate (3.80 mmol, 1.44 g), DIPEA (3.80 mmol, 0.66 mL), and

piperazine-1-carboxylic acid tert-butyl ester (2.85 mmol, 0.53 g) were

P

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combined in 50 mL of anhydrous acetonitrile and stirred at room

temperature overnight. The reaction was diluted with EtOAc and

washed with 100 mL of 1 N NaOH. The organics were then dried over

MgSO₄and ﬁltered through a silica gel plug, and the volatiles were

removed under reduced pressure. The residue was puriﬁed by column

chromatography (SiO₂, hexanes/ethyl acetate gradient (100% hexanes

to 50% hexanes over 30 min)) to produce the title compound (0.60 g,

69% yield) as a tan solid. ¹H NMR (300 MHz, CDCl₃) δ 0.74 (dd, J =

7.7, 4.6 Hz, 1H), 1.19−1.27 (m, 2H), 1.40 (s, 9H), 1.45−1.62 (m,

4H), 3.18−3.69 (m, 12H), 5.06 (s, 2H), 7.34 (s, 5H). MS (ES+) m/z

[M + Na]⁺= 480.2.

tert-Butyl 4-(6-Azaspiro[2.5]octane-1-carbonyl)piperazine-

1-carboxylate (9). Benzyl 1-(4-(tert-butoxycarbonyl)piperazine-1-

carbonyl)-6-azaspiro[2.5]octane-6-carboxylate (1.31 mmol, 0.60 g)

was dissolved in 100 mL of EtOH and purged with N₂for 15 min.

This was followed by the addition of 3 drops of glacial acetic acid and

5% Pd/C (0.06 g, 10% by weight of piperazine), and the reaction was

placed under a balloon of H₂. The reaction was then stirred overnight

at room temperature. The reaction was ﬁltered through a Celite plug,

and the volatiles were removed under reduced pressure. The residue

was puriﬁed via column chromatography and eluted with a CH₂Cl₂/

CH₂Cl₂containing 10% (2 M NH₃/MeOH) gradient over 30 min to

give the title compound as clear oil (0.42 g, 99% yield). ¹H NMR (300

MHz, CD₃OD) δ 0.79 (dd, J = 7.8, 4.3 Hz, 1H), 1.15 (t, J = 4.8 Hz,

1H), 1.32−1.44 (m, 2H), 1.47 (s, 9H), 1.50−1.68 (m, 2H), 1.83 (dd, J

= 7.8, 5.3 Hz, 1H), 2.72−2.76 (m, 2H), 2.83−2.89 (m, 2H), 3.37−3.78

(m, 8H). MS (ES+) m/z [M + H]⁺= 324.3.

high vacuum at 50 °C overnight to give the title compound as a white

solid (0.44 g, 11%). H NMR (500 MHz, CDCl₃) δ 1.02 (br s, 1H),

1

1.27−1.33 (m, 3H), 1.59−1.61 (m, 1H), 1.70−1.71 (m, 1H), 1.82−

1.84 (m, 1H), 1.99−2.25 (m, 4H), 2.94 (br s, 1H), 3.22 (br s 1H),

3.22−3.25 (m, 3H), 3.54−3.57 (m, 3H), 3.63−3.72 (m, 3H), 3.92−

4.10 (m, 4H), 4.29−4.41 (m, 3H), 4.83 (br s, 1H), 5.15−5.17 (m,

2H), 7.33−7.48 (m, 5H). MS (ES+) m/z [M + H]⁺= 442.2.

Benzyl 1-(4-Cyclohexylpiperazine-1-carbonyl)-6-

azaspiro[2.5]octane-6-carboxylate (13). To 6-(benzyloxycarbon-

yl)-6-azaspiro[2.5]octane-1-carboxylic acid 7 (2.99 g, 10.3 mmol) were

added DMF (40 mL), HBTU (3.92 g, 10.3 mmol), and DIPEA (5.41

mL, 31.0 mmol), and the mixture was stirred for 5 min. To this was

added 1-cyclohexylpiperazine (1.91 g, 11.4 mmol), and the reaction

was stirred for 2 h. The DMF was partially removed under vacuum,

and the remaining material was partitioned between EtOAc/NaHCO₃.

The organic layer was washed with NaHCO₃(aq. sat.) and NaCl (aq.

sat.) and dried over Na₂SO_4.The solvent was removed, and the

material was chromatographed (SiO₂, 2.5% MeOH/CH₂Cl₂).

Fractions were combined, and the solvent was evaporated to give

1

the title compound (3.21 g 70%). H NMR (500 MHz, DMSO-d₆) δ

086−0.87 (m, 1H), 1.10−1.30 (m, 8H), 1.40−1.43 (m, 1H), 1.59−

1.64 (m, 4H), 1.78−1.83 (m, 4H), 2.25−2.29 (m, 1H), 2.37 (s, 1H),

2.45−2.49 (m, 2H), 2.64−2.65 (m, 1H), 3.28−3.30 (m, 1H), 3.30−

3.38 (m, 1H), 3.41−3.48 (m, 1H), 3.60−3.69 (m, 2H), 3.69−3.72 (m,

2H), 5.13 (s, 2H), 7.29−7.32 (m, 1H), 7.32−7.36 (m, 4H).

(R)-Benzyl 1-(4-Isopropylpiperazine-1-carbonyl)-6-

azaspiro[2.5]octane-6-carboxylate (14a). The procedure was

identical to 14b except (R)-6-(benzyloxycarbonyl)-6-azaspiro[2.5]-

octane-1-carboxylic acid 7a was used to give the title compound as a

white solid (3.7 g, 100%). ¹H NMR (400 MHz, DMSO-d₆) δ 0.75 (br

s, 1H), 0.87−1.13 (m, 7H), 1.20−1.35 (m, 2H), 1.39 (br s, 2H), 1.50

(br s, 1H), 1.90 (br s, 1H), 2.37 (br s, 2H), 3.20−3.42 (m, 6H), 3.50

(br s, 4H), 5.07 (s, 2H), 7.27−7.42 (m, 5H). MS (ES+) m/z [M +

tert-Butyl 4-(6-(Tetrahydro-2H-pyran-4-yl)-6-azaspiro[2.5]-

octane-1-carbonyl)piperazine-1-carboxylate (10). tert-Butyl 4-

(6-azaspiro[2.5]octane-1-carbonyl)piperazine-1-carboxylate 9 (1.30

mmol, 0.42 g), dihydro-2H-pyran-4(3H)-one (1.95 mmol, 0.13 mL),

3 drops of glacial acetic acid, and NaBH₃CN (2.92 mmol, 0.183 g)

were combined in 60 mL of anhydrous EtOH and stirred for 12 h at

room temperature followed by heating to reﬂux for 6 h. The reaction

was cooled to room temperature, diluted with EtOAc, and washed

with 50 mL of 1 N NaOH. The organics were dried over Na₂SO₄and

ﬁltered through a glass ﬁlter frit, and the volatiles were removed under

reduced pressure. The residue was puriﬁed by column chromatog-

raphy (SiO₂, CH₂Cl₂/CH₂Cl₂containing 10% (2 M NH₃/MeOH)

gradient over 30 min) to produce the title compound (0.44 g, 83%

20

H]⁺= 400.3. [α]_D= +13.1 (c 2.034, MeOH).

(S)-Benzyl 1-(4-Isopropylpiperazine-1-carbonyl)-6-

azaspiro[2.5]octane-6-carboxylate (14b). HBTU (3.98 g, 10.5

mmol) was added to a solution of (S)-6-(benzyloxycarbonyl)-6-

azaspiro[2.5]octane-1-carboxylic acid 7b (2.76 g, 9.54 mmol) in DMF

(65 mL). Upon complete dilution, DIEA (5.00 mL, 28.6 mmol) was

added, and the reaction mixture was stirred for 5 min. 1-(2-Propyl)-

piperazine (1.85 g, 14.3 mmol) was then added, the reaction mixture

was stirred for 1 h, and the solvent was concentrated. The residue was

recovered in EtOAc (200 mL) and washed with saturated NaHCO₃

solution (3 × 50 mL) and brine. The solution was dried over

anhydrous Na₂SO₄and concentrated. The product was puriﬁed on

silica gel (120 g, using 5% MeOH, 10% acetone in DCM as eluent) to

1

yield) as a clear oil. H NMR (300 MHz, CDCl₃) δ 0.82 (dd, J = 8.0,

4.6 Hz, 2H), 1.23 (t, J = 4.8 Hz, 2H), 1.44 (s, 9H), 1.39−1.55 (m,

4H), 1.55−1.75 (m, 4H), 1.80−1.97 (m, 2H), 2.72−2.97 (m, 5H),

3.34−3.58 (m, 6H), 4.03 (dd, J = 11.4, 3.5 Hz, 2H). MS (ES+) m/z

[M + H]⁺= 408.3.

Piperazin-1-yl(6-(tetrahydro-2H-pyran-4-yl)-6-azaspiro[2.5]-

octan-1-yl)methanone (11). tert-Butyl-4-(6-(tetrahydro-2H-pyran-

4-yl)-6-azaspiro[2.5]octane-1-carbonyl)piperazine-1-carboxylate 10

(1.08 mmol, 0.44 g) was dissolved in 25 mL of CH₂Cl₂followed by

the addition of 5 mL of TFA, and the reaction was stirred at room

temperature for 1.5 h. The volatiles were removed under reduced

pressure, and the product was dried overnight under high vacuum to

give the title compound (0.57 g as a tan TFA salt, 99% yield), which

was used without further puriﬁcation. ¹H NMR (300 MHz, CD₃OD) δ

0.97−1.10 (m, 2H), 1.24−1.65 (m, 4H), 1.71−1.89 (m, 3H), 1.95−

2.15 (m, 4H), 3.06−3.49 (m, 5H), 3.55−3.69 (m, 6H), 3.71−4.13 (m,

4H), MS (ES+) m/z [M + H]⁺= 308.3.

Benzyl 1-(4-(Tetrahydro-2H-pyran-4-yl)piperazine-1-carbon-

yl)-6-azaspiro[2.5]octane-6-carboxylate (12). To 6-(benzyloxy-

carbonyl)-6-azaspiro[2.5]octane-1-carboxylic acid 7 (1.95 g, 6.74

mmol) were added DMF (50 mL), O-benzotriazol-1-yl-tetramethy-

luronium hexaﬂuorophosphate (2.56 g, 6.74 mmol), and DIPEA (5.89

mL, 33.7 mmol). After stirring for 5 min, to this was added 1-

(tetrahydro-2H-pyran-4-yl)piperazine dihydrochloride (1.72 g, 7.08

mmol). The reaction was stirred for 1 h, and the solvent was removed.

The material was partitioned between EtOAc/NaHCO₃. The organic

layer was washed with NaHCO₃(2×) and NaCl (1×) and dried over

Na₂SO₄, and the solvent was removed. The material was chromato-

graphed (330 g SiO₂, with gradient running from pure CH₂Cl₂to 5%

MeOH). The material was converted to the TFA salt and placed under

1

provide the title compound (3.80 g, 100%) as a white solid. H NMR

(400 MHz, DMSO-d₆) δ 0.75 (br s, 1H), 0.84−1.22 (m, 7H), 1.21−

1.35 (m, 2H), 1.39 (br s, 2H), 1.50 (br s, 1H), 1.91 (br s, 1H), 2.38

(br s, 2H), 3.33 (br s, 6H), 3.50 (br s, 4H), 5.07 (s, 2H), 7.23−7.44

(m, 5H). MS (ES+) m/z [M + H]⁺= 400.4. [α]_D²⁰= −12.9 (c 2.052,

MeOH).

Methyl 2-(Bi(cyclohexan)-4-ylidene)acetate (16). To pentane-

washed NaH 60% in mineral oil (0.31 g, 7.77 mmol) were added THF

(25 mL) and methyl 2-(dimethoxyphosphoryl)acetate (1.20 mL, 8.32

mmol) dropwise over 20 min, resulting in a very thick suspension.

After the reaction ceased bubbling, bi(cyclohexan)-4-one 15 (1.00 g,

5.55 mmol) (5 mL THF solution) was added. After 1 h, the reaction

was ﬁltered and washed with cold EtOAc. The ﬁltrate was removed of

solvent and put under high vacuum. The material was chromato-

graphed (80 g SiO₂, hexanes to 75% CH₂Cl₂/hexanes to give the title

compound as a clear oil (0.950 g, 72%). The material was used without

further puriﬁcation.

6-Methyl 6-Cyclohexylspiro[2.5]octane-1-carboxylate (17).

To unwashed NaH 60% in mineral oil (0.040 g, 1.02 mmol) was

added DMF (2 mL) followed by portionwise addition of solid

trimethylsulfoxonium iodide (0.233 g, 1.06 mmol). The reaction was

stirred for 1.5 h. To this was added methyl 2-(bi(cyclohexan)-4-

ylidene)acetate 16 (0.10 mg, 0.42 mmol) in 1 mL of DMF. A fresh

suspension of NaH/DMF (0.04 g, 1.02 mmol) and trimethylsulfoxo-

Q

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nium iodide (0.23 g, 1.06 mmol) was prepared and stirred for 10 min,

which was then added to the ﬁrst reaction. The reaction was stirred at

room temperature for 48 h, and the solvent was removed. The white

solids were triturated with CH₂Cl₂, and salts were ﬁltered oﬀ. The

crude material was chromatographed (SiO₂20 g, gradient from pure

hexanes to 1:1 hexanes/CH₂Cl₂) to give the title compound (0.52 g

49%). ¹H NMR (500 MHz, CDCl₃) δ 0.80−0.95 (m, 4H), 1.05−1.27

(m, 7H), 1.40−1.43 (m, 1H), 1.71−1.79 (m, 11H), 3.83 (s, 3H). ¹³C

MNR δ 20.57, 25.63, 26.86, 28.53, 28.94, 30.34,31.02, 37.29, 42.88,

42.97, 51.47, 173.49.

tert-Butyl 1,1-Dichloro-2-oxo-7-azaspiro[3.5]nonane-7-car-

boxylate (19). To a suspension of zinc−copper couple (35.4 g)

and tert-butyl 4-methylenepiperidine-1-carboxylate 18 (8.0 g, 40

mmol) in ether (150 mL) was added a solution of trichloroacetyl

chloride (24.32 g, 130 mmol) in 1,2-dimetoxy ethane (40 mL)

dropwise at room temperature under a nitrogen atmosphere. After

stirring the mixture at room temperature for 6 h, the reaction mixture

was poured into a saturated solution of NaHCO₃(150 mL) at 0 °C

and ﬁltered. The ﬁltrate was extracted with EtOAc (300 mL × 2), and

the combined organic layer was washed with brine (150 mL), dried,

and evaporated. The crude material was puriﬁed by column

chromatography over silica gel using 20% of EtOAc in petroleum

ether as an eluent to yield the title dichloro ketone (5.3 g, 40%) as a

pale brown oil, which was used in the next step without further

characterization.

tert-Butyl 2-Oxo-7-azaspiro[3.5]nonane-7-carboxylate (20).

To a solution of tert-butyl 1,1-dichloro-2-oxo-7-azaspiro[3.5]nonane-7-

carboxylate (5.3 g, mmol) in saturated NH₄Cl and MeOH (80 mL)

was added zinc (6.0 g) portionwise at room temperature. The reaction

mixture was stirred at rt for 10 h and ﬁltered. The ﬁltrate was

concentrated under vacuum, and the crude material was puriﬁed by

column chromatography over silica gel using 20% of EtOAc in

petroleum ether as an eluent to yield the title compound (4.0 g, 97%)

as a solid. ¹H NMR (400 MHz, CDCl₃) δ 1.46 (s, 9H), 1.70 (t, J = 5.6

Hz, 4H), 2.81 (s, 4H), 3.41 (t, J = 5.6 Hz, 4H).

7-carboxylate 22 (1.4 g, mmol) in ethanol (25 mL) was slowly added

sulfuric acid (10 mL), and the mixture was reﬂuxed for 16 h. The

reaction mixture was cooled to room temperature, and the solvent was

removed under reduced pressure. The residue was added to ice water

(20 mL) and treated with sat. NaHCO₃until basic, and a solution of

Boc anhydride (1.54 g, 7.1 mmol) in THF (25 mL) was added at 5−

10 °C. The mixture was allowed to come to room temperature and

stirred for 16 h. The reaction mixture was extracted with EtOAc (50

mL × 2). The combined organic layer was washed with water and

brine, dried, and concentrated. The crude material was puriﬁed by

column chromatography over silica gel using 0−10% of EtOAc in

petroleum ether as an eluent to aﬀord the title compound (0.7 g, 41%)

1

as a colorless liquid. H NMR (400 MHz, CDCl₃) δ 1.26 (t, 3H, J =

7.12 Hz), 1.45 (s, 9H), 1.51−1.62 (m, 4H), 2.06 (d, J = 8.76 Hz, 4H),

3.08 (m, 1H), 3.27 (m, 2H), 3.35 (m, 2H), 4.14 (q, J = 7.12 Hz, 1H).

Benzyl 1,1-Dichloro-2-oxo-7-azaspiro[3.5]nonane-7-carbox-

ylate (24). In a 500 mL round-bottomed ﬂask were added benzyl 4-

methylenepiperidine-1-carboxylate (11.0 g, 47.5 mmol) and zinc−

copper couple (41.9 g, 642.0 mmol) combined in ether (150 mL) to

give a gray suspension. The reaction suspension was cooled to 0 °C. In

a separate ﬂask were combined trichloroacetyl chloride (28.7 mL,

256.8 mmol) and DME (85 mL), which was then transferred to a

dropping funnel. This solution was added dropwise to the round-

bottomed ﬂask reaction over 30 min. Moderate gas evolution with an

exotherm was noted during the acid chloride addition. The reaction

was stirred at 0 °C for 1 h and allowed to warm to room temperature

by removing the ice bath. The reaction was stirred at room

temperature for an additional 18 h. The reaction was quenched by

slowly pouring the reaction mixture into a saturated NaHCO₃solution

(500 mL), which was kept at 0 °C. During the addition, vigorous gas

evolution occurred. The quenched reaction was ﬁltered through a bed

of Celite, and all solids were removed. The ﬁltrate was transferred to a

separatory funnel and partitioned with EtOAc (1 L). The aqueous

layer was back-extracted with EtOAc (2 × 250 mL). The combined

organic layers were washed with brine (2 × 500 mL) and dried over

MgSO₄. The organic layer was concentrated, and the resultant material

was puriﬁed by SiO₂chromatography (SiO₂, 330 g column, 100%

hexane to 50% EtOAc/hexane over 30 min). The fractions were

collected and evaporated to give the title compound (6.50 g, 40%). MS

(ES+) (m/z) = 342, (m/z + 2) = 344. The material was used as is

without further characterization.

tert-Butyl 2-(Methylsulfonyloxy)-7-azaspiro[3.5]nonane-7-

carboxylate (21). To a solution of tert-butyl 2-oxo-7-azaspiro[3.5]-

nonane-7-carboxylate 20 (3.00 g, 12.5 mmol) in MeOH (30 mL) was

added NaBH₄(0.57 g 15.0 mmol) at 0 °C, and the mixture was stirred

at room temperature for 1 h. The reaction mixture was concentrated

under reduced pressure. The residue was dissolved in EtOAc (80 mL),

washed with water (30 mL) and brine (30 mL), dried over Na₂SO₄,

and concentrated to give the intermediate alcohol (3.0 g, 99%) as a

white solid, which was used without further puriﬁcation. To a solution

of tert-butyl 2-hydroxy-7-azaspiro[3.5]nonane-7-carboxylate (3.50 g,

14.5 mmol) and Et₃N (4 mL, 29 mmol) in CH₂Cl₂(50 mL) was

added methane sulphonyl chloride (1.35 mL, 17.4 mmol) at 0−5 °C,

and the mixture was stirred at room temperature for 1 h. The reaction

mixture was poured onto water (50 mL) and extracted with CH₂Cl₂.

The combined organic layer was washed with water (20 mL) and brine

(20 mL) and dried over Na₂SO_4.The solvent was removed under

reduced pressure to give the title compound (4.5 g, 97%) as a yellow

Benzyl 2-Oxo-7-azaspiro[3.5]nonane-7-carboxylate (25). In a

1 L round-bottomed ﬂask were added benzyl 1,1-dichloro-2-oxo-7-

azaspiro[3.5]nonane-7-carboxylate (6.45 g, 18.8 mmol) and MeOH

(50 mL) to give an orange solution. To this was added a solution of

saturated NH₄Cl/MeOH (125 mL) followed by zinc dust (12.94 g,

197.9 mmol) in 6 equal portions over 15 min. The reaction was stirred

at room temperature overnight. The solvent was removed by rotary

evaporation, leaving behind a thick, yellow syrup. To this was added

CH₂Cl₂(100 mL), and the solids were ﬁltered oﬀ followed by further

concentration of the organics to give a yellow syrup. The residue was

puriﬁed via SiO₂chromatography (120 g column, 100% hexane to 50%

EtOAc/hexane over 30 min) to give 3.85 g (74%) of the title

1

solid. H NMR (400 MHz, CDCl₃) δ 1.46 (s, 9H), 1.52−1.63 (m,

1

compound as a pale oil. H NMR (400 MHz, CDCl₃-d) δ 1.71 (m,

4H), 2.04−2.12 (m, 2H), 2.40−2.45 (m, 2H), 3.00 (s, 3H), 3.25−3.37

(m, 4H), 5.00−5.08 (m, 1H).

4H), 2.90 (s, 4H), 3.48 (m, 4H), 5.13 (s, 2H), 7.28−7.36 (m, 5H).

Benzyl 2-Formyl-7-azaspiro[3.5]nonane-7-carboxylate (26).

Potassium tert-butoxide (1 M in THF, 77 mL, 0.77 mol) was loaded

into an oven-dried ﬂask under a nitrogen atmosphere. To this were

added t-BuOH (7.38 mL, 77.16 mmol) and THF (70 mL). The

mixture was cooled to −78 °C, and methoxymethyltriphenylphospho-

nium chloride (26.50 g, 77.16 mmol) was added portionwise. The

reaction mixture was allowed to warm to room temperature, stirred for

1 h, and cooled back to −78 °C. A solution of benzyl 2-oxo-7-

azaspiro[3.5]nonane-7-carboxylate 25 (7.03 g, 25.72 mmol) in THF

(70 mL) was added dropwise to the reaction mixture over a period of

30 min. The reaction mixture was allowed to warm to room

temperature and stirred for 1 h. The reaction was quenched with water

(150 mL), and the product was extracted using EtOAc. The combined

organic phases were washed with brine and dried over anhydrous

MgSO₄, and the solvent was concentrated. The product was puriﬁed

tert-Butyl 2-Cyano-7-azaspiro[3.5]nonane-7-carboxylate

(22). A mixture of tert-butyl 2-(methylsulfonyloxy)-7-azaspiro[3.5]-

nonane-7-carboxylate (4.50 g, 14 mmol), sodium cyanide (1.35 g, 28

mmol), TBAB (20 mg), and DMF (40 mL) was heated at 120 °C and

stirred for 16 h. The reaction mixture was poured into water (200 mL)

and extracted with EtOAc (100 mL × 2). The combined organic layer

was washed with water and brine, dried, and ﬁltered, and the solvent

was removed under reduced pressure. The crude material was puriﬁed

by column chromatography over silica gel using 20% of EtOAc in

petroleum ether as an eluent to yield the title compound (2.0 g, 57%)

as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 1.45 (s, 9H), 1.55 (m,

2H), 1.64 (m, 2H), 2.19−2.25 (m, 4H), 3.1 (m, 1H), 3.30−3.32 (m,

4H).

7-tert-Butyl 2-Ethyl 7-azaspiro[3.5]nonane-2,7-dicarboxy-

late (23). To a solution of tert-butyl 2-cyano-7-azaspiro[3.5]nonane-

R

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on SiO₂(330 g, 20% EtOAc in heptane as eluent) to provide the title

compound (3.53 g, 47.8%) as a colorless oil. H NMR (400 MHz,

CDCl₃-d) δ 1.48 (br s, 2H), 1.62 (br s, 2H), 1.91−2.16 (m, 4H), 3.16

(quin, J = 8.6 Hz, 1H), 3.31−3.41 (m, 2H), 3.41−3.50 (m, 2H), 5.12

(s, 2H), 7.28−7.44 (m, 5H), 9.76 (d, J = 1.56 Hz, 1H). MS (ES+) m/z

[M + H]⁺= 288.1.

were put under high vacuum for 12 h. The material was subjected to

SiO₂chromatography (80 g, using a gradient of 0−10% 7 N NH₃/

MeOH in CH₂Cl₂). The impure fractions were collected and further

puriﬁed with preparatory HPLC using procedure A to provide the title

1

compound as a white solid (0.112 g, 76%). H NMR (300 MHz,

CDCl₃) δ 1.68−1.83 (m, 12H), 1.85−2.05 (m, 7H), 2.23−2.28 (m,

4H), 2.35−2.47 (m, 4H), 2.70 (ddd, J = 11.4, 7.8, 7.8 Hz, 1H), 3.13

(ddd, J = 13.2, 9, 9 Hz, 1H), 3.31−3.38 (m, 4H), 3.59−3.62 (m, 2H),

3.99 (dd, J = 10.9, 3.9 Hz, 2H). HRMS (TOF) m/z calcd for

C₂₁H₃₇N₃O₂(M + H)⁺, 376.2986; found, 376.2986.

(4-Cyclohexylpiperazin-1-yl)(7-(tetrahydro-2H-pyran-4-yl)-

7-azaspiro[3.5]nonan-2-yl)methanone (29c). To ethyl 7-(tetrahy-

dro-2H-pyran-4-yl)-7-azaspiro[3.5]nonane-2-carboxylate 28 (0.395 g,

1.40 mmol) were added MeOH (10 mL) and NaOH (1 N, 1.68 mL,

1.68 mmol). The reaction was heated to reﬂux for 2 h. The reaction

was quenched by adding HCl (1 N, 1.684 mL, 1.68 mmol), and then

the solvent was removed. The tan foam was placed under high vacuum

at 50 °C for 12 h to give the intermediate acid in quantitative yield,

which was used as is in the next reaction without further puriﬁcation.

¹H NMR (500 MHz, DMSO-d₆) δ 1.45−1.69 (m, 8H), 1.86−1.88 (m,

2H), 1.94−1.96 (m, 2H), 2.45−2.50 (m, 3H), 2.98−3.02 (m, 1H),

3.22−3.26 (m, 4H), 3.78−3.88 (m, 2H). MS (ES+) m/z [M]⁺= 254.3.

To 7-(tetrahydro-2H-pyran-4-yl)-7-azaspiro[3.5]nonane-2-carboxylic

acid (309 mg, 1.22 mmol) was added thionyl chloride (10 mL), and

the cloudy reaction was stirred at room temperature. After 30 min, the

solvent was removed under vacuum, and the resulting white solid was

put under high vacuum for 4 h. A sample of this was taken (0.050 g) to

make the title compound as follows. To 7-(tetrahydro-2H-pyran-4-yl)-

7-azaspiro[3.5]nonane-2-carbonyl chloride (0.050 mg, 0.18 mmol)

was added a solution of 1-cyclohexylpiperazine (46.4 mg, 0.28 mmol)

and DIPEA (0.129 mL, 0.74 mmol) in CH₂Cl₂(2M, 2.0 mL), and the

reaction was stirred for 2 h. The solvent was removed, and the residue

was dried under high vacuum at 50 °C overnight. The material was

puriﬁed by reverse-phase HPLC using procedure A. The fractions were

collected, the solvent was removed, and the product was dried under

high vacuum at 50 °C overnight (0.074 g, 61%). ¹H NMR (500 MHz,

DMSO-d₆) δ 1.14−1.18 (m, 4H), 1.37−1.42 (m, 4H), 1.57−1.59 (m,

5H), 1.61−1.63 (m, 4H), 1.70−1.73 (m, 4H), 1.86 (app t, J = 10 Hz,

4H), 2.22−2.34 (m, 4H), 2.33−2.39 (m, 6H), 3.17−3.21 (m, 2H),

3.32−3.39 (m, 2H), 3.84 (d, J = 5.0 Hz, 1H), 3.85 (d, J = 5 Hz, 1H).

HRMS (TOF) m/z calcd for C₂₄H₄₂N₃O₂(M + H)⁺, 404.3272; found,

404.3267.

7-(Benzyloxycarbonyl)-7-azaspiro[3.5]nonane-2-carboxylic

Acid (27). A solution of 6% aqueous sodium hypochlorite (84.0 g,

1.12 mol) was added to a solution of benzyl 2-formyl-7-azaspiro[3.5]-

nonane-7-carboxylate (3.53 g, 12.28 mmol), NaHCO₃(saturated) (8

mL), sodium bromide (253 mg, 2.46 mmol), and TEMPO (6 mg, 0.04

mmol, in CH₂Cl₂6 mL solution) in water (30 mL) over a period of 2

h. The reaction mixture was stirred for 2 h, and the pH was adjusted to

∼1 by addition of concentrated hydrochloric acid. The product was

extracted using Et₂O (3 × 70 mL). The combined organic phases were

washed with brine and dried over anhydrous Na₂SO₄. The solvent was

concentrated, and the product was puriﬁed on SiO₂(120 g, eluting

with a 3:2 mixture of heptane/EtOAc) to aﬀord 7-(benzyloxycarbon-

yl)-7-azaspiro[3.5]nonane-2-carboxylic acid (2.69 g, 72.2%) as a white

solid. ¹H NMR (400 MHz, CDCl₃) δ 1.58 (d, J = 16.8 Hz, 4H), 2.10

(d, J = 8.9 Hz, 4H), 3.13 (quin, J = 8.8 Hz, 1H), 3.33−3.41 (m, 2H),

3.41−3.48 (m, 2H), 5.12 (s, 2H), 7.28−7.41 (m, 5 H). MS (ES+) m/z

[M + H]⁺= 304.2.

Ethyl 7-(Tetrahydro-2H-pyran-4-yl)-7-azaspiro[3.5]nonane-

2-carboxylate (28). To 7-tert-butyl 2-ethyl 7-azaspiro[3.5]nonane-

2,7-dicarboxylate 23 (0.815 g, 2.74 mmol) in CH₂Cl₂(2.7 mL) was

added TFA (2.7 mL) to give an amber solution. The reaction was

stirred at room temperature for 3 h, and the solvent was removed. The

residue was triturated with ether (1 × 25 mL) and placed on the

vacuum manifold for 1 h to give roughly 0.95 g of crude TFA salt. The

salt was redissolved in CH₂Cl₂(20 mL), and to this was added Si-

Amine resin (∼2g, Silicycle, loading of 1.67 mmol/g). The

heterogeneous mixture was stirred for 30 min at room temperature.

The silica gel was ﬁltered and washed with CH₂Cl₂, and the solvent

was removed. The compound was placed under high vacuum for 8 h

to give the intermediate piperidine as a waxy solid (0.541 g, 83%). ¹H

NMR (300 MHz, CDCl₃) δ 1.25 (t, J = 7.2 Hz, 3H), 1.69 (m, 4H),

2.06 (d, J = 8.7 Hz, 4H), 2.41 (br s, N-H/TFA-H), 2.82 (app t, J = 5.7

Hz, 2H), 2.88 (app t, J = 5.4 Hz, 2H), 3.08 (quintet, J = 8.7 Hz, 1H),

4.14 (q, J = 7.2 Hz, 2H). MS (ES+) m/z [M]⁺= 198.0. To ethyl 7-

azaspiro[3.5]nonane-2-carboxylate (0.47 g, 2.38 mmol) were added

MeOH (20 mL), dihydro-2H-pyran-4(3H)-one (0.80 mL, 9.53

mmol), and Pd/C (0.05 g, 0.05 mmol), and a balloon was attached

to the reaction ﬂask ﬁlled with H₂. The reaction was stirred at room

temperature overnight. The reaction was ﬁltered, and the solvent was

removed in vacuo. The material was puriﬁed using SiO₂chromatog-

raphy (80 g, solvent gradient of 0−10% MeOH in CH₂Cl₂) to give the

title compound (0.39 g, 1.40 mmol, 58%). ¹H NMR (500 MHz,

CDCl₃) δ 1.25 (t, J = 7 Hz, 3H), 1.56−1.86 (br m, 10H), 2.01 (d, J =

8.5 Hz, 4H), 3.03 (m, 1H), (br m, 4H), 3.36 (app dt, J = 12.0, 1.5 Hz,

2H), 4.01 (dd, J = 11.0, 4.0 Hz, 2H), 4.12 (q, J = 7.0 Hz, 2H). MS (ES

+) m/z [M]⁺= 282.4.

(Dihydro-1H-pyrido[1,2-a]pyrazin-2(6H,7H,8H,9H,9aH)-yl)(7-

(tetrahydro-2H-pyran-4-yl)-7-azaspiro[3.5]nonan-2-yl)-

methanone (29d). The material was prepared in an analogous

manner to 29c using octahydro-1H-pyrido[1,2-a]pyrazine to give the

1

title compound as a white solid (0.69 g, 32%). H NMR (500 MHz,

CDCl₃) δ 1.40−1.35 (m, 3H), 1.73−1.81 (m, 9H), 2.02−2.07 (m,

4H), 2.06−2.11 (m, 4H), 2.34−2.47 (m, 6H), 2.72−2.77 (m, 2H),

2.77−2.83 (m, 2H), 3.11 (t, J = 8.5 Hz, 1H), 3.20 (t, J = 12.0 Hz, 0.5

H), 3.42 (d, J = 13.5 Hz, 2H), 3.99 (d, J = 11.5 Hz, 0.5 H), 4.01 (dd, J

= 11.0 Hz, 4.0 Hz, 2H), 4.30 (d, J = 13.0 Hz, 0.5 H), 4.51 (d, J = 13.5

Hz, 0.5 H). HRMS (TOF) m/z calcd for C₂₂H₃₇N₃O₂(M + H)⁺,

376.2958; found, 376.2954. The compound appears to exist as

rotational isomers based on multiple signals that integrate to less than

1H. These signals coalesce upon heating in DMSO-d₆, which supports

this hypothesis.

(4-Isopropyl-1,4-diazepan-1-yl)(7-(tetrahydro-2H-pyran-4-

yl)-7-azaspiro[3.5]nonan-2-yl)methanone dihydrochloride

(29e). The material was prepared in an analogous manner to 29c

using 1-isopropyl-1,4-diazepane to give the title compound in 34%

yield. The material was converted to the di-HCl salt by addition of aq.

0.2 M HCl and lyophilization. ¹H NMR (400 MHz, DMSO-d₆) δ 1.25

(d, J = 6.6 Hz, 6H), 1.61−1.80 (m, 2H), 1.78−2.14 (m, 9H), 2.69−

3.20 (m, 3H), 3.20−3.44 (m, 8H), 3.43−3.81 (m, 10H), 3.95 (dd, J =

11.33, 3.91 Hz, 2H), 10.45 (br s, 1H). HRMS (TOF) m/z calcd for

C₂₂H₄₀N₃O₂(M + H)⁺, 378.3115; found, 378.3108.

(4-Cyclobutyl-1,4-diazepan-1-yl)(7-(tetrahydro-2H-pyran-4-

yl)-7-azaspiro[3.5]nonan-2-yl)methanone (29a). The title com-

pound was prepared as in example 29c in 39% yield as a ﬁlm. ¹H NMR

(500 MHz, DMSO-d₆) δ 0.97−0.99 (m, 6H), 1.56−1.61 (m, 6H),

1.61−1.68 (m, 2H), 1.71−1.76 (m, 2H), 1.78−1.81 (m, 2H), 1.94−

1.97 (m, 2H), 2.07−2.11 (m, 2H), 2.40−2.47 (m, 2H), 2.55 (s, 1H),

2.55−2.58 (m, 2H), 2.62−2.66 (m, 2H), 2.85−2.91 (quin, J = 7.0 Hz,

1H), 3.12−3.17 (m, 1H), 3.36−3.40 (m, 4H), 3.57−3.60 (m, 2H),

3.99 (d, 1H, J = 4.5 Hz), 4.00 (d, 1H, J = 4.5 Hz). HRMS (TOF) m/z

calcd for C₂₃H₄₀N₃O₂(M + H)⁺, 390.3115; found, 390.3114.

(4-Cyclobutylpiperazin-1-yl)(7-(tetrahydro-2H-pyran-4-yl)-7-

azaspiro[3.5]nonan-2-yl)methanone (29b). To 7-(tetrahydro-2H-

pyran-4-yl)-7-azaspiro[3.5]nonane-2-carboxylic acid (as prepared in

example 29c) (0.128 g, 0.39 mmol) were added DMF (3 mL), HBTU

(0.150 g, 0.390 mmol), and then DIPEA (0.345 mL, 1.97 mmol). The

reaction was stirred for 5 min, and 1-cyclobutylpiperazine dihydro-

chloride (0.126 g, 0.59 mmol) was added. The reaction was stirred at

room temperature for 2 h, and the solvent was removed. The solids

(4-Isopropylpiperazin-1-yl)(7-(tetrahydro-2H-pyran-4-yl)-7-

azaspiro[3.5]nonan-2-yl)methanone (29f). To 7-(tetrahydro-2H-

pyran-4-yl)-7-azaspiro[3.5]nonane-2-carboxylic acid (as prepared in

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example 29c) (0.309 g, 1.22 mmol) was added thionyl chloride (10

mL), and the cloudy reaction was stirred at room temperature. After

30 min, the solvent was removed, and the white solid was placed under

high vacuum for 4 h. A sample of this was taken (0.050 g) to make the

title compound as follows. To 7-(tetrahydro-2H-pyran-4-yl)-7-

azaspiro[3.5]nonane-2-carbonyl chloride (0.050g, 0.18 mmol) was

added a solution of 1-isopropylpiperazine (0.039 mL, 0.28 mmol) and

DIPEA (0.129 mL, 0.74 mmol) in CH₂Cl₂(2.0 mL). The reaction was

stirred at room temperature for 2 h. The solvent was removed, and the

gum was put under high vacuum at 50 °C overnight. The material was

puriﬁed by reverse-phase HPLC using the exact methods as for

previous compounds. The resulting gum was placed under high

vacuum at 50 °C overnight to give the title compound as a white solid

reaction mixture was stirred overnight, and the solvent was

concentrated. The crude material was puriﬁed on preparative HPLC

using procedure B to provide the title compound as a white solid

(0.082 g, 64%). ¹H NMR (400 MHz, CDCl₃) δ 1.53 (br s, 1H), 1.56−

1.72 (m, 4H), 1.72−1.95 (m, 5H), 1.96−2.11 (m, 4H), 2.17 (br s,

2H), 2.35−2.45 (m, 2H), 2.45−2.54 (m, 2H), 2.76−2.91 (m, 1H),

3.26 (br s, 2H), 3.34 (br s, 1H), 3.42 (t, J = 5.86 Hz, 2H), 3.55−3.68

(m, 3H), 3.72, (br s, 1H), 7.31−7.49 (m, 5H). HRMS (TOF) m/z

calcd for C₂₅H₃₅N₃O₂(M + H)⁺, 410.2802; found, 410.2797.

Benzyl 2-(4-Cyclobutylpiperazine-1-carbonyl)-7-

azaspiro[3.5]nonane-7-carboxylate (30g). HBTU (1.018 g, 2.68

mmol) and 1-cyclobutylpiperazine (342 mg, 2.44 mmol) were added

to a solution of 7-(benzyloxycarbonyl)-7-azaspiro[3.5]nonane-2-

carboxylic acid (0.74 g, 2.44 mmol) and DIEA (0.511 mL, 2.93

mmol) in DMF (60 mL). The reaction mixture was stirred for 3 h, and

the solvent was concentrated. The product was puriﬁed on silica gel by

using 3% MeOH and 5% acetone in CH₂Cl₂with 0.1 N ammonia as

the eluent (80 g column; 20 mL/min then 40 mL/min) to provide the

1

(0.043 g, 65%). H NMR (300 MHz, CDCl₃) δ 1.02 (d, J = 6.5 Hz,

6H), 1.58 (m, 4H), 1.65−1.67 (m, 2H), 1.71−1.74 (m, 2H), 1.96−

1.98 (m, 2H), 2.08 (m, 2H), 2.39- 2.47 (br m, 9H), 3.11 (quintet, J =

9.0 Hz, 1H), 3.33−3.38 (m, 4H), 3.59−3.61 (m, 2H), 3.99 (d, J = 4.0

Hz, 1H), 4.01 (d, J = 4.0 Hz, 1H). HRMS (TOF) m/z calcd for

C₂₁H₃₇N₃O₂(M + H)⁺, 364.2958; found, 364.2957.

1

title compound (0.81 g, 78%) as a white solid. H NMR (400 MHz,

CDCl₃) δ 1.43−1.57 (m, 3H), 1.56−1.67 (m, 2H), 1.67−1.82 (m,

2H), 1.82−1.98 (m, 3H), 1.98−2.08 (m, 3H), 2.08−2.18 (m, 2H),

2.26−2.35 (m, 3H), 2.69−2.80 (m, J = 8.20, 7.91, 7.76, 7.76 Hz, 1H),

3.18 (quin, J = 8.50 Hz, 1H), 3.33−3.42 (m, 4H), 3.41−3.50 (m, 2H),

3.58−3.69 (m, 2H), 5.12 (s, 2H), 7.29−7.41 (m, 5H). MS m/z (ES+)

[M + H]⁺= 426.3.

(4-Cyclobutylpiperazin-1-yl)(7-(methylsulfonyl)-7-azaspiro-

[3.5]nonan-2-yl)methanone (29g). Methanesulfonyl chloride

(0.032 mL, 0.41 mmol) was added to a solution of (4-cyclo-

butylpiperazin-1-yl)(7-azaspiro[3.5]nonan-2-yl)methanone (0.100 g,

0.34 mmol) and triethylamine (52.1 mg, 0.51 mmol) in dichloro-

methane (10 mL) at 0 °C. The reaction mixture was allowed to warm

to room temperature and stirred overnight. The solvent was

concentrated. The product was puriﬁed on preparative HPLC using

procedure B to provide the title compound (49.6 mg, 39.1%) as a

white solid. ¹H NMR (400 MHz, CDCl₃) δ 1.59−1.72 (m, 3H), 1.72−

1.80 (m, 3H), 1.79−1.94 (m, J = 10.1, 9.7, 9.5, 9.5 Hz, 2H), 1.95−2.10

(m, 4H), 2.09−2.20 (m, 2H), 2.22−2.34 (m, 4H), 2.66−2.74 (m, 1H),

2.76 (s, 3H), 3.05−3.15 (m, 2H), 3.14−3.26 (m, 3H), 3.30−3.38 (m,

2H), 3.62 (t, J = 4.8 Hz, 2H). HRMS (TOF) m/z calcd for

C₁₈H₃₁N₃O₃S (M + H)⁺, 370.2158; found, 370.2160.

(4-Cyclobutylpiperazin-1-yl)(7-phenyl-7-azaspiro[3.5]-

nonan-2-yl)methanone (29h). Cesium carbonate (0.123 g, 0.38

mmol) was added to a solution of (4-cyclobutylpiperazin-1-yl)(7-

azaspiro[3.5]nonan-2-yl)methanone 31g (0.100 g, 0.340 mmol),

Pd(OAc)₂(0.007 g, 0.03 mmol), BINAP (0.042 g, 0.07 mmol), and

bromobenzene (0.056 g, 0.36 mmol) in toluene (5 mL). The reaction

mixture was heated at 110 °C for 18 h. The reaction was cooled to

room temperature and ﬁltered over Celite. The solvent was

concentrated, and the product was puriﬁed by preparative HPLC

using procedure B to provide the title compound as white solid (59.0

mg, 46.8%). ¹H NMR (400 MHz, CDCl₃) δ 1.60−1.75 (m, 4H),

1.75−1.81 (m, 2H), 1.81−1.95 (m, 2H), 1.96−2.10 (m, 4H), 2.10−

2.20 (m, 2H), 2.21−2.35 (m, 4H), 2.71 (dq, J = 8.0, 7.7 Hz, 1H),

3.00−3.11 (m, 2H), 3.11−3.26 (m, 3H), 3.31−3.42 (m, 2H), 3.56−

3.70 (m, 2H), 6.82 (t, J = 7.2 Hz, 1H), 6.93 (d, J = 7.81 Hz, 2H),

7.18−7.29 (m, 2H). HRMS (TOF) m/z calcd for C₂₃H₃₃N₃O (M +

H)⁺, 368.2696; found, 368.2698.

Benzyl 2-(4-Cyclobutyl-1,4-diazepane-1-carbonyl)-7-

azaspiro[3.5]nonane-7-carboxylate (30j). Oxalyl chloride (0.970

mL, 11.08 mmol) was slowly added to a solution of 7-

(benzyloxycarbonyl)-7-azaspiro[3.5]nonane-2-carboxylic acid (1.12 g,

3.69 mmol) in dichloromethane (50 mL) at 0 °C under a nitrogen

atmosphere. The solution was stirred for 4 h while gradually warming

to 15 °C. The solvent was concentrated under vacuum, and the residue

was recovered in CH₂Cl₂(10 mL). The resulting solution was added

dropwise over 15 min to a solution of 1-cyclobutyl-1,4-diazepane (0.62

g, 4.06 mmol) and DIEA (1.934 mL, 11.08 mmol) in CH₂Cl₂(50 mL)

at 0 °C under a nitrogen atmosphere. The reaction mixture was stirred

for 30 min and concentrated. The product was puriﬁed on SiO₂

(gradient of MeOH 3% in CH₂Cl₂(500 mL) to MeOH 5% in CH₂Cl₂

(500 mL)). Impure fractions were resubjected to puriﬁcation under

the same conditions to give several batches of pure compound, which

were combined to provide the title compound as a white solid (0.681

g, 42.0%). t_R= 1.80 min (5 min run). MS m/z (ES+) [M + H]⁺=

440.39. The material was used is as without further characterization.

(4-Cyclobutylpiperazin-1-yl)(7-azaspiro[3.5]nonan-2-yl)-

methanone (31g). A mixture of benzyl 2-(4-cyclobutylpiperazine-1-

carbonyl)-7-azaspiro[3.5]nonane-7-carboxylate (0.80 g, 1.88 mmol),

Pd/C (0.01 g, 0.09 mmol), and ethanol (100 mL) was shaken in a Parr

apparatus under a 50 psi atmosphere of hydrogen for 4 h. The mixture

was ﬁltered over a Celite bed, and the solvent was concentrated to

provide (4-cyclobutylpiperazin-1-yl)(7-azaspiro[3.5]nonan-2-yl)-

1

methanone (480 mg, 88%) as a white solid. H NMR (400 MHz,

CDCl₃) δ 1.49−1.62 (m, 2H), 1.62−1.78 (m, 3H), 1.79−1.95 (m, J =

9.4, 9.4, 9.2, 8.9 Hz, 2H), 1.95−2.16 (m, 5H), 2.27 (q, J = 5.1 Hz, 4H),

2.60 (br s, 3H), 2.65−2.74 (m, 1H), 2.74−2.81 (m, 2H), 2.81−2.93

(m, 2H), 3.16 (quin, J = 8.7 Hz, 1H), 3.28−3.40 (m, 2H), 3.62 (t, J =

4.9 Hz, 2H). MS m/z (ES+) [M + H]⁺= 292.2.

(7-Benzoyl-7-azaspiro[3.5]nonan-2-yl)(4-cyclobutylpipera-

zin-1-yl)methanone (29i). Benzoyl chloride (0.035 mL, 0.30 mmol)

was added to a solution of (4-cyclobutylpiperazin-1-yl)(7-

azaspiro[3.5]nonan-2-yl)methanone 31g (80 mg, 0.27 mmol) and

DIPEA (0.058 mL, 0.33 mmol) in dichloromethane (8 mL). The

reaction mixture was stirred for 3 days, and the solvent was

concentrated. The crude material was puriﬁed on preparative HPLC

MS using procedure B to provide the title compound as white solid

(4-Cyclobutyl-1,4-diazepan-1-yl)(7-azaspiro[3.5]nonan-2-yl)-

methanone (31j). The title compound was prepared in an analogous

1

manner to 31g to yield a white solid (1.62 g, 95%). H NMR (400

1

MHz, CDCl₃) δ 1.49−1.58 (m, 2H), 1.58−1.74 (m, 3H), 1.74−1.93

(m, 4H), 1.93−2.17 (m, 8H), 2.36−2.45 (m, 2H), 2.48 (dt, J = 4.98,

2.15 Hz, 2H), 2.67−2.78 (m, 2H), 2.78−2.91 (m, 3H), 3.17 (quin, J =

8.79 Hz, 1H), 3.37−3.47 (m, 2H), 3.5 −3.67 (m, 2H).

tert-Butyl 2-(4-Isopropylpiperazine-1-carbonyl)-2,7-

diazaspiro[3.5]nonane-7-carboxylate (33). To a −78 °C cooled

solution of 4-nitrophenyl carbonochloridate (0.384 g, 1.90 mmol) in

THF (5 mL) was added DIPEA (1.00 mL, 5.71 mmol) followed by 1-

isopropylpiperazine (0.28 mL, 1.90 mmol). This cloudy mixture was

stirred 5 min, and then tert-butyl 2,7-diazaspiro[3.5]nonane-7-

carboxylate hydrochloride (0.50 g, 1.90 mmol) was added. The

(36.5 mg, 33.6%). H NMR (400 MHz, CDCl₃) δ 1.51 (br s, 1H),

1.62−1.68 (m, 2H), 1.68−1.80 (m, 3H), 1.80−1.95 (m, 2H), 1.96−

2.11 (m, 4H), 2.16 (br s, 2H), 2.27 (q, J = 5.47 Hz, 4H), 2.71 (dq, J =

8.0, 7.7 Hz, 1H), 3.11−3.30 (m, 2H), 3.35 (br s, 3H), 3.55−3.67 (m,

3H), 3.72 (br s, 1H), 7.32−7.44 (m, 5H). HRMS (TOF) m/z calcd

for C₂₄H₃₃N₃O₂(M + H)⁺, 396.2645; found, 396.2650.

(7-Benzoyl-7-azaspiro[3.5]nonan-2-yl)(4-cyclobutyl-1,4-dia-

zepan-1-yl)methanone (29j). Benzoyl chloride (0.040 mL, 0.34

mmol) was added to a solution of (4-cyclobutyl-1,4-diazepan-1-yl)(7-

azaspiro[3.5]nonan-2-yl)methanone 31g (0.095 g, 0.31 mmol) and

DIEA (0.081 mL, 0.47 mmol) in dichloromethane (10 mL). The

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reaction was warmed to room temperature and then reﬂuxed for 12 h.

The crude reaction was partitioned between EtOAc/NaHCO₃(sat.).

The organic layer was dried over Na₂SO₄, and the solvent was

removed to give an amber gum. The material was chromatographed

with SiO₂chromatography (80 g, using a gradient of 0−10% MeOH to

(4-Cyclobutyl-1,4-diazepan-1-yl)(7-(tetrahydro-2H-pyran-4-

yl)-2,7-diazaspiro[4.5]decan-2-yl)methanone (37a). tert-Butyl 2-

(4-cyclobutyl-1,4-diazepane-1-carbonyl)-2,7-diazaspiro[4.5]decane-7-

carboxylate 36a (400 mg, 0.95 mmol) was dissolved in 6 N HCl

iPrOH (5 mL) and stirred overnight at room temperature. The

reaction mixture was concentrated to dryness, CH₂Cl₂(10 mL) was

added followed by Et₃N (0.29 mL, 2.08 mmol), and the reaction

mixture was stirred 10 min. Dihydro-2H-pyran-4(3H)-one (156 mg,

1.56 mmol) and AcOH (56 μL, 0.95 mmol) were added. After 15 min

of stirring, NaBH(OAc)₃(331 mg, 1.56 mmol) was added, and the

reaction mixture was left stirring overnight at room temperature. The

reaction mixture was washed with 5% NaHCO₃, extracted with

CH₂Cl₂(3×), dried over Na₂SO₄, ﬁltered, and concentrated in vacuo.

The residue was puriﬁed via preparatory HPLC using procedure C to

1

CH₂Cl₂) and dried to give a tan solid (0.525 g, 72%). H NMR 500

MHz, CDCl₃) δ 1.03 (d, J = 6.5 Hz, 6H), 1.45 (s, 9H), 1.69 (app t, J =

5.5 Hz, 4H), 2.47 (br s, 4H), 2.65 (m, 1H), 3.35−3.33 (m, 8H), 3.70

(s, 4H). MS m/z (ES+) [M + H]⁺= 381.3.

(4-Isopropylpiperazin-1-yl)(7-(tetrahydro-2H-pyran-4-yl)-

2,7-diazaspiro[3.5]nonan-2-yl)methanone (34). To tert-butyl 2-

(4-isopropylpiperazine-1-carbonyl)-2,7-diazaspiro[3.5]nonane-7-car-

boxylate (0.51 g, 1.34 mmol) was added MeOH (20 mL) that had

been saturated with HCl gas. The reaction was stirred 1 h, the solvent

was removed, and the white solid was put under high vacuum for 12 h

(0.52 g). MS m/z (ES+) [M + H]⁺= 281.2. The material contained

∼50% impurity that was not removed but was used as is in the next

reaction without further puriﬁcation. To impure (4-isopropylpiperazin-

1-yl)(2,7-diazaspiro[3.5]nonan-2-yl)methanone dihydrochloride (0.52

g, 1.49 mmol) were added MeOH (10 mL), TEA (0.621 mL, 4.46

mmol), Pd/C (63.3 mg, 0.06 mmol), and dihydro-2H-pyran-4(3H)-

one (0.55 mL, 5.94 mmol). The reaction was ﬁtted with a balloon

ﬁlled with H₂and stirred for 24 h. The catalyst was ﬁltered oﬀ, the

solvent was removed under reduced pressure, and the resulting solids

were collected. The material was puriﬁed with preparatory HPLC

procedure A to provide the title compound as a white solid (0.155 g,

1

produce the title compound (150 mg, 36%) as a clear oil. H NMR

(300 MHz, CDCl₃) δ 1.18−2.01 (m, 19H), 2.18 (dd, J = 28.1, 10.9

Hz, 2H), 2.28−2.53 (m, 6H), 2.79 (m, 1H), 2.99 (d, J = 10.3 Hz, 1H),

3.16−3.40 (m, 7H), 3.66 (s, 2H), 3.82−3.93 (m, 2H). HRMS (TOF)

m/z calcd for C₂₃H₄₀N₄O₂(M + H)⁺, 405.3229; found, 405.3224.

(4-Isopropylpiperazin-1-yl)(7-(tetrahydro-2H-pyran-4-yl)-

2,7-diazaspiro[4.5]decan-2-yl)methanone (37b). Material was

prepared in an analogous manner to 37a to provide the title

1

compound (167 mg, 44%). H NMR (300 MHz, CDCl₃) δ 1.04 (d, J

= 6.5 Hz, 6H), 1.29−1.69 (m, 10H), 1.77−1.88 (m, 1H), 2.28 (q, J =

11.0 Hz, 2H), 2.38−2.58 (m, 6H), 2.61−2.74 (m, 1H), 3.12 (d, J =

10.4 Hz, 1H), 3.22 −3.52 (m, 9H), 3.98 (d, J = 9.4 Hz, 2H). HRMS

(TOF) m/z calcd for C₂₁H₃₈N₄O₂(M + H)⁺, 379.3073; found,

379.3080.

1

28%). H NMR (500 MHz, CDCl₃) δ 1.06 (s, 6H), 1.76−1.80 (m,

2H), 1.79−1.87 (m, 2H), 2.07−2.09 (2H), 2.34−2.35 (m, 2H), 2.47

(app t, J = 5 Hz, 4H), 2.64 (quintet, J = 6.5 Hz, 1H), 3.48−3.52 (m,

8H), 3.58−3.61 (m, 6H), 3.91−3.94 (m, 1H), 4.08 (dd, J = 11.7, 4.5

Hz, 2H). HRMS (TOF) m/z calcd for C₂₀H₃₆N₄O₂(M + H)⁺,

365.2911; found, 365.2913.

(7-Benzoyl-2,7-diazaspiro[4.5]decan-2-yl)(4-cyclobutyl-1,4-

diazepan-1-yl)methanone (37c). (4-Cyclobutyl-1,4-diazepan-1-yl)-

(2,7-diazaspiro[4.5]decan-2-yl)methanone 36a (200 mg, 0.51 mmol)

was dissolved in CH₂Cl₂(3 mL). Triethylamine (0.21 mL, 1.53 mmol)

was added, and the solution was stirred at room temperature for 10

min. Benzoyl chloride (0.07 mL, 0.61 mmol) was then added, and the

mixture was stirred at room temperature for 4 h. The solution was

diluted with CH₂Cl₂(10 mL) and washed with 5% aq. NaHCO₃(1 ×

10 mL) and brine (1 × 10 mL). The organic layer was dried over

Na₂SO₄, ﬁltered, and evaporated. The product was puriﬁed through

ﬂash-chromatography (SiO₂, gradient of CH₂Cl₂/MeOH 100:0 to

80:20) and then on reverse phase (ﬂow: 30 mL/min, H₂O/CH₃CN

100:0 to 5:95) to provide the title compound (103 mg, 48%) as an oﬀ-

tert-Butyl 2-(4-Cyclobutyl-1,4-diazepane-1-carbonyl)-2,7-

diazaspiro[4.5]decane-7-carboxylate (36a). tert-Butyl 2,7-

diazaspiro[4.5]decane-7-carboxylate hydrochloride 35 (2.30 g, 8.32

mmol) was dissolved in dry THF (40 mL), and to this was added

freshly recrystallized carbonyl diimidazole (1.48 g, 9.15 mmol). The

reaction mixture was heated at reﬂux for 24 h, and then THF was

removed in vacuo. The residue was dissolved in EtOAc (75 mL),

washed with 5% NaHCO₃and brine, dried over anhydrous Na₂SO₄,

and concentrated under reduced pressure to aﬀord the title imidazole

1

white solid. H NMR (300 MHz, CDCl₃) δ 1.44−2.16 (m, 13H),

intermediate (3.13 g, 113%) as a yellowish oil. H NMR (300 MHz,

2.21−2.65 (m, 6H), 2.75−2.92 (m, 1H), 2.99−3.71 (m, 10H), 3.72−

3.94 (m, 1H), 7.30−7.45 (m, 5H). ¹³C NMR (75 MHz, CDCl₃) δ

13.4, 22.4, 23.6, 27.9, 33.9, 42.1, 46.6, 47.6, 47.8, 49.0, 51.4, 52.2, 54.2,

56.4, 56.7, 59.7, 126.8, 128.4, 129.4, 136.0, 163.0, 170.7. HRMS

(TOF) m/z calcd for C₂₅H₃₆N₄O₂(M + H)⁺, 425.2911; found,

425.2909.

CDCl₃) δ 1.39 (s, 9H), 1.49−1.98 (m, 6H), 3.12−3.83 (m, 8H), 7.08

(s, 1H), 7.33 (s, 1H), 8.00 (s, 1H). This intermediate (2.78 g, 8.31

mmol) was dissolved in acetonitrile (20 mL), MeI (4.72 g, 33.25

mmol) was added, and the reaction mixture was stirred at room

temperature for 24 h. The reaction mixture was concentrated to

dryness to yield the intermediate imidazolium salt as a yellow solid

1

(4-Cyclobutyl-1,4-diazepan-1-yl)(7-(phenylsulfonyl)-2,7-

diazaspiro[4.5]decan-2-yl)methanone (37d). (4-Cyclobutyl-1,4-

diazepan-1-yl)(2,7-diazaspiro[4.5]decan-2-yl)methanone (200 mg,

0.51 mmol) was dissolved in CH₂Cl₂(3 mL). Triethylamine (0.21

mL, 1.53 mmol) was added, and the solution was stirred at room

temperature for 10 min. Benzenesulfonyl chloride (0.078 mL, 0.61

mmol) was then added, and the mixture was stirred at room

temperature for 4 h. The solution was then was diluted with CH₂Cl₂

(10 mL) and washed with 5% aq. NaHCO₃(1 × 10 mL) and brine (1

× 10 mL). The organic layer was dried over Na₂SO₄, ﬁltered, and

evaporated. The product was puriﬁed through ﬂash-chromatography

(SiO₂, gradient of CH₂Cl₂/MeOH 100:0 to 80:20) and then on

reverse phase (ﬂow: 30 mL/min, H₂O/CH₃CN 100:0 to 5:95) to

(4.59 g, 158%). H NMR (300 MHz, CD₃OD) δ 1.45 (s, 9H), 1.51−

2.03 (m, 6H), 3.34−3.91 (m, 8H), 7.72 (s, 1H), 8.04 (s, 1H), 9.52 (s,

1H). In a round-bottomed 50 mL ﬂask were added the imidazolium

salt (738 mg, 2.11 mmol), CH₂Cl₂(10 mL), and Et₃N (0.24 mL, 1.72

mmol). Another ﬂask was charged with 1-cyclobutyl-1,4-diazepane

dihydrochloride (400 mg, 1.76 mmol), CH₂Cl₂(10 mL), and Et₃N

(0.50 mL, 3.56 mmol). Both mixtures were stirred 15 min, and the

amine solution was transferred via syringe into the ﬁrst ﬂask. The

reaction mixture was stirred 24 h at room temperature and then

washed with NaHCO₃and brine, dried over anhydrous Na₂SO₄, and

concentrated under reduced pressure. The residue was puriﬁed via

preparatory HPLC using procedure C to produce the title compound

(392 mg, 40%) as a yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ 1.38

(s, 9H), 1.33−1.91 (m, 12H), 1.91−2.05 (m, 2H), 2.27−2.59 (m, 4H),

2.73−2.88 (m, 1H), 2.98−3.54 (m, 12H).

1

provide the title compound (117 mg, 50%) as an oﬀ-white solid. H

NMR (300 MHz, CDCl₃) δ 1.38−1.44 (m, 2H), 1.52−1.73 (m, 5H),

1.77−1.94 (m, 4H), 1.98−2.07 (m, 2H), 2.17−2.28 (m, 1H), 2.35−

2.48 (m, 2H), 2.50−2.57 (m, 2H), 2.66 (d, J = 11.4 Hz, 1H), 2.79−

2.86 (m, 2H), 2.95 (d, J = 11.5 Hz, 1H), 3.05−3.10 (m, 1H), 3.18 (q, J

= 10.9 Hz, 2H), 3.35−3.51 (m, 6H), 7.49−7.61 (m, 3H), 7.73 (d, J =

8.1 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 13.5, 22.6, 27.9, 28.1,

33,0, 33.8, 41.5, 46.6, 46.8, 47.6, 48.0, 51.5, 52.1, 53.1, 57.1, 59.7,

127.4, 129.0, 132.7, 136.3, 163.0. MS m/z (ES+) [M + H]⁺= 461.25.

tert-Butyl 2-(4-Isopropylpiperazine-1-carbonyl)-2,7-

diazaspiro[4.5]decane-7-carboxylate (36b). The title compound

was prepared in an analogous fashion to 36a beginning with 1-

isopropylpiperazine dihydrochloride to produce the title compound as

a yellowish oil (392 mg, 40%). ¹H NMR (300 MHz, CDCl₃) δ 0.94 (d,

J = 6.5 Hz, 6H), 1.33 (s, 9H), 1.33−1.54 (m, 5H), 1.57−1.73 (m, 1H),

2.30−2.45 (m, 4H), 2.51−2.64 (m, 1H), 2.94−3.47 (m, 12H).

U

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Journal of Medicinal Chemistry

Article

HRMS (TOF) m/z calcd for C₂₄H₃₆N₄SO₃(M + H)⁺, 461.2580;

found, 461.2576.

(m, 4H), 1.75−1.77 (m, 2H), 2.98 (app t, J = 11.5 Hz, 2H), 2.98−3.00

(m, 4H), 3.15−3.18 (m, 2H), 3.22 (s, 2H), 3.40−3.48 (m, 5H), 3.49−

3.57 (m, 1H), 3.75 (app d, J = 14.0 Hz, 2H). MS m/z (ES+) [M + H]⁺

= 295.3. To (4-isopropylpiperazin-1-yl)(2,8-diazaspiro[4.5]decan-2-

yl)methanone dihydrochloride (0.20 g, 0.54 mmol) were added

MeOH (5 mL), dihydro-2H-pyran-4(3H)-one (0.20 mL, 2.18 mmol),

TEA (0.23 mL, 1.63 mmol), and Pd/C (23.18 mg, 0.02 mmol), and a

balloon ﬁlled with H₂gas was ﬁtted to the reaction. After stirring for

24 h, the catalyst was ﬁltered oﬀ, the solvent was removed under

reduced pressure, and solids were put under high vacuum. The

material was puriﬁed with preparatory HPLC using procedure A to

tert-Butyl 2-(4-Isopropylpiperazine-1-carbonyl)-2,8-

diazaspiro[4.5]decane-8-carboxylate (40). To a −78 °C cooled

solution of 4-nitrophenyl carbonochloridate (0.36 g, 1.81 mmol) in

THF (10 mL) was added DIPEA (0.95 mL, 5.42 mmol) and then 1-

isopropylpiperazine (0.26 mL, 1.81 mmol). After stirring the mixture

for 5 min, tert-butyl 2,8-diazaspiro[4.5]decane-8-carboxylate hydro-

chloride (0.500 g, 1.81 mmol) was added, and the reaction was

warmed to room temperature. The reaction was reﬂuxed overnight for

12 h. The material was chromatographed with SiO₂chromatography

(80 g, using a gradient of 0−10% MeOH to CH₂Cl₂) and dried to give

the title compound as white solid (0.20 g, 28%). ¹H NMR (500 MHz,

CDCl₃) δ 1.05 (d, J = 6.5 Hz, 6H), 1.45 (s, 9H), 1.49−1.52 (m, 4H),

1.71 (t, 2H, J = 7 Hz, 2H), 2.48−2.50 (m, 4H), 2.67−2.69 (m, 1H),

3.22 (s, 2H), 3.31−3.36 (m, 6H), 3.46−3.42 (m, 4H). MS m/z (ES+)

[M + H]⁺= 395.3.

1

provide the title compound as a white solid (0.205 g, 62%). H NMR

(500 MHz, CDCl₃) δ 1.03 (d, J = 6.5 Hz, 6H), 1.57−1.61 (m, 7H),

1.72 (t, J = 7.0 Hz, 2H), 1.70−1.75 (m, 1H), 2.42−2.49 (m, 7H),

2.57−2.58 (m, 2H), 2.67 (quin., J = 6.5 Hz, 1H), 3.19 (s, 2H), 3.30−

3.28 (m, 4H), 3.36 (dt, J = 12, 2, Hz, 2H), 3.42 (t, J = 7.0 Hz, 2H),

4.00−4.03 (m, 2H). HRMS (TOF) m/z calcd for C₂₁H₃₈N₄O₂(M +

H)⁺, 379.3067; found, 379.3055.

tert-Butyl 2-(4-Cyclobutyl-1,4-diazepane-1-carbonyl)-2,8-

diazaspiro[4.5]decane-8-carboxylate (40b). tert-Butyl 2,8-

diazaspiro[4.5]decane-8-carboxylate hydrochloride 39 (1.0 g, 3.61

mmol) was dissolved in dry THF (20 mL), and then freshly

recrystallized carbonyl diimidazole (644 mg, 3.97 mmol) was added.

The reaction mixture was heated at reﬂux for 24 h, and THF was

removed in vacuo. The residue was dissolved in EtOAc (50 mL),

washed with NaHCO₃(5%) and brine, dried over anhydrous Na₂SO₄,

and concentrated under reduced pressure to yield the intermediate

(8-Benzoyl-2,8-diazaspiro[4.5]decan-2-yl)(4-cyclobutyl-1,4-

diazepan-1-yl)methanone (41b). To tert-butyl 2-(4-cyclobutyl-1,4-

diazepane-1-carbonyl)-2,8-diazaspiro[4.5]decane-8-carboxylate 40b

(742 mg, 1.76 mmol) was added 6 N HCl in iPrOH (5 mL). The

reaction mixture was stirred 4 h, and then Et₂O (50 mL) was added.

The salt was ﬁltered under N₂and dried in vacuo to make the

1

intermediate amine (755 mg, 108%) as a white solid. H NMR (300

MHz, CD₃OD) δ 1.72−2.01 (m, 7H), 2.15−2.43 (m, 6H), 2.92−3.28

(m, 7H), 3.35−3.90 (m, 11H). (4-Cyclobutyl-1,4-diazepan-1-yl)(2,8-

diazaspiro[4.5]decan-2-yl)methanone (200 mg, 0.51 mmol) and Et₃N

(212 μL, 1.52 mmol) were mixed in a ﬂask in CH₂Cl₂(3 mL) and

stirred 10 min. Benzoyl chloride (73 mg, 0.52 mmol) was added, and

the reaction mixture was stirred for 2 h. The reaction mixture was

diluted with CH₂Cl₂and washed with 5% NaHCO₃and brine, then

dried over anhydrous Na₂SO₄, ﬁltered, and concentrated in vacuo. The

residue was puriﬁed via preparatory HPLC using procedure C and

then reverse-phase preparatory HPLC (H₂O/ACN, ﬂow: 30 mL/min,

12 g column, 100:0 to 0:100 over 30 min) to produce the title

compound (200 mg, 85%) as a clear oil after lyophilization (H₂O/

1

imidazole (1.45 g, 120%) as a yellowish oil. H NMR (300 MHz,

CDCl₃) δ 1.46 (s, 9H), 1.50−1.65 (m, 4H), 1.89 (t, J = 7.1 Hz, 2 H),

3.28−3.57 (m, 6H), 3.73 (t, J = 7.1 Hz, 2H), 7.09 (s, 1H), 7.34 (s,

1H), 8.01 (s, 1H). This material (1.13 g, 2.71 mmol) was dissolved in

acetonitrile (8 mL), MeI (1.54 g, 10.8 mmol) was added, and the

reaction mixture was stirred at room temperature for 24 h. The

reaction mixture was concentrated to dryness, and to this were added

CH₂Cl₂(8 mL), Et₃N (1.13 mL, 8.13 mmol), and 1-cyclobutyl-1,4-

diazepane (616 mg, 2.71 mmol). The reaction mixture was stirred 24 h

at room temperature, washed with NaHCO₃(sat.) brine, dried over

anhydrous Na₂SO₄, and concentrated under reduced pressure. The

residue was puriﬁed via preparatory HPLC using procedure C and

then reverse-phase preparatory HPLC (H₂O/ACN, Flow: 30 mL/min,

12 g column, 100:0 to 0:100 over 30 min) to produce the title

1

ACN). H NMR (300 MHz, CDCl₃) δ 1.36−1.93 (m, 12H), 1.93−

2.10 (m, 2H), 2.28−2.47 (m, 3H), 2.47−2.57 (m, 2H), 2.74−2.90 (m,

1H), 3.25 (s, 2H), 3.25−3.50 (m, 7H), 3.50−3.72 (m, 1H), 3.72−3.94

(m, 1H), 7.32−7.44 (m, 5H). ¹³C NMR (75 MHz, CDCl₃) δ 13.5,

27.9, 28.2, 34.0, 35.0, 35.8, 39.7, 40.2, 45.4, 46.7, 47.6, 48.0, 51.4, 52.1,

57.8, 59.7, 126.8, 128.4, 129.5, 136.0, 163.1, 170.3. HRMS (TOF) m/z

calcd for C₂₅H₃₆N₄O₂(M + H)⁺, 425.2911; found, 425.2910.

1

compound (742 mg, 65%) as a yellowish oil. H NMR (300 MHz,

CDCl₃) δ 1.37−1.56 (m, 13H), 1.56−1.76 (m, 6H), 1.76−1.95 (m,

4H), 1.95−2.09 (m, 2H), 2.37−2.57 (m, 4H), 2.77−2.91 (m, 1H),

3.21 (s, 2H), 3.24−3.37 (m, 2H), 3.37−3.54 (m, 6H).

(4-Cyclobutyl-1,4-diazepan-1-yl)(8-(phenylsulfonyl)-2,8-

diazaspiro[4.5]decan-2-yl)methanone (41c). (4-Cyclobutyl-1,4-

diazepan-1-yl)(2,8-diazaspiro[4.5]decan-2-yl)methanone 40b (200

mg, 0.510 mmol) and Et₃N (212 μL, 1.52 mmol) were mixed in a

ﬂask in CH₂Cl₂(3 mL) and stirred 10 min. Benzenesulfonyl chloride

(92 mg, 0.52 mmol) was added, and the reaction mixture was stirred 2

h. The reaction mixture was diluted with CH₂Cl₂, washed with 5%

NaHCO₃and brine, dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated in vacuo. The residue was puriﬁed via preparatory HPLC

using procedure C and then reverse-phase preparatory HPLC (H₂O/

ACN, ﬂow: 30 mL/min, 12 g column, 100:0 to 0:100 over 30 min) to

produce the title compound (180 mg, 77%) as a white solid after

tert-Butyl 2-(4-Cyclobutylpiperazine-1-carbonyl)-2,8-

diazaspiro[4.5]decane-8-carboxylate (40e). Crude tert-butyl 2-

(1H-imidazole-1-carbonyl)-2,8-diazaspiro[4.5]decane-8-carboxylate

(1.45 g, 3.61 mmol) was dissolved in acetonitrile (8 mL), MeI (2.05 g,

14.4 mmol) was added, and the reaction mixture was stirred at room

temperature for 24 h. The reaction mixture was concentrated to

dryness, and to this were added CH₂Cl₂(8 mL), Et₃N (1.51 mL, 10.83

mmol), and 1-cyclobutylpiperazine (808 mg, 3.79 mmol). The

reaction mixture was stirred 24 h at room temperature and then

washed with NaHCO₃and NaCl (sat.), dried over anhydrous Na₂SO₄,

and concentrated under reduced pressure. The residue was puriﬁed via

preparatory HPLC using procedure C and then reverse-phase

preparatory HPLC (H₂O/ACN, ﬂow: 30 mL/min, 12 g column,

100:0 to 0:100 over 30 min) to produce the title compound (980 mg,

1

lyophilization (H₂O/ACN). H NMR (300 MHz, CDCl₃) δ 1.51−

1.72 (m, 8H), 1.72−1.89 (m, 4H), 1.96−2.08 (m, 2H), 2.34−2.43 (m,

2H), 2.46−2.53 (m, 2H), 2.72−2.86 (m, 3H), 3.07 (s, 2H), 3.18−3.29

(m, 2H), 3.29−3.44 (m, 6H), 7.50−7.65 (m, 3H), 7.72−7.77 (m, 2H).

¹³C NMR (75 MHz, CDCl₃) δ 13.5, 28.0, 28.2, 33.7, 36.2, 39.1, 43.7,

1

67%) as a white solid. H NMR (300 MHz, CDCl₃) δ 1.37−1.54 (m,

13H), 1.60−1.78 (m, 4H), 1.78−1.95 (m, 2H), 1.95−2.09 (m, 2H),

2.24−2.36 (m, 4H), 2.64−2.78 (m, 1H), 3.22 (s, 2H), 3.25−3.39 (m,

6H), 3.39−3.52 (m, 4H).

46.7, 47.6, 48.1, 51.4, 52.1, 56.8, 59.7, 127.5, 129.0, 132.8, 136.0, 163.1.

HPLC: 99%. MS m/z (ES+) [M + H]⁺= 461.25. HRMS (TOF) m/z

calcd for C₂₃H₃₆N₄SO₃(M + H)⁺, 461.2580; found, 461.2581.

(4-Cyclobutyl-1,4-diazepan-1-yl)(8-(isopropylsulfonyl)-2,8-

diazaspiro[4.5]decan-2-yl)methanone (41d). The title compound

was made in an analogous manner to 41c using isopropyl sulfonyl

choride to give the title compound in 80% yield. ¹H NMR (300 MHz,

CDCl₃) δ 1.33 (d, J = 6.8 Hz, 6H), 1.52−1.95 (m, 12H), 1.95−2.10

(m, 2H), 2.39−2.48 (m, 2H), 2.48−2.59 (m, 2H), 2.77−2.91 (m, 1H),

(4-Isopropylpiperazin-1-yl)(8-(tetrahydro-2H-pyran-4-yl)-

2,8-diazaspiro[4.5]decan-2-yl)methanone (41a). To tert-butyl 2-

(4-isopropylpiperazine-1-carbonyl)-2,8-diazaspiro[4.5]decane-8-car-

boxylate (0.185 g, 0.47 mmol) was added MeOH (20 mL) that had

been saturated with HCl gas. The reaction was stirred 1 h, the solvent

was removed, and the white solid was put under high vacuum

1

overnight to give the intermediate amine (0.120 g, 75%). H NMR

(500 MHz, DMSO-d₆, TFA shake) δ 1.24−1.25 (m, 6H), 1.61−1.67

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3.11−3.31 (m, 5H), 3.35−3.50 (m, 8H). ¹³C NMR (75 MHz, CDCl₃)

δ 13.4, 16.7, 27.9, 28.2, 34.6, 35.9, 39.6, 43.8, 46.6, 47.6, 48.0, 51.4,

52.1, 53.2, 57.4, 59.6, 163.1. HRMS (TOF) m/z calcd for

C₂₁H₃₈N₄O₃S (M + H)⁺, 427.2737; found, 427.2738.

pressure. The residue was diluted in EtOAc, washed with brine, and

concentrated under reduced pressure. The crude material was puriﬁed

by preparative HPLC using procedure B to provide the title

compound (7.0 mg, 37%) as a white solid. ¹H NMR (400 MHz,

CD₃OD) δ 0.86−0.99 (m, 2H), 1.24−1.49 (m, 6H), 1.52−1.64 (m,

1H), 1.64−1.77 (m, 1H), 1.80−2.13 (m, 5H), 2.15−2.28 (m, 1H),

2.28−2.43 (m, 1H), 2.77−2.93 (m, 1H), 3.41−3.62 (m, 5H), 3.62−

3.89 (m, 6H), 4.19−4.26 (m, 1H), 7.35−7.44 (m, 2H), 7.44−7.53 (m,

3H). HRMS (TOF) m/z calcd for C₂₅H₃₆N₃O₂(M + H)⁺, 410.2802;

found, 410.2807.

(9-Benzoyl-2,9-diazaspiro[5.5]undecan-2-yl)(4-cyclobutyl-

1,4-diazepan-1-yl)methanone (41h). This compound was made in

an analogous manner to 41b with commercially available tert-butyl 2,9-

diazaspiro[5.5]undecane-9-carboxylate to give the title compound in

80% yield. ¹H NMR (300 MHz, CDCl₃) δ 1.21−1.97 (m, 14H), 1.97−

2.13 (m, 2H), 2.35−2.67 (m, 4H), 2.76−2.94 (m, 1H), 2.94−3.06 (m,

1H), 3.06−3.24 (m, 3H), 3.32−3.52 (m, 6H), 3.52−3.73 (m, 1H),

3.73−3.94 (m, 1H), 7.35−7.43 (m, 5H). HRMS (TOF) m/z calcd for

C₂₆H₃₈N₄O₂(M + H)⁺, 439.3067; found, 439.3077.

tert-Butyl 8-Benzoyl-2,8-diazaspiro[4.5]decane-2-carboxy-

late (43). A solution of tert-butyl 2,8-diazaspiro[4.5]decane-2-

carboxylate·HCl (0.13 g, 0.47 mmol), DIPEA (0.164 mL, 0.94

mmol), and benzoic acid (0.057 g, 0.47 mmol) in 2-methyl

tetrahydrofuran (5 mL) was stirred for 1 h at room temperature. 4-

(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium (0.125 g,

0.52 mmol) was then added, and the mixture was stirred overnight.

The mixture was quenched with water, concentrated under reduced

pressure, diluted in EtOAc, washed with NaHCO₃(sat.) and brine,

and then concentrated under reduced pressure. The crude material

was puriﬁed on SiO₂(40 g), eluting with 5−80% EtOAc in heptane to

provide tert-butyl 8-benzoyl-2,8-diazaspiro[4.5]decane-2-carboxylate

(0.100 g, 61.8%) as an oil. The product was analyzed by analytical

HPLC MS using procedure D. MS m/z (ES+) [M + H]⁺= 345.3. t_R=

2.23 min.

tert-Butyl 4-(8-Benzoyl-2,8-diazaspiro[4.5]decane-2-

carbonyl)piperidine-1-carboxylate (44). tert-Butyl 8-benzoyl-2,8-

diazaspiro[4.5]decane-2-carboxylate (0.10 g, 0.29 mmol) was solubi-

lized in MeOH (2 mL), and a 4 M solution of HCl (0.508 mL, 2.03

mmol) in dioxane was added. The mixture was then stirred at room

temperature for 2 days. The mixture was concentrated under reduced

pressure to provide phenyl(2,8-diazaspiro[4.5]decan-8-yl)methanone·

HCl (0.120 g, 147%) as an oil. The product was analyzed by analytical

HPLC-MS using procedure D. MS m/z (ES+) [M + H]⁺= 245.4. t_R=

1.42 min. A mixture of phenyl(2,8-diazaspiro[4.5]decan-8-yl)-

methanone·HCl (0.12 g, 0.43 mmol), DIPEA (0.136 mL, 0.78

mmol), and 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (0.089

g, 0.39 mmol) in methyl THF (5 mL) was stirred at room temperature

for 15 min. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-

ium (0.094 g, 0.39 mmol) was then added, and the mixture was stirred

for 1 h. The solvent was removed under reduced pressure. The

mixture was diluted in ethyl acetate, washed with a saturated solution

of NaHCO₃and brine, and then concentrated under reduced pressure.

The crude material was puriﬁed on SiO₂, eluting with 5−60% EtOAc

in heptane to provide tert-butyl 4-(8-benzoyl-2,8-diazaspiro[4.5]-

decane-2-carbonyl)piperidine-1-carboxylate (0.040 g, 22.60%) as an

oil. The product was analyzed by analytical HPLC MS using procedure

D. MS m/z (ES+) [M + H]⁺= 456.3 (major peak: 356.3). t_R= 2.14

min.

(8-Benzoyl-2,8-diazaspiro[4.5]decan-2-yl)(4-cyclobutylpi-

perazin-1-yl)methanone (41e). To tert-butyl 2-(4-cyclobutylpiper-

azine-1-carbonyl)-2,8-diazaspiro[4.5]decane-8-carboxylate 40e (980

mg, 2.33 mmol) was added 6 N HCl in iPrOH (5 mL). The reaction

mixture was stirred 4 h, and then Et₂O (50 mL) was added. The salt

was ﬁltered under N₂and dried in vacuo to yield the intermediate

amine (800 mg, 91%) as a white solid. ¹H NMR (300 MHz, CD₃OD)

δ 1.71−1.98 (m, 8H), 2.22−2.41 (m, 4H), 2.89−3.03 (m, 2H), 3.12−

3.28 (m, 6H), 3.37 (s, 2H), 3.40−3.49 (m, 2H), 3.54 (t, J = 7.1 Hz,

2H), 3.65−3.80 (m, 1H), 3.87−3.97 (m, 2H). This intermediate (200

mg, 0.53 mmol) and Et₃N (294 μL, 2.11 mmol) were mixed in a ﬂask

in CH₂Cl₂(3 mL) and stirred 10 min. Benzoyl chloride (76 mg, 0.53

mmol) was added, and the reaction mixture was stirred for 2 h. The

reaction mixture was diluted with CH₂Cl₂and washed with 5%

NaHCO₃and brine and then dried over anhydrous Na₂SO₄. The

organic phase was ﬁltered and concentrated in vacuo. The residue was

puriﬁed via preparatory HPLC using procedure C and then reverse-

phase preparatory HPLC (H₂O/ACN, ﬂow: 30 mL/min, 12 g column,

100:0 to 0:100 over 30 min) to give the title compound (190 mg,

88%) as a white solid after lyophilization (H₂O/ACN). ¹H NMR (300

MHz, CDCl₃) δ 1.41−1.96 (m, 10H), 1.96−2.10 (m, 2H), 2.25−2.37

(m, 4H), 2.65−2.79 (m, 1H), 3.23−3.51 (m, 10H), 3.54−3.74 (m,

1H), 3.74−3.92 (m, 1H), 7.36−7.44 (m, 5H). ¹³C NMR (75 MHz,

CDCl₃) δ 14.3, 26.9, 34.0, 35.0, 35.6, 39.6, 40.5, 45.2, 45.8, 46.4, 49.2,

57.8, 60.2, 126.8, 128.4, 129.6, 136.0, 162.6, 170.3. MS m/z (ES+) [M

+ H]⁺= 411.31. HRMS (TOF) m/z calcd for C₂₄H₃₄N₄O₂(M + H)⁺,

411.2754; found, 411.2752.

(4-Cyclobutylpiperazin-1-yl)(8-(phenylsulfonyl)-2,8-

diazaspiro[4.5]decan-2-yl)methanone (41f). (4-Cyclobutylpiper-

azin-1-yl)(2,8-diazaspiro[4.5]decan-2-yl)methanone (200 mg, 0.53

mmol) and Et₃N (220 μL, 1.58 mmol) were mixed in a ﬂask in

CH₂Cl₂(3 mL) and stirred 10 min. Benzenesulfonyl chloride (95 mg,

0.54 mmol) was added, and the reaction mixture was stirred for 2 h.

The reaction mixture was diluted with CH₂Cl₂and washed with 5%

NaHCO₃and brine, dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated in vacuo. The residue was puriﬁed via preparatory HPLC

using procedure C and then reverse-phase preparatory HPLC (H₂O/

ACN, ﬂow: 30 mL/min, 12 g column, 100:0 to 0:100 over 30 min) to

produce the title compound (200 mg, 85%) as a white solid after

1

lyophilization (H₂O/ACN). H NMR (300 MHz, CDCl₃) δ 1.55−

1.76 (m, 8H), 1.76−1.93 (m, 2H), 1.93−2.07 (m, 2H), 2.21−2.31 (m,

4H), 2.63−2.75 (m, 1H), 2.77−2.90 (m, 2H), 3.08 (s, 2H), 3.14−3.29

(m, 6H), 3.37 (t, J = 7.1 Hz, 2H), 7.50−7.65 (m, 3H), 7.71−7.79 (m,

2H). ¹³C NMR (75 MHz, CDCl₃) δ 14.3, 26.9, 33.7, 35.9, 39.3, 43.6,

45.7, 46.4, 49.2, 56.9, 60.2, 127.5, 129.1, 132.8, 136.0, 162.5. Analytical

HPLC method D was employed. MS m/z (ES+) [M + H]⁺= 447.24.

(8-Benzoyl-2,8-diazaspiro[4.5]decan-2-yl)(1-cyclobutylpi-

peridin-4-yl)methanone (41g). A mixture of tert-butyl 4-(8-

benzoyl-2,8-diazaspiro[4.5]decane-2-carbonyl)piperidine-1-carboxylate

(40 mg, 0.09 mmol) and a 4 M solution of HCl (0.2 mL, 0.80 mmol)

in dioxane was stirred at room temperature for 2 days. The mixture

was then concentrated under reduced pressure, basiﬁed with a solution

of NaHCO₃(sat.), and extracted with EtOAc (3×). The combined

organic phases were then dried and concentrated under reduced

pressure. The crude material was loaded on a 4 g silica gel column and

puriﬁed on SiO₂, eluting with 5−60% EtOAc in heptane and then

ﬂushed with MeOH to provide (8-benzoyl-2,8-diazaspiro[4.5]decan-2-

yl)(piperidin-4-yl)methanone (20 mg, 64.1%) as an oil. The product

was analyzed by analytical HPLC MS using procedure D. MS m/z (ES

+) [M + H]⁺= 356.3. t_R= 1.60 min.

GTPγS Assay. Functional antagonism of the human H₃R long form

(445 amino acids) was quantiﬁed using an agonist-induced

[³⁵S]GTPγS scintillation proximity binding assay.²⁹Brieﬂy, H₃R-

CHO-K1 membranes were incubated for 3 h with test compound, 25

nM R-α-methyl histamine, 10 μM GDP, 0.2 nM [³⁵S]GTPγS, and 150

μg of WGA PVT SPA beads in a 100 μL total volume. The amount of

[³⁵S]GTPγS binding to the SPA beads was measured on a TopCount

(PerkinElmer).

Binding Assay. The aﬃnity of test compounds to recombinant rat

and human H₃R was determined using a scintillation proximity

binding assay with [³H]-N-α-methylhistamine as ligand. Human H₃R-

CHO-K1 membranes were incubated for 1.5 h with test compound,

To a solution of (8-benzoyl-2,8-diazaspiro[4.5]decan-2-yl)-

(piperidin-4-yl)methanone (15 mg, 0.04 mmol) in DMF (3 mL)

was added cyclobutanone (2.96 mg, 0.04 mmol), and the mixture was

stirred for 30 min. NaCNBH₄(2.65 mg, 0.04 mmol) was then added,

and the mixture was stirred overnight. The mixture was then quenched

with a saturated solution of NaHCO₃and concentrated under reduced

W

dx.doi.org/10.1021/jm4014828 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1.4 nM radioligand, and 1750 μg of WGA PVT SPA beads in a 200 μL

total volume. Imetit (10 μM) was used to deﬁne nonspeciﬁc binding.

The amount of radioligand binding to the SPA beads was measured on

a TopCount (PerkinElmer). Binding to H₃R in rat brain membranes

was performed using the same radioligand but in a ﬁltration format

using GF/B ﬁlters. Bound radioligand was determined by liquid

scintillation counting on a TopCount.

ABBREVIATIONS USED

■

ADHD, attention deﬁcit hyperactivity disoreder; AUC, area

under the curve; CDI, carbonyldiimidazole; CSF, cerebrospinal

ﬂuid; CNS, central nervous system; DMPK, drug metabolism

and pharmacokinetics; DIPEA, diisopropyl ethyl amine; EDCI,

1-ethly-3-(3-dimethylaminopropyl)carbodiimide; HOBT, hy-

VCD Experimental. For each compound, ∼20 mg of sample was

dissolved in 0.5 mL of CDCl₃. Each solution was then loaded into a

0.1 mm BaF₂infrared cell for analysis 4 cm⁻¹resolution using a 5 h,

dual source, VCD scan protocol. All analyses were conducted using the

BioTools ChiralIR instrument. The instrument incorporated a single

photoelastic modulator set for polarization modulation at 37.024 kHz

with λ/4 retardation (optimized for acquisition of the spectral region

centered around 1400 cm⁻¹). Lock-in ampliﬁcation with a 30 μs time

constant, a 20 kHz high-pass ﬁlter, and a 4 kHz low-pass ﬁlter was

used. Polarity was veriﬁed with α-pinene.

̀

droxy benzotriazole; hERG, human ether-a-go-go-related gene

encoding for potassium ion channel; HBTU, O-benzotriazole-

N,N,N′,N′-tetramethyl-uronium-hexaﬂuoro-phosphate;

MDCK, Madin-Darby canine kidney; PD, pharmacodynamic;

PK, pharmacokinetic; SAR, structure−activity relationships;

SFC, supercritical ﬂuid chromatography; TBAB, tetrabutylam-

monium bromide; TEMPO, (2,2,6,6-tetramethylpiperidin-1-

yl)oxidanyl; TFA, triﬂuoracetic acid; THF, tetrahydrofuran;

VCD, vibrational circular dichroism

Computational Spectral Simulations. A MonteCarlo molecular

mechanics search of low-energy conformers for the (R) conﬁguration

of the Figure 5 compounds was conducted using MacroModel³⁵

within the Maestro graphical interface. The 32 lowest-energy

conformers identiﬁed were used as starting points and minimized

using density functional theory (DFT) within Gaussian 03. Optimized

structures, harmonic vibrational frequencies/intensities, VCD rota-

tional strengths, and free energies at standard temperature and

pressure (STP, including zero-point energies) were determined for

each conformer. In these calculations, the B3LYP generalized gradient

approximation (GGA) exchange-correlation density functional was

used in conjunction with the cc-pVTZ basis set. Simulations of

infrared and VCD spectra for each conformer were generated using an

in-house written program to ﬁt Lorentzian line shapes (16 cm⁻¹line

width) to the computed spectra. In this manner, direct comparisons

between simulated and experimental spectra were made.

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ASSOCIATED CONTENT

■

S

* Supporting Information

Secondary pharmacological targets and data for Cpd 6s. This

material is available free of charge via the Internet at http://

pubs.acs.org.

AUTHOR INFORMATION

■

Corresponding Author

*E-mail: dean.brown@astrazeneca.com.

̈

Present Address

P.; Mir, P.; Muller, N.; Barr, C.; Paschou, P. Support of the

̈

^∥AstraZeneca Pharmaceuticals, 35 Gatehouse Drive, Waltham,

Massachusetts 02451, United States.

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The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

Notes

The authors declare no competing ﬁnancial interest.

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Kang, C. H.; Denny, L. I.; Witte, D. G.; Yao, B. B.; Pan, L.; Wetter, J.;

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ACKNOWLEDGMENTS

■

We acknowledge help from Francis Beaulieu, Thalia Daniels,

James Hall, Helene Desfosses, Sandrine Leclerc, Bernard A.

Lanoue, Ying Zhou, Ying Liu, Eric Miele, Erika Manyak,

Debora Roaquin, Torcan Chemical, and Don Pivonka.

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Komater, V. A.; Miner, H.; Zhang, M.; Faghih, R.; Rueter, L. E.; Bitner,

R.; Drescher, K. U.; Wetter, J.; Marsh, K.; Lemaire, M.; Porsolt, R. D.;

Benneni, Y. L.; Sullivan, J. P.; Cowart, M. D.; Decker, M. W.; Hancock,

A. A. Pharmacological properties of ABT-239 [4-(2{2-[(2R)-2-

DEDICATION

■

^⊥We dedicate this manuscript to the memory of Thierry

Groblewski, who passed away during the time of this eﬀort. His

leadership was critical to the advancement of the project, and

he will be greatly missed by his friends and colleagues.

X

dx.doi.org/10.1021/jm4014828 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Discovery of spirofused piperazine and diazepane amides as selective histamine-3 antagonists with in vivo efficacy in a mouse model of cognition

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