ST7612AA1, a thioacetate-ω(γ-lactam carboxamide) derivative selected from a novel generation of oral HDAC inhibitors

Article

pubs.acs.org/jmc

ST7612AA1, a Thioacetate-ω(γ-lactam carboxamide) Derivative

Selected from a Novel Generation of Oral HDAC Inhibitors

Giuseppe Giannini,*^,†Loredana Vesci,^†Gianfranco Battistuzzi,^†Davide Vignola,^†

Ferdinando M. Milazzo,^†Mario Berardino Guglielmi,^†Marcella Barbarino,^†Mose Santaniello,^†

̀

Nicola Fanto,^†Marco Mor,^‡Silvia Rivara,^‡Daniele Pala,^‡Maurizio Taddei,^§Claudio Pisano,^†,∥

̀

and Walter Cabri^†,⊥

^†R&D Sigma-Tau Industrie Farmaceutiche Riunite SpA, Via Pontina Km 30,400, I-00040 Pomezia, Roma, Italy

^‡Dipartimento di Farmacia, Universita

̀

degli Studi di Parma, Parco Area delle Scienze 27/A, I-43124 Parma, Italy

^§Dipartimento di Biotecnologie, Chimica e Farmacia, Universita

̀

degli Studi di Siena, Via A. Moro 2, I-53100 Siena, Italy

S

* Supporting Information

ABSTRACT: A systematic study of medicinal chemistry

aimed at identifying a new generation of HDAC inhibitors,

through the introduction of a thiol zinc-binding group (ZBG)

and of an amide-lactam in the ω-position of the polyethylene

chain of the vorinostat scaﬀold, allowed the selection of a new

class of potent pan-HDAC inhibitors (pan-HDACis). Simple,

highly versatile, and eﬃcient synthetic approaches were used

to synthesize a library of these new derivatives, which were

then submitted to a screening for HDAC inhibition as well as to a preliminary in vitro assessment of their antiproliferative

activity. Molecular docking into HDAC crystal structures suggested a binding mode for these thiol derivatives consistent with the

stereoselectivity observed upon insertion of amide-lactam substituents in the ω-position. ST7612AA1 (117), selected as a drug

candidate for further development, showed an in vitro activity in the nanomolar range associated with a remarkable in vivo

antitumor activity, highly competitive with the most potent HDAC inhibitors, currently under clinical trials. A preliminary study

of PK and metabolism is also illustrated.

deacetylation of histone proteins but also, and perhaps

primarily, of a large number of other nonhistone (cytoplasmic

and nuclear) proteins. For this reason much still remains to be

elucidated, and impressive opportunities await pharmaceutical

companies and clinicians committed to ﬁnding novel

therapeutic solutions.

Compounds 1 (SAHA, vorinostat, Zolinza) and 2 (depsipep-

tide; FK-228, Istodax) are the two HDAC inhibitors currently

used in the clinical practice. Both compounds were licensed by

the FDA, in 2006 and 2009, respectively, for the treatment of

the rare cancer, cutaneous T-cell lymphoma. In 2011, 2 was

approved for an additional indication, the peripheral T-cell

lymphoma.

In addition to these two ﬁrst-in-class compounds, more than

20 new HDAC inhibitors are currently under preclinical and

clinical investigation against diﬀerent cancers as well as for

several other novel therapeutic indications. For an overview of

this topic, the reader is encouraged to consult a recent review

published by some of us⁵and other interesting reviews covering

this area.⁶⁻⁹

INTRODUCTION

■

Since 1964, when Allfrey, Faulkner, and Mirsky discovered the

reversible acetylation of histone proteins,¹much has been

learned on their role and importance in pathophysiological

conditions. Over these 50 years, therapeutic prospects for

epigenetic modulation have become increasingly concrete, even

if a growing body of literature is highlighting the complexity of

the histone proteins that should be rather considered as a

cluster of proteins. Histone deacetylase inhibitors represent one

of the most advanced tools for epigenetic modulation, although

a plethora of other nonhistone proteins are modulated by this

class of compounds. After recent important proteomic

studies,²⁻⁴we can now refer to a more appropriate deﬁnition

of “lysine deacetylases” instead of “histone deacetylases”.

However, so far the attention has been focused mainly on

histone deacetylases (HDACs), which play an important role in

reversing aberrant epigenetic changes associated with cancer as

well as with noncancer diseases (i.e., neurological and

inﬂammatory pathologies). Overexpression of these enzymes

has been shown in several types of cancer with a diﬀerent

behavior from isoform to isoform, hence generating the interest

toward HDAC inhibitors in various oncological clinical

applications. All HDAC inhibitors studied so far, including

the ﬁrst two FDA approved, vorinostat (1) and romidepsin (2)

depicted in Figure 1, work by inhibiting not only the

Human HDACs are divided into four classes (I−IV) based

on their homology to yeast HDACs, their subcellular

Received: May 29, 2014

A

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Figure 1. Chemical structure of the two FDA approved HDAC inhibitors. Romidepsin (2) is a prodrug, with a disulﬁde bridge reduced by

glutathione upon uptake into the cell, allowing the release of the drug (thiol free).

localization, and their enzymatic activities. Classes I−II and IV

are Zn²⁺-dependent amidohydrolases, while class III enzymes,

also called sirtuins, greatly diﬀer from the previous ones

because they use NAD as a cofactor. So far, 11 HDAC isoforms

have been identiﬁed in humans belonging to the Zn²⁺-

dependent group. Class I HDAC family consists of HDAC1,

2, 3, and 8, primarily detected in the nucleus, and with isoform

8 also found in the cytoplasm. These are small proteins

expressed ubiquitously in various human cell types and tissues

displaying high enzymatic activity toward histone and other

protein substrates with a primary role in controlling and

regulating cell survival, proliferation, and diﬀerentiation. Class

II HDAC family, which in turn is further divided into two

subclasses, class IIa (HDAC4, 5, 7, and 9) and class IIb

(HDAC6 and 10), encompasses larger proteins expressed in a

limited number of cell types. They either shuttle between the

nucleus and cytoplasm (i.e., class IIa) or are mainly located

within the cytoplasm (i.e., class IIb), with more tissue-speciﬁc

regulatory function than class I HDACs. HDAC11 is the sole

isoform of class IV, the least investigated so far, although it has

recently been reported that a selective depletion of this isoform,

overexpressed in several carcinomas, is suﬃcient to cause cell

death and to inhibit metabolic activity in various cancer cell

lines.¹⁰

A kink atom completes the model, as connection unit (CU)

between linker and CAP (Figure 2).⁵

Figure 2. Pharmacophore model for HDAC inhibitors applied to the

structure of compound 1.

Our interest was mainly focused on two elements of this

model, the ZBG and CU. Numerous studies have been made to

identify new groups, metabolically stable, able to chelate the

Zn²⁺ion. To date, the ZBGs introduced in the most

preclinically and clinically advanced HDAC inhibitors are

carboxylic acid, a weak Zn-chelating group present in

compounds active in the millimolar range (i.e., valproic acid),

hydroxamic acid, a powerful Zn-chelating group found in

compounds active in the range between submicromolar to one-

digit nanomolar concentrations (i.e., vorinostat, panobinostat,

belinostat, etc.), and benzamide (i.e., entinostat, mocetinostat,

tacedinaline, etc.). Only one natural derivative with a thiol

residue, masked as a disulﬁde moiety undergoing in vivo

reduction (2), has successfully passed clinical trials getting to

the FDA approval.

Since 1996, when the ﬁrst HDAC inhibitor, trapoxin,¹¹was

identiﬁed, much progress has been made in the knowledge of

the biology and the structural aspects of the diﬀerent HDAC

isoforms. Moreover, new assays have been set up, allowing the

evaluation of the selectivity proﬁle of the newly synthesized

inhibitors. Nevertheless, the scientiﬁc community has not yet

been able to prove the advantage, in terms, for example, of

increased eﬃcacy and/or decreased adverse events of selective

HDAC inhibitors (toward a single-isoform or single-class) over

pan-inhibitors, also because of the expanding interest of these

inhibitors in other therapeutic applications, diﬀerent from

cancer.¹²However, an increasingly greater characterization of

individual tumors to the overexpression of single isoforms or

class of HDACs is available.^5,13But, so far, these observations

have not been translated into any therapeutic advantage,

rendering selective inhibitors superior to pan-inhibitors.

We started our project with reference to the widely accepted

pharmacophore model for HDAC inhibitors, represented by a

zinc-binding group (ZBG) able to complex the Zn²⁺ion at the

bottom of the catalytic cavity, opposite to a capping group

(CAP), able to interact with the rim of the catalytic tunnel of

the enzyme, and a hydrophobic linker connecting the two parts.

A recent paper by Grassadonia et al.¹⁴analyzed the role of

hydroxamate-based HDAC inhibitors, highlighting that these

drugs seem to be more active in hematological malignancies

than in solid tumors. A possible explanation might involve the

pharmacokinetic proﬁle of hydroxamate-based HDAC inhib-

itors. Therefore, the availability of novel inhibitors with

nonhydroxamate ZBGs represents now a highly desirable

condition.¹⁵The thiol group has been successfully introduced

in drugs like the antidiarrheal Racecadotril (Acetorphan)¹⁶and

the antihypertensive angiotensin-converting enzyme inhibitor

Captopril (Capoten).¹⁷It has been also widely investigated in

HDAC inhibitors,¹⁸⁻²⁰including cyclic tetrapeptides,²¹and has

been found to be a potent Zn-chelating group. In fact,

replacement of the hydroxamate portion of 1 with a thiol group

provided compounds with comparable HDAC inhibitory

potency and cancer cell growth inhibition for their S-modiﬁed

B

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Figure 3. Examples of ω-substituents on the aliphatic chain of suberoylanilide hydroxamic acid (1) and ω-substituted thioacetates described in this

work.

a

Scheme 1. Synthesis of Linear Reference Compounds 9−23

a

Reagents and conditions: (a) R¹NH₂, PyBOP, NEt₃, DMF, and DCM, RT; (b) AcSH, AIBN, dioxane, 75 °C; (c) CH₃SNa, MeOH, RT; (d) R²Cl,

Et₃N, DCM, RT.

prodrugs. Until now, no synthetic thiol derivative has been

included in a clinical trial because of the thiol reactivity and too

easy capability of oxidation. However, given the interesting

clinical proﬁle of 2, we wanted to further explore the potential

of the thiol group in a new class of synthetic HDAC inhibitors.

A second point where we focused our interest was the kink

atom or connecting unit (CU, Figure 2). The introduction of

substituents on the methylene chain, in ω-position, has been

extensively studied, leading to more potent and/or more

selective derivatives. The ﬁrst attempts were made by Breslow

and co-workers, with a series of 7-substituted ethers, amines,

and amide derivatives of 1 with excellent inhibitory activities.²²

Eﬀorts in the same direction were made also in our laboratory,

studying diﬀerent 7-alkoxy derivatives alone^23,24or inserted in a

macrocyclic fraction,²⁵and more recently, introducing lactam-

carboxamide substituents (Figure 3).²⁶In all these examples,

the introduction of substituents on the methylene chain

showed an enhancement of the activity compared to the

parent compound, leading to a strong inhibition of diﬀerent

HDAC isoforms associated with a high cytotoxic potency

against selected tumor cell lines.

lactam stereocenters was evaluated. The importance of the

phenyl ring in the CAP region was also investigated, preparing

benzyl, phenethyl, and cyclopentyl derivatives. An enhance-

ment of HDAC inhibitory activity was observed not only versus

the parent compounds, lacking substituents on the methylene

chain, but also toward the corresponding hydroxamic acid

derivatives. Docking studies into HDAC crystal structures gave

results consistent with structure−activity relationships (SAR).

Docking poses for diﬀerent stereoisomers of lactam carbox-

amide inhibitors into HDAC3 correctly predicted the highest

inhibitory potency observed with ω-(S)-derivatives. Moreover,

docking solutions provided an explanation for the highest

inhibitory activity on HDAC8 found with benzyl and phenethyl

derivatives compared to their phenyl analogues.

We report here multiple chemical approaches for the

preparation of these lactam carboxamide inhibitors and their

characterization as HDAC inhibitors. The thioacetyl derivative

ST7612AA1 (117) showed a high cytotoxic activity on NCI-

H460 and HCT116 cell lines and a high inhibitory potency

against tubulin and histone H4 acetylation in cellular assays.

This compound was also investigated for its metabolic stability

and antitumor activity against two human solid tumors, a colon

carcinoma HCT116 and an ovarian A2780 tumor xenograft

models. As a potent HDAC inhibitor, promising results in

terms of in vitro proﬁle, formulation, metabolic stability, cell

permeability, and in vivo activity are herein described.

In this article, we present new HDAC inhibitors based on a

protected thiol group replacing the hydroxamate ZBG of 1,

containing lactam carboxamide substituents in ω-position of

the methylene chain (Figure 3). β-, γ-, and δ-lactams were

inserted in ω-position of the carboxamide fragment, and the

eﬀect of diﬀerent conﬁgurations of both C7 carbon atom and

C

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a

Scheme 2. Synthesis of Thioacetates 37−44, Thiols 45−52, and the Derivative 53

a

Reagents and conditions: (a) (1) EtOH, EtONa, reﬂux, 30 min, (2) Br(CH₂)₅Br, reﬂux, 3 h; (b) NaOH aq, EtOH, 0 °C, 5 h; (c) toluene, reﬂux, 4

h; (d) NaOH aq, EtOH, 0 °C, 4 h; (e) aminoacylase, H₂O, pH 7, CoCl₂·6H₂O, 38 °C, 24 h; (f) (Boc)₂O, Et₃N, THF, H₂O, rt, 20 h; (g) R₁NH₂,

EDCI, HOBt.H₂O, THF, RT; (h) KSAc, EtOH, RT; (i) CF₃COOH, CH₂Cl₂, RT, 5 h; (l) R²COOH, DMF, PyBOP, DIPEA, rt, 20 h; (m) 2N

NaOH, EtOH, RT; (n) R³Cl, Et₃N, DCM, RT.

a

Scheme 3. Synthesis of Thioacetates 69−79 and Thiols 80−89

a

Reagents and conditions: (a) R¹NH₂, PyBOP, NEt₃, DMF and DCM, RT; (b) AcSH, AIBN, dioxane, 75 °C; (c) TFA, DCM, 0 °C to RT; (d)

R²COOH, PyBOP, NEt₃, DMF and DCM, RT; (e) CH₃SNa, MeOH, RT, 0.1 M HCl.

product (S−S). Condensation of compound 15 with

appropriate chlorides (i.e., ethyl chloroformate, isobutyryl

chloride, and (2R)-2-amino-3-methyl-butanoyl chloride) af-

forded thioesters 21−23. Compound 24 is derived from

oxidation of thiol 9 as side product after hydrolysis.

The remaining molecules were prepared following four

diﬀerent synthetic approaches. Compounds 37−53, including

the three reference ones 44, 52, and 53, were synthesized

following the synthetic method described in the literature^20,28

except for the Boc protection of the amino acid 29 (Scheme 2).

As an alternative (Scheme 3), commercially available (S)-2-tert-

butoxycarbonylamino-hept-6-enoic acid was coupled with the

proper aromatic amines to obtain compounds 54−58, then

CHEMISTRY

■

Syntheses of thiol HDAC inhibitors were carried out as

outlined in Schemes 1−5. The route for synthesis of

compounds 9−23 is described in Scheme 1. Condensation of

hept-6-enoic acid with appropriate amines aﬀorded amides 3−

8, which in their turn were treated with thioacetic acid and

AIBN in dioxane to yield the desired thioesters 9−14.

Compounds 15−20 were synthesized by hydrolysis of thioester

with sodium thiomethoxide in MeOH under dry nitrogen

atmosphere. It is noteworthy that the classical procedure of

hydrolysis with NaOH/H₂O, reﬂux (or with KOH, K₂CO₃,

NaOMe),²⁷led to a signiﬁcant proportion of the oxidized

D

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a

Scheme 4. Synthesis of Thioacetates 115−125, Thiols 126−133, and Thio-derivatives 134−137

a

Reagents and conditions: (a) R¹NH₂, PyBOP, NEt₃, DMF and DCM, RT; (b) TFA, DCM, 0 °C to RT; (c) R²COOH, PyBOP, NEt₃, DMF and

DCM, RT; (d) AcSH, AIBN, dioxane, 75 °C; (e) CH₃SNa, MeOH, RT, 0.1 M HCl; (f) R³Cl, Et₃N, DCM, RT.

a

Scheme 5. Synthesis of Derivatives with R Chirality in ω-Position

a

Reagents and conditions: (a) PhNH₂, PyBOP, NEt₃, DMF and DCM, RT; (b) TFA, DCM, 0 °C to RT; (c) R₂COOH, PyBOP, NEt₃, DMF and

DCM, RT; (d) AcSH, AIBN, dioxane, 75 °C; (e) CH₃SNa, MeOH, RT, 0.1 M HCl.

thioacetylation was achieved by AIBN with thioacetic acid

followed by Boc removal with TFA. The obtained thioesters

64−68 were reacted with the proper lactam carboxylic acid to

aﬀord compounds 69−79 that were subsequent hydrolyzed

with sodium thiomethoxide in MeOH to obtain thiols 80−89.

Thioacetate derivatives 115−125 and thiols 126−133 were

synthesized according to Scheme 4, similar to the above

synthesis except for the coupling with the proper lactam

carboxylic acid carried out before the thioacetylation with AIBN

and thioacetic acid. Condensation of compounds 127 and 128

with the appropriate acid or ethylchloroformate aﬀorded

thioesters 134−136. Dithio compound 137 was obtained as

the side-product from the hydrolysis step of 116 to give 127.

Compounds 143−145 were synthesized according to Scheme

5. Commercially available (R)-2-tert-butoxycarbonylamino-

hept-6-enoic acid was coupled with aniline to obtain compound

138, then Boc removal with TFA, followed by coupling with

the proper lactam carboxylic acid to aﬀord compounds 140−

141. Then thioacetylation was achieved by AIBN with

thioacetic acid. The obtained thioesters 142−143 were

hydrolyzed with sodium thiomethoxide in MeOH to obtain

thiols 144−145. Recently, a diﬀerent synthetic approach to

obtain thiol derivatives analogues to ours has been reported.²⁹

through their ability to directly inactivate speciﬁc transcription

factors.^30,31Indeed, when tested on class IIa isoforms, our thiol

inhibitors usually gave IC₅₀values in the micromolar range,

with no apparent correlation with their structure. The

inhibitory activities of the newly synthesized compounds on

class IIa isoforms are reported in Supporting Information Table

S2.

We started our investigation on thiol derivatives of

compound 1 by verifying the optimal length of the methylene

chain connecting the amide function to the terminal thiol

group. In fact, HDAC activity of compounds with diﬀerent

chain lengths had been tested only on HeLa nuclear extracts¹⁹

and, in the literature, data on the diﬀerent isoforms have not

been reported so far. Compounds 15 and 147 with 6 and 7

methylene units, respectively, were generally more potent than

the 5 methylene-derivative 146. We decided to focus on the 6

methylene chain (also present in reference compound 1) given

the superior activity of its acetyl prodrug in in vitro

antiproliferative studies on NCI-H460 cell line (IC₅₀= 14.8

μM measured for 15 and IC₅₀> 30 μM for 147).

The ﬁrst ω-lactam carboxamide substituent to be evaluated

was the six-membered one, for which the importance of

stereochemistry of both the lactam ring and the ω position of

the amide chain was investigated. We found that the

conﬁguration of the stereocenter in ω position is crucial for

good inhibitory potency, while conﬁguration of the lactam

substituent is irrelevant. In fact, compound 144 with ω-(R)

chirality was practically inactive on all HDAC isoforms, while

its enantiomer 126 and the diastereoisomer 127, having both S

conﬁguration in the ω position, had comparable and good

inhibitory potencies. With the lactam carboxamide in the ω

position having the most favorable S conﬁguration, the

substituent improved inhibitory potency on all isoforms

compared to the unsubstituted 15. This eﬀect reﬂects the

RESULTS AND DISCUSSION

■

We systematically investigated several ω-lactam-carboxamide

thiols for their in vitro inhibitory activity on diﬀerent HDAC

isoforms and for their antiproliferative activity on tumor cell

lines. In Table 1, the inhibitory activities on class I (1−3, 8), IIb

(6, 10), and IV (11) HDAC isoforms are reported. Class IIa (4,

5, 7, 9) enzymes were not considered for structure−activity

relationships evaluation and compound optimization. In fact, it

is known that class IIa enzymes are very ineﬃcient on histone

substrates and exert a transcriptional repression function mainly

E

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a

Table 1. In Vitro Inhibitory Activity (IC₅₀) of Thiol Derivatives Against Isoforms HDAC1−3, 6, 8, 10, 11

F

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Table 1. continued

a

b

Values are the means of a minimum of three experiments. No inhibition at concentration <50 μM.

Figure 4. Docking solutions for ω-(S)-127 (orange carbons) and its diastereoisomer ω-(R)-144 (green carbons) into HDAC3 active site.

Compound 1 (yellow carbons) cocrystallized with HDAC2 (PDB 4LHZ) is depicted for comparison.

results already reported for lactam carboxamide derivatives of

1.²⁶On the other hand, the increase of inhibitory potency given

by the lactam carboxamide substituent was greater for these

thiol derivatives than for hydroxamic acid counterparts. The

highest potency of the (S)-ω-amide derivative was consistent

with the presence of the same conﬁguration within the natural

peptide substrate, and it can be rationalized on the basis of the

putative binding mode. To evaluate the accommodation of

lactam carboxamide derivatives into the enzyme active site,

HDAC3 obtained from the crystal structure of its complex with

an acetate product was selected as a model structure and it was

used for docking studies. The docking poses found for

compounds 127 and 144 are represented in Figure 4.

Compound 127 was strongly bound to Asp93 in the rim of

the active site, while the thiol group was deeply inserted into

the active site and coordinated the zinc ion. A similar binding

mode was also observed for reference compound 1 in its

crystallized complex with HDAC (Figure 4, left panel). The

lactam ring was exposed to the solvent and was involved in

limited interactions with amino acids in the rim of the binding

site. This could explain the lack of stereoselectivity observed for

the lactam stereocenter and the limited eﬀect on potency of

lactam ring modiﬁcations (ring contraction, opening, sub-

stitution, i.e., see next SAR discussion). Compound 144

showed the same polar interactions as compound 127 even

if, given the opposite stereochemistry, its ω-hydrogen atom

G

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Table 2. In Vitro Inhibitory Activity (IC₅₀) of Thiol Derivatives with Modiﬁed CAP Group Against Isoforms HDAC1−3, 6, 8,

a

10, 11

a

b

c

Values are the means of a minimum of three experiments. Reference 20. No inhibition at concentration <50 μM.

points toward the carboxylic group of Asp93 (Figure 4, right

panel). The binding modes of compounds 127 and 144

resembled those of a P53-derived diacetylated peptide substrate

and of a peptide inhibitor with R stereochemistry,³²

respectively, crystallized into HDAC8 (Supporting Information

Figure S1). The opposite stereochemistry of the ω-carbon atom

had a strong impact on the stability of the corresponding

complexes, as evaluated by molecular dynamics (MD)

simulations into an explicit water box. Compound 127

remained stably bound to the active site amino acids during

the 20 ns long MD simulations, while compound 144 lost its

interactions with HDAC residues in the ﬁrst steps of the

simulation (Supporting Information Table S3).

Modiﬁcations of the lactam ring, such as N-methylation (82)

and inversion of the lactam function (50, 51), did not

signiﬁcantly aﬀect the inhibitory potency of the compounds

compared to the reference 127. On the other hand, the

presence of a methyl group on the phenyl ring had a favorable

eﬀect, leading to an increase of inhibitory potency on all

isoforms when inserted in para-position (130) and, particularly,

in meta-position (129). The positive eﬀect of a meta-methyl

had already been observed for hydroxamate-based inhibitors on

total HDAC activity in Caco-2 colon cancer cells, meanwhile

para-methyl analogues demonstrated slightly reduced inhibitory

activity.³³

Ring contraction to γ-lactam (81, 128) aﬀorded compounds

with HDAC inhibitory potency comparable to those with larger

δ-lactams, with similar SARs. Indeed, the presence of an (R)-ω-

amide stereocenter signiﬁcantly lowered the potency (145),

even if to a lower extent than in the δ-lactam series.

Methylation of the lactam nitrogen on the 7S/2′R derivative

led to a modest reduction of inhibitory potency on all HDAC

isoforms (83). As observed for δ-lactam derivatives, the

presence of a methyl group in meta-position of the phenyl

ring (86, 87, 88) led to an increase of inhibitory potency,

particularly for the 7S/2′R conﬁguration. Replacement of the

methyl substituent with a triﬂuoromethyl (89) did not bring

any further improvement. Four γ-lactam derivatives were also

prepared (45, 46, 47, 48) with inverted amide function and

insertion of a phenyl substituent close to the lactam connection

point. The phenyl substituent was not detrimental to the

activity, particularly on lactams with 3′S chirality (45, 47), with

the highest inhibitory potency observed with compound 45

having 7S/3′S/4′R stereochemistry. A further contraction of

the lactam ring, the β-lactam 80, maintained good inhibitory

potencies, comparable to the γ-lactam analogue 81.

An exploration of the CAP region was also done with the

synthesis of diﬀerent aliphatic amides (Table 2). We ﬁrst

evaluated the cyclopentyl ring which conferred selectivity for

HDAC6 to thiol-based HDAC inhibitors.²⁰Replacement of the

H

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Figure 5. Docking poses of compound 132 into HDAC8 (left) and HDAC3 (right) crystal structures. Loop L1 is depicted as an orange tube and L2

in light blue.

phenyl ring with a cyclopentyl moiety (19) maintained the 10-

fold selectivity for HDAC6 already observed for the phenyl

derivative 15. Insertion of the lactam carboxamide substituent

on the cyclopentyl derivative 19 produced compound 133,

which showed improved inhibitory potency and selectivity for

HDAC6. Carbamate derivative 52, reported as an HDAC6-

selective inhibitor,²⁰in our hands gave poor results, with very

limited inhibitory activity on HDAC6 isoform. An inferior

potency of carbamoyl derivative 52 compared to lactam

carboxamide 133 could be explained on the basis of their

accommodation within the HDAC catalytic site. In fact, while

docking studies for 52 showed a pose similar to that of lactam

carboxamide derivatives, the carbamic oxygen of 52 was close

to the carboxylic group of Asp93, exerting an unfavorable

repulsive eﬀect (Supporting Information Figure S2).

Phenylalkyl substituents were also investigated, such as in p-

triﬂuoromethylbenzyl (18, 131), benzyloxy-benzyl (17, 132),

and m-methylphenylethyl derivatives (20, 84), in which the

lactam carboxamide substituent always improved the inhibitory

potencies of the unsubstituted parent counterparts (as detailed

in Table 2). Interestingly, these phenylalkyl derivatives showed

a certain selectivity for HDAC8 compared to the other class I

enzymes, while their phenyl analogue 127 had its lowest

inhibitory potency on this isoform. Docking studies with

compound 132 into a HDAC8 crystal structure evidenced that

the benzyloxy-benzyl substituent can be inserted into a pocket

lined by the N-terminal L1 loop (Figure 5). The same

accommodation was not possible in the case of HDAC3. In

fact, HDAC8 L1 loop is two residues shorter than that of the

other class I HDACs, resulting in a wider active-site pocket with

a larger surface opening and higher ﬂexibility.³⁴The region

occupied by the benzyl fragment was very close to the

secondary pocket observed in the HDAC8 crystal structure in

complex with selective inhibitors.³⁴The benzyloxy-benzyl

substituent adopted a diﬀerent conformation in the complex

with HDAC3, being essentially solvent exposed. Finally, a

tetrahydroisoquinoline, incorporating the amide nitrogen atom,

was also evaluated. The poor inhibitory potencies of

compounds 16 and 85 supported the need of the free amide

NH group for HDAC binding and inhibition.

various derivatives (acetyl, thiocarbonate, isobutyryl, aminoacyl,

and the dimeric oxidized form) of the linear compound 15. The

results obtained were in line with what was described in the

literature and are reported in Supporting Information Table

S4a. The acetyl (9), isobutyryl (22), and thiocarbonate (21)

derivatives were soluble in H₂O (>1 mg/mL) and, with the

exception of the acetyl one, even at pH = 7.4. These

compounds were stable in aqueous solution at pH = 1.2 (3

h) and pH = 7.4 (24 h), contrarily to aminoacyl (23) and

disulﬁde (24) derivatives. In addition, the acetyl and isobutyryl

prodrugs tested on NCI-H460 nonsmall cell lung carcinoma

(NSCLC) cell line showed IC₅₀values in the range of 15−20

μM (Supporting Information Table S5a). The other thiol

derivatives 23, 21, and 24 had IC₅₀values >30 μM. A similar

study was conducted on ω-functionalized compounds, and

solubility and stability data for derivatives of compound 127 are

reported in Supporting Information Table S4b. In this case, the

isobutyryl derivative 135 was unstable, while the thiocarbonate

(136) and the acetyl (116) analogues were stable. Table 3

reports the results of cytotoxicity assays for these prodrugs and

for the most potent prodrugs of other inhibitors (i.e., those

with IC₅₀≤ 0.1 μM; the complete list of cytotoxic activity data

can be found in Supporting Information Tables 5a,5b). On

NCI-H460 cell line, the acetyl derivative was found to be more

active than the thiocarbonate analogue (IC₅₀0.1 vs 0.5 μM).

For this reason, the acetyl was chosen as the protecting group

for prodrug preparation.

The acetyl derivatives of our thiol inhibitors showed

cytotoxicity potencies on NCI-H460 cell line generally

consistent with their inhibitory activity on HDAC isoforms

(Tables 3 and Supporting Information Tables S5a, S5b). The

highest activities were seen for meta-substituted phenyl

derivatives 119, 76, 77, and 79 and for unsubstituted δ-, γ-,

and β-lactams 116, 115, 117, and 69. In these assay conditions,

reference compound 1 was signiﬁcantly less potent.

The eﬃcacy of δ-lactam 116, γ-lactam 117, and β-lactam 69

acetyl prodrugs was further evaluated in a cytotoxicity assay on

HCT116 colon cancer cells. Moreover, their ability to aﬀect

acetylation of tubulin and histone H4 substrates, which is

mainly dependent on HDAC6 and class I HDACs, respectively,

was assessed through Western Blot analysis on NCI-H460

NSCLC cells (Table 4). Cytotoxic activity on HCT116 cells

was in line with that seen on NCI-H460 cells, with similar IC₅₀

values and with the highest activity observed for compounds

The antiproliferative activity of thiol inhibitors greatly

improved when their sulfhydryl group was transiently masked

as a prodrug.¹⁸To select the most eﬀective protective group for

our thiols, we started studying the solubility and stability of

I

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117 and 69. Compound 117 showed its highest inhibitory

activity on acetylation of histone H4 (IC₅₀= 0.005 μM), with a

40-fold higher potency with respect to that on acetylation of

tubulin (IC₅₀= 0.20 μM). It is noteworthy that 117 was very

eﬀective at inhibiting histone deacetylation at concentrations

lower than those resulting as cytotoxic on the same cell lines.

On the contrary, both 69 and 116 revealed similar inhibitory

potency with respect to acetylation of tubulin and histone H4,

with IC₅₀values resembling those measured in the cytotoxic

assay on the same cells. These in vitro tests highlighted

compound 117 as the best one in terms of HDAC inhibition

eﬃcacy and cytotoxic activity. It was logically selected as the

drug candidate for the development of a novel oral HDAC

inhibitor, and its preliminary pharmacokinetic characterization

is here reported. Metabolic stability in human hepatocytes and

permeability in Caco-2 cells were evaluated for prodrug 117

and for its thiol analogue 128 (Table 5). The acetyl prodrug

117 showed limited stability in human hepatocytes (t_1/2∼ 2.5

min), likely due to its hydrolysis, while the free thiol 128 was

signiﬁcantly more stable, with a half-life comparable to that of

the reference compound 1 (∼45 min). Permeability experi-

ments on Caco-2 cells suggest the contribution of an active

transport process to the net ﬂux of both compounds, and the

presence of digoxin reduced cellular uptake of compounds 117

and 128 by 40% and 30%, respectively. The selectivity proﬁle of

117 and 128 was evaluated on a panel of 40 diﬀerent receptors,

ion channels, and enzymes. Both compounds did not

signiﬁcantly interact with any of the targets at the concentration

of 10⁻⁵M, with the only exception of compound 128, causing

45% displacement of radiolabeled reference compound at the

CCK1 receptor. Such results suggest a substantial safety of

compound 117, while the corresponding drug 128 could have a

low probability of gastrointestinal disturbance due to the

modest aﬃnity versus the colecystokinin receptor CCK1

(Supporting Information Table S6). Plasma stability for

prodrug 117 was also measured, having t_1/2= 26 min, with

10 1.6% compound left after 2 h.

Table 3. In Vitro Cytotoxic Activity (IC₅₀) of Selected

Lactam-Carboxamide Thiol Prodrugs on H460 Lung Cancer

Cells

a

A systematic exploration was performed with the aim to

identify a suitable vehicle to both oral and parenteral

administration of compound 117. A series of surfactants and

cosolvents miscible or soluble in water were investigated. The

cosolvents and surfactants evaluated in this study are all

excipients normally used in the development of a formulation.³⁵

Compound 117 was soluble (>10 mg/mL) in a few of

excipients tested (Supporting Information Table S7). These

organic solutions were then diluted in water (1/20) in order to

verify their stability under these conditions. The ability or

inability to form micellar systems or microemulsions was also

taken as an additional selection criterion. The vehicle chosen

was the Solutol HS15, diluted in water (1/20). This solution

did not interfere with membrane glycoproteins nor did it

inﬂuence the oral bioavailability of the compound. Further-

more, the same vehicle is suitable for a parenteral formulation.

The eﬃcacy of compound 117 was tested in vivo, assessing

its antitumor activity in two human solid tumors: a colon

carcinoma HCT116 and an ovarian A2780 tumor xenograft

models. The compound was administered by the oral route

once daily according to the schedules qdx5/wx3w or qdx5/

wx2w. Against HCT116 colon cancer, compound 117 at the

dose of 60 mg/kg showed to signiﬁcantly inhibit the tumor

volume (TVI = 62%, P < 0.05) without any sign of toxicity

(Table 6 and Figure 6). The free drug 128 was less active (TVI

= 27%), supporting the importance of the prodrug derivatiza-

a

Values are the means of a minimum of three experiments.

Table 4. In Vitro Cytotoxic Activity (IC₅₀) of Selected Acetyl

Prodrugs on HCT116 Colon Cancer Cell Line, and

Assessment (through Western Blot) of Their Inhibitory

Activity on Acetylation of Tubulin and Histone H4 Target

a

Proteins in NCI-H460 Cells

tubulin

HCT116

cytotoxicity

IC₅₀(μM)

acetylation

(WB) IC₅₀

(μM)

histone H4

acetylation (WB)

IC₅₀(μM)

ST code

compd number

116

117

69

ST7572

ST7612

ST7876

0.130

0.075

0.086

0.10

0.20

0.05

0.100

0.005

0.031

a

Values are the means of a minimum of two experiments.

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Table 5. Metabolic Stability of Selected HDAC Inhibitors in Human Hepatocytes and Permeability in Caco-2 Cells with and

a

without Digoxin

Cl_int

(mL/min/kg)

b

c

d

e

compd ST code number T_1/2(min) Cl_int(μL/min/1 × 10⁶cells)

AB/BA eﬄux ratio AB/BA eﬄux ratio with digoxin coincubated

1

47.5

2.7

14.7

252.3

15.4

35.3

605.6

37.0

1.1

117

128

ST7612

ST7464

6.9

5.2

4.2

3.6

45.0

a

b

c

Values are the means of a minimum of three experiments. Cl_intvalues are calculated considering 1 × 10⁶cells/mL. Cl_intvalues are calculated

d

considering 120 × 10⁶cells/g liver and 20 g liver/kg body weight. Ratio of ﬂux in the apical:basolateral (AB) direction to ﬂux in the

basolateral:apical (BA) direction. Eﬄux ratio (AB/BA) values with digoxin coincubated with test and reference compounds.

e

Table 6. Antitumor Activity of Compounds 117 and 128,

delivered by the Oral Route According to the Schedule

(qdx5/wx3w), against HCT116 Colon Carcinoma Cells, In

Comparison to Carboplatin, and of Compound 117 in

A2780 Ovarian Carcinoma Cells, in Comparison to

Compound 1 (SAHA, Vorinostat), Xenografted in CD1

Nude Mice

a

b

ST code

number

dose

(mg/kg)

TVI

BWL

(%)

compd

117

(%)

HCT116 colon

cancer

ST7612

60

62

1

128

ST7464

100

50

27

43

3

c

carboplatin

Figure 7. Antitumor activity of compound 117 (ST7612AA1) and 1

(SAHA, vorinostat) against A2780 ovarian carcinoma xenografted in

CD1 nude mice. TVI = tumor volume inhibition. Schedule qdx5/

wx2w where blue triangles indicate ﬁrst and last day of treatments.

A2780 ovarian

cancer

117

1

ST7612

60

89

53

7

4

100

a

b

c

Tumor volume inhibition. Body weight loss. Carboplatin was

administered according to the schedule q4d/wx3w.

ﬁndings of overexpression of certain HDAC isoforms, diﬀering

in each tumor, might suggest a greater therapeutic eﬃcacy for

selective HDAC inhibitors or mainly selective toward a single

class, even if so far the most successful HDAC inhibitors are

pan-inhibitors. From a medicinal chemistry point of view, the

challenge is to identify new small molecules as new eﬀective

inhibitors, and a lot of attention has been focused on speciﬁc

changes of the classic pharmacophore model.

We reported a systematic study aimed at evaluating a new

class of pan-HDAC inhibitors, having a thiol as ZBG and a

lactam carboxamide substituent in ω position of the poly-

methylene chain, and their prodrugs as in vivo agents. This

combination signiﬁcantly increased HDAC inhibition as well as

the antiproliferative activity on various cancer cell lines when

compared to unsubstituted counterparts. The inhibitory

activities shown were compared with those from both their

linear analogues and their hydroxamic acid analogues counter-

parts. It was observed that the in vitro activity did not change

much going through β-, γ-, and δ-lactam, whereas stereo-

chemistry in the ω position was very signiﬁcant, being the S-

conﬁguration of the lactam carboxamide greatly favored over

the R-conﬁguration. A selection of the best protective group for

the thiol ZBG was also made. The acetyl-derivatives were stable

when evaluated under diﬀerent pH conditions, being active in

cytotoxicity studies on cancer cell lines. A graphical summary of

the most relevant SARs observed for this class of compounds is

depicted in Figure 8.

Figure 6. Antitumor activity of compound 117 (ST7612AA1) and

carboplatin against HCT116 colon carcinoma xenografted in CD1

nude mice. TVI = tumor volume inhibition. Schedule qdx5/wx3w,

where red triangles indicate ﬁrst and last day of treatments.

tion to achieve an improved distribution. Carboplatin, used as

reference compound, administered at the dose of 50 mg/kg

intraperitoneally (q4d/w×3w), was characterized by a lower

eﬃcacy, having a TVI of 43%. On A2780 ovarian cancer,

compound 117 revealed a potent and signiﬁcant antitumor

eﬀect (TVI = 89%, P < 0.05) when administered at the dose of

60 mg/kg, higher than that found with the reference compound

1 (TVI = 53%) at the dose of 100 mg/kg (Figure 7).

After a primary extensive screening in vitro, followed by the

assessment of eﬃcacy for selected compounds through in vivo

studies, we selected a potential drug candidate, ST7612AA1

(117), and its preclinical assessment is currently in a phase of

completion. This thioderivative may represent a progenitor of a

new class of synthetic compounds that, through the

CONCLUSIONS

■

Extensive research on HDAC’s structure and function has led

to a progressively deeper knowledge of their role in

pathophysiological conditions and has revealed wider areas

for therapeutic applications of HDAC inhibitors. The recent

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N-Phenylhept-6-enamide (3). A solution of hept-6-enoic acid (1.88

g, 14.7 mmol), DIPEA (7.68 mL, 44 mmol), and aniline (1.47 mL,16.2

mmol) was stirred at RT in DCM (100 mL) for 20 min before adding

PyBOP (7.65 g, 14.7 mmol) and anhydrous DMF (10 mL). The

reaction mixture was stirred for 2 h at RT. The solvent was removed

under reduced pressure, and the crude reaction mixture was diluted

with AcOEt, washed with 5% Na₂CO₃, water, and then with 5%

aqueous citric acid, and ﬁnally with brine. After removal of the solvent

under reduced pressure and puriﬁcation on silica gel (n-hexane/

1

AcOEt: 9/1), the desired adduct 3 was obtained (85%). H NMR

(300 MHz, DMSO-d₆) δ: 1.32−1.43 (m, 2H), 1.53−1.63 (m, 2H),

2.03 (q, J = 6.9 Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H), 4.91−5.03 (m, 2H),

5.73−5.86 (m, 1H), 7.00 (t, J = 7.3 Hz, 1H), 7.26 (t, J = 7.8 Hz, 2H),

7.57 (d, J = 8.3 Hz, 2H), 9.84 (s, 1H). ESI-MS m/z 204.1 (M + H)⁺.

Compounds 4−8 were prepared similarly from hept-6-enoic acid

and the corresponding amines.

(S)-(7-Anilino-7-oxo-heptyl) ethanethioate (9). To a stirred

solution of N-phenylhept-6-enamide 3 (250 mg, 0.78 mmol) and

thioacetic acid (564 μL, 7.8 mmol) at 75 °C in degassed dioxane was

added AIBN (129 mg, 0.78 mmol). The reaction mixture was stirred

for 1 h. The reaction mixture was cooled to 0 °C, and an excess of

cyclohexene was added under stirring, the latter being maintained for

20 min. The reaction mixture was concentrated under reduced

pressure, and the resulting crude product was rinsed more times with

Figure 8. Graphical representation of structure−activity relationships

for thiol HDAC inhibitors.

replacement of the phenyl ring with bulkier residues on CAP,

could lead to more selective derivatives toward a single class or

a single isoform, in an area of great therapeutic importance as

well as of pharmaceutical interest.

Development of new generation HDAC inhibitors (isoform-

selective or pan-inhibitors) remains an important medical need

for single-agent and/or combination chemotherapy. Their

association with other epigenetic target modulators could be

also exploited according to what recent therapeutic approaches

are suggesting.¹²The drug candidate 117 could represent a

novel therapeutic agent useful in all those diseases where an

epigenetic modulation is required. Compound 117 is actually

under preclinical investigation studies to become a candidate to

phase I clinical trials.

1

hexane to aﬀord the desired adduct 9 (81%). H NMR (300 MHz,

DMSO-d₆) δ: 1.40−1.20 (m, 4H), 1.44−1.60 (m, 4H), 2.26 (t, J = 7.3

Hz, 2H), 2.29 (s, 3H), 2.80 (t, J = 7.2 Hz, 2H), 6.99 (t, J = 7.4 Hz,

1H), 7.25 (t, J = 8.2 Hz, 2H), 7.57 (d, J = 7.6 Hz, 2H), 9.81 (s, 1H).

ESI-MS m/z 280.3 (M + H)⁺.

Compounds 10−14 were prepared similarly from 5−8.

Thioacetic Acid S-[7-(3,4-Dihydro-1H-isoquinolin-2-yl)-7-oxo-

1

heptyl] Ester (10). H NMR (300 MHz, DMSO-d₆) δ: 1.20−1.34

(m, 4H), 1.35−1.55 (m, 4H), 2.29 (s, 3H), 2.36 (t, J = 6.4 Hz, 2H),

2.65−2.85 (m, 4H), 3.64 (t, J = 6.0 Hz, 2H), 4.59 (d, J = 15.5 Hz, 2H),

7.16 (m, 4H). ESI-MS m/z 320.1 (M + H)⁺.

Thioacetic Acid S-[6-(3-Benzyloxy-benzylcarbamoyl)-hexyl] Ester

1

(11). H NMR (300 MHz, DMSO-d₆) δ: 1.20−1.28 (m, 4H), 1.43−

1.57 (m, 4H), 2.11 (t, J = 7.3 Hz, 2H), 2.29 (s, 3H), 2.79 (t, J = 7.1

Hz, 2H), 4.21 (d, J = 5.9 Hz, 2H), 5.06 (s, 2H), 6.78−6.87 (m, 3H),

7.21 (t, J = 7.5 Hz, 1H), 7.31−7.43 (m, 5H), 8.26 (t, J = 5.6 Hz, 1H).

ESI-MS m/z 422.3 (M + Na)⁺.

EXPERIMENTAL SECTION

■

General Procedures. Reagents were purchased from commercial

suppliers and used without further puriﬁcation. All nonaqueous

reactions were run in ﬂame-dried glassware under a positive pressure

of argon. The exclusion of moisture from reagents and glassware was

performed following standard techniques for manipulating air-sensitive

compounds. Anhydrous THF, toluene, Et₂O, and DCM were obtained

by ﬁltration through drying columns (Solvent Delivery System); other

solvents were distilled under positive pressure of dry argon before use

and dried by standard methods. Flash chromatography was performed

on 230−400 mesh silica gel with the indicated solvent systems. Thin

layer chromatographies (TLCs) were performed on precoated, glass-

backed silica gel plates (Merck 60F254). Visualization was performed

under short-wavelength ultraviolet light and/or by dipping the plates

in an aqueous H₂SO₄solution of cerium sulfate/ammonium

molybdate, potassium permanganate, or ethanolic solution of

anisaldehyde, followed by charring with a heat gun. Alternatively,

TLC can be stained by exposing them to iodine vapor into a iodine

development chamber. Nuclear magnetic resonance spectra were

recorded on Gemini spectrometers (Varian) at 300 or 500 MHz or on

Bruker spectrometer at 300 MHz. Peak positions are given in parts per

million downﬁeld from tetramethylsilane as the internal standard; J

values are expressed in hertz. Mass analyses were performed on Waters

ZQ2000 spectrometer using electrospray (ES) technique. LCMS

analyses were performed on a LC-Waters apparatus (HPLC Waters

Alliance 2695, ZQ2000 MS, and PDA-UV detector 2996) equipped

with a Phenomenex Gemini C6-Phenyl (3 μm, 150 mm × 4.6 mm),

and all compounds tested were determined to be >98% pure using this

method.

Thioacetic Acid S-[6-(4-Triﬂuoromethyl-benzylcarbamoyl)-hexyl]

1

Ester (12). H NMR (300 MHz, DMSO-d₆) δ: 1.34−1.21 (m, 4H),

1.55−1.42 (m, 4H), 2.13 (t, J = 7.3 Hz, 2H), 2.30 (s, 3H), 2.80 (t, J =

7.2 Hz, 2H), 4.32 (d, J = 5.9 Hz, 2H), 7.44 (d, J = 7.9 Hz, 2H), 7.66

(d, J = 8.3 Hz, 2H), 8.37 (t, J = 6.0 Hz, 1H). ESI-MS m/z 362.3 (M +

H)⁺.

S-[7-(Cyclopentylamino)-7-oxoheptyl] Ethanethioate (13). ¹H

NMR (300 MHz, DMSO-d₆) δ: 1.16−1.37 (m, 6H), 1.38−1.52 (m,

6H), 1.54−1.65 (m, 2H), 1.68−1.80 (m, 2H), 1.99 (t, J = 7.5 Hz, 2H),

2.30 (s, 3H), 2.36 (t, 2H), 2.80 (t, J = 7.2 Hz, 2H), 3.88−4.01 (m,

1H), 7.67 (d, J = 6.9 Hz, 1H). ESI-MS m/z 272.26 (M + Na)⁺.

Thioacetic Acid S-[6-(2-m-Tolyl-ethylcarbamoyl)-hexyl] Ester

1

(14). H NMR (300 MHz, DMSO-d₆) δ: 1.10−1.34 (m, 4H), 1.37−

1.52 (m, 4H), 2.00 (t, J = 7.4 Hz, 2H), 2.26 (s, 3H), 2.30 (s, 3H), 2.63

(t, J = 7.0 Hz, 2H), 2.80 (t, J = 7.3 Hz, 2H), 3.17−3.27 (m, 2H), 6.93−

7.02 (m, 3H), 7.11 (t, J = 7.7 Hz, 1H), 7.80 (t, J = 5.2 Hz, 1H). ESI-

MS m/z 344.13 (M + Na)⁺.

N-Phenyl-7-sulfanyl-heptanamide (15). To a stirred solution of

(S)-(7-anilino-7-oxo-heptyl) ethanethioate 9 (322 mg, 1 mmol) in

methanol (10 mL) under nitrogen at 23 °C was added sodium

thiomethoxide (70 mg, 1 equiv 1 M solution in MeOH). The reaction

mixture was stirred at 23 °C for 30 min. The solution was then added

to aqueous HCl 20 mL/0.1M). The aqueous solution was extracted

with CH₂Cl₂. The combined organic layers were washed with brine,

dried over MgSO₄, ﬁltered, and concentrated to obtain the compound

1

15, which was puriﬁed through HPLC (27%). H NMR (300 MHz,

DMSO-d₆) δ: 1.32 (m, 4H), 1.54 (m, 4H), 2.20 (t, J = 7.4 Hz, 1H),

2.27 (t, J = 7.5 Hz, 2H), 2.45 (m, 2H), 6.99 (t, J = 7.4 Hz, 1H), 7.26 (t,

HRMS for 117 and 128 were performed on a Thermo Scientiﬁc

Orbitrap Exactive equipped with an ESI source.

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J = 7.6 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 9.82 (s, 1H). ESI-MS m/z

260.15 (M + Na)⁺.

7.5 Hz, 2H), 7.27 (t, J = 8.0 Hz, 4H), 7.58 (d, J = 7.5 Hz, 4H), 9.84 (s,

2H). ESI-MS m/z 445.25 (M + H)⁺.

Thiols 45−48 were isolated by HPLC, from hydrolysis mixture of

their corresponding stereoisomers 37−40, and characterized (see

below). As thioacetates, only 38−39 were easily isolated.

Compounds 16−20 were prepared similarly from 10−14.

1-(3,4-Dihydro-1H-isoquinolin-2-yl)-7-mercapto-heptan-1-one

1

(16). H NMR (300 MHz, DMSO-d₆) δ: 1.20−1.41 (m, 4H), 1.43−

1.63 (m, 4H), 2.31−2.42 (m, 2H), 2.44−2.50 (m, 1H), 2.72 (t, J = 6.1

Hz, 1H), 2.82 (t, J = 6.1 Hz, 2H), 3.64 (t, J = 6.1 Hz, 2H), 4.59 (d, J =

15.5 Hz, 2H), 7.16 (bs, 4H). ESI-MS m/z 278.25 (M + H)⁺.

7-Mercapto-heptanoic Acid 3-Benzyloxy-benzylamide (17). ¹H

NMR (300 MHz, DMSO-d₆) δ: 1.20−1.40 (m, 4H), 1.44−1.54 (m,

4H), 2.11 (t, J = 7.4 Hz, 2H), 2.19 (t, J = 7.5 Hz, 1H), 2.37−2.48 (m,

2H), 4.21 (d, J = 5.9 Hz, 2H), 5.06 (s, 2H), 6.76−6.90 (m, 3H), 7.16−

7.26 (m, 1H), 7.28−7.46 (m, 5H), 8.26 (bt, J = 5.8 Hz, 1H). ESI-MS

m/z 358.22 (M + H)⁺.

Thioacetic Acid S-{(S)-6-[((3R,4S)-2-Oxo-4-phenyl-pyrrolidine-3-

carbonyl)-amino]-6 phenylcarbamoyl-hexyl} Ester (38). ¹H NMR

(300 MHz, acetone-d₆) δ: 1.30−1.80 (m, 8H), 2.28 (s, 3H), 2.81 (t, J

= 7.5 Hz, 2H), 3,51 (t, J = 9.6 Hz, 1H), 3.75 (d, J = 10.7 Hz, 1H),

3.82−3.92 (m, 1H), 4.25−4.37 (m, 1H), 4.47−4.60 (m, 1H), 7.00−

7.40 (m, 8H), 7.53 (bs, 1H), 7.81−7.88 (m, 2H), 7.89 (d, J = 6.6 Hz,

1H), 9.80 (bs, 1H). ESI-MS m/z 482.1 (M + H)⁺.

Thioacetic Acid S-{(S)-6-[((3S,4S)-2-Oxo-4-phenyl-pyrrolidine-3-

carbonyl)-amino]-6-phenylcarbamoyl-hexyl} Ester (39). ¹H NMR

(300 MHz, acetone-d₆) δ: 1.35−2.00 (m, 8H), 2.28 (s, 3H), 2.84 (t, J

= 7.5 Hz, 2H), 3.40 (t, J = 9.6 Hz, 1H), 3.59 (d, J = 10.7 Hz, 1H),

3.75−3.82 (m, 1H), 4.08−4.19 (m, 1H), 4.42−4.52 (m, 1H), 7.00−

7.40 (m, 9H), 7.58−7.67 (m, 2H), 7.89 (d, J = 6.6 Hz, 1H), 9.25 (bs,

1H). ESI-MS m/z 482.1 (M + H)⁺.

7-Mercapto-heptanoic Acid 4-Triﬂuoromethyl-benzylamide (18).

¹H NMR (500 MHz, DMSO-d₆) δ: 1.20−1.30 (m, 2H), 1.30−1.38

(m, 2H), 1.47−1.57 (m, 4H), 2.15 (t, J = 7.6 Hz, 2H), 2.20 (bs, 1H),

2.42−2.48 (m,2H), 4.32 (d, J = 5.8 Hz, 2H), 7.45 (d, J = 7.9 Hz, 2H),

7.68 (d, J = 7.9 Hz, 2H), 8.39 (t, J = 5.5 Hz, 1H). ESI-MS m/z 319.9

(M + H)⁺.

Thioacetic Acid S-{(S)-6-[(R/S)-(2-Oxo-piperidine-3-carbonyl)-

1

N-Cyclopentyl-7-sulfanyl-heptanamide (19). ¹H NMR (300 MHz,

DMSO-d₆) δ: 1.12−1.38 (m, 6H), 1.39−1.67 (m, 8H), 1.67−1.83 (m,

2H), 2.00 (t, J = 7.2 Hz, 2H), 2.21 (t, J = 7.6 Hz, 1H), 2.38−2.50 (m,

2H), 3.83−4.02 (m, 1H), 7.67 (d, J = 6.7 Hz, 1H). ESI-MS m/z 230.3

(M + H)⁺.

amino]-6-phenyl carbamoyl-hexyl} Ester (41−42). H NMR (500

MHz, CD₃OD) δ: 1.42−1,99 (m, 10H), 2.29 (s, 3H), 2.55 (t, J = 7.2

Hz, 2H), 2,80 (t, J = 7.6 Hz, 2H), 3.35 (m, 2H), 3.40 (m, 2H), 4.53

(m, 2H), 7.12 (t, J = 7.0 Hz, 1H), 7.33 (t, J = 7.5 Hz, 2H), 7.60 (d, J =

8.5 Hz, 2H). ESI-MS m/z 442.1 (M + Na)⁺.

7-Mercapto-heptanoic Acid (2-m-Tolyl-ethyl)-amide (20). ¹H

NMR (300 MHz, DMSO-d₆) δ: 1.12−1.37 (m, 4H), 1.38−1.61 (m,

4H), 2.01 (t, J = 7.4 Hz, 2H), 2.20 (t, J = 6.9 Hz, 1H), 2.26 (s, 3H),

2.38−2.47 (m, 2H), 2.63 (t, J = 7.1 Hz, 2H), 3.17−27 (m, 2H), 6.93−

7.01 (m, 3H), 7.11−7.19 (m, 1H), 7.81 (bt, J = 5.4 Hz, 1H). ESI-MS

m/z 280.31 (M + H)⁺.

(3S,4R)-2-Oxo-4-phenyl-pyrrolidine-3-carboxylic Acid ((S)-6-Mer-

1

capto-1-phenylcarbamoyl-hexyl)-amide (45). H NMR (500 MHz,

CD₃OD) δ: 1.24−1.50 (m, 4H), 1.50−1.61 (m, 2H), 1.64−1.74 (m,

1H), 1.95−2.05 (m, 1H), 2.42 (t, J = 7.3 Hz, 2H), 3.51 (t, J = 9.6 Hz,

1H), 3.72−3.85 (m, 3H), 4.49−4.55 (m, 1H), 7.10−7.16 (m, 1H),

7.26−7.40 (m, 7H), 7.64−7.69 (m, 2H). ESI-MS m/z 462.3 (M +

Na)⁺.

Ethyl (7-Anilino-7-oxo-heptyl)sulfanylformate (21). To a solution

of N-phenyl-7-sulfanyl-heptanamide 15 (50 mg, 0.21 mmol) in DCM

(20 mL) were added NEt₃(32 μL, 0.23 mmol) and ethyl-

chloroformate (21 mg, 0.23 mmol). The reaction mixture was stirred

at RT for 2 h. The reaction mixture was concentrated under reduced

pressure, and the crude product was puriﬁed through chromatography

on silica gel using AcOEt/MeOH 80/20 as eluent to obtain the

compound 21 (74%). ¹H NMR (300 MHz, acetone-d₆) δ: 1.25 (t, J =

7.0 Hz, 3H), 1.38−1.43 (m, 4H), 1.61−1.71 (m, 4H), 2.36 (t, J = 7.3

Hz, 2H), 2.86 (t, J = 7.3 Hz, 2H), 4.24 (q, J = 7.0 Hz, 2H), 7.02 (t, J =

7.3 Hz, 1H), 7.27 (t, J = 7.9 Hz, 2H), 7.65 (d, J = 7.6 Hz, 2H), 9.04 (s,

1H). ESI-MS m/z 332.1 (M + Na)⁺.

(3R,4S)-2-Oxo-4-phenyl-pyrrolidine-3-carboxylic Acid ((S)-6-Mer-

1

capto-1-phenylcarbamoyl-hexyl)-amide (46). H NMR (500 MHz,

CD₃OD) δ: 1.25−1.50 (m, 4H), 1.50−1.60 (m, 2H), 1.65−1.75 (m,

1H), 1.95−2.05 (m, 1H), 2.44 (t, J = 7.3 Hz, 2H), 3.49 (t, J = 9.6 Hz,

1H), 3.72−3.85 (m, 2H), 4.16−4.25 (m, 1H), 4.49−4.55 (m, 1H),

7.07−7.14 (m, 1H), 7.26−7.40 (m, 7H), 7.64−7.69 (d, J = 7.9 Hz,

2H). ESI-MS m/z 462.3 (M + Na)⁺.

(3S,4S)-2-Oxo-4-phenyl-pyrrolidine-3-carboxylic Acid ((S)-6-Mer-

1

capto-1-phenylcarbamoyl-hexyl)-amide (47). H NMR (500 MHz,

CD₃OD) δ: 1.25−1.50 (m, 4H), 1.50−1.70 (m, 3H), 1.95−2.05 (m,

1H), 2.40 (t, J = 7.3 Hz, 2H), 3.45 (t, J = 9.6 Hz, 1H), 3.71−3.84 (m,

3H), 4.49−55 (m, 1H), 7.10−7.20 (m, 1H), 7.24−7.37 (m, 7H),

7.65−7.70 (m, 2H). ESI-MS m/z 462.3 (M + Na)⁺.

Compounds 22−23 were prepared similarly from 15 and isobutyryl

choride and (2R)-2-amino-3-methyl-butanoyl chloride, respectively.

For disulﬁde 24, oxidation side product of thioacetate 9 hydrolysis,

see below.

(3R,4R)-2-Oxo-4-phenyl-pyrrolidine-3-carboxylic Acid ((S)-6-Mer-

1

capto-1-phenylcarbamoyl-hexyl)-amide (48). H NMR (500 MHz,

CD₃OD) δ: 1.24−1.49 (m, 4H), 1.50−1.61 (m, 2H), 1.65−1.75 (m,

1H), 1.90−2.00 (m, 1H), 2.46 (t, J = 7.3 Hz, 2H), 3.47 (t, J = 9.6 Hz,

1H), 3.72−3.84 (m, 2H), 4.16−4.25 (m, 1H), 4.49−55 (m, 1H),

7.07−7.14 (m, 1H), 7.26−7.40 (m, 7H), 7.63−7.70 (m, 2H). ESI-MS

m/z 462.3 (M + Na)⁺.

1

Thioisobutyric Acid S-(6-Phenylcarbamoyl-hexyl) Ester (22). H

NMR (300 MHz, acetone-d₆) δ: 1.14 (d, J = 7.0 Hz, 6H), 1.36−1.45

(m, 4H), 1.53−1.60 (m, 2H), 1.64−1.72 (m, 2H), 2.35 (t, J = 7.3 Hz,

2H), 2.68−2.78 (m, 1H), 2.85 (t, J = 7.3 Hz, 2H), 7.02 (t, J = 7.6 Hz,

1H), 7.26 (t, J = 7.6 Hz, 2H), 7.65 (d, J = 8.2 Hz, 2H), 9.03 (s, 1H).

ESI-MS m/z 308.19 (M + H)⁺.

(3R)-2-Oxo-pyrrolidine-3-carboxylic Acid ((S)-6-Mercapto-1-phe-

1

nylcarbamoyl-hexyl)-amide (49). H NMR (500 MHz, CD₃OD) δ:

(R)-2-Amino-3-methyl-thiobutyric Acid S-(6-Phenylcarbamoyl-

1.40−1.60 (m, 4H), 1.66−1.85 (m, 4H), 1.85−2.08 (m, 1H), 2.27−

2.48 (m, 2H), 2.52−2.62 (m, 1H), 2.70 (m, 2H), 3.31−3.49 (m, 4H),

3.54 (t, J = 9.2 Hz, 1H), 4.45−4.55 (m, 1H), 7.07−7.13(m, 1H),

7.27−7.34 (m, 2H), 7.56 (m, J = 7.9 Hz, 1H), 7.65 (d, J = 7.6 Hz, 1H).

ESI-MS m/z 364.2 (M + H)⁺.

(S)-2-Oxo-piperidine-3-carboxylic Acid ((S)-6-Mercapto-1-phenyl-

carbamoyl-hexyl)-amide (50). ¹H NMR (500 MHz, CD₃OD) δ:

1.40−1,55 (m, 4H), 1.56−1.69 (m, 2H), 1.70−1.85 (m, 2H), 1.95−

2.10 (m, 3H), 2.10−2.21 (m, 1H),2.55 (t, J = 7.0 Hz, 2H), 3.41−3.46

(m, 1H), 4.44−4.49 (m, 1H), 7.09 (t, J = 7.6 Hz, 1H), 7.29 (t, J = 8.2

Hz, 2H), 7.68 (d, J = 7.9 Hz, 2H). ESI-MS m/z 400.4 (M + Na)⁺.

(R)-2-Oxo-piperidine-3-carboxylic Acid ((S)-6-Mercapto-1-phenyl-

carbamoyl-hexyl)-amide (51). ¹H NMR (500 MHz, CD₃OD) δ:

1.40−1,60 (m, 4H), 1.60−1.70 (m, 2H), 1.72−1.85 (m, 2H), 1.76−

2.00 (m, 2H), 2.04−2.20 (m, 2H), 2.55 (t, J = 7.0 Hz, 2H), 3.40 (t, J =

7.0 Hz, 1H), 4.44−4.79 (m, 1H), 7.12 (t, J = 7.3 Hz, 1H), 7.33 (t, J =

8.2 Hz, 2H), 7.60 (d, J = 7.9 Hz, 2H). ESI-MS m/z 400.4 (M + Na)⁺.

1

hexyl) Ester (23). H NMR (300 MHz, DMSO-d₆) δ: 0.77 (d, J =

6.8 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H), 1.20−1.40 (m, 4H), 1.41−1.62

(m, 4H), 1.91−2.01 (m, 3H), 2.27 (t, J = 7.5 Hz, 2H), 2.75 (t, J = 7.2

Hz, 2H), 3.18 (d, J = 4.8 Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H), 7.26 (t, J =

8.4 Hz, 2H), 7.56 (d, J = 7.6 Hz, 2H), 9.83 (s, 1H). ESI-MS m/z

337.28 (M + H)⁺.

6-(5-Phenylcarbamoyl-pentyldisulfanyl)-hexanoic Acid Phenyl-

amide (24). A 2N solution of NaOH (343 mg, 7.0 mmol) was

added to a solution of (S)-(7-anilino-7-oxo-heptyl) ethanethioate 9

(265 mg, 0.95 mmol) in EtOH (13 mL). The reaction mixture was

stirred at RT overnight and then poured into water and extracted with

AcOEt, washed with water, brine, and ﬁnally dried over Na₂SO₄.

Removal of the solvent under reduced pressure led to the desired

adduct as a side product 24 (20%), which was puriﬁed through HPLC.

¹H NMR (500 MHz, DMSO-d₆) δ: 1.34−1.42 (m, 4H), 1.56−1.68

(m, 8H), 2.29 (t, J = 7.0 Hz, 4H), 2.70 (t, J = 7.0 Hz, 4H), 7.01 (t, J =

M

dx.doi.org/10.1021/jm5008209 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

((S)-1-Cyclopentylcarbamoyl-6-mercapto-hexyl)-carbamic acid tert-

butyl ester (52) and thioisobutyric acid S-((S)-6-tert-butoxycarbony-

lamino-6-cyclopentylcarbamoyl-hexyl) ester (53) were synthesized

following the synthetic method described in the literature.²⁰

1H), 8.19 (d, J = 8.1 Hz, 1H), 10.03 (s, 1H). ESI-MS m/z 406.4 (M +

H)⁺.

Thioacetic acid S-{(S)-6-[((S)-1-Methyl-6-oxo-piperidine-2-car-

bonyl)-amino]-6-phenylcarbamoyl-hexyl} Ester (71). ¹H NMR

(300 MHz, DMSO-d₆) δ: 1.20−1.41 (m, 4H), 1.42−1.78 (m, 5H),

1.79−2.01 (m, 2H), 2.17 (bt, J = 6.6 Hz, 2H), 2.29 (s, 3H), 2.68 (s,

3H), 2.79 (t, J = 7.0 Hz, 2H), 4.04−4.07 (m, 1H), 4.39−4.49 (m, 1H),

7.03 (t, J = 7.2 Hz, 1H), 7.29 (t, J = 7.3 Hz, 2H), 7.57 (d, J = 8.2 Hz,

2H), 8.34 (d, J = 7.6 Hz, 1H), 10.06 (s, 1H). ESI-MS m/z 433.9 (M +

H)⁺.

((S)-1-Phenylcarbamoyl-hex-5-enyl)-carbamic Acid tert-Butyl

Ester (54). A solution of (S)-2-tert-butoxycarbonylamino-hept-6-

enoic acid (745 mg, 5.24 mmol), DIPEA (2.74 mL, 15.7 mmol),

and aniline (525 μL, 5.76 mmol) was stirred at RT in DCM (70 mL)

for 20 min before adding PyBOP (2.73 g, 5.24 mmol) and anhydrous

DMF (5 mL). The reaction mixture was stirred for 2 h at RT. The

solvent was removed under reduced pressure, and the crude reaction

mixture was diluted with AcOEt, washed with 5% Na₂CO₃, water, and

then with 5% aqueous citric acid and ﬁnally with brine. After removal

of the solvent under reduced pressure and puriﬁcation on silica gel (n-

Thioacetic Acid S-{(S)-6-[((S)-6-Oxo-1,2,3,6-tetrahydro-pyridine-

1

2-carbonyl)-amino]-6-phenylcarbamoyl-hexyl} Ester (72). H NMR

(300 MHz, DMSO-d₆) δ: 1.18−1.38 (m, 4H), 1.40−1.75 (m, 4H),

2.28 (s, 3H), 2.50−2.58 (m, 2H), 2.78 (t, J = 7.2 Hz, 2H), 4.04−4.12

(m, 1H), 4.33−4.43 (m, 1H), 5.61−5.68 (m, 1H), 6.45−6.55 (m, 1H),

7.03 (t, J = 7.3 Hz, 1H), 7.28 (t, J = 8.4 Hz, 2H), 7.43−4.9 (m, 1H),

7.56 (d, J = 8.7 Hz, 2H), 8.01 (d, J = 7.9 Hz, 1H), 10.03 (s, 1H). ESI-

MS m/z 440.1 (M + Na)⁺.

Thioacetic Acid S-{(S)-6-[((R)-1-Methyl-5-oxo-pyrrolidine-2-car-

bonyl)-amino]-6-phenylcarbamoyl-hexyl} Ester (73). ¹H NMR

(300 MHz, DMSO-d₆) δ: 1.20−1.41 (m, 4H), 1.42−1.56 (m, 2H),

1.57−1.75 (m, 2H), 1.76−1.92 (m, 1H), 2.07−2.29 (m, 3H), 2.31 (s,

3H), 2.61 (s, 3H), 2.82 (t, J = 7.1 Hz, 2H), 4.09−4.18 (m, 1H), 4.36−

4.45 (m, 1H), 7.05 (t, J = 7.4 Hz, 1H), 7.31 (t, J = 8.2 Hz, 2H), 7.9 (d,

J = 7.6 Hz, 2H), 8.50 (d, J = 7.8 Hz, 1H), 10.10 (s, 1H). ESI-MS m/z

420.2 (M + H)⁺.

Thioacetic Acid S-[(S)-6-[((S)-6-Oxo-piperidine-2-carbonyl)-

amino]-6-(2-m-tolyl-ethylcarbamoyl)-hexyl] Ester (74). ¹H NMR

(300 MHz, DMSO-d₆) δ: 1.06−1.32 (m, 4H), 1.36−1.76 (m, 7H),

1.76−1.92 (m, 1H), 2.10 (t, J = 6.5 Hz, 2H), 2.26 (s, 3H), 2.30 (s,

3H), 2.65 (t, J = 7.1 Hz, 2H), 2.78 (t, J = 7.2 Hz, 2H), 3.12−3.30 (m,

2H), 3.85−4.95 (m, 1H), 4.10−4.22 (m, 1H), 6.93−7.03 (m, 3H),

7.14 (t, J = 7.9 Hz, 1H), 7.50 (d, J = 2.5 Hz, 1H), 7.85−8.00 (m, 2H).

ESI-MS m/z 462.1 (M + H)⁺.

Thioacetic Acid S-{(S)-7-(3,4-Dihydro-1H-isoquinolin-2-yl)-7-oxo-

6-[((S)-6-oxo-piperidine-2-carbonyl)-amino]-heptyl} Ester (75). ¹H

NMR (300 MHz, DMSO-d₆) δ: 1.20−1.36 (m, 4H), 1.37−1.90 (m,

9H), 2.07 (m, 2H), 2.29 (s, 3H), 2.71−2.90 (m, 3H), 3.64−3.75 (m,

2H), 3.81−3.95 (m, 1H), 4.49−4.70 (m, 2H), 4.71−4.83 (m, 1H),

7.17 (s, 4H) 7.45−7.50 (m, 1H), 8.06−8.16 (m, 1H). ESI-MS m/z

460.2 (M + H)⁺.

1

hexane/AcOEt: 9/1), the desired adduct 54 was obtained (85%). H

NMR (300 MHz, DMSO-d₆) δ: 1.20−1.72 (m, 13H), 1.93−2.10 (m,

2H), 3.98−4.12 (m, 1H), 4.88−5.05 (m, 2H), 5.70−5.85 (m, 1H),

7.02 (t, J = 7.4 Hz, 2H), 7.29 (t, J = 7.5 Hz, 2H), 7.59 (d, J = 8.6 Hz,

2H), 9.92 (s, 1H). ESI-MS m/z 341.2 (M + Na)⁺.

Compounds 55−58 were prepared similarly from the correspond-

ing amines.

Thioacetic Acid S-((S)-6-tert-Butoxycarbonylamino-6-phenylcar-

bamoyl-hexyl) Ester (59). To a stirred solution of ((S)-1-phenyl-

carbamoyl-hex-5-enyl)-carbamic acid tert-butyl ester 54 (250 mg, 0.78

mmol) and thioacetic acid (564 μL, 7.8 mmol) at 75 °C in degassed

dioxane was added AIBN (129 mg, 0.78 mmol). The reaction mixture

was stirred for 1 h. The reaction mixture was cooled to 0 °C, and an

excess of cyclohexene was added under stirring, the latter being

maintained for 20 min. The reaction mixture was concentrated under

reduced pressure, and the resulting crude product was rinsed more

1

times with hexane to aﬀord the desired adduct 59 (81%). H NMR

(300 MHz, DMSO-d₆) δ: 1.20−1.40 (m, 13H), 1.40−1.70 (m, 4H),

2.29 (s, 3H), 2.79 (t, J = 7.2 Hz, 2H), 3.95−4.09 (m, 1H), 6.96 (d, J =

7.9 Hz, 1H), 7.02 (t, J = 7.3 Hz, 1H), 7.28 (t, J = 8.4 Hz, 2H), 7.57 (d,

J = 8.6 Hz, 2H), 9.89 (s, 1H). ESI-MS m/z 417.2 (M + Na)⁺.

Compounds 60−63 were prepared similarly 55−58.

Thioacetic Acid S-((S)-6-Amino-6-phenylcarbamoyl-hexyl) Ester

(64). To a stirred solution in DCM (20 mL) of thioacetic acid S-((S)-

6-tert-butoxycarbonylamino-6-phenylcarbamoyl-hexyl) ester 59 (103

mg, 0.35 mmol) at 0 °C was added TFA (4 mL) slowly. The reaction

mixture was then allowed to warm to RT and stirred overnight. The

solvent was removed under reduced pressure to aﬀord the desired

adduct 64 as the triﬂuoroacetate salt, which was used without any

puriﬁcation in the next step.

Thioacetic Acid S-{(S)-6-[((S)-5-Oxo-pyrrolidine-2-carbonyl)-

1

amino]-6-m-tolylcarbamoyl-hexyl} Ester (76). H NMR (300 MHz,

DMSO-d₆) δ: 1.20−1.43 (m, 4H), 1.44−1.76 (m, 4H), 1.83−1.96 (m,

1H), 2.00−2.2.26 (m, 3H), 2.27 (s, 3H), 2.31 (s, 3H), 2.81 (t, J = 7.2

Hz, 2H), 4.07−4.14 (m, 1H), 4.33−4.43 (m, 1H), 6.87 (d, J = 7.5 Hz,

1H), 7.18 (t, J = 7.8 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.42 (s, 1H),

7.79 (s, 1H), 8.19 (d, J = 7.8 Hz, 1H), 9.96 (s, 1H). ESI-MS m/z 420.3

(M + H)⁺.

Compounds 65−68 were prepared similarly from 60−63.

Thioacetic Acid S-{(S)-6-[((S)-4-Oxo-azetidine-2-carbonyl)-

amino]-6-phenylcarbamoyl-hexyl} Ester (69). To a solution of the

triﬂuoroacetate salt 64 as obtained in step C (305 mg, 0.75 mmol) in

DCM (10 mL) were added NEt₃(312 μL, 2.24 mmol), (S)-4-oxo-

azetidine-2-carboxylic acid (90 mg, 0.79 mmol), PyBOP (408 mg, 0.79

mmol), and DMF (1.7 mL). The reaction mixture was stirred

overnight and then diluted with AcOEt, washed with water, 5% aq

Na₂CO₃, brine 5% citric acid solution, and brine again. The crude

material was puriﬁed through chromatography on silica gel using

AcOEt as eluent to allow the desired adduct 69 as a white solid (42%).

¹H NMR (300 MHz, DMSO-d₆) δ: 1.20−1.40 (m, 4H), 1.40−1.54

Thioacetic Acid S-{(S)-6-[((R)-5-Oxo-pyrrolidine-2-carbonyl)-

1

amino]-6-m-tolylcarbamoyl-hexyl} Ester (77). H NMR (300 MHz,

DMSO-d₆) δ: 1.20−1.40 (m, 4H), 1.41−1.76 (m, 4H), 1.78−1.91 (m,

1H), 1.99−2.24 (m, 3H), 2.25 (s, 3H), 2.29 (s, 3H), 2.79 (t, J = 7.2

Hz, 2H), 4.05−4.13 (m, 1H), 4.34−4.45 (m, 1H), 6.85 (d, J = 7.5 Hz,

1H), 7.16 (t, J = 7.9 Hz, 1H), 7.36 (d, J = 7.9 Hz, 1H), 7.40 (s, 1H),

7.80 (s, 1H), 8.15 (d, J = 8.1 Hz, 1H), 9.96 (s, 1H). ESI-MS m/z 420.3

(M + H)⁺.

(m, 2H), 1.55−1.78 (m, 2H), 2.29 (s, 3H), 2.67 (dt, J₁= 14.5 Hz, J₂=

2.1 Hz, 1H), 2.79 (t, J = 7.2 Hz, 2H), 3.09 (dd, J₁= 14.4 Hz, J₂= 5.4

Hz, 1H), 4.06 (dd, J₁= 5.4 Hz, J₂= 2.4 Hz, 1H), 4.37−4.47 (m, 1H),

7.03 (t, J = 7.5 Hz, 1H), 7.28 (t, J = 8.4 Hz, 2H), 7.57 (d, J = 8.7 Hz,

2H), 8.12 (s, 1H), 8.38 (d, J = 8.1 Hz, 1H), 10.07 (s, 1H). ESIMS m/z

392.0 (M + H)⁺. ESI-MS m/z 504.2 (M + CF₃COO)⁻.

Thioacetic Acid S-{(S)-6-[((R)-1-Methyl-5-oxo-pyrrolidine-2-car-

bonyl)-amino]-6-m-tolylcarbamoyl-hexyl} Ester (78). ¹H NMR

(300 MHz, DMSO-d₆) δ: 1.20−1.40 (m, 4H), 1.41−1.87 (m, 5H),

2.03−2.24 (m, 3H), 2.25 (s, 3H), 2.28 (s, 3H), 2.58 (s, 3H), 2.80 (t, J

= 7.2 Hz, 2H), 4.09−4.18 (m, 1H), 4.31−4.43 (m, 1H), 6.85 (d, J =

7.5 Hz, 1H), 7.16 (t, J = 7.8 Hz, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.40 (s,

1H), 8.47 (d, J = 7.6 Hz, 1H), 9.99 (s, 1H). ESI-MS m/z 434.4 (M +

H)⁺; 546.5 (M + CF₃COO)⁻.

Thioacetic Acid S-[(S)-6-[((R)-5-Oxo-pyrrolidine-2-carbonyl)-

amino]-6-(3-triﬂuoromethyl-phenylcarbamoyl)-hexyl] Ester (79).

¹H NMR (300 MHz, DMSO-d₆) δ: 1.20−1.40 (m, 4H), 1.42−1.78

(m, 4H), 1.78−1.90 (m, 1H), 2.28 (s, 3H), 2.00−2.34 (m, 3H), 2.79

(t, J = 7.2 Hz, 2H), 4.05−4.13 (m, 1H), 4.32−4.42 (m, 1H), 7.39 (d, J

= 7.8 Hz, 1H), 7.54 (t, J = 8.2 Hz, 1H), 7.74−7.84 (m, 2H), 8.08 (s,

Compounds 70−79 were prepared similarly from triﬂuoroacetate

salts 64−68 and the corresponding carboxylic acids.

Thioacetic Acid S-{(S)-6-[((S)-5-Oxo-pyrrolidine-2-carbonyl)-

1

amino]-6-phenylcarbamoyl-hexyl} Ester (70). H NMR (300 MHz,

DMSO-d₆) δ: 1.20−1.40 (m, 4H), 1.41−1.54 (m, 2H), 1.55−1.75 (m,

2H), 1.80−1.92 (m, 1H), 1.95−2.27 (m, 3H), 2.28 (s, 3H), 2.79 (t, J =

7.2 Hz, 2H), 4.05−4.11 (m, 1H), 4.32−4.42 (m, 1H), 7.02 (t, J = 7.3

Hz, 1H), 7.28 (t, J = 7.8 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H), 7.77 (s,

N

dx.doi.org/10.1021/jm5008209 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1H), 8.26 (d, J = 8.1 Hz, 1H), 10.43 (s, 1H). ¹⁹F NMR (282 MHz,

DMSO-d₆) δ: −62.97. ESI-MS m/z 474.3 (M + H)⁺; 586.3 (M +

CF₃COO)⁻.

(2R)-1-Methyl-N-[(1S)-1-(m-Tolylcarbamoyl)-6-sulfanyl-hexyl]-5-

1

oxo-pyrrolidine-2-carboxamide (88). H NMR (300 MHz, DMSO-

d₆) δ: 1.20−1.42 (m, 4H), 1.45−1.90 (m, 5H), 2.10−2.30 (m, 3H),

2.26 (s, 3H), 2.39−2.50 (m, 2H), 2.60 (s, 3H), 2.65 (m, 1H), 4.05−

4.18 (m, 1H), 4.33−4.44 (m, 1H), 6.86 (d, J = 7.6 Hz, 1H), 7.17 (t, J =

7.8 Hz, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.41 (s, 1H), 8.46 (d, J = 7.9 Hz,

1H), 9.99 (s, 1H). ESI-MS m/z 392.2 (M + H)⁺; 390.2 (M − H)⁻.

(2R)-5-Oxo-N-[(1S)-6-Sulfanyl-1-[[3-(triﬂuoromethyl)phenyl]-

carbamoyl]hexyl]pyrrolidine-2-carboxamide (89). ¹H NMR (300

MHz, DMSO-d₆) δ: 1.20−1.43 (m, 4H), 1.45−1.97 (m, 5H), 2.00−

2.35 (m, 4H), 2.39−2.50 (m, 2H), 4.06−4.14 (m, 1H), 4.34−4.45 (m,

1H), 7.40 (d, J = 7.6 Hz, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.78 (m, 2H),

7.80 (s, 1H), 8.09 (s, 1H), 8.23 (d, J = 7.8 Hz, 1H), 10.41 (s, 1H). ESI-

MS m/z 432.1 (M + H)⁺; 430.1 (M − H)⁻.

((S)-1-Phenylcarbamoyl-hex-5-enyl)-carbamic acid tert-butyl ester

(54) and compounds 57 and 90−95 were prepared from

corresponding amines (the ﬁrst step for route depicted in Scheme 4

is the same for route in Scheme 3, see above for synthesis and

characterization).

(S)-4-Oxo-azetidine-2-carboxylic Acid ((S)-6-Mercapto-1-phenyl-

carbamoyl-hexyl)-amide (80). To a stirred solution of thioacetate 69

(392 mg, 1 mmol) in methanol (10 mL) under nitrogen at 23 °C was

added sodium thiomethoxide (70 mg, 1 equiv 1 M solution in

MeOH). The reaction mixture was stirred at 23 °C for 30 min. The

solution was then added to aqueous HCl 20 mL/0.1M). The aqueous

solution was extracted with CH₂Cl₂. The combined organic layers

were washed with brine, dried over MgSO₄, ﬁltered, and concentrated

to obtain the compound 80, which was puriﬁed through HPLC (27%).

¹H NMR (300 MHz, DMSO-d₆) δ: 1.20−1.41 (m, 4H), 1.43−1.80

(m, 4H), 2.22 (t, J = 7.0 Hz, 1H), 2.40−2.50 (m, 1H), 2.60−2.73 (m,

2H), 3.10 (dd, J₁= 14.3 Hz, J₂= 5.3 Hz, 1H), 4.07 (dd, J₁= 5.3 Hz, J₂

= 2.3 Hz, 1H), 4.37−4.50 (m, 1H), 7.04 (t, J = 7.4 Hz, 1H), 7.29 (t, J

= 7.7 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 8.16 (s, 1H), 8.43 (d, J = 8.0

Hz, 1H), 10.11 (s, 1H). ESI-MS m/z 372.2 (M + Na)⁺; 348.2 (M −

H)⁻.

(S)-2-Amino-hept-6-enoic Acid Phenylamide (96). To a stirred

solution in DCM (20 mL) of ((S)-1-phenylcarbamoyl-hex-5-enyl)-

carbamic acid tert-butyl ester 54 (76 mg, 0.35 mmol) at 0 °C was

added TFA (4 mL) slowly. The reaction mixture was then allowed to

warm to RT and stirred for 2 h. The solvent was removed under

reduced pressure to aﬀord the desired adduct quantitatively as the

triﬂuoroacetate salt 96, which was used without any puriﬁcation in the

next step.

Compounds 81−89 were prepared similarly from 70, 71, and 73−

79.

(2S)-5-Oxo-N-[(1S)-1-(phenylcarbamoyl)-6-sulfanyl-hexyl]-

1

pyrrolidine-2-carboxamide (81). H NMR (300 MHz, DMSO-d₆) δ:

1.20−1.45 (m, 4H), 1.47−1.80 (m, 4H), 1.81−1.97 (m, 1H), 2.01−

2.25 (m, 4H), 2.41−2.50 (m, 2H), 4.07−4.15 (m, 1H), 4.35−4.47 (m,

1H), 7.05 (t, J = 7.4 Hz, 1H), 7.31 (t, J = 8.3 Hz, 2H), 7.61 (d, J = 8.1

Hz, 2H), 7.80 (s, 1H), 8.20 (d, J = 7.9 Hz, 1H), 10.05 (s, 1H). ESI-MS

m/z 364.2 (M + H)⁺.

(S)-1-Methyl-6-oxo-piperidine-2-carboxylic Acid ((S)-6-Mercapto-

1-phenylcarbamoyl-hexyl)-amide (82). ¹H NMR (300 MHz,

DMSO-d₆) δ: 1.20−1.45 (m, 4H), 1.46−1.80 (m, 6H), 1.80−2.03

(m, 2H), 2.15−2.25 (m, 3H), 2.40−2.50 (m, 2H), 2.69 (s, 3H), 4.03−

5.02 (m, 1H), 4.40−4.50 (m, 1H), 7.05 (t, J = 7.4 Hz, 1H), 7.30 (t, J =

7.6 Hz, 2H), 7.58 (d, J = 8.1 Hz, 2H), 8.35 (d, J = 7.9 Hz, 1H), 10.08

(s, 1H). ESI-MS m/z 392.0 (M + H)⁺.

(2R)-1-Methyl-5-oxo-N-[(1S)-1-(phenylcarbamoyl)-6-sulfanyl-

hexyl]pyrrolidine-2-carboxamide (83). ¹H NMR (300 MHz, DMSO-

d₆) δ: 1.20−1.45 (m, 4H), 1.45−1.90 (m, 5H), 2.20−2.35 (m, 4H),

2.35−2.50 (m, 2H), 2.61 (s, 3H), 4.11−4.20 (m, 1H), 4.35−4.47 (m,

1H), 7.05 (t, J = 7.4 Hz, 1H), 7.30 (t, J = 7.4 Hz, 2H), 7.59 (d, J = 8.3

Hz, 2H), 8.50 (d, J = 7.7 Hz, 1H), 10.09 (s, 1H). ESI-MS m/z 378.2

(M + H)⁺.

(S)-6-Oxo-piperidine-2-carboxylic Acid [(S)-6-Mercapto-1-(2-m-

tolyl-ethylcarbamoyl)-hexyl]-amide (84). ¹H NMR (300 MHz,

DMSO-d₆) δ: 1.10−1.34 (m, 4H), 1.42−1.53 (m, 3H), 1.54−1.76

(m, 4H), 1.80−1.89 (m, 1H), 2.11 (t, J = 6.4 Hz, 2H), 2.21 (t, J = 7.9

Hz, 1H), 2.27 (s, 3H), 2.40−2.48 (m, 2H), 2.66 (t, J = 7.0 Hz, 2H),

3.16−3.26 (m, 2H), 3.90−3.95 (m, 1H), 4.15−4.25 (m, 1H), 6.95−

7.05 (m, 3H), 7.16 (t, 1 J = 7.9 Hz, 1H), 7.51 (d, J = 2.1 Hz, 1H), 7.89

(d, J = 8.2 Hz, 1H), 7.95 (t, J = 5.5 Hz, 1H). ESI-MS m/z 420.1 (M +

H)⁺.

(S)-6-Oxo-piperidine-2-carboxylic Acid [(S)-1-(3,4-Dihydro-1H-

isoquinoline-2-carbonyl)-6-mercapto-hexyl]-amide (85). ¹H NMR

(300 MHz, DMSO-d₆) δ: 1.10−1.90 (m, 13H), 2.05−2.23 (m, 2H),

2.35−3.00 (m, 4H), 3.60−4.10 (m, 3H), 4.48−4.86 (m, 3H), 7.17 (s,

4H), 7.49 (m, 1H), 8.10 (m, 4H). ESI-MS m/z 418.1 (M + H)⁺.

(2S)-N-[(1S)-1-(m-Tolylcarbamoyl)-6-sulfanyl-hexyl]-5-oxo-pyrro-

lidine-2-carboxamide (86). ¹H NMR (300 MHz, DMSO-d₆) δ: 1.20−

1.42 (m, 4H), 1.45−1.78 (m, 4H), 1.80−1.93 (m, 1H), 1.95−2.25 (m,

3H), 2.26 (s, 3H), 2.40−2.50 (m, 2H), 4.06−4.13 (m, 1H), 4.33−4.43

(m, 1H), 6.86 (d, J = 7.3 Hz, 1H), 7.17 (t, J = 7.9 Hz, 1H), 7.36 (d, J =

8.1 Hz, 1H), 7.42 (s, 1H), 7.78 (s, 1H), 8.15 (d, J = 7.7 Hz, 1H), 9.95

(s, 1H). ESI-MS m/z 378.2 (M + H)⁺.

(2R)-N-[(1S)-1-(m-Tolylcarbamoyl)-6-sulfanyl-hexyl]-5-oxo-pyrro-

lidine-2-carboxamide (87). ¹H NMR (300 MHz, DMSO-d₆) δ: 1.20−

1.41 (m, 4H), 1.42−1.72 (m, 4H), 1.75−1.97 (m, 1H), 2.00−2.40 (m,

6H), 2.38−2.50 (m, 2H), 4.06−4.14 (m, 1H), 4.34−4.46 (m, 1H),

6.86 (d, J = 7.7 Hz, 1H), 7.17 (t, J = 7.8 Hz, 1H), 7.37 (d, J = 8.4 Hz,

1H), 7.41 (s, 1H), 7.81 (s, 1H), 8.15 (d, J = 7.9 Hz, 1H), 9.96 (s, 1H).

ESI-MS m/z 378.1 (M + H)⁺; 376.1 (M − H)⁻.

Compounds 97−103 were prepared similarly from 57 and 90−95.

(R)-6-Oxo-piperidine-2-carboxylic Acid ((S)-1-Phenylcarbamoyl-

hex-5-enyl)-amide (104). A solution of DCM/DMF (10 mL, 10/2) of

the triﬂuoroacetate salt was reacted with (R)-6-oxo-piperidine-2-

carboxylic acid (263 mg, 0.79 mmol) and PyBOP (411 mg, 0.79

mmol) in the presence of NEt₃(316 μL, 2.25 mmol) for 2 h. The

reaction mixture was diluted with DCM and washed with 5% Na₂CO₃,

brine, 5% citric acid, and brine. The organic phase was dried over

Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The crude

reaction mixture was puriﬁed through chromatography on silica gel

using AcOEt/MeOH (9/1) as eluent to allow the desired adduct as a

white solid 104 (85%). ¹H NMR (300 MHz, DMSO-d₆) δ: 1.28−1.50

(m, 2H), 1.52−1.78 (m, 5H), 1.80−1.95 (m, 1H), 1.97−2.08 (m, 2H),

2.11 (t, J = 6.6 Hz, 2H), 3.92−4.00 (m, 1H), 4.38−4.49 (m, 1H),

4.90−5.04 (m, 2H), 5.70−5.85 (m, 1H), 7.04 (t, J = 7.5 Hz, 1H), 7.29

(t, J = 8.4 Hz, 2H), 7.47 (d, J = 2.6 Hz, 1H), 7.57 (d, J = 8.6 Hz, 2H),

8.13 (d, J = 8.1 Hz, 1H), 10.03 (s, 1H). ESI-MS m/z 366.3 (M + Na)⁺;

342.2 (M − H)⁻.

Compounds 105−114 were prepared similarly from triﬂuoroacetate

salts 96−103 and corresponding carboxylic acids.

Thioacetic Acid S-{(S)-6-[((R)-6-Oxo-piperidine-2-carbonyl)-

amino]-6-phenylcarbamoyl-hexyl} Ester (115). To a stirred solution

of (R)-6-oxo-piperidine-2-carboxylic acid ((S)-1-phenylcarbamoyl-hex-

5-enyl)-amide 104 (220 mg, 0.64 mmol), and thioacetic acid (460 μL,

6.4 mmol) at 75 °C in degassed dioxane (7 mL) was added AIBN

(105 mg, 0.64 mmol). The reaction mixture was stirred until complete

conversion of the starting material as monitored by TLC analysis. The

reaction mixture was cooled to 0 °C and quenched with an excess of

cyclohexene under stirring, the latter being maintained for 20 min.

Concentration under reduced pressure and puriﬁcation through

chromatography on silica gel using hexane/DCM/IPA (50/40/10)

as eluent aﬀorded the desired adduct 115 (53%). ¹H NMR (300 MHz,

DMSO-d₆) δ: 1.18−1.40 (m, 4H), 1.40−1.80 (m, 7H), 1.80−1.95 (m,

1H), 2.11 (t, J = 6.6 Hz, 2H), 2.29 (s, 3H), 2.79 (t, J = 7.2 Hz, 2H),

3.91−3.99 (m, 1H), 4.35−4.47 (m, 1H), 7.03 (t, J = 7.5 Hz, 1H), 7.29

(t, J = 8.4 Hz, 2H), 7.48 (d, J = 2.4 Hz, 1H), 7.57 (d, J = 8.5 Hz, 2H),

8.14 (d, J = 8.2 Hz, 1H), 10.05 (s, 1H). ESI-MS m/z 420.1 (M + H)⁺;

532.2 (M + CF₃COO)⁻.

Compounds 116−125 were prepared similarly from 105−114.

Thioacetic Acid S-{(S)-6-[((S)-6-Oxo-piperidine-2-carbonyl)-

amino]-6-phenylcarbamoyl-hexyl} Ester (116). ¹H NMR (300

MHz, DMSO-d₆) δ: 1.20−1.40 (m, 4H), 1.41−1.87 (m, 8H), 2.10

(t, J = 6.6 Hz, 2H), 2.29 (s, 3H), 2.80 (t, J = 7.1 Hz, 2H), 3.90−4.01

O

dx.doi.org/10.1021/jm5008209 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(m, 1H), 4.30−4.45 (m, 1H), 7.04 (t, J = 7.4 Hz, 1H), 7.29 (t, J = 8.2

Hz, 2H), 7.49 (d, J = 2.3 Hz, 1H), 7.57 (d, J = 7.7 Hz, 2H), 8.11 (d, J

= 7.7 Hz, 1H), 9.99 (s, 1H). ESI-MS m/z 420.0 (M + H)⁺; 532.2 (M +

CF₃COO)⁻.

MHz, DMSO-d₆) δ: 1.10−1.90 (m, 20H), 2.10 (t, J = 6.5 Hz, 2H),

2.30 (s, 3H), 2.79 (t, J = 7.1 Hz, 2H), 3.86−4.02 (m, 2H), 4.13−4.24

(m, 1H), 7.50 (d, J = 2.5 Hz, 1H), 7.82 (s, 1H), 7.84 (s,1H). ESI-MS

m/z 434.4 (M + Na)⁺.

Thioacetic Acid S-{(S)-6-[((R)-5-Oxo-pyrrolidine-2-carbonyl)-

amino]-6-phenylcarbamoyl-hexyl} Ester (117). ¹H NMR (500

MHz, CD₃OD) δ: 1.20−1.40 (m, 4H), 1.41−1.76 (m, 4H), 1.77−

1.91 (m, 1H), 1.99−2.34 (m, 3H), 2.29 (s, 3H), 2.79 (t, J = 7.2 Hz,

2H), 4.04−4.14 (m, 1H), 4.35−4.47 (m, 1H), 7.03 (t, J = 7.3 Hz, 1H),

7.28 (t, J = 8.4 Hz, 2H), 7.57 (d, J = 7.6 Hz, 2H), 7.80 (s, 1H), 8.19 (d,

J = 7.9 Hz, 1H), 10.06 (s, 1H). ESI-MS m/z 428.17 (M + Na)⁺.

HRMS (Orbitrap, ESI+): C₂₀H₂₇N₃O₄S, [M + Na]⁺: found 428.1602,

calculated 428.1614. HRMS (Orbitrap, ESI−): C₂₀H₂₇N₃O₄S, [M −

H]⁻: found 404.1656, calculated 404.1650.

(R)-6-Oxo-piperidine-2-carboxylic Acid ((S)-6-Mercapto-1-phenyl-

carbamoyl-hexyl)-amide (126). To a stirred solution of thioacetate

115 (420 mg, 1 mmol) in methanol (10 mL) under nitrogen at 23 °C

was added sodium thiomethoxide (70 mg, 1 equiv 1 M solution in

MeOH). The reaction mixture was stirred at 23 °C for 30 min. The

solution was then added to aqueous HCl 20 mL/0.1M. The aqueous

solution was extracted with CH₂Cl₂. The combined organic layers

were washed with brine, dried over MgSO₄, ﬁltered, and concentrated

to obtain compound 126 which was puriﬁed through HPLC (27%).

¹H NMR (300 MHz, DMSO-d₆) δ: 1.18−1.42 (m, 4H), 1.45−1.79

S-{(6S)-7-Oxo-7-(phenylamino)-6-[(N-propanoyl-^D-alanyl)-

(m, 7H), 1.80−1.95 (m, 1H), 2.11 (t, J = 6.4 Hz, 2H), 2.22 (t, J = 7.9

Hz, 1H), 2.38−2.49 (m, 2H), 3.90−4.01 (m, 1H), 4.38−4.48 (m, 1H),

7.04 (t, J = 7.4 Hz, 1H), 7.29 (t, J = 8.2 Hz, 2H), 7.49 (d, J = 2.3 Hz,

1H), 7.57 (d, J = 7.6 Hz, 2H), 8.13 (d, J = 8.0 Hz, 1H), 10.04 (s, 1H).

ESI-MS m/z 400.2 (M + Na)⁺; 376.2 (M − H)⁻.

1

amino]heptyl} Ethanethioate (118). H NMR (300 MHz, DMSO-

d₆) δ: 0.97(t, J = 7.6 Hz, 3H), 1.18 (d, J = 7.1 Hz, 3H), 1.20−1.40 (m,

4H), 1.40−1.78 (m, 4H), 2.11 (q, J = 7.6 Hz, 2H), 2.29 (s, 3H), 2.80

(t, J = 7.1 Hz, 2H), 4.19−4.40 (m, 2H), 7.03 (t, J = 7.2 Hz, 1H), 7.28

(t, J = 7.9 Hz, 2H), 7.60 (d, J = 8.1 Hz, 2H), 7.97 (d, J = 7.1 Hz, 2H),

9.86 (s, 1H). ESI-MS m/z 422.7 (M + H)⁺.

Thioacetic Acid S-{(S)-6-[((S)-6-Oxo-piperidine-2-carbonyl)-

amino]-6-m-tolylcarbamoyl-hexyl} Ester (119). ¹H NMR (300

MHz, DMSO-d₆) δ: 1.20−1.40 (m, 4H), 1.41−1.79 (m, 6H), 1.79−

1.96 (m, 2H), 2.09 (t, J = 6.7 Hz, 2H), 2.25 (s, 3H), 2.29 (s, 3H), 2.79

(t, J = 7.2 Hz, 2H), 3.90−3.98 (m, 1H), 4.31−4.42 (m, 1H), 6.85 (d, J

= 7.5 Hz, 1H), 7.16 (t, J = 7.9 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.40

(s, 1H), 7.47 (d, J = 2.3 Hz, 1H), 8.09 (d, J = 7.8 Hz, 1H), 9.90 (s,

1H). ESI-MS m/z 456.4 (M + H)⁺.

Thioacetic Acid S-{(S)-6-[((S)-6-Oxo-piperidine-2-carbonyl)-

amino]-6-p-tolylcarbamoyl-hexyl} Ester (120). ¹H NMR (300

MHz, DMSO-d₆) δ: 1.20−1.40 (m, 4H), 1.41−1.79 (m, 7H), 1.79−

1.92 (m, 1H), 2.10 (t, J = 6.6 Hz, 2H), 2.23 (s, 3H), 2.29 (s, 3H), 2.79

(t, J = 7.3 Hz, 2H), 3.91−3.99 (m, 1H), 4.31−4.42 (m, 1H), 7.09 (d, J

= 8.2 Hz, 2H), 7.41−7.49 (m, 3H), 8.06 (d, J = 7.9 Hz, 1H), 9.88 (s,

1H). ESI-MS m/z 456.6 (M + H)⁺.

Compounds 127−133 were prepared from the corresponding

thioacetates 116−117, 119−120, and 123−125.

(S)-6-Oxo-piperidine-2-carboxylic Acid ((S)-6-Mercapto-1-phenyl-

1

carbamoyl-hexyl)-amide (127). H NMR (300 MHz, DMSO-d₆) δ:

1.20−1.42 (m, 4H), 1.45−1.80 (m, 7H), 1.80−1.95 (m, 1H), 2.10 (t, J

= 6.5 Hz, 2H), 2.21 (t, J = 7.1 Hz, 1H), 2.37−2.50 (m, 2H), 3.92−4.02

(m, 1H), 4.33−4.47 (m, 1H), 7.04 (t, J = 7.4 Hz, 1H), 7.29 (t, J = 8.2

Hz, 2H), 7.49 (d, J = 2.3 Hz, 1H), 7.58 (d, J = 7.6 Hz, 2H), 8.10 (d, J

= 7.9 Hz, 1H), 9.99 (s, 1H). ESI-MS m/z 400.4 (M + Na)⁺; 376.3 (M

− H)⁻.

(R)-5-Oxo-pyrrolidine-2-carboxylic Acid ((S)-6-Mercapto-1-phe-

1

nylcarbamoyl-hexyl)-amide (128). H NMR (300 MHz, DMSO-d₆)

δ: 1.20−1.45 (m, 4H), 1.46−1.93 (m, 5H), 2.00−2.39 (m, 4H), 2.40−

2.5 (m, 2H), 4.08−4.17 (m, 1H), 4.37−4.49 (m, 1H), 7.05 (t, J = 7.4

Hz, 1H), 7.31 (t, J = 7.4 Hz, 2H), 7.59 (d, J = 7.6 Hz, 2H), 7.83 (s,

1H), 8.19 (d, J = 8.1 Hz, 1H), 10.07 (s, 1H). ESI-MS m/z 364.3 (M +

H)⁺. HRMS (Orbitrap, ESI+): C₁₈H₂₅N₃O₃S. [M + Na]⁺: found

386.1509, calculated 386.1509. HRMS (Orbitrap, ESI−)

C₁₈H₂₅N₃O₃S, [M − H]⁻: found 362.1548, calculated 362.1544.

(S)-6-Oxo-piperidine-2-carboxylic Acid ((S)-6-Mercapto-1-m-tol-

ylcarbamoyl-hexyl)-amide (129). ¹H NMR (300 MHz, DMSO-d₆) δ:

1.20−1.41 (m, 4H), 1.44−1.91 (m, 8H), 2.10 (t, J = 6.7 Hz, 2H), 2.20

(t, J = 7.9 Hz, 1H) 2.26 (s, 3H), 2.39−2.47 (m, 2H), 3.92−3.99 (m,

1H), 4.32−4.43 (m, 1H), 6.85 (d, J = 7.3 Hz, 1H), 7.16 (t, J = 7.9 Hz,

1H), 7.36 (d, J = 7.9 Hz, 1H), 7.41 (s, 1H), 7.48 (d, J = 2.6 Hz, 1H),

8.06 (d, J = 7.9 Hz, 1H), 9.89 (s, 1H). ESI-MS m/z 414.3 (M + Na)⁺;

390.4 (M − H)⁻.

9H-Fluoren-9-ylmethyl-4-(3-{[(2S)-7-(acetylsulfanyl)-2-{[(6-oxopi-

peridin-2-yl)carbonyl]-amino}heptanoyl]amino}phenyl)piperidine-

1

1-carboxylate (121). H NMR (300 MHz, DMSO-d₆) δ: 1.20−1.40

(m, 4H), 1.41−1.95 (m, 12H), 2.10 (t, J = 6.6 Hz, 2H), 2.28 (s, 3H),

2.55−2.70 (m, 1H), 2.70−2.92 (m, 4H), 3.80−4.15 (m, 3H), 4.24−

4.33 (m, 1H), 4.34−4.55 (m, 3H), 6.89 (d, J = 7.5 Hz, 1H), 7.22 (t, J =

7.8 Hz, 1H), 7.30−7.51 (m, 7H), 7.62 (d, J = 7.2 Hz, 2H), 7.87 (d, J =

6.6 Hz, 2H), 8.07 (d, J = 7.8 Hz, 1H), 9.93 (s, 1H). ESI-MS m/z 725.4

(M + H)⁺; 837.3 (M + CF₃COO)⁻.

9H-Fluoren-9-ylmethyl-4-(4-{[(2S)-7-(acetylsulfanyl)-2-{[(6-oxopi-

peridin-2-yl)carbonyl]-amino}heptanoyl]amino}phenyl)piperidine-

1

(S)-6-Oxo-piperidine-2-carboxylic Acid ((S)-6-Mercapto-1-p-tolyl-

1-carboxylate (122). H NMR (300 MHz, DMSO-d₆) δ: 1.20−1.40

1

carbamoyl-hexyl)-amide (130). H NMR (500 MHz, DMSO-d₆) δ:

(m, 6H), 1.42−1.93 (m, 10H), 2.10 (t, J = 6.6 Hz, 2H), 2.29 (s, 3H),

2.50−2.68 (m, 1H), 2.69−2.90 (m, 4H), 3.75−4.15 (m, 3H), 4.20−

4.55 (m, 4H), 7.12 (d, J = 8.4 Hz, 2H), 7.29−7.45 (m, 4H), 7.46−7.53

(m, 3H), 7.63 (d, J = 7.2 Hz, 2H), 7.87 (d, J = 7.2 Hz, 2H), 8.08 (d, J

= 7.5 Hz, 1H), 9.93 (s, 1H). ESI-MS m/z 725.4 (M + H)⁺; 837.3 (M +

CF₃COO)⁻.

1.33 (m, 4H), 1.51 (m, 2H), 1.60 (m, 5H), 1.83 (m, 1H), 2.10 (t, J =

6.6 Hz, 2H), 2.21 (t, J = 7.0 Hz, 1H), 2.23 (s, 3H), 2.40−2.49 (m,

2H), 3.91−3.90 (m, 1H), 4.33−4.42 (m, 1H), 7.09 (d, J = 8.4 Hz,

2H), 7.45 (d, J = 8.2 Hz, 2H), 7.48 (s, 1H), 8.08 (d, J = 7.9 Hz, 1H),

9.89 (s, 1H). ESI-MS m/z 414.4 (M + Na)⁺; 390.3 (M − H)⁻.

(S)-6-Oxo-piperidine-2-carboxylic Acid [(S)-6-Mercapto-1-(4-tri-

Thioacetic Acid S-[(S)-6-[((S)-6-Oxo-piperidine-2-carbonyl)-

amino]-6-(4-triﬂuoromethyl-benzylcarbamoyl)-hexyl] Ester (123).

¹H NMR (500 MHz, CD₂Cl₂) δ: 1.30−1.47 (m, 4H), 1.52−1.62

(m, 2H), 1.64−1.94 (m, 5H), 2.00−2.10 (m, 1H), 2.17−2.30 (m, 2H),

2.32 (s, 3H), 2.84 (t, J = 7.0 Hz, 2H), 3.95−4.05 (m, 1H), 4.40−4.51

(m, 3H), 7.16−7.25 (m, 1H), 7.30−7.47 (m, 4H), 7.57−7.64 (m, 2H).

ESI-MS m/z 502.1 (M + H)⁺.

Thioacetic Acid S-{(S)-6-(3-Benzyloxy-benzylcarbamoyl)-6-[((S)-6-

oxo-piperidine-2-carbonyl)-amino]-hexyl} Ester (124). ¹H NMR

(500 MHz, CD₂Cl₂) δ: 1.25−1.45 (m, 4H), 1.50−1.60 (m, 2H),

1.62−1.95 (m, 5H), 1.97−2.07 (m, 1H), 2.15−2.30 (m, 2H), 2.30 (s,

3H), 2.83 (t, J = 7.1 Hz, 2H), 3.95−4.01 (m, 1H), 4.30−4.42 (m, 2H),

4.42−4.50 (m, 1H), 5.06 (m, 2H), 6.82−6.92 (m, 3H), 6.97−7.07 (m,

1H), 7.24 (t, J = 7.8 Hz, 1H), 7.32−7.50 (m, 5H). ESI-MS m/z 540.2

(M + H)⁺.

1

ﬂuoromethyl-benzylcarbamoyl)-hexyl]-amide (131). H NMR (500

MHz, CD₂Cl₂) δ: 1.38 (m, 5H), 1.52−2.12 (m, 8H), 2.18−2.28 (m,

2H), 2.45−2.56 (m, 2H), 3.94−4.03 (m, 1H), 4.34−4.53 (m, 3H),

7.20 (t, J = 5.7 Hz, 1H), 7.38 (d, J = 7.8 Hz, 3H), 7.48 (s, 1H), 7.58 (d,

J = 8.1 Hz, 2H). ESI-MS m/z 460.1 (M + H)⁺.

(S)-6-Oxo-piperidine-2-carboxylic Acid [(S)-1-(3-Benzyloxy-ben-

zylcarbamoyl)-6-mercapto-hexyl]-amide (132). ¹H NMR (500

MHz, CD₂Cl₂) δ: 1.24−1.46 (m, 5H), 1.54−2.02 (m, 8H), 2.13−

2.30 (m, 2H), 2.45−3.53 (m, 2H), 3.91−3.97 (m, 1H), 4.25−4.43 (m,

2H), 4.47−4.55 (m, 1H), 5.04 (s, 2H), 6.81−6.91 (m, 3H), 7.23 (t, J =

7.9 Hz, 1H), 7.32−7.38 (m, 2H), 7.38−7.47 (m, 3H), 7.64 (s, 1H),

7.75 (d, J = 8.2 Hz, 1H). ESI-MS m/z 498.4 (M + H)⁺.

(S)-6-Oxo-piperidine-2-carboxylic Acid ((S)-1-Cyclopentylcarba-

moyl-6-mercapto-hexyl)-amide (133). ¹H NMR (500 MHz, DMSO-

d₆) δ: 1.10−1.90 (m, 20H), 2.12 (t, J = 6.4 Hz, 2H), 2.21 (t, J = 7.9

Hz, 1H), 2.40−2.50 (m, 2H), 3.88−4.02 (m, 2H), 4.18−4.28 (m, 1H),

Thioacetic Acid S-{(S)- 6-[((S)-6-Oxo-piperidine-2-carbonyl)-

1

amino]-6-cyclopentylcarbamoyl-hexyl} Ester (125). H NMR (300

P

dx.doi.org/10.1021/jm5008209 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

7.55 (d, J = 2.3 Hz, 1H), 7.80 (s, 1H), 7.82 (s,1H). ESI-MS m/z 392.2

(M + Na)⁺; 368.1 (M − H)⁻.

quantitatively as the triﬂuoroacetate salt 139, which was used without

any puriﬁcation in the next step.

Ethyl [(6S)-7-Anilino-7-oxo-6-[[(2R)-5-oxopyrrolidine-2-

carbonyl]amino]heptyl]sulfanylformate (134). To a solution of

(R)-5-oxo-pyrrolidine-2-carboxylic acid ((S)-6-mercapto-1-phenylcar-

bamoyl-hexyl)-amide 128 (76 mg, 0.21 mmol) in DCM (20 mL) were

added NEt₃(32 μL, 0.23 mmol) and ethylchloroformate (22 μL, 0.23

mmol). The reaction mixture was stirred at RT for 2 h. The reaction

mixture was concentrated under reduced pressure, and the crude

product was puriﬁed through chromatography on silica gel using

AcOEt/MeOH 80/20 as eluent to obtain the compound 134 (74%).

¹H NMR (300 MHz, DMSO-d₆) δ:1.19 (t, J = 7.0 Hz, 3H), 1.25−1.45

(2S)-6-Oxo-N-[(1R)-1-(phenylcarbamoyl)hex-5-enyl]piperidine-2-

carboxamide (140). A solution of DCM/DMF (45 mL, 8/1) of the

triﬂuoroacetate salt 139 was reacted with (R)-6-oxo-piperidine-2-

carboxylic acid (1.78 mmol) and PyBOP (1.78 mmol) in the presence

of DIPEA (16.2 mmol) for 2 h. The reaction mixture was diluted with

DCM and washed with 5% Na₂CO₃, brine, 5% citric acid, and brine.

The organic phase was dried over Na₂SO₄, ﬁltered, and concentrated

under reduced pressure. The crude reaction mixture was puriﬁed

through chromatography on silica gel using AcOEt/MeOH (9/1) as

1

eluent to allow the desired adduct as a white solid 140 (80%). H

NMR (300 MHz, DMSO-d₆) δ: 1.27−1.52 (m, 2H), 1.52−1.79 (m,

5H), 1.80−1.95 (m, 1H), 1.95−2.15 (m, 4H), 3.92−4.00 (m, 1H),

4.35−4.48 (m, 1H), 4.90−5.04 (m, 2H), 5.70−5.83 (m, 1H), 7.04 (t, J

= 7.3 Hz, 1H), 7.30 (t, J = 8.1 Hz, 2H), 7.49 (s, 1H), 7.58 (d, J = 8.0

Hz, 2H), 8.14 (d, J = 8.2 Hz, 1H), 10.05 (s, 1H). ESI-MS m/z 366.3

(M + Na)⁺; 342.2 (M − H)⁻.

(m, 4H), 1.45−1.95 (m, 5H), 2.00−2.35 (m, 3H), 2.80 (t, J = 7.3 Hz,

2H), 4.05−4.11 (m, 1H), 4.16 (q, J = 7.0 Hz, 2H), 4.35−4.48 (m,

1H), 7.03 (t, J = 7.3 Hz, 1H), 7.28 (t, J = 8.2 Hz, 2H), 7.57 (d, J = 7.6

Hz, 2H), 7.81 (s, 1H), 8.18 (d, J = 8.2 Hz, 1H), 10.05 (s, 1H). ESI-MS

m/z 436.2 (M + H)⁺.

Compounds 135−136 were prepared similarly from 128 and

isobutyryl chloride and ethylchloroformate, respectively.

Compound 141 was prepared similarly from 139 and (2R)-5-

oxopyrrolidine-2-carboxylic acid.

Thioisobutyric Acid S-{(S)-6-[((S)-6-Oxo-piperidine-2-carbonyl)-

amino]-6-phenylcarbamoyl-hexyl} Ester (135). ¹H NMR (500

MHz, DMSO-d₆) δ: 1.00−1.07 (m, 6H), 1.22−1.44 (m, 4H), 1.46−

1.77 (m, 6H), 1.88−1.97 (m, 1H), 2.02−2.10 (m, 1H), 2.20 (t, J = 7.6

Hz, 1H), 2.34−2.41 (m, 1H), 2.43−54 (m, 4H), 3.42−3.52 (m, 1H),

4.28−4.36 (m, 1H), 4.79−4.85 (m, 1H), 7.04 (t, J = 7.3 Hz, 1H), 7.29

(t, J = 8.2 Hz, 2H), 7.58 (d, J = 7.6 Hz, 2H), 8.33 (d, J = 7.6 Hz, 1H),

9.99 (s, 1H). ESI-MS m/z 448.2 (M + H)⁺.

S-[(6R)-7-Anilino-7-oxo-6-[[(2S)-6-oxopiperidine-2-carbonyl]-

amino]heptyl] Ethanethioate (142). To a stirred solution of (2S)-6-

oxo-N-[(1R)-1-(phenylcarbamoyl)hex-5-enyl]piperidine-2-carboxa-

mide 140 (343 mg, 1.0 mmol) and thioacetic acid (715 μL, 10.0

mmol) at 75 °C in degassed dioxane (40 mL) was added AIBN (328

mg, 0.5 mmol). The reaction mixture was stirred until complete

conversion of the starting material as monitored by TLC analysis. The

reaction mixture was cooled to 0 °C and quenched with an excess of

cyclohexene under stirring, the latter being maintained for 20 min.

Concentration under reduced pressure and puriﬁcation through

chromatography on silica gel using hexane/DCM/IPA 50/40/10 as

Thiocarbonic Acid Ethyl Ester {(S)-6-[((S)-6-Oxo-piperidine-2-

1

carbonyl)-amino]-6-phenylcarbamoyl-hexyl} Ester (136). H NMR

(500 MHz, DMSO-d₆) δ: 1.20 (t, J = 7.0 Hz, 3H), 1.25−1.45 (m, 4H),

1.52−1.80 (m, 7H), 1.83−1.93 (m, 1H), 2.11 (t, J = 6.5 Hz, 2H), 2.82

(t, J = 7.0 Hz, 2H), 3.88−4.01 (m, 1H), 4.21 (q, J = 7.1 Hz, 2H),

4.35−4.45 (m, 1H), 7.05 (t, J = 7.5 Hz, 1H), 7.30 (t, J = 8.0 Hz, 2H),

7.48 (d, J = 2.5 Hz, 1H), 7.58 (d, J = 8.0 Hz, 2H), 8.10 (d, J = 7.5 Hz,

1H), 9.99 (s, 1H). ESI-MS m/z 450.1 (M + H)⁺.

1

eluent aﬀorded the desired adduct 142 (50%). H NMR (300 MHz,

DMSO-d₆) δ: 1.20−1.40 (m, 4H), 1.40−1.53 (m, 2H), 1.53−1.78 (m,

5H), 1.78−1.95 (m, 1H), 2.12 (t, J = 6.4 Hz, 2H), 2.30 (s, 3H), 2.79

(t, J = 7.1 Hz, 2H), 3.92−4.00 (m, 1H), 4.35−4.47 (m, 1H), 7.04 (t, J

= 7.4 Hz, 1H), 7.30 (t, J = 8.1 Hz, 2H), 7.49 (d, J = 2.4 Hz, 1H), 7.57

(d, J = 7.7 Hz, 2H), 8.13 (d, J = 8.0 Hz, 1H), 10.04 (s, 1H). ESI-MS

m/z 420.1 (M + H)⁺; 532.2 (M + CF₃COO)⁻.

(2S,2′S)-N,N′-{Disulfanediylbis[(2S)-1-oxo-1-(phenylamino)-

heptane-7,2-diyl]}bis(6-oxopiperidine-2-carboxamide) (137). A 2N

solution of NaOH (343 mg, 7.0 mmol) was added to a solution of

thioacetic acid S-{(S)-6-[((S)-6-oxo-piperidine-2-carbonyl)-amino]-6-

phenylcarbamoyl-hexyl} ester 116 (399 mg, 0.95 mmol) in EtOH (13

mL). The reaction mixture was stirred at RT overnight and then

poured into water and extracted with AcOEt, washed with water and

brine, and ﬁnally dried over Na₂SO₄. Removal of the solvent under

reduced pressure led to the desired adduct as a side product 137

(20%), which was puriﬁed through HPLC. ¹H NMR (300 MHz,

DMSO-d₆) δ: 1.18−1.93 (m, 24H), 2.09 (t, J = 6.5 Hz, 4H), 2.65 (t, J

= 7.0 Hz, 4H), 3.90−4.01 (m, 2H), 4.30−4.43 (m, 2H), 7.04 (t, J = 7.3

Hz, 2H), 7.28 (t, J = 8.0 Hz, 4H), 7.51 (m, 2H), 7.59 (d, 4H), 8.35 (d,

J = 7.7 Hz, 2H), 10.13 (s, 2H). ESI-MS m/z 753.4 (M + H)⁺.

((R)-1-Phenylcarbamoyl-hex-5-enyl)-carbamic Acid tert-Butyl

Ester (138). A solution of (R)-2-tert-butoxycarbonylamino-hept-6-

enoic acid (292 mg, 2.05 mmol), DIPEA (1.07 mL, 6.15 mmol), and

aniline (205 μL, 2.25 mmol) was stirred at RT in DCM (30 mL) for

20 min before adding PyBOP (1.07 g, 2.05 mmol) and anhydrous

DMF (2 mL). The reaction mixture was stirred for 2 h at RT. The

solvent was removed under reduced pressure, and the crude reaction

mixture was diluted with AcOEt, washed with 5% Na₂CO₃, water, and

then with 5% aqueous citric acid and ﬁnally with brine. After removal

of the solvent under reduced pressure and puriﬁcation on silica gel (n-

Compound 143 was prepared similarly from 141.

(2R)-5-Oxo-N-[(1R)-1-(phenylcarbamoyl)-6-sulfanyl-hexyl]-

1

pyrrolidine-2-carboxamide (143). H NMR (300 MHz, DMSO-d₆)

δ: 1.20−1.40 (m, 4H), 1.41−1.80 (m, 4H),1.80−1.95 (m, 1H), 2.00−

2.32 (m, 3H), 2.30 (s, 3H), 2.80 (t, J = 7.2 Hz, 2H), 4.06−4.13 (m,

1H), 4.32−4.43 (m, 1H), 7.04 (t, J = 7.4 Hz, 1H), 7.29 (t, J = 8.3 Hz,

2H), 7.58 (d, J = 7.6 Hz, 2H), 7.79 (s, 1H), 8.21 (d, J = 7.9 Hz, 1H),

10.06 (s, 1H). ESI-MS m/z 428.1 (M + H)⁺.

(2S)-6-Oxo-N-[(1R)-1-(phenylcarbamoyl)-6-sulfanyl-hexyl]-

piperidine-2-carboxamide (144). To a stirred solution of thioacetate

142 (101 mg, 0.24 mmol) in methanol (2 mL) under nitrogen at 23

°C was added sodium thiomethoxide (71 mg, 1 equiv 1 M solution in

MeOH). The reaction mixture was stirred at 23 °C for 30 min. The

solution was then added to aqueous HCl 4 mL/0.1M). The aqueous

solution is extracted with CH₂Cl₂. The combined organic layers were

washed with brine, dried over MgSO₄, ﬁltered, and concentrated to

obtain the compound 144, which was puriﬁed through HPLC (25%).

¹H NMR (300 MHz, DMSO-d₆) δ:1.20−1.45 (m, 4H), 1.45−1.80 (m,

7H), 1.80−1.97 (m, 1H), 2.12 (t, J = 6.4 Hz, 2H), 2.22 (t, J = 7.6 Hz,

1H), 2.37−2.50 (m, 2H), 3.92−4.00 (m, 1H), 4.38−4.48 (m, 1H),

7.04 (t, J = 7.2 Hz, 1H), 7.30 (t, J = 7.9 Hz, 2H), 7.49 (d, J = 2.3 Hz,

1H), 7.58 (d, J = 8.0 Hz, 2H), 8.13 (d, J = 8.2 Hz, 1H), 10.04 (s, 1H).

ESIMS m/z 400.1 (M + Na)⁺.

1

hexane/AcOEt 9/1), the desired adduct 138 was obtained (83%). H

NMR (300 MHz, DMSO-d₆) δ: 1.25−1.50 (m, 11H), 1.50−1.71 (m,

2H), 1.97−2.07 (m, 2H), 4.00−4.11 (m, 1H), 4.87−5.05 (m, 2H),

5.70−5.85 (m, 1H), 7.03 (m, J = 7.4 Hz, 2H), 7.29 (t, J = 8.3 Hz, 2H),

7.58 (d, J = 7.6 Hz, 2H), 9.93 (s, 1H). ESI-MS m/z 341.1 (M + Na)⁺.

(R)-2-Amino-hept-6-enoic Acid Phenylamide (139). To a stirred

solution of ((R)-1-phenylcarbamoyl-hex-5-enyl)-carbamic acid tert-

butyl ester at 0 °C was added TFA slowly. The reaction mixture was

then allowed to warm to RT and stirred for 2 h. The solvent was

removed under reduced pressure to aﬀord the desired adduct

Compound 145 was prepared similarly from 143.

(2R)-5-Oxo-N-[(1R)-1-(phenylcarbamoyl)-6-sulfanyl-hexyl]-

1

pyrrolidine-2-carboxamide (145). H NMR (300 MHz, DMSO-d₆)

δ: 1.20−1.40 (m, 4H), 1.45−1.79 (m, 4H),1.80−1.94 (m, 1H), 1.97−

2.12 (m, 3H), 2.40−2.50 (m, 2H), 4.05−4.13 (m, 1H), 4.32−4.44 (m,

1H), 7.04 (t, J = 7.4 Hz, 1H), 7.29 (t, J = 8.3 Hz, 2H), 7.58 (d, J = 8.1

Hz, 2H), 7.79 (s, 1H), 8.18 (d, J = 7.8 Hz, 1H), 10.04 (s, 1H). ESI-MS

m/z 364.0 (M + H)⁺.

Q

dx.doi.org/10.1021/jm5008209 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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HDAC Inhibition Assay. The newly synthesized thiol derivatives

reported in Tables 1 and 2 were evaluated for their activity against

class I (1−3, 8), IIa (4, 5, 7, 9), IIb (6, 11), and IV (11) HDAC

isoforms. HDAC proﬁling was performed in the presence of a 50 μM

solution of the ﬂuorogenic tetrapeptide RHKK(Ac) substrate (from

p53 residues 379−382) for all isoforms, with the exception of HDAC8,

which was assayed in the presence of a 50 μM solution of its

diacetylated analogue RHK(Ac)K(Ac). Class IIa isoforms were

assayed either applying the standard substrate or in the presence of

a 50 μM solution of the ﬂuorogenic Boc-Lys(triﬂuoroacetyl)-AMC

(Supporting Information Table S2).³⁶

Cell Culture. HCT116 (human colon carcinoma) and NCI-H460

(human nonsmall cell lung carcinoma) cells were obtained from

American Type Culture Collection and were grown, respectively, in

McCoy’s 5A and RPMI-1640 medium containing ^L-glutamine and

supplemented with 10% heat-inactivated fetal calf serum (FCS, Life

Technologies) and 50 μg/mL gentamicin.

(GE Healthcare) and analyzed by a phosphoimaging system (STORM,

Molecular Dynamics). Following densitometry analysis and normal-

ization, the IC₅₀values were ﬁnally calculated by the “ALLFIT”

computer program.

Plasma Stability. Test compounds were added to human plasma to

get a ﬁnal concentration of 5 μM and incubated at 37 °C at diﬀerent

times (until 120 min). Then 0.1 mL of each sample was taken, added

in a corresponding 2-fold amount of chilled acetonitrile, and

centrifuged at +4 °C. The supernatant was then submitted to LC-

MS/MS analysis. At the end of incubation, % of intact compound

remaining was calculated.

Animals. In vivo experiments were carried out using female athymic

nude mice, 5−6 weeks old (Harlan). Mice were maintained in laminar

ﬂow rooms keeping temperature and humidity constant. Mice had free

access to food and water. Experiments were approved by the Ethics

Committee for Animal Experimentation of Sigma-Tau according to

institutional guidelines.

Cell Proliferation Assay. HCT-116 and NCI-H460 tumor cell lines

were grown in a volume of 200 μL, at approximately 10% conﬂuence,

in 96-well Multi-Tier plates and were allowed to recover for an

additional 24 h. Tumor cells were treated with either varying

concentrations of drugs or solvent for 24 h. After treatment, the

plates were washed to remove drug and incubated for further other 48

h. The fraction of cells surviving after compound treatment was

determined using the SRB assay.³⁷IC₅₀was deﬁned as the drug

concentration causing a 50% reduction in cell number compared with

that of vehicle-treated cells and was calculated by the “ALLFIT”

computer program by analyzing dose−response inhibition curves.

Western Blot Analysis for H4-Histone and α-Tubulin Acetylation.

The day before the experiment NCI-H460 NSCLC cells were plated in

complete culture medium. Cells were then treated for 3 h with test

compounds at various concentrations (dose−response curves),

depending on their relative potency. Compound 1 was used as

reference inhibitor. After treatment cells were washed twice with

DPBS supplemented with 5 mM NaB and then were collected and

resuspended in HNB buﬀer (0.5 M sucrose, 15 mM Tris/HCl pH 7.5,

60 mM KCl, 0.25 mM EDTA pH 8, 0.125 mM EGTA pH 8, 0.5 mM

spermidine, 0.15 mM spermine, 1 mM DTT, 0.5 mM PMSF, 5 μg/mL

pepstatin, 5 μg/mL leupeptin, 5 μg/mL aprotinin) supplemented with

1% NP-40. After 5 min of incubation at 4 °C, nuclei were isolated by

centrifugation at 2000g for 3 min. Cytoplasm proteins isolated at this

step were collected and then separated on SDS-PAGE for the analysis

of α-tubulin acetylation.

Histones, acid soluble proteins, were isolated from nuclei by

addition of 0.4 equiv/L H₂SO₄and incubation at 4 °C for 1 h. After

centrifugation, histones were precipitated from the supernatant by

addition of prechilled acetone and incubation overnight at −20 °C and

then air-dried. After solvent removal, proteins were resuspended in

distilled water, and the protein content was determined by the classical

colorimetric Bradford method (Coomassie Bradford protein assay kit;

Pierce, cat. 23200), according to the manufacturer’s instruction.

To assess the extent of acetylation, equal amounts of histones or

cytoplasmic proteins for each sample were resolved by SDS-PAGE and

transferred onto a nitrocellulose membrane (Hybond-C extra,

Amersham-GE Healthcare). Molecular weights were estimated based

upon the relative migration with molecular weight protein markers

(Prestained Kaleidoscope Standards; cat. 161-0324, Bio-Rad). Non-

speciﬁc binding sites were then blocked by incubation of the

membranes with 5% nonfat dry milk in TBS, overnight at 4 °C.

Speciﬁc primary antibodies (rabbit antiacetyl-histone H4 polyclonal

antibody, cat. 06-598, and mouse anti-histone H4 monoclonal

antibody, cat. 07-108, from Upstate; mouse antiacetylated α-tubulin

monoclonal antibody, cat. T6793, mouse anti-α-tubulin monoclonal

antibody, cat. T5168, and mouse anti-β-actin monoclonal antibody,

cat. A5316, from Sigma), were added to the membranes at the optimal

dilution in 5% nonfat dry milk/TBST overnight at 4 °C. Following

four washes in TBST, membranes were incubated for 1 h with HRP-

conjugated secondary antibodies in 5% nonfat dry milk/TBST.

Immunoreactive bands were ﬁnally visualized by enhanced chem-

iluminescence with the ECLplus Western blotting detection reagent

Xenograft Tumor Models. For subcutaneous (sc) tumor model,

exponentially growing tumor cells (5 × 10⁶/100 μL) were sc

inoculated in the right ﬂank of nude mice. Mice were treated starting

3 days after tumor injection. Tumor volume (TV) was calculated

according to the formula: TV (mm³) = d²× D/2 where d and D are

the shortest and the longest diameter, respectively. HDAC inhibitors

were delivered by oral route according to the schedule qdx5/wx3w or

qdx5/wx2w. Carboplatin was given intraperitoneally according to the

schedule q4d/wx3w. Compound 1 was administered by oral route

according to the schedule qdx5w/x2w. Drug eﬃcacy was assessed as

tumor volume inhibition percentage (TVI%) in drug-treated versus

control mice, expressed as TVI% = 100 − [(mean TV treated/mean

TV control) × 100]. Toxic eﬀects of the drug treatment were assessed

as body weight loss percentage (BWL%), calculated as BWL% = 100 −

[(mean BW day x/mean BW day 1) × 100], where day 1 was the ﬁrst

day of treatment and day x was any day thereafter. The highest (max)

BWL% is reported in Table 6.

Molecular Modeling. Molecular modeling studies were conducted

with the Schrodinger software suite. Ligand molecules were built in

Maestro 9.6³⁸and prepared with Ligprep 2.8,³⁹while protein

structures were reﬁned using the Protein Preparation Wizard.⁴⁰

Docking studies were carried out with Glide 6.1^41,42using the SP

scoring function. Default settings were used, unless stated otherwise.

Protein Preparation. The crystal structures of human HDAC3

(PDB 4A69⁴³(chain B)) and HDAC8 (PDB 2V5X⁴⁴(chain A)) were

used for docking studies. The protein C- and N-termini were capped

with acetyl and methylamino groups, respectively, and missing

hydrogen atoms were added. The overall hydrogen bonding network

was optimized by sampling the orientations of hydroxyl and thiol

groups, the conformations of asparagine, glutamine, and histidine side

chains and by adjusting the protonation state of glutamate and

aspartate residues. At the end of the protein preparation, all histidine

residues were in their neutral form except for H142 in HDAC8 and

H134 in HDAC3 which were protonated. Zero-order bonds

connecting the potassium and zinc ions to their coordinating atoms

were built. The resulting structures were submitted to a ﬁrst energy

minimization procedure with the OPLS2005 force ﬁeld⁴⁵in which

only hydrogen atoms were free to move. A second minimization run

was then performed on all atoms, restraining the positions of protein

heavy atoms until a root-mean-square deviation (RMSD) value of 0.3

Å was reached. All crystallized water molecules were removed before

docking studies.

Docking Calculations. Glide grids were built with Glide 6.1 and

were centered on the acetate and inhibitor molecules cocrystallized

with HDAC3 and HDAC8, respectively. The dimensions of bounding

and enclosing boxes were set to 16 and 31 Å, respectively; no van der

Waals scaling was applied to nonpolar protein atoms. The zinc ion in

HDAC8 adopts a tetrahedral coordination geometry to interact with

the thiolate group of the cocrystallized inhibitor.⁴⁶For this reason, a

positional constraint was applied during grids generation to force the

thiol group of docked compounds to occupy the free coordination site

of the tetrahedrally coordinated zinc ion. Ligands were initially

prepared with Ligprep 2.8³⁹to generate suitable protonation states and

R

dx.doi.org/10.1021/jm5008209 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

were then subjected to energy minimization with Macromodel 10.2,⁴⁷

applying the OPLS2005 force ﬁeld to an energy gradient of 0.05 kJ

mol⁻¹Å⁻¹. 100 Poses were collected for each docking run and

subsequently ranked according to their Emodel values. The top-ranked

pose for each ligand was retained and further analyzed.

To allow the accommodation of the benzyloxy-benzyl substituent of

compound 132 in the rim of HDAC8 structure, the side chain of K33

which occludes the binding site was rotated in the same conformation

as that observed in another HDAC8 crystal structure, in complex with

a hydroxamate-based inhibitor (PDB 3F07).⁴⁸

Molecular Dynamics (MD) Simulations. The best-ranked HDAC3-

127 and HDAC3-144 complexes obtained from molecular docking

were submitted to MD simulations. The cocrystallized water molecules

removed prior to docking studies were reintroduced. Resulting

complexes were then solvated with ∼11600 TIP3P water molecules

in a box of 75 × 72 × 79 Å³and neutralized by adding 7 sodium ions.

Systems were ﬁrst equilibrated for 3 ns using a modiﬁed version of the

relaxation protocol implemented in Desmond 3.6.^49,50The equilibra-

tion phase was followed by 20 ns long MD simulations performed at

310 K and 1 atm in the NPT ensemble applying the Langevin coupling

scheme⁵¹and the OPLS2005 force ﬁeld. The M-SHAKE algorithm⁵²

was applied to constrain all bond lengths to hydrogen atoms. Long-

range electrostatic interactions were calculated by applying the Smooth

Particle Mesh Ewald method,⁵³while short-range electrostatic

interactions were cut oﬀ at 9 Å. A RESPA integrator⁵⁴was used

with a time-step of 2 fs, and long-range electrostatics were computed

every 6 fs. Three independent simulations were conducted for each

system using diﬀerent initial random velocity seeds.

midehydrochloride; HDAC, histone deacetylase; HNB, hydrox-

ynaphthol blue; pan-HDACis, pan-HDAC inhibitors; PK,

pharmacokinetics; PMSF, phenylmethylsulfonyl ﬂuoride;

THF, tetrahydrofuran; TFA, triﬂuoroacetic acid; ZBG, zinc-

binding group

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ASSOCIATED CONTENT

* Supporting Information

■

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List of all mentioned compounds; inhibitory activity (IC₅₀) on

class IIa HDAC isoforms; distances monitored during

molecular dynamics simulations; stability and solubility of

thiol prodrugs; cytotoxicity of thiol prodrugs; selectivity proﬁles

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AUTHOR INFORMATION

Corresponding Author

*Phone: +39-0691394441. Fax: +39-0691393638. E-mail:

giuseppe.giannini@sigma-tau.it.

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^⊥For W.C.: Fresenius Kabi, Piazza Maestri del Lavoro 7, I-

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Notes

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ACKNOWLEDGMENTS

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ST7612AA1, a thioacetate-ω(γ-lactam carboxamide) derivative selected from a novel generation of oral HDAC inhibitors

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