Oxime amides as a novel zinc binding group in histone deacetylase inhibitors: Synthesis, biological activity, and computational evaluation

ARTICLE

pubs.acs.org/jmc

Oxime Amides as a Novel Zinc Binding Group in Histone Deacetylase

Inhibitors: Synthesis, Biological Activity, and Computational

Evaluation

Cinzia B. Botta,^‡Walter Cabri,*^{,^}Elena Cini,^†Lucia De Cesare,^||Caterina Fattorusso,*^,||Giuseppe Giannini,^{^}

Marco Persico,^||Antonello Petrella,^‡Francesca Rondinelli,^||Manuela Rodriquez,*^,‡,§Adele Russo,^†and

Maurizio Taddei^†

^†Dipartimento Farmaco Chimico Tecnologico, Universitꢀa degli Studi di Siena, Via A. Moro 2, I-53100 Siena, Italy

^‡Dipartimento di Scienze Farmaceutiche e Biomediche, Universitꢀa di Salerno, Via Ponte don Melillo, I-84084 Fisciano (SA), Italy

^§Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, United

States

Dipartimento di Chimica delle Sostanze Naturali, Universitꢀa di Napoli, Via D. Montesano, 49 I-80131 Napoli, Italy

^{^}Chemistry and Analytical Development, R&D Sigma-Tau S.p.A., Via Pontina, km 30,400 I-00040 Pomezia (RM), Italy

S Supporting Information

b

ABSTRACT: Several oxime containing molecules, characterized by a SAHA-like struc-

ture, were explored to select a potentially new biasing binding element for the zinc in

HDAC catalytic site. All compounds were evaluated for their in vitro inhibitory acti-

vity against the 11 human HDACs isoforms. After identiﬁcation of a “hit” molecule, a

programmed variation at the cap group and at the linker was carried out in order to

increase HDAC inhibition and/or paralogue selectivity. Some of the new derivatives

showed increased activity against a number of HDAC isoforms, even if their overall activity

range is still far from the inhibition values reported for SAHA. Moreover, diﬀerent from

what was reported for their hydroxamic acid analogues the new R-oxime amide derivatives

do not select between class I and class II HDACs; rather they target speciﬁc isoforms in

each class. These somehow contradictory results were ﬁnally rationalized by a computa-

tional assisted SAR, which gave us the chance to understand how the oxime derivatives interact with the catalytic site and justify the

observed activity proﬁle.

’ INTRODUCTION

acid (SAHA) from Merck and depsipeptide (FK228) from

Gloucester Pharmaceuticals, both for use in cutaneous T-cell

lymphoma (CTCL).²Moreover, the prospects of using HDACIs

as therapeutic interventions for diseases other than cancer are

evidenced by the growing number of papers detailing the use of

HDACIs in immunomodulation, neurodegeneration, protozoan

infections, inﬂammatory, and heart diseases.³

The largely accepted pharmacophore model for most of the

known hydroxamic acid derivatives inhibitors consists of (a) a

capping group that interacts with the residues at the active site

entrance (cap), (b) a zinc binding group (ZBG) that coordinates

to the catalytic metal atom within the active site, and (c) a linker

that binds to the hydrophobic channel and helps Cap and ZBG to

ﬁnd the correct position. Although SAHA contains a hydroxamic

acid moiety and is considered a pan-inhibitor, some data suggest

that selectivity toward a speciﬁc HDAC might be useful to ﬁnd

more eﬀective inhibitors with limiting side eﬀects. Modiﬁcations

Histone deacetylases (HDACs) are enzymes involved in the

remodeling of chromatin and have a key role in the epigenetic

regulation of gene expression, as they remove the acetyl moiety

from the ε-amino group of lysine side chains of histone and non-

histone proteins. High levels of histone acetylation are associated

with increased transcriptional activity, whereas low levels of

acetylation are associated with repression of gene expression.

Eighteen HDACs, subdivided into four classes, have been identi-

ﬁed in humans. Class I (HDACs 1, 2, 3, and 8), class IIa (HDACs

4, 5, 7, and 9), class IIb (HDACs 6 and 10), and class IV (HDAC

11) operate as Zn dependent enzymes, have distinct gene

expression patterns, and have diﬀerent cellular location and

function. Inhibition of HDACs causes histone hyperacetylation

with contemporary transcriptional activation of genes associated

with cell cycle arrest or apoptosis in tumor cells. Indeed, several

types of HDAC inhibitors have been found to have potential

anticancer activity with remarkable tumor speciﬁcity.¹A number

of HDAC inhibitors (HDACIs) reached clinical trials, and two of

them were lately approved by FDA: suberoylanilide hydroxamic

Received: October 21, 2010

Published: March 18, 2011

r

2011 American Chemical Society

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Chart 1. SAHA (1) and Oxime Derivatives 2-5

Scheme 1. Synthesis of Keto Oxime Ester 2^a

^aReagents and conditions: (i) (1) Ac₂O, 140 ꢀC, 39%; (2) aniline, THF,

room temp, 74%; (ii) CDI, room temp, THF, CH₂(CO₂Et)₂, Mg-

(OEt)₂, room temp, THF, 91%; (iii) NaNO₂, AcOH, room temp, 80%.

a “hit” molecule, a programmed variation at the cap group and at

the linker was carried out in order to increase the activity and/or

paralogue selectivity. The results were ﬁnally rationalized by a

computational assisted SAR, which gave us the chance to under-

stand how the oxime derivatives interact with the catalytic site

and justify the observed activity proﬁle.

of the Cap, linker, or ZBG have been demonstrated to increase

selectivity.⁴Moreover, hydroxamic acid, a strong zinc chelator,

presents metabolic and pharmacokinetic issues such as glucur-

onidation, sulfation, and enzymatic hydrolysis that result in a

short in vivo half-life.⁵

’ CHEMISTRY

Consequently, there are many ongoing research activities to

ﬁnd replacement groups of hydroxamic acid with the dual target

of reducing side eﬀects and increasing paralogue selectivity.

Many diﬀerent moieties have been proposed as hydroxamic acid

surrogates yielding encouraging results.⁶

Oxime derivatives employed in this study were prepared

following the diﬀerent routes described in Schemes 1-4. Suberic

acid (6) was acetylated with Ac₂O and further reacted with

aniline to generate monoamide 7 (Scheme 1). Activation of the

carboxylic group with CDI followed by reaction with the

magnesium salt of diethyl malonate gave keto ester 8 which

was eventually oxidized with NaNO₂in acetic acid to form keto

oxime ester 2 in good overall yields.

Following our interest in this ﬁeld,⁷we focused on the replace-

ment of the hydroxamic group to ﬁnd some potent non-hydro-

xamate small-molecule HDAC inhibitors and to investigate on

the paralogue selectivity in order to identify key elements for

selective HDAC inhibition. In addition, in terms of chemical

biology research, the discovery of novel ZBGs may lead to new

types of HDAC isozyme selective inhibitors that are useful tools

for probing the biology of the enzyme.⁸

For the synthesis of other oximes correlated to structures 3-5,

commercially available ethyl 7-bromo-heptanoate 9 underwent

hydrolysis, benzylation, and direct halogen exchange, yielding

benzyliodo derivative 10 (Scheme 2). Reaction with diethyl mal-

onate and NaH aﬀorded compound 11 in 88% yield. The benzyl

protection was removed by hydrogenolysis (Pd/C, H₂, 1 atm) to

give acid 12. This compound was the key intermediate for the

synthesis of 3-5 and 14-24, as it enables the introduction of

diﬀerent arylamides at the carboxylic position in order to optimize

the Cap. After transformation of 12 into the corresponding acyl

chloride, reaction with aniline generated compound 13 in overall

80% yield. The amide was then transformed into the corresponding

R-oxime ester 3 with ethyl nitrite and sodium ethoxide. This

transformation passes through the formation of the nitroso deriva-

tive, which is further cleaved by sodium ethoxide to give the sodium

salt of the oxime ester and diethyl carbonate.¹⁴This reaction gave

exclusively one isomer (NMR and HPLC analysis). X-ray analysis of

the crystals obtained from compound 3 showed that the oxime was

in the E conﬁguration (Figure 1SI in Supporting Information).

Since all the other oximes were obtained as single diastereomers, the

conﬁguration of the oxime moiety of all the other compounds was

consequently assigned. The ethyl ester 3 was then transformed into

the corresponding amide 4 or methylamide 5 in good yields by

reaction with aqueous ammonia or methylamine in ethanol respec-

tively. Amides 14-19 were then prepared by coupling of 12 with

the corresponding arylamines followed by transformation of the

malonic ester into the oxime ester and ﬁnal amidation.

Oxime-containing molecules caught our attention, as they

appear to be amenable to biotransformation and conjugations

with organic and inorganic molecules. The properties of these

classes of compounds have been recently exploited with the aim

to design and develop novel therapeutic agents that can display

acyl group transfer capabilities and serve for the evaluation of

novel candidate drugs for the treatment of various diseases. For

example, furan oximes were found to inhibit DNA, RNA, and

protein synthesis in lipoid leukemia cells.⁹Derivatives of quino-

line oximes were also shown to possess antitumor activity,¹⁰and

glucosinolates were suggested as cancer preventive agents. The

stability of oxime complexes with various metals has been shown

to result in promising compounds with antitumor activity, such

as cis and trans platinum complexes¹¹and homo- and hetero-

nuclear Cu(II) and Mn(II) oxime complexes.¹²A complex of

technetium with hexamethylpropyleneamine oxime was also

used to monitor photodynamic therapy of prostate tumors.¹³

On the basis of these considerations, several oxime containing

molecules characterized by a SAHA-like structure were explored

selecting those where a mimetic of the natural substrates chelat-

ing features were possible, such as R-oxime esters and R-oxime

amides (compounds 2-5 in Chart 1). After identiﬁcation of 5 as

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Scheme 2. Synthesis of Oximes 3-5 and 14-24^a

^aReagents and conditions: (i) (1) LiOH, EtOH/THF/H₂O 1:1:1, room temp; (2) BnOH, EDC, DMAP, 0 ꢀC to room temp; (3) NaI, acetone, room

temp, overall 87%; (ii) NaCH(CO₂Et)₂, THF, 0 ꢀC to room temp, 88%; (iii) H₂(1 atm), Pd/C, CHCl₃, 96%; (iv) (1) (COCl)₂, DMF, CH₂Cl₂, room

temp, 2 h; (2) aniline, Et₃N, CH₂Cl₂, 0 ꢀC to room temp, overall 80%; (v) C₂H₅NO₂, NaOEt, EtOH, -5 ꢀC, 92%. (vi) 4: NH₄OH conc, room temp,

90%. 5: MeNH₂(33 wt % in ethanol), room temp, 80%.

Amide 19 was used to introduce a substituent in the meta

position through a Suzuki coupling in order to increase the size of

the cap group and to get more data useful for a comprehensive

SAR study. It was possible to carry out the coupling on the free

oxime amide with a selected catalyst and conditions as reported

in Table 1SI in Supporting Information.

It is interesting to note that the use of microwave dielectric

heating (in combination with Pd(PPh₃)₂Cl₂) provides exclu-

sively compounds 20-22, while for the preparation of amides

23-24 conventional long time heating is required.

Oxime amides 30-33 were prepared by coupling of amino

derivative 29 with arylisoxazolecarboxylic acids¹⁵as reported in

Scheme 3. Commercially available 6-aminohexanol 25 was ﬁrst

transformed into the aldehyde 26 and immediately submitted to

Knoevenagel reaction with diethyl malonate with piperidine/DMF

followed by hydrogenation of the double bond giving 27 in 53%

overall yield. The malonic ester was treated with ethyl nitrite and

sodium ethoxide, aﬀording the R-oxime ester 28. After transformation

of the ethyl ester into the corresponding methylamide, the Boc

protecting group was removed with TFA to give the amine 29 in good

yields. In order to carry out the reaction between the previously

described 5-(3-tert-butoxycarbonylaminophenyl)isoxazole-3-carbox-

ylic acid and 5-(4-tertbutoxycarbonylaminophenyl)isoxazole-3-

carboxylic acid with the amine 29, carrying unprotected oxime

group, several coupling agents were then explored.¹⁵

The most selective reaction occurred using DMTMM¹⁶in the

presence of 1-methylmorpholine, with the formation of com-

pounds 30 and 31 in good yields. Final removal of the Boc with

TFA gave the aniline derivatives 32 and 33 in good yields.

The introduction of a stereocenter (with S conﬁguration)

close to the carboxyamido group was often required in order to

obtain a more potent inhibitor.¹⁷This goal was accomplished

starting from (S)-glutamic acid (Scheme 4) protected as the

tetrabenzyl derivative and further transformed into aldehyde 34

as described in the literature.¹⁸

Treatment with vinylmagnesium bromide gave the allylic

alcohol with contemporary formation of lactone 35 as a mixture

of diastereoisomers. The lactone was opened with aniline in the

presence of AlMe₃and the hydroxyl group acetylated. The allyl

acetate 36 was submitted to a Tsuji-Trost reaction with sodium

malonate in the presence of Pd(PPh₃)₂Cl₂that gave compounds

37a and 37b in a 2:1 ratio. Complete hydrogenation/hydro-

genolysis of the mixture followed by reductive amination

with p-methoxybenzaldehyde in the presence of NaBH(OAc)₃

yielded compound38, which was separated from the corresponding

regioisomer originating from 37b and isolated in 46% overall yield.

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Scheme 3. Synthesis of Oxime Amides 30-33^a

^aReagents and conditions: (i) 1) Boc₂O, Et₃N, DCM, room temp, 12 h, 88%; (2) (COCl)₂, DMSO, Et₃N, CH₂Cl₂, -78 ꢀC, 30 min, 82%; (ii) (1)

CH₂(COOEt)₂DMF, piperidine, room temp, 1 h, 65%; (2) H₂(1 atm), PtO₂, MeOH, room temp, 1 h, 92%; (iii) C₂H₅NO₂, NaOEt, EtOH, -15 ꢀC,

5 h, 61%; (iv) (1) NH₂Me, EtOH, room temp, 12 h, 75%; (2) TFA, DCM, room temp, 12 h, 67%; (v) 5-(3-tert-butoxycarbonylaminophenyl)isoxazole-

3-carboxylic acid and 5-(4-tert-butoxycarbonylaminophenyl)isoxazole-3-carboxylic acid, respectively, DMTMM, NMM, THF, room temp, 12 h.

Scheme 4. Synthesis of Chiral Oxime Amides 40, 41, and 43^a

^aReagents and conditions: (i) see ref 18; (ii) CH₂CHMgBr, THF, 0 ꢀC to room temp, 60%; (iii) (1) aniline, AlMe₃, CH₂Cl₂, 0 ꢀC to room temp, 95%;

(2) Ac₂O, NEt₃, DMAP, CH₂Cl₂, 0 ꢀC to room temp, 92%; (iv) CH₂(CO₂Et)₂, NaH, Pd[PPh₃]₂Cl₂, PPh₃, THF, 0 ꢀC to room temp, 80%; (v) (1) H₂

(1 atm), Pd/C, CH₃OH, room temp; (2) 4-methoxybenzaldehyde, NaBH(OAc)₃, AcOH_cat, CH₂Cl₂, room temp, chromatographic separation, overall

72%; (vi) (1) Boc₂O, NEt₃, H₂O/THF, room temp; (2) C₂H₅NO₂, NaOEt, EtOH, -5 ꢀC, 50%; (vii) (1) MeNH₂(33 wt % in ethanol), room temp,

12 h, 70%; (2) CH₂Cl₂/TFA (4:1), 82%; (viii) H₂(1 atm), PtO₂, CH₃OH, room temp, chromatographic separation 60%; (ix) (1) C₂H₅NO₂, NaOEt,

EtOH, -5 ꢀC, 76%; (2) MeNH₂(33 wt % in ethanol), room temp, 84%.

Attempts to carry out the direct transformation of 38 into the

corresponding oxime ester gave exclusively degradation of the

starting material. Consequently, the amino group was protected

with Boc₂O and compound 39 was then transformed into oxime

amide 41 after deprotection.

Mosher acids conﬁrmed that racemization did not occur along the full

synthetic pathway of chiral oxime amides (Scheme 4).

’ RESULTS AND DISCUSSION

Alternatively, double bond reduction with Pt₂O in MeOH was

carried out in a mixture of 37a and 37b, giving the malonate 42 in

60% yield as a single isomer after column chromatography separation.

On 42 the standard sequence of oximation and transformation of the

ethyl ester into methylamide gave compound 43 in 84% isolated

yields (Scheme 4). HPLC/MS analysis (Chiralpack column) and ¹⁹F

NMR analysis of the amides derived from 41 with (S)- and (R)-

HDAC Isoform Inhibition Assay. All the compounds pre-

pared in this study were evaluated for their in vitro inhibitory

activity against the 11 human HDACs isoforms in order to obtain

a complete potency profile. The inhibitory activity was carried

out as previously described using SAHA (1) as the reference

compound.^17a,19

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Table 1. Activity of r-Oxime Amides against the 11 Human HDAC Isoforms (IC₅₀, μM)^a

^aThe asterisk (/) represents IC₅₀> 100.

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series of SAHA analogues characterized by the R-keto amide

function as ZBG.²⁰Since the HDAC natural substrate also

contains an acetamide moiety, it is likely that a methyl substituted

amide is able to reproduce substrate interaction with the catalytic

site, thus leading to a gain of inhibitory activity (4 vs 5, Table 1).

Although not comparable with SAHA, these data were encoura-

ging, suggesting an effective binding of the R-oxime methylamide

to the catalytic zinc. Consequently, with the aim of increasing

inhibitory activity, different cap groups on the molecular skeleton

of compound 5 were explored (14-24, Scheme 2). Some

derivatives of this series showed increased activity against a

number of HDAC isoforms (20-23, Table 1), even if their

activity range is still far from the inhibition values of the reference

compound. The shape of the steric hindrance around the phenyl

ring was critical for inhibitory activity (compounds 14-19 and

24 vs compounds 20-23, Table 1), while the presence of an acid

or a basic substituent modulated activity (23 > 20 > 22 > 21,

Table 1) and selectivity (21, 22 > 20 > 23, Table 1). Still in the

attempt to increase inhibitory activity, a Cap containing an

arylisoxazole moiety was introduced (30-33, Scheme 3), as this

modification has been described to generate very potent and

selective hydroxamic acid inhibitors.¹⁵Compounds 30 and 31

carrying a hindered N-Boc group did not show any activity,

whereas the m- and p-aniline derivatives 32 and 33 were more

active, the former being the most active compound of the whole

series. Thus, different from what was reported for their hydro-

xamic acid analogues,¹⁵the activity and the selectivity of com-

pounds 30-33 were driven by the steric hindrance at the Cap

region. Finally, to further explore SARs in the Cap, a stereogenic

center carrying a nitrogen atom was introduced, leading to

branched cap groups (40, 41, and 43; Scheme 4). Compound

41 still retained activity on HDACs 1, 3, and 9, while a wider loss

of activity was again observed by further increasing the steric

hindrance at the Cap, as in the case of the N-Boc derivative 40

and the bis-benzyl derivative 43 (Table 1).

Figure 1. 5/Zn^2þcomplexes optimized at DFT level: (a) TC-NO

complex; (b) TT-NN complex; (c) CC-OO complex; (d) CT-ON

complex. Only zinc binding groups are shown for the sake of clarity. The

conformers are colored by atom type [C = (a) yellow, (b) orange, (c)

green, and (d) cyan; H = white; N = blue; O = red; Zn = magenta].

Table 2. Dihedral Angles and ZBG Distances of the r-Oxime

Methylamide/Zn Complex

τ₁(deg)

τ₂(deg)

D₁(Å)

D₂(Å)

DFT

Thus, in the search of new ZBGs, the R-oxime amide function

on one hand showed an overall poorer ability to fulﬁll HDAC

catalytic site pharmacophore compared to hydroxamic acid. On

the other hand, it conferred to our compounds a peculiar

paralogue selectivity proﬁle. In order to rationalize these some-

how contradictory results in terms of HDAC activity and

selectivity, a computational approach was carried out.

TC-NO^a

TT-NN^a

CC-OO^a

CT-ON^a

-0.37^b

-39.13^f

32.17^b

-1.29^c

7.74^c

41.56^h

18.95^h

2.02^d

2.20^g

2.00^d

2.27^g

2.23^e

2.22^e

2.15ⁱ

77.49^f

Docking

90.74^h

17.00^c

32^j

32^k

41

1.75^b

7.84^b

10.49^b

2.63^d

2.47^d

2.68^d

2.49ⁱ

2.99^e

2.49ⁱ

Computational Studies. The accommodation of the ZBG

into the HDAC catalytic site is a crucial step of the inhibition

process and is finely controlled by its zinc coordination ability

and by key interactions with the surrounding protein residues.

First, we analyzed the ability of the R-oxime amide function to

coordinate the zinc atom by using a computational protocol,

which included a dynamic (SA) conformational search followed

by molecular mechanic (MM) and semiempirical (PM6) geo-

metry optimization to generate input structures for calculations

at the density functional theory (DFT) level (see Experimental

Section for details). Our results indicated the putative formation

of several zinc coordination complexes, involving different R-

oxime amide heteroatoms. In particular, our analysis identified

four putative complexes, two of which were characterized by a

five-membered metal coordination ring and two by a six-mem-

bered metal coordination ring, named according to their O_ox-

N_ox-C_ox-C_amand O_am-C_am-C_ox-N_oxdihedral angles values

(C = 0ꢀ; T = 180ꢀ) and zinc coordination atoms (NO = Nox and

Oam; NN = Nox and Nam; OO = Oox and Oam; ON = Oox and

Nam) (Figure 1, Table 2).

86.73^h

^aDFT complexes as deﬁned in the text and Figure 1. ^bC_ox-C_am-O_am-Zn

dihedral angle values. C_am-C_ox-N_ox-Zn dihedral angle values. ^dDis-

c

tance between the O_amand Zn. ^eDistance between the N_oxand Zn. ^fC_ox-

C_am-N_am-Zn dihedral angle values. ^gDistance between N_amand

Zn. C_ox-N_ox-O_ox-Zn dihedral angle values. ⁱDistance between

h

the O_oxand Zn. First set of docking results. ^kSecond set of docking

j

results.

Structure-Activity Relationships (SARs). At first, com-

pounds 2-5 (Chart 1) were tested to select a potentially new

biasing binding element for the zinc in HDAC catalytic site

(namely, ZBG). The R-keto oxime ester function of 2, as well as

the R-oxime ester of 3, was inactive, while the comparison of the

relative activities of compounds 4 and 5 indicated that the ability

of the R-oxime amide function to inhibit HDAC catalytic site is

related to the nature of the substituent on the amide nitrogen

(Table 1). Interestingly, the same SAR trend was reported for a

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conformer, and (d) a ﬁve-membered bidentate N-N interaction

mode, (e) a monodentate interaction mode by the oxime

nitrogen, and (f) a monodentate interaction mode by the amide

nitrogen were considered for the TT-NN DFT conformer (see

Experimental Section for details). All the other DFT conformers

(Figure 1, Table 2) when inserted in the enzyme catalytic site

caused a steric clash with enzyme residues and/or zinc. Each of

the six docking starting structures produced 20 results, which

were selected on the bases of inhibitor conformations (solutions

presenting cis alkyl chain or amide bond were discarded) and

grouped according to enzyme-inhibitor interactions. In parti-

cular, the coordination geometry of the ZBG and the positions of

(i) the hydroxyl group, (ii) the methyl group, (iii) the alkyl chain,

(iv) the H-bond group at the end of the alkyl chain, and (v) the

aromatic Cap have been analyzed and compared to those

reported in all known inhibitors/HDAC X-ray complexes pre-

sent in the Brookhaven PDB (Table 2SI in the Supporting

information). This analysis provided an explanation for the

inhibitory activity proﬁle of our R-oxime amide derivatives on

classes I and IIa HDAC enzymes.

Compound 32 produced two main sets of results (Figures 2a,b

and 3a,b). In the ﬁrst set of docking results (binding mode 1,

Figures 2a and 3a) the ZBG binds His669 through its hydroxyl

hydrogen and interacts with the zinc atom through the amide

oxygen and the oxime oxygen, thus forming a six-membered

metal coordination ring. The resulting coordination geometry is

very diﬀerent from that calculated through DFT methods (CC-

OO complex, Figure 1, Table 2), revealing a reduced zinc binding

ability of the R-oxime amide function when introduced into

HDAC catalytic site. The N-methylamide group reproduces

triﬂuoromethyl ketone (TFMK, PDB code 2VQJ; Table 2SI in

the Supporting information) interactions with Pro667, Gly678,

Phe679, and Gly841 (human HDAC 7 numbering), while the

volume of the aliphatic linker occupies a region similar to that

covered by X-ray determined inhibitors in complex with HDAC

(Figure 3a, Table 2SI in the Supporting information). Finally, the

amide function at the end of the linker interacts with Thr625

through its nitrogen and the aromatic cap group is surrounded by

Cys535, Asp537, Pro617, Cys618 (structural Zn coordination

site; Figures 2a and 3a).

The second set of docking results obtained for compound

32 (binding mode 2, Figures 2b and 3b) still presents the N-

methylamide group positioned in the same enzyme cleft occu-

pied by TFMK (Table 2SI in the Supporting information), as

occurred for binding mode 1. Nevertheless, the R-oxime amide

function is diﬀerently oriented with the hydroxyl hydrogen

binding Asp801 and the amide oxygen and oxime nitrogen

interacting with zinc through a ﬁve-membered ring coordination

geometry (Figure 3b). In this case, the docked structure is similar

to the one obtained by DFT calculations (TC-NO complex,

Figure 1) even if, because of the presence of the protein Zn

coordinators, it showed an increased distance between the oxime

nitrogen and the zinc atom (D2, Table 2). The aromatic amide

function at the end of the linker interacts through an H-bond

with the backbone of Ala808, while the Cap is positioned within

the region Asp801-Gly812 and loop Arg731-Pro739 (named

pocket 2 in Figure 2b). A comparison of this set of docking results

with X-ray determined HDAC inhibitor structures (Table 2SI in

the Supporting information) revealed that the only X-ray deter-

mined HDAC complex in which the ligand projects the cap

region in pocket 2 (Figure 2) is that of APHA in complex with

HDAC 8 (PDB code 3F07). All the other cocrystallized ligands

Figure 2. Overall view of HDAC 7 in complex with compound 32

[binding mode 1 (a) and binding mode 2 (b)] and compound 41 (c).

vdW volumes of the ligands (colored in green) are displayed. Connolly

surface of the enzyme is shown and colored on the base of active site

substructures. The region V623-I628 (blue) and loop H732-S741

(yellow) set up pocket 1. Loop H732-S741 (yellow) and the region

E804-G812 (red) set up pocket 2. The region H531-H544 (orange)

and region E804-G812 (red) set up pocket 3.

All structures presented a planar coordination geometry in

which the atoms involved in zinc coordination tend to lie on the

same plane of the metal. A fragment based search was performed

in the Cambridge Structural Database (CSD) using as query an

R-oxime amide function in complex with zinc, and the structural

analysis of the results (CSD codes BICFUP, CABTUU, GIFSAF,

GEPMET, IXEBIW, LOVQUI, MEHZUU, WASXEV, ZUDL-

UF) conﬁrmed our computational data. Thus, starting from the

calculated zinc coordination geometries (Figure 1), compounds

32 and 41 were introduced into the human HDAC 7 (PDB code

3C0Y) active site and several dynamic docking procedures were

carried out considering either a bi- or a monodentate zinc

interaction mode. In particular, (a) a six-membered bidentate

O-O interaction mode, (b) a monodentate interaction mode by

the amide oxygen, and (c) a monodentate interaction mode by

the oxime oxygen were considered for the CC-OO DFT

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Figure 4. Superimposition of cap groups of low energy conformers of

14 (yellow), 18 (pink), 30 (cyan), and 31 (orange) by ﬁtting the

carbonyl group atoms and the amide and isoxazole nitrogens. Structures

are displayed as sticks (a) and as Connolly surfaces (b). In (a) all

hydrogens, except those of amide function, have been omitted for the

sake of clarity.

mode 2, the R-oxime amide function is better accommodated

in the catalytic site. However, the volume of the alkyl chain of 32

does not ﬁt the volume occupied by any ligand cocrystallized in

complex with HDAC (Figure 2b, Table 2SI in the Supporting

information), thus projecting the Cap in a surface pocket

diﬀerent from that occupied by SAHA and accounting for the

peculiar SARs and selectivity proﬁle of the R-oxime amide

derivatives. Indeed, the oxazole ring of 32 is involved in π-π

interaction with Phe737 (loop Arg731-Pro739) and the aniline

moiety is embedded between Asn736 and Leu765, Asp766 (loop

Gly763-Pro767) (Figure 3b). The side chains of Leu765 and

Asp766 surround the aromatic amine, thus explaining the

inactivity of sterically hindered derivatives, such as 30 and 31

(Scheme 3, Table 1). The presence of the bulky Met274 in

HDAC 8 (Table 3SI in the Supporting information), replacing

HDAC 7 Leu810 (Figure 3b), may account for the inactivity of

32 against this HDAC isoform. Indeed, the less hindered

monophenyl analogue 5 resulted the most active derivative of

the series on HDAC 8 (Table 1). The structural superimposition

of low energy conformers of 5 and 14-24 on low energy

conformers of 30-33 pointed out that the steric bulk that

unfavorably aﬀects their HDAC inhibitory activity (14-19, 24

vs 5, 20-23; 30, 31 vs 32 and 33; Table 1) partially overlays

(Figure 4), suggesting a similar binding mode for the two series

of compounds.

In this view, the complete loss of activity of compound 21

bearing a p-carboxylic function on the phenyl ring (Scheme 2,

Table 1) is in agreement with the presence of a negatively

charged residue (Asp766) on the loop Gly763-Gly770, con-

served in all HDAC isoforms (Table 3SI and Figure 2SI in the

Supporting Information). Moreover, the activity of the mono-

phenyl derivative 5 toward HDAC 3 could be explained by the

diﬀerent amino acid composition of pocket 2 (Figure 2b).

Indeed, only HDAC 3 presents an additional aromatic residue

in this region (Tyr198, human HDAC 3 numbering), which,

replacing HDAC 7 Asn736 (Figure 3b), could establish favorable

π-π interactions with the phenyl ring of 5. On the contrary, all

other HDAC isoforms lack residues able to interact with the Cap

of 5 (Table 3SI in the Supporting information).

In the case of compound 41 all input structures led to only one

set of docking results (Figures 2c and 3c). Compared to 32

compound 41 loses activity against HDAC 7, accordingly; the

strong (ionic) interaction between the protonated nitrogen at

the Cap level and Asp624 in region Pro617-Thr627 (loop 2,

Figure 2c) seems to drive enzyme binding, inducing a distortion

Figure 3. (a) Binding mode 1 of 32 in HDAC 7. (b) Binding mode 2 of

32 in HDAC 7. (c) Docked complex of 41/HDAC 7. HDAC 7 active site

substructures are colored: orange (D537-R547); blue (P617-T627);

yellow (loop R731-P739); red (D801-G812). Carbon atoms of the

ligands are colored in green. vdW volumes of catalytic and structural zinc

(magenta) are displayed. Heteroatoms are colored by atom type (O =

red; N = blue). Hydrogen bonds are highlighted by green dashed lines.

Hydrogens and water molecules are omitted for clarity except those

involved in hydrogen bond interactions.

occupy the regions between loop Arg731-Pro739 and region

Pro617-Thr627 (human HDAC 7 numbering; pocket 1 in

Figure 2) and/or between loop region Asp537-Arg547 and loop

Asp801-Gly812 (human HDAC 7 numbering; pocket 3 in

Figure 2). Interestingly, similar to 32, APHA is characterized

by a lower (i.e., in the micromolar range) in vitro HDAC

inhibition constant²¹compared to other classes of known

inhibitors, including SAHA. Thus, with adoption of binding

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in 41 alkyl chain conformation and reducing ZBG accommoda-

tion in the catalytic site. The zinc atom in the catalytic site is

coordinated through the amide oxygen and the oxime oxygen, as

in the case of binding mode 1 of 32, presenting a six-membered

metal coordination geometry, which was very diﬀerent from that

calculated through DFT methods (CC-OO complex, Figure 1,

Table 2). The amide methyl group of 41 accommodates in the

cleft formed by Pro667, Phe679, and Gly841, reproducing

HDAC cocrystallized ligand positioning, as was also for com-

pound 32 in both sets of docking results. Moreover, the

threonine residue present in HDAC 7 (Thr625) on loop 2, just

after Asp624, hinders the accommodation of the branched cap

group of 41. This residue is replaced by a negatively charged

residue in HDACs 1 and 3 (Glu98 and Asp92, respectively). This

could account for the higher inhibitory activity of 41 toward

these enzyme isoforms and for the complete lack of activity of

compounds 40 and 43, bearing a bulky N-Boc group and a benzyl

group on the amine nitrogen, respectively (Scheme 4, Table 1).

The p-OMe phenyl ring is accommodated in the same region

(pocket 2 in Figure 2c) occupied by the 32 cap group in the

second set of docking results (Figure 2b). On the other hand, the

phenyl ring of 41 interacts with region Asp537-Arg547 (loop 1;

Figures 2c and 3c) similar to 32 Cap, according to the binding

mode obtained in the ﬁrst set of docking results (Figure 2a).

The hypothesized binding modes also relate with the low

activity of all our derivatives toward HDAC 8. Indeed, in this

isoform, HDAC 7 Pro667 and Pro542 (Figure 3) are replaced by

Trp141 and Lys33, respectively (Table 3SI in the Supporting

information), thus preventing the accommodation of the R-

oxime amide group in the catalytic site. On the other hand, the

lack of activity against HDAC 4 can be due to the presence of

Pro809, corresponding to Ala676 in HDAC 7 (Table 3SI in the

Supporting information), which limits the structural ﬂexibility of

a loop undergoing a signiﬁcant conformational change during

our docking calculations (Ala676-Cys680 loop, rms value on all

CR atoms of 2.6 Å).

In summary, our computational results illustrated how the R-

oxime amide moiety scarcely adapts into HDAC catalytic site,

being not able to reproduce any known Zn binding mode except

for the position of the amide methyl group, which indeed was

critical for the inhibitory activity (4 vs 5, Table 1). The posi-

tioning of the ZBG aﬀects the placement of the Cap on the

enzyme surface pockets, whose diﬀerent amino acid composition

among HDAC isoforms is mainly responsible for inhibitor

selectivity. Accordingly, the introduced modiﬁcations at the

Cap of our R-oxime amide derivatives did not produce the same

HDAC inhibitor proﬁle reported for their hydroxamic acid

analogues.

The rationalization of the peculiar binding proﬁle of oxime

derivatives provides us with a useful tool to continue our

investigation on HDAC inhibitors. Indeed, the information on

the mutual relation between ZBG and Cap positioning will be

exploited to sample the enzyme pockets at the entrance of the

channel where isoform selectivity stands. Moreover, our study

disclosed interesting insights into the molecular determinants for

ZBG accommodation into the catalytic site, which will be further

explored through the synthesis of new optimized derivatives.

inhibitors, R-oxime amide derivatives show in vitro inhibitory

activity in the micromolar range and a peculiar paralogue

selectivity proﬁle. Diﬀerent from results observed for some class

I selective inhibitors, our R-oxime amides do not select between

class I and class II HDAC; rather they target speciﬁc isoforms in each

class. Our computational results indicate that despite its ability to

coordinate the zinc ion, the R-oxime amide function of compounds

32 and 41 was able to reproduce known inhibitor-substrate ZBG

interactions with the HDAC catalytic site. The unusual accom-

modation of the ZBG was also critical for the positioning of the

linker and the projection of the Cap toward the diﬀerent surface

pockets of the enzyme. Accordingly, the SARs of the new

derivatives diﬀer from those of other classes of active com-

pounds. The rationalization of the activity proﬁle of the new

oxime amide derivatives lays the base for future structural

modiﬁcations.

’ EXPERIMENTAL SECTION

Chemistry. Reagents were purchased from commercial suppliers

and used without further purification. Anhydrous reactions were run

under (a positive pressure) dry N₂. Flash chromatography employed

Merck silica gel 60 (23-400 mesh). Microwave dielectric heating was

applied using a CEM Discover microwave oven for chemical synthesis.

NMR spectra were acquired at 300 K using Bruker AC200F and Bruker

Advance DPX400 spectrometers. Data are reported as chemical shifts (δ

ppm), multiplicity (s = singlet, bs = broad singlet, d = doublet, dd =

doublet of doublets, t = triplet, m = multiplet), coupling constants (J,

Hz), and relative integral. Chemical shifts are reported relative to

tetramethylsilane at 0.00 ppm. Mass spectral (MS) data were obtained

using an Agilent 1100 LC/MSD VL system (G1946C) with a 0.4 mL/

min flow rate using a binary solvent system of 95:5 methanol/water. UV

detection was monitored at 254 nm. Mass spectra were acquired either in

positive or in negative mode scanning over the mass range of 105-1500.

Microwave irradiations were conducted using a CEM Discover synthesis

unit (CEM Corp., Matthews, NC). Elemental analyses were performed

on a Perkin-Elmer PE 2004 elemental analyzer, and the data for C, H,

and N are within 0.4% of the theoretical values. The chemical purity of

the target compounds was determined using the following conditions:

an Agilent 1100 series LC/MS with a EC 125/4.6 NUCLEODUR 100-5

C18 reversed phase column. The binary solvent system (A/B) was as

follows: 0.1% TFA in water (A) and acetonitrile (B). The absorbance

was detected at 254 nm, and the flow rate was 1 mL/min. The purity of

each compound was g95% in either analysis.

Ethyl 2-(Hydroxyimino)-3,10-dioxo-10-(phenylamino)de-

canoate (2). β-Keto ester 8 (0.207 g, 0.649 mmol) was dissolved in

glacial acetic acid (5 mL) and cooled to 0 ꢀC in ice bath. A solution of

NaNO₂(0.089 g, 1.30 mmol) in H₂O (2 mL) was added. The reaction

mixture was allowed to warm to room temperature and stirred overnight.

Dry Et₂O was added, and the organic layer was separated, washed with

saturated NaHCO₃, dried (anhydrous Na₂SO₄), filtered, and concen-

trated in vacuo. The crude residue was purified by flash chromatography

(99:1 dichloromethane/methanol) to afford the title compound 2 as a

colorless oil (0.180 g, 80%). ¹H NMR (400 MHz, CDCl₃): δ 1.3 (t, J =

6.8 Hz, 3H), 1.34 (m, 4H), 1.59 (m, 2H), 1.72 (m, 2H), 2.3 (t, J = 7.6 Hz,

2H), 2.71 (t, J = 6.8 Hz, 2H), 4.31 (q, J = 7.2 Hz, 2H), 7.05 (t, J= 8.8 Hz,

1H), 7.25 (m, 2H), 7.5 (2H, J = 8 Hz, 2H). MS(ESI) m/z: 371.1 [M þ

Na]^þ. Anal. Calcd for C₁₈H₂₄N₂O₅: C, 63.73; H, 7.55; N, 8.74. Found:

C, 63.93; H, 7.49; N, 8.69.

General Procedure To Convert Alkylated Malonic Ester to

r-Oxime Ester. Ethyl 2-(Hydeoxyimino)-9-oxo-9, 8-phenyla-

mino)nonanoate (3). Diester 13 (0.473 g, 1.30 mmol) was placed in

a 50 mL flask equipped with a magnetic stirring bar and N₂inlet. The

’ CONCLUSIONS

Adopting a diﬀerent binding mode with respect to that

reported for hydroxamic acid and substrate-like HDAC

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flask was immersed in an ice bath, and a solution of ethyl nitrite in

ethanol (1.31 mL, 2.61 mmol) was added to the stirred solution. The

mixture was then cooled to -5 ꢀC in ice-salt bath, and 1.31 mmol of

sodium ethoxide (prepared from 0.030 g of sodium in 2 mL of dry

ethanol) was added slowly with stirring. The resulting reaction mixture

was allowed to warm to room temperatue during the night. The solvent

was concentrated under reduced pressure. To the residue equal volumes

of ice-water and cold concentrated hydrochloric acid were added until

pH 4 was obtained. The aqueous solution was extracted three times with

diethyl ether. The combined ether extracts were dried (anhydrous

Na₂SO₄), filtered, and evaporated to dryness. The crude was purified

by flash chromatography (100% dichloromethane) to afford the title

compound 3 as a white solid (0.384 g, 92%). Mp 118-120 ꢀC. ¹H NMR

(400 MHz, CDCl₃): δ 1.3 (t, J = 7.2 Hz, 3H), 1.35 (m, 4H), 1.52

(m, 2H), 1.70 (m, 2H), 2.32 (t, J = 3.8 Hz, 2H), 2.59 (t, J = 7.2 Hz, 2H),

4.28 (q, J = 7.2 Hz, 2H), 7.05 (t, J = 8.8 Hz, 1H), 7.25 (m, 2H), 7.5 (d,

J = 8 Hz, 2H). MS(ESI) m/z: 343.1 [M þ Na]^þ. Anal. Calcd for

C₁₇H₂₄N₂O₄: C, 63.73; H, 7.55; N, 8.74. Found: C, 63.79; H, 7.51;

N, 8.70.

7.25 (m, 2H), 7.5 (d, J = 8.0 Hz, 2H). MS(ESI) m/z: 272.1 [M þ Na]^þ.

Anal. Calcd for C₁₄H₁₉NO₃: C, 67.45; H, 7.68; N, 5.62. Found: C, 67.40;

H, 7.65; N, 5.60.

Ethyl 3,10-Dioxo-10-(phenylamino)decanoate (8). To a

solution of 7 (1.08 g, 4.36 mmol) in anhydrous THF (25 mL) was

added carbonyldiimidazole (0.777 g, 4.79 mmol) at room temperature.

The reaction mixture was stirred for 6 h. Magnesium salt of monoethyl

malonate (prepared from 4.03 g of monoethyl malonate and 1.74 g of

Mg(OEt)₂) was added, and the resulting mixture was stirred overnight.

After concentration under reduced pressure, the residue was purified by

flash chromatography (petroleum ether/diethyl ether 1:9) to afford 8

(1.27 g, 91%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃): δ 1.25 (t,

J = 6.8 Hz, 3H), 1.34 (m, 4H), 1.58 (m, 2H), 1.68 (m, 2H), 2.3 (t, J =

7.6 Hz, 2H), 2.49 (t, J = 6.8 Hz, 2H), 3.4 (s, 2H), 4.15 (q, J = 7.2 Hz, 2H),

7.05 (t, J = 8.8 Hz, 1H), 7.25 (m, 2H), 7.5 (d, J = 8 Hz, 2H). MS(ESI)

m/z: 342.1 [M þ Na]^þ. Anal. Calcd for C₁₈H₂₅NO₄: C, 67.69; H, 7.89;

N, 4.39. Found: C, 67.72; H, 7.84; N, 4.34.

Benzyl 7-Iodoheptanoate (10). To a solution of ethyl 7-bro-

moheptanoate (1.3 g, 6.3 mmol) in THF/H₂O/EtOH 1:1:1 (15 mL),

LiOH (0.343 g, 8.19 mmol) was added. The reaction mixture was stirred

for 3 h. The solvent was removed, and the residue was dissolved in

CH₂Cl₂. The reaction mixture was acidified with 4 M HCl until pH 1 was

obtained. The organic layer was separated, washed with NaCl_ss, dried

over sodium sulfate, filtered, and concentrated in vacuo. The crude was

submitted to the next step without additional purification (98%). The

crude 7-bromoheptanoic acid (1.3 g, 6.3 mmol) and DMAP (0.077 g,

0.63 mmol) were then added to a solution of benzyl alcohol (1.3 mL,

12.6 mmol) and EDC (2.4 g, 12.6 mmol) in CH₂Cl₂(3 mL) at 0 ꢀC. The

resulting mixture was stirred at room temperature for 7 h. Then it was

poured into water and extracted with CH₂Cl₂ꢀ 3. The organic layer was

washed with saturated aqueous NaHCO₃ꢀ 2, water, and brine, dried

(anhydrous Na₂SO₄), filtered, and concentrated. The crude was purified

by flash column chromatography (petroleum ether/ethyl acetate 9:1) to

afford the benzyl 7-bromoheptanoate as a colorless oil (91%). A solution

of this bromide (0.3 g, 1.0 mmol) in acetone (1 mL) was added to a

solution of NaI (0.45 g, 3.01 mmol) in acetone (1 mL), and the resulting

mixture was stirred overnight (18 h) at room temperature. The pre-

cipitate NaBr was filtered off, and the filtrate was concentrated under

reduced pressure. The pulpy yellow residue was purified by flash chro-

matography (petroleum ether/ethyl acetate 9:1) to yield iodide 10

(0.336 g, 97%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃): δ 1.49-

1.22 (m, 4H), 1.70-1.55 (m, 4H), 2.29 (t, J = 7.6 Hz, 2H), 3.41 (t, J = 6.8

Hz, 2H), 5.06 (s, 2H), 7.29-7.24 (m, 5H). ¹³C NMR (100 MHz,

CDCl₃): δ 24.3, 26.0, 27.8, 33.9, 33.6, 44.4, 65.5, 127.7 (ꢀ3), 128.05

(ꢀ2), 135.8, 172.6. MS(ESI) m/z: 368.7 [M þ Na]^þ

8-(Hydroxyimino)-N¹-phenylnonanediamide (4). To a solu-

tion of 3 (0.050 g, 0.16 mmol) in ammonium hydroxide solution (28-

30%, 5 mL) a catalytic amount of ammonium chloride was added. The

resulting mixture was stirred at room temperature for 18 h. The reaction

mixture was diluted with ethyl acetate, and the organic layer was

separated, dried (anhydrous Na₂SO₄), filtered, and concentrated in

vacuo. The crude was purified by flash chromatography (ethyl acetate/

petroleum ether 6:4) to afford the desired compound 4 as a yellow solid

(0.042 g, 90%). Mp 134-136 ꢀC. ¹H NMR (400 MHz, CD₃OD): δ 1.36

(m, 4H), 1.48 (m, 2H), 1.66 (m, 2H), 2.32 (t, J = 3.8 Hz, 2H), 2.52 (t, J =

7.2 Hz, 2H), 7.05 (t, J = 8.8 Hz, 1H), 7.25 (m, 2H), 7.5 (d, J = 8 Hz, 2H).

MS(ESI) m/z: 314.1 [M þ Na]^þ. Anal. Calcd for C₁₅H₂₁N₃O₃: C,

61.84; H, 7.27; N, 14.42. Found C, 61.79; H, 7.23; N, 14.40.

General Procedure To Convert the Oximino Ester to

r-Oxime Amide. 2-(Hydroxyimino)-N¹-methyl-N⁹-phenylno-

nanediamide (5). Compound 3 (0.170 g, 0.302 mmol) was dissolved

in 7 mL of methylamine solution (33 wt % in ethanol), and the reaction

mixture was stirred at room temperature overnight. The solvent was

evaporated to dryness and the residue purified by flash chromatography

(dichloromethane/methanol 95:5) to afford the desired compound 5 as a

1

white solid (0.074 g, 80%). Mp 138-140 ꢀC. H NMR (200 MHz,

CDCl₃): δ 1.34 (m, 4H), 1.50 (m, 2H) 1.72 (m, 2H), 2.24 (t, J = 6.8 Hz,

2H), 2.7 (t, J = 7.2 Hz, 2H), 2.75 (s, 3H), 7.03 (t, J = 8.8 Hz, 1H), 7.13

(m, 2H), 7.48 (d, J = 8 Hz, 2H). MS(ESI) m/z: 328.2 [M þ Na]^þ. Anal.

Calcd for C₁₆H₂₃N₃O₃: C, 62.93; H, 7.59; N, 13.76. Found: C, 62.88; H,

7.55; N, 13.73.

7-Benzyl 1,1-Diethylheptane-1,1,1-tricarboxylate (11). NaH

(1.05 g, 26.2 mmol, 60% dispersion in oil) was added to a two-

necked round-bottom flask closed with a septum to which dry hexane

(4 mL) was added, and the mixture was stirred for 5 min to dissolve the

mineral oil. The hexane was removed by a syringe, THF (10 mL) was

added, and the resulting mixture was stirred at 0 ꢀC for 15 min. A

solution of diethyl malonate (6.7 mL, 44 mmol) in THF (12 mL) was

then slowly added over 30 min at 0 ꢀC to the resulting suspension. The

solution became clear after 10 min of stirring. The stirring was continued

for another 30 min. Iodide 10 (5.05 g, 14.6 mmol) in THF (15 mL) was

added dropwise over 30 min to the stirred solution, and this mixture was

stirred at room temperature for 4 h. The reaction mixture was quenched

with NH₄Cl_ss. The aqueous phase was extracted three times with ether,

and the combined organic phases were dried (anhydrous Na₂SO₄),

filtered and evaporated in vacuo. The crude residue was purified by flash

chromatography (hexanes/ethyl acetate 9:1) to afford 11 as a colorless

oil (4.84 g, 88%). ¹H NMR (200 MHz, CDCl₃): δ 1.23 (t, J = 6.9 Hz,

6H), 1.30 (m, 4H), 1.7 (m, 4H), 1.87 (m, 2H), 2.3 (t, J = 3.8 Hz, 2H),

3.30 (t, J = 7.2 Hz, 1H), 4.20 (q, J = 6.8 Hz, 4H), 5.09 (s, 2H), 7.05 (t, J =

8-Oxo-8-(phenylamino)octanoic Acid (7). Acetic anhydride

(25 mL) was placed in a 250 mL flask equipped with a magnetic stirrer

and a refrigerator and warmed to 180 ꢀC. While the mixture was

refluxing, suberic acid (10 g, 57 mmol) was added. The resulting solution

was warmed for 2 h and then concentrated. After concentration, the

product was crystallized from DCM. After drying the product weighed

3.1 g and a second crop was obtained from the mother liquor to obtain

the intermediate anhydride in 39% of combined yield. To a solution of

acetylated suberic acid in anhydrous THF (20 mL) was added aniline

(0.56 mL, 6.2 mmol) at room temperature. The reaction mixture was

stirred for 2 h and then treated with 150 mL of H₂O and 3 g of K₂CO₃.

The basic resulting aqueous solution was stirred for 20 min and then

extracted three times with EtOAc. The combined organic phases

contained only the secondary product bis-anilide, whereas the aqueous

phase was treated with HCl until pH 3-4 was obtained. The mono-

anilide 7, precipitating during the night at 4 ꢀC, was filtered and dried

over P₂O₅(74% yield, the product is a white solid). Mp 114-116 ꢀC. ¹H

NMR (200 MHz, CDCl₃): δ 1.29 (m, 4H), 1.55 (m, 2H), 1.65 (m, 2H),

2.30 (t, J = 3.8 Hz, 2H), 2.35 (t, J = 3.8 Hz, 2H), 7.05 (t, J = 8.8 Hz, 1H),

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8.8 Hz, 1H), 7.25 (m, 2H), 7.5 (d, J =8.0 Hz, 2H). ¹³C NMR (50 MHz,

CDCl₃): δ 13.7, 24.5, 26.9, 28.3, 28.4, 28.5, 33.8, 41.3, 51.6, 60.9 (ꢀ2),

61.1, 65.7, 127.6, 127.6, 128.2, 135.6, 166.3, 169.1 (ꢀ2), 173.1. MS(ESI)

m/z: 386.1 [M þ Na]^þ. Anal. Calcd for C₂₁H₃₀O₆: C, 66.65; H, 7.99.

Found: C, 66.60; H, 7.95.

9-Ethoxy-8-(ethoxycarbonyl)-9-oxononanoic Acid (12).

Pd-C (0.32 g, 10% in mmol, 10% w/w) was added to a solution of

11 (0.993 g, 2.6 mmol) in CHCl₃(30 mL), and the mixture was stirred

under H₂(1 bar) for 15 h. After full conversion, the reaction mixture was

filtered and concentrated in vacuo. Flash chromatography (petroleum

ether/ethyl acetate 7:3) gave 12 (96%) as a colorless oil. ¹H NMR (400

MHz, CDCl₃): δ 1.21 (t, J = 7.2 Hz, 6H), 1.57 (m, 2H), 1.29 (m, 6H),

1.83-1.82 (m, 2H), 2.28 (t, J = 7.2 Hz, 2H), 3.25 (t, J = 7.6 Hz, 1H), 4.15

(q, J = 7.2 Hz, 4H). ¹³CNMR (100 MHz, CDCl₃) δ: 13.6 (ꢀ2), 24.0,

26.6, 28.2, 28.2, 28.4, 33.5, 51.5, 60.8 (ꢀ2), 169.1 (ꢀ2), 179.5. MS(ESI)

m/z: 310.8 [M þ Na]^þ. Anal. Calcd for C₁₄H₂₄O₆: C, 58.32; H, 8.39.

Found: C, 58.27; H, 8.35.

General Amidation Reaction Procedure via Acyl Chloride

Intermediate. Diethyl 2-(7-Oxo-7-(phenylamino)heptyl)ma-

lonate (13). To a suspension of 12 (0.3 g, 1.04 mmol) in CH₂Cl₂

(2.5 mL) were added oxalyl chloride (0.13 mL, 1.5 mmol) and a catalytic

amount of DMF. The mixture was stirred at room temperature for 2 h.

The solvent was removed by evaporation in vacuo to give acid chloride

intermediate. A solution of this chloride in CH₂Cl₂(1.3 mL) was added

dropwise to a solution of aniline (0.53 g, 5.7 mmol) and triethylamine

(0.43 mL, 3.12 mmol) in CH₂Cl₂(3.2 mL) cooled in an ice-water bath.

The mixture was stirred at room temperature for 18 h. Then it was

diluted with CH₂Cl₂and washed with HCl 1 M, water, and brine, before

being dried over anhydrous Na₂SO₄. Filtration and concentration in

vacuo and purification by flash chromatography (petroleum ether/ethyl

acetate 8:2) gave the title compound 13 as a white solid (80%). Mp 65-

67 ꢀC. ¹H NMR (400 MHz, CDCl₃): δ 1.23 (t, J = 6.8 Hz, 6H), 1.33

(m, 6H), 1.67 (m, 2H), 1.85 (m, 2H), 2.28 (t, J = 7.2 Hz, 2H), 3.27 (t, J =

7.2 Hz, 1H), 4.14 (q, J = 6.8 Hz, 4H), 7.04 (m, 1H), 7.24 (t, J = 7.6 Hz,

2H), 7.48 (d, J = 8.0 Hz, 2H), 7.68 (bs, 1H). ¹³C NMR (100 MHz,

CDCl₃): δ 14.06 (ꢀ2), 25.2, 26.9, 29.1, 28.5, 28.7, 37.3, 51.9, 61.3 (ꢀ2),

119.9 (ꢀ2), 124.0, 128.9 (ꢀ2), 138.1, 169.5 (ꢀ2), 171.5. MS(ESI) m/z:

371.8 [M þ Na]^þ. Anal. Calcd for C₂₀H₂₉NO₅: C, 66.09; H, 8.04; N,

3.85. Found: C, 66.01; H, 8.00; N, 3.83.

R-oxime ester using the procedure described for 5 in 70% yield as a

yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 1.42-1.19 (m, 4H), 1.47-

1.43 (m, 2H), 1.66-1.60 (m, 2H), 2.22 (t, J = 7.6 Hz, 2H), 2.50 (t, J = 7.2

Hz, 2H), 2.73 (s, 3H), 6.14 (bs, 1H), 7.26-7.22 (m, 2H), 7.35-7.31

(m, 2H), 7.48 (d, J = 7.2 Hz, 2H), 7.62 (d, J = 7.2 Hz, 2H). ¹³C NMR

(50 MHz, CDCl₃): δ 23.1, 25.6, 28.5, 28.9, 36.6 (ꢀ2), 54.5, 119.9 (ꢀ3),

125.0 (ꢀ4), 127.7 (ꢀ2), 128.6 (ꢀ2), 140.5 (ꢀ2), 144.3 (ꢀ2), 174.7.

MS(ESI) m/z: 392.1 [M - H]^-. Anal. Calcd for C₂₃H₂₇N₃O₃: C, 70.21;

H, 6.92; N, 10.68. Found; C, 70.14; H, 6.90; N, 10.65.

N⁹-(4-(Dimethylamino)phenyl)-2-(hydroxyimino)-N¹-me-

thylnonanediamide (17). Compound 17 was prepared from the

corresponding R-oxime ester using the procedure described for 5 in 87%

yield as a yellow oil. ¹H NMR (400 MHz, CD₃OD): δ 1.36 (m, 4H),

1.48 (m, 2H), 1.65 (m, 2H), 2.29 (t, J = 7.5 Hz, 2H), 2.53 (t, J = 7.2 Hz,

2H), 2.7 (s, 3H), 2.85 (s, 6H), 6.72 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 8.8

Hz, 2H). ¹³C NMR (50 MHz; CDCl₃): δ 24.2, 26.1, 26.9, 30.0, 30.4,

37.8, 41.4 (ꢀ2), 114.5 (ꢀ2), 123.0 (ꢀ2), 130.0, 149.5, 150.2, 155.1,

166.9, 174.3. MS(ESI) m/z: 370.8 [M þ Na]^þ. Anal. Calcd for

C₁₈H₂₈N₄O₃: C, 62.05; H, 8.10; N, 16.08. Found: C, 61.98; H, 8.07;

N, 16.01.

N⁹-([1,1⁰-Biphenyl]-4-yl)-2-(hydroxyimino)-N¹-methylno-

nanediamide (18). Compound 18 was prepared from the corre-

sponding R-oxime ester using the procedure described for 5 in 52% yield

as a white solid. Mp 177-180 ꢀC. ¹HNMR (400 MHz, CD₃OD): δ 1.36

(m, 4H), 1.47 (m, 2H), 1.67 (m, 2H), 2.33 (t, J = 7.6 Hz, 2H), 2.52 (t, J =

7.2 Hz, 2H), 2.76 (s, 3H), 7.25 (t, J = 7.2 Hz, 1H), 7.35 (t, J = 7.2 Hz,

2H), 7.50 (t, J = 7.6 Hz, 4H), 7.57 (d, J = 7.6 Hz, 2H). ¹³C NMR (100

MHz, CDCl₃/CD₃OD) δ: 22.6, 24.8, 25.1 (ꢀ2), 28.3, 28.7, 36.4, 119.7,

120.0, 125.9, 126.2, 126.4, 126.5, 126.8 (ꢀ2), 128.0, 136.3, 137.4, 140.1,

153.4, 164.9, 172.9. MS(ESI) m/z: 403.8 [M þ Na]^þ. Anal. Calcd for

C₂₂H₂₇N₃O₃: C, 69.27; H, 7.13; N, 11.02. Found: C, 69.20; H, 7.10;

N, 10.99.

N⁹-(3-Bromophenyl)-2-(hydroxyimino)-N¹-methylnona-

nediamide (19). Compound 19 was prepared from the corresponding

R-oxime ester using the procedure described for 5 in quantitative yield as

a white solid. Mp 142-145 ꢀC. ¹H NMR (400 MHz, CD₃OD): δ 1.35

(m, 4H), 1.47 (m, 2H), 1.66-1.64 (m, 2H), 2.32 (t, J = 7.2 Hz, 2H),

2.53 (t, J = 6.8 Hz, 2H), 2.69 (s, 3H), 7.17-7.13 (m, 2H), 7.43-7.41

(m, 1H), 7.85 (bs, 1H). ¹³C NMR (100 MHz, CD₃OD): δ 22.4, 24.4,

24.8, 25.1, 28.2, 28.6, 36.2, 117.7, 121.4, 122.0, 129.5, 125.9, 139.7, 153.3,

165.1, 172.9. MS(ESI) m/z: 405.7 [M þ Na]^þ. Anal. Calcd for

C₁₆H₂₂BrN₃O₃: C, 50.01; H, 5.77; Br, 20.79; N, 10.94. Found: C,

49.95; H, 5.74; N, 10.90.

2-(Hydroxyimino)-N¹-methyl-N⁹-(naphthalene-2-yl)non-

anediamide (14). Compound 14 was prepared from the correspond-

ing R-oxime ester using the procedure described for 5 in 76% yield as a

yellow solid. Mp 153-155 ꢀC. ¹H NMR (400 MHz, CD₃OD): δ 1.57-

1.23 (m, 9H), 1.72-1.67 (m, 2H), 2.39 (t, J = 7.2 Hz, 2H), 2.54 (t, J = 7.6

Hz, 2H), 2.75 (s, 3H), 7.42-7.30 (m, 2H), 7.52 (d, J = 8.8 Hz, 1H),

7.77-7.72 (m, 3H), 8.17 (s, 1H). ¹³C NMR (50 MHz, CD₃OD): δ 22.4,

24.5, 24.9, 25.0, 28.1, 28.5, 28.7, 36.2, 115.9, 119.4, 123.9, 125.4, 126.6,

127.5, 129.9, 133.2, 135.3, 153.1, 165.0, 172.9. MS(ESI) m/z: 355.8

[M þ H]^þ. Anal. Calcd for C₂₀H₂₅N₃O₃: C, 67.58; H, 7.09; N, 11.82.

Found: C, 67.50; H, 7.04; N, 11.80.

General Procedure of Suzuki Coupling with Pd[PPh₃]₂Cl₂

as Catalyst. N⁹-([1,1⁰-Biphenyl]-3-yl)-2-(hydroxyimino)-N¹-

methylnonanediamide (20). To a solution of 19 (0.026 g, 0.068

mmol) in THF (2 mL), phenylboronic acid (0.025 g, 0.204 mmol),

K₂CO₃(0.056 g, 0.41 mmol), and Pd(PPh₃)₂Cl₂(0.002 g, 0.034 mmol)

were added. The suspension obtained was submitted to microwave

dielectric heating 2 ꢀ 20 min at 110 ꢀC at 200 W. H₂O was used for

quenching, and the organic layer was separated. The aqueous phase was

extracted several times with ethyl acetate; the organic fractions were

collected and dried over anhydrous Na₂SO₄. The solvent was then

removed in vacuo. The residue was purified by flash chromatography

(petroleum ether/ethyl acetate 4:6) to give the title compound 20 as a

2-(Hydroxyimino)-N¹-methyl-N⁹-(quinolin-3-yl)nonaned-

iamide (15). Compound 15 was prepared from the corresponding

R-oxime ester using the procedure described for 5 in 80% yield as a white

solid. Mp 208-210 ꢀC. ¹H NMR (400 MHz, CD₃OD): δ 1.22 (m, 4H),

1.34 (m, 2H), 1.57 (m, 2H), 2.26 (t, J = 7.2 Hz, 2H), 2.39 (t, J = 7.6 Hz,

2H), 2.64 (s, 3H), 7.36 (t, J = 7.6 Hz, 1H), 7.45 (t, J = 6.8 Hz, 1H), 7.63

(d, J = 8 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 8.61 (bs, 2H). ¹³C

NMR (50 MHz; CDCl₃): δ 22.9, 25.1, 25.4 (ꢀ2), 28.4, 28.9, 36.7,

124.1, 127.0, 127.5 (ꢀ2), 128.1, 128.2, 132.3, 143.7, 143.8, 153.7,

164.9, 173.6. MS(ESI) m/z: 378.8 [M þ Na]^þ. Anal. Calcd for C₁₉-

H₂₄N₄O₃: C, 64.03; H, 6.79; N, 15.72. Found: C, 64.95; H, 6.73;

N, 15.68.

1

white solid (0.015 g, 60%). Mp 128-130 ꢀC. H NMR (200 MHz,

CDCl₃): δ 1.47-1.25 (m, 6H), 1.67 (m, 2H), 2.32 (t, J = 7.6 Hz, 2H),

2.56 (t, J = 7.6 Hz, 2H), 2.76 (d, J = 4.8 Hz, 3H), 5.90 (bs, 1H), 6.82-

6.79 (m, 1H), 7.63-7.24 (m, 4H), 7.89-7.69 (m, 2H), 8.26-8.19 (m,

2H), 9.68 (bs, 1H). ¹³C NMR (50 MHz, CD₃OD): δ 25.0, 26.9, 27.6,

27.7, 30.9, 31.2, 38.8, 120.6, 120.9, 124.5, 128.8 (ꢀ2), 129.3, 130.6 (ꢀ3),

131.1, 135.6, 141.2, 143.9, 155.9, 175.6. MS(ESI) m/z: 403.8 [M þ

Na]^þ. Anal. Calcd for C₂₂H₂₇N₃O₃: C, 69.27; H, 7.13; N, 11.02. Found:

C, 69.21; H, 7.09; N, 11.00.

N⁹-(9H-Fluoren-9-yl)-2-(hydroxyimino)-N¹-methylnona-

nediamide (16). Compound 16 was prepared from the corresponding

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3⁰-(8-(Hydroxyimino)-9-(methylamino)-9-oxononanami-

do)-[1,1⁰-biphenyl]-4-carboxylic Acid (21). Compound 21 was

prepared from 19 using the procedure described for 20 and obtained in

45% yield as a yellow oil. ¹H NMR (400 MHz, CD₃OD): δ 1.55-1.24

(m, 6H), 1.70-1.65 (m, 2H), 2.37 (t, J = 7.2 Hz, 2H), 2.54 (t, J = 7.2 Hz,

2H), 2.79 (s, 3H), 7.38 (d, J = 8.4 Hz, 2H), 7.56-7.53 (m, 2H), 7.98 (d,

J = 8.4 Hz, 2H), 8.19 (d, J = 8.4 Hz, 2H). ¹³C NMR (100 MHz,

CD₃OD): δ 22.3, 24.3, 24.9, 25.1, 28.2, 28.6, 36.2, 118.1, 119.0, 122.0,

126.1 (ꢀ2), 127.8, 128.6, 129.5 (ꢀ2), 132.6, 138.7, 140.2, 144.5,

153.3, 165.1, 173.0. MS(ESI) m/z: 424.1 [M - H]^-. Anal. Calcd for

C₂₃H₂₇N₃O₅: C, 64.93; H, 6.40; N, 9.88. Found: C, 64.99; H, 6.35;

N, 9.84.

stirred at room temperature for 1 h. The solvent was removed in vacuo

and the mixture was purified by flash chromatography (petroleum

ether/ethyl acetate 70:30) to give the expected compound as a colorless

oil (2.21 g, 65% yield). This latter was dissolved in MeOH (10 mL), and

PtO₂(28 mg, 0.120 mmol) was added. The mixture was stirred under H₂

at room temperature for 30 min. The catalyst was filtered off and washed

1

with MeOH to give 27 as colorless oil (2.05 g, 92% yield). H NMR

(CDCl₃, 200 MHz): δ 1.12-1.44 (m, 23H), 1.71-1.89 (m, 2H), 2.94-

1.05 (m, 2H), 3.21 (t, J = 7.6 Hz, 1H), 4.12 (q, J = 7.2 Hz, 4H). ¹³C NMR

(50 MHz, CDCl₃): δ 14.0 (ꢀ2), 26.3, 27.1, 28.3 (ꢀ3), 28.5, 28.8, 29.8,

51.9 (ꢀ2), 61.1 (ꢀ2), 78.9, 157.7, 169.4 (ꢀ2). MS(ESI) m/z: 382.1 [M

þ Na]^þ. Anal. Calcd for C₁₈H₃₃NO₆: C, 60.14; H, 9.25; N, 3.90. Found:

C, 60.10; H, 9.23; N, 3.88.

Ethyl 8-(tert-Butoxycarbonylamino)-2-(hydroxyimino)oc-

tanoate (28). Compound 28 was prepared from 27 using the

procedure described for 3 in 61% yield (the product is a colorless oil).

¹H NMR (CDCl₃, 200 MHz): δ 1.25-1.46 (m, 20H), 2.53 (t, J =

7.6 Hz, 2H), 3.02 (t, J = 6.8 Hz, 2H), 4.22 (q, J = 7.2 Hz, 2H). ¹³C NMR

(100 MHz, CDCl₃): δ 13.6, 24.1, 25.3, 25.9, 27.9 (ꢀ3), 28.7, 29.4, 40.2,

61.1, 78.4, 152.2, 157.1, 163.3. MS(ESI) m/z: 339.1 [M þ Na]^þ. Anal.

Calcd for C₁₅H₂₈N₂O₅: C, 56.94; H, 8.92; N, 8.85. Found: C, 56.99; H,

8.95; N, 8.83.

8-Amino-2-(hydroxyimino)-N-methyloctanamide (29). The

R-oxime ester 28 was first converted to the corresponding R-oxime amide

using the procedure described for 5 in 75% yield. The product (0.40 g, 1.33

mmol) was then dissolved in CH₂Cl₂(4 mL), and TFA (1 mL, 13.1 mmol)

was added. The mixture was stirred at room temperature for 12 h, the

solvent was removed in vacuo, and the mixture was purified by flash

chromatography (CHCl₃/MeOH 9:1) to give 29 as a yellow oil (0.240 g,

67% yield). ¹H NMR (CDCl₃, 400 MHz): δ 1.33-1.40 (m, 4H), 1.46-

1.50 (m, 2H), 1.59-1.64 (m, 2H), 2.53 (t, J = 7.6 Hz, 2H), 2.76 (s, 3H),

2.89 (t, J = 7.6 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 22.2, 24.4, 24.9,

25.2, 26.5, 28.2, 38.9, 153.1, 165.1. MS(ESI) m/z: 202.1 [M þ H]^þ. Anal.

Calcd for C₉H₁₉N₃O₂: C, 53.71; H, 9.52; N, 20.88. Found: C, 53.77; H,

9.55; N, 20.84.

2-(Hydroxyimino)-N¹-methyl-N⁹-(3-(pyridine-3-yl)phen-

yl)nonanediamide (22). Compound 22 was prepared from 19 using

the procedure described for 20 in 50% yield (the product is a yellow oil).

¹H NMR (400 MHz, CD₃OD): δ 1.52-1.43 (m, 6H), 1.72-1.65 (m,

2H), 2.37 (t, J = 7.6 Hz, 2H), 2.53 (t, J = 7.6 Hz, 2H), 2.75 (s, 3H), 7.36-

7.34 (m, 1H), 7.43-7.39 (m, 1H), 7.51-7.48 (m, 1H), 7.57-7.55 (m,

1H), 7.90 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 8.50 (s, 1H), 8.76 (s, 1H).

¹³C NMR (100 MHz, CD₃OD): δ 22.4, 24.3, 24.9, 25.1, 28.2, 28.6, 36.2,

117.9, 119.3, 121.8, 123.7, 128.9, 134.7, 136.5, 137.2, 139.0, 146.5, 147.1,

153.3, 165.0, 173.0. MS(ESI) m/z: 404.8 [M þ Na]^þ. Anal. Calcd for

C₂₁H₂₆N₄O₃: C, 65.95; H, 6.85; N, 14.65. Found: C, 65.90; H, 6.82;

N, 14.68.

N⁹-(3-(6-Chloropyridin-3-yl)phenyl)-2-(hydroxyimino)-

N¹-methylnonanediamide (23). Compound 23 was prepared from

19 using the procedure described for 24 in 56% yield (the product is a

1

yellow oil). H NMR (400 MHz; CD₃OD): δ 1.38 (m, 4H), 1.48

(m, 2H), 1.68 (m, 2H), 2.37 (t, J = 7.2 Hz, 2H), 2.53 (t, J = 7.6 Hz, 2H),

3.28 (s, 3H), 7.35-7.51 (m, 5H), 7.9 (bs, 1H), 8.2 (m, 1H), 8.6 (bs,

1H). ¹³C NMR (100 MHz; CDCl₃): δ 22.4, 24.3, 24.9, 25.1, 28.1, 28.6,

36.2 117.8, 119.4, 121.8, 123.8 (ꢀ2), 129.0 (ꢀ2), 135.4, 136.3, 137.3,

146.4, 153.3, 165.0 172.9. MS(ESI) m/z: 415.1 [M - H]^-. Anal. Calcd

for C₂₁H₂₅ClN₄O₃: C, 60.50; H, 6.04; Cl, 8.50; N, 13.44. Found C,

60.57; H, 6.01; N, 13.40.

General Procedure of Suzuki Coupling with Pd₂(dba)₃as

Catalyst. N⁹-(3-(7-Chloroisoquinolin-4-yl)phenyl)-2-)hydro-

xyimino)-N¹-methylnonanediamide (24). A vial containing a

magnetic stir bar was charged with Pd₂(dba)₃(0.012 g, 1.0 mol %),

P(^tBu)₃(0.01 g, 2 mol %), 7-chloroquinoline-4-boronic acid pinacol

(0.075 g, 0.26 mmol), and K₃PO₄(0.055 g, 0.26 mmol). The vial was

sealed with a Teflon-coated cap and then evacuated and backfilled with

argon (three times). The aryl halide 19 (0.05 g, 0.13 mmol) and ^tBuOH

were added sequentially via syringe through the septum. Then the vial

was hermetically sealed and the reaction mixture was vigorously stirred

at 100 ꢀC for 18 h. The solvent was removed by evaporation in vacuo and

the residue was purified by flash chromatography (dichloromethane/

methanol 9:1) to yield 24 as a yellow oil (0.028 g, 47%). ¹HNMR (400

MHz, CDCl₃): δ 1.44-1.31 (m, 4H), 1.49-1.45 (m, 2H), 1.69-1.65

(m, 2H), 2.36 (t, J = 7.2 Hz, 2H), 2.52 (t, J = 7.2 Hz, 2H), 7.21 (d, J = 7.2

Hz, 1H), 2.74 (s, 3H), 7.5-7.45 (m, 3H), 7.52 (d, J= 2 Hz, 1H), 7.55 (d,

J= 1.6 Hz, 1H), 7.65 (m, 1H), 7.78 (bs, 1H), 7.94 (d, J= 9.2 Hz, 1H), 8.06

(d, J= 1.6 Hz, 1H), 8.86 (d, J= 4.4 Hz, 1H). ¹³C NMR (100 MHz;

CDCl₃): δ 22.4, 24.3, 24.9, 25.1, 28.1, 28.5, 36.1, 119.7, 120.4, 121.1,

124.2, 126.6, 127.1, 127.2, 128.6, 135.1, 137.1, 138.6, 147.8, 148.9, 150.3

(ꢀ2), 153.3, 165.1, 173.1. MS(ESI) m/z: 489.2 [M þ Na]^þ. Anal. Calcd

for C₂₅H₂₇ClN₄O₃: C, 64.30; H, 5.83; Cl, 7.59; N, 12.00. Found: C,

64.34; H, 5.80; N, 11.97.

tert-Butyl 3-(3-(7-(Hydroxyimino)-8-(methylamino)-8-ox-

ooctylcarbamoyl)isoxazol-5-yl)phenylcarbamate (30). To a

solution of isoxazolecarboxylic acid¹⁵(0.101 g, 0.33 mmol) in dry THF

(3 mL), the amine 29 (0.100 g, 0.49 mmol) was added. The reaction

mixture was cooled to 0 ꢀC, and DMTMM (0.164 g, 0.59 mmol) and

NMM (0.100 g, 102 μL, 0.99 mmol) were added. The cold bath was

removed and the mixture allowed to warm up to room temperature and

stirred for 12 h. The white solid was filtered off and the solvent removed

in vacuo. The crude mixture was then purified by flash chromatography

(CHCl₃/MeOH 9:1) to give the title compound 30 as a white solid

(0.097 g, 60% yield). Mp 110-112 ꢀC. ¹H NMR (CDCl₃, 400 MHz): δ

1.37-1.61 (m, 17H), 2.53 (t, J = 7.6 Hz, 2H), 2.75 (s, 3H), 3.27-3.37

(m, 2H), 6.98 (s, 1H), 7.34-7.48 (m, 3H), 7.94 (m, 1H). ¹³C NMR

(100 MHz, CDCl₃): δ 24.1, 26.2, 26.9, 27.7, 28.7 (ꢀ3), 30.2, 30.3, 40.5,

81.2, 99.9, 116.5, 120.9, 121.7, 128.5, 130.7, 141.6, 155.0, 155.1, 160.6,

161.2, 166.9, 172.7. MS(ESI) m/z: 510.1 [M þ Na]^þ. Anal. Calcd for

C₂₄H₃₃N₅O₆: C, 59.12; H, 6.82; N, 14.36. Found: C, 59.19; H, 6.80;

N, 14.34.

tert-Butyl 4-(3-(7-Hydroxyimino-8-(methylamino)-8-oxo-

octylcarbamoyl)isoxazol-5-yl)phenylcarbamate (31). Com-

pound 31 was prepared from isoxazole carboxylic acid¹⁵and 29 using the

procedure described for 30 in 65% yield (the product is a white solid). Mp

170-172 ꢀC). ¹H NMR (CDCl₃, 400 MHz): δ 1.38-1.61 (m, 17H), 2.54

(t, J = 7.2 Hz, 2H), 2.75 (s, 3H), 3.35 (t, J = 7.2 Hz, 2H), 6.98 (m, 1H), 7.54

(d, J= 8.8 Hz, 2H), 7.73 (d, J= 8.8 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃):

δ 24.2, 26.2, 26.9, 27.7, 28.6 (ꢀ3), 30.2, 30.3, 40.5, 81.3, 98.6, 116.5, 119.6,

122.1, 127.7 (ꢀ2), 143.3, 154.8, 155.1, 160.6, 161.4, 166.9, 172.7. MS(ESI)

m/z: 510.1 [M þ Na]^þ. Anal. Calcd for C₂₄H₃₃N₅O₆: C, 59.12; H, 6.82; N,

14.36. Found: C, 59.07; H, 6.80; N, 14.34.

tert-Butyl 6-Oxohexylcarbamate (26). Compound 26 was

prepared following a reported procedure.²²

Diethyl 2-(6-(tert-Butoxycarbonylamino)hexyl)malonate

(27). To a solution of the aldehyde 26 (2.05 g, 9.52 mmol) in DMF

(10 mL), dimethyl malonate (3.05 g, 2.89 mL, 19.04 mmol) and

piperidine (81 mg, 94 μL, 0.952 mmol) were added. The mixture was

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5-(3-Aminophenyl)-N-(7-(hydroxyimino)-8-(methylami-

no)-8-oxooctyl)isoxazole-3-carboxamide (32). Compound 30

(0.050 g, 0.102 mmol) was dissolved in CH₂Cl₂(400 μL), and TFA

(100 μL, 1.31 mmol) was added. The mixture was stirred at room

temperature for 12 h, the solvent was removed in vacuo, and the mixture

was purified by flash chromatography (CHCl₃/MeOH 9:1) to give 32 as

a yellow oil (0.033 g, 85%). ¹H NMR (CDCl₃, 400 MHz): δ 1.35-1.60

(m, 8H), 2.53 (t, J = 7.6 Hz, 2H), 2.75 (s, 3H), 3.28-3.36 (m, 2H),

6.77-6.79 (m, 1H), 6.91 (s, 1H), 7.08-7.20 (m, 3H). ¹³C NMR (100

MHz, CDCl₃): δ 22.3, 24.3, 25.1, 25.8, 28.4, 28.5, 38.7, 97.6, 111.0,

114.3, 116.7, 126.9, 129.1, 148.1, 153.4, 158.7, 159.6, 165.1, 171.6.

MS(ESI) m/z: 410.1 (M þ Na)^þ. Anal. Calcd for C₁₉H₂₅N₅O₄: C,

58.90; H, 6.50; N, 18.08. Found: C, 58.93; H, 6.49; N, 18.06.

5-(4-Aminophenyl)-N-(7-(hydroxyimino)-8-(methylamin-

o)-8-oxooctyl)isoxazole-3-carboxamide (33). Compound 33

was prepared from 31 using the procedure described for 32 in 81%

yield (the product is a yellow solid). Mp 114-116 ꢀC. ¹H NMR (CD-

Cl₃, 400 MHz): δ 1.36-1.60 (m, 8H), 2.54 (t, J = 7.6 Hz, 2H), 2.75

(s, 3H), 3.34 (t, J = 7.2 Hz, 2H), 6.73 (s, 1H), 6.74 (t, J = 9.6 Hz, 2H),

7.55 (d, J = 8.8 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 22.3, 24.3,

25.1, 25.8, 28.4, 28.5, 38.7, 94.9, 114.1 (ꢀ2), 115.4, 126.5 (ꢀ2), 149.7,

153.3, 158.6, 159.8, 165.1, 171.9. MS(ESI) m/z: 410.1 [M þ Na]^þ.

Anal. Calcd for C₁₉H₂₅N₅O₄: C, 58.90; H, 6.50; N, 18.08. Found: C,

58.96; H, 6.52; N, 18.06.

(3S)-3-(Dibenzylamino)-6-vinyltetrahydro-2H-pyran-2-

one (35). To a solution of aldehyde 34 (1.4 g, 3.5 mmol) in THF

(28 mL) vinylmagnesium bromide (1.9 mL, 1.9 mmol, solution 1 M in

THF) was added dropwise cooling in an ice-water bath. After the

addition, the reaction mixture was allowed to slowly warm to room

temperature overnight. The mixture was quenched with saturated

aqueous NH₄Cl solution and diluted with ethyl acetate. The organic

layer was separated and washed with brine, dried (anhydrous Na₂SO₄),

filtered, concentrated, and purified by flash chromatography (petroleum

ether/ethyl acetate 9:1) to give a mixture of diastereomeric lactones 35

as colorless oil in 60% yield. ¹H NMR (400 MHz, CDCl₃): δ 2.04-1.58

(m, 4H^Rþβ), 3.52-3.38 (m, 1H^Rþβ), 3.79 (d, J = 14 Hz, 2H^Rþβ), 4.07-

3.99 (m, 2H^Rþβ), 4.69 (m, 1H^Rþβ), 5.34-5.15 (m, 2H^Rþβ), 5.83-5.76

(m, 1H^Rþβ), 7.40-7.23 (m, 10H^Rþβ). ¹³C NMR (400 MHz, CDCl₃): δ

22.4, 26.3, 27.5, 29.2, 54.9, 55.4, 56.6, 58.1, 81.1, 116.9, 117.2, 127.1,

128.4, 128.5, 128.7, 136.0, 136.1, 139.7, 104.0, 171.1, 172.3. MS(ESI)

m/z: 343.8 [M þ Na]^þ. Anal. Calcd for C₂₁H₂₃NO₂: C, 78.47; H, 7.21;

N, 4.36. Found: C, 78.40; H, 7.18; N, 4.34.

Hz, 2H), 4.15 (m, 1H^Rþβ), 5.33-5.20 (m, 2H), 5.84 (m, 1H), 7.10

(m, 1H), 7.40-7.24 (m, 12H), 7.53 (m, 2H), 8.90 (d, J = 16 Hz, 1H).

¹³C NMR (400 MHz, CDCl₃): δ 14.0, 19.9, 20.4, 20.5, 20.9, 21.1, 21.1,

32.6, 32.6, 54.4, 60.3, 62.3, 62.5, 74.4, 74.6, 116.8, 116.9, 119.0, 119.0,

123.8, 127.4, 127.4, 127.8, 128.0, 128.3, 128.6, 128.9, 136.0, 136.1, 137.6,

137.6, 138.4, 138.5, 170.3, 170.4, 171.1, 171.2, 171.3, 175.6. MS(ESI)

m/z: 478.8 [M þ Na]^þ. Anal. Calcd for C₂₉H₃₂N₂O₃: C, 76.29; H, 7.06;

N, 6.14. Found: C, 76.36; H, 7.04; N, 6.10.

(S)-Diethyl 2-(6((4-Methoxybenzyl)amino)-7-oxo-7-(phe-

nylamino)heptyl)malonate (38). NaH (0.29 g, 7.6 mmol, 60%

dispersion in oil) was added to a two-necked round-bottom flask closed

with a septum to which dry hexane (2 mL) was added, and the mixture

was stirred for 5 min to dissolve the mineral oil. The hexane was removed

by a syringe, THF (15 mL) was added, and the resulting mixture was

stirred at 0 ꢀC for 10 min. A solution of diethyl malonate (1.7 mL,

11.5 mmol) in THF (15 mL) was then slowly added over 30 min at 0 ꢀC

to the resulting suspension. The solution became clear after 10 min of

stirring. The stirring was continued for another 30 min. A solution of

PdCl₂(PPh₃)₂and PPh₃in THF (7 mL) was added dropwise at room

temperature. After 20 min of stirring, a solution of 36 in THF (15 mL)

was slowly added. This mixture was stirred at room temperature

overnight. The reaction mixture was concentrated and the residue

dissolved in ethyl acetate. A solution of aqueous NH₄Cl was added,

and the organic phase was separated and washed with water and brine.

The organic extracts were dried (anhydrous Na₂SO₄), filtered, and

concentrated in vacuo to give the desired product as a mixture of two

regioisomers 37a and 37b as colorless oil (80%, ratio 37a/37b is equal to

1

66:34 from H NMR integration analysis of distinct vinyl protons). The

product was dissolved in methanol (5 mL) and submitted to H₂(1 atm)

over Pd/C (5%) at room temperature for 2 h. The solvent was evaporated

and the residue dissolved in CH₂Cl₂(6 mL). NaBH(OAc)₃(0.054 g, 0.40

mmol) and acetic acid (few drops) were added, and the mixture was stirred

at room temperature for 3 h. The reaction mixture was quenched with H₂O,

extracted with CH₂Cl₂, and the organic layer was dried (anhydrous

Na₂SO₄), filtered, and concentrated in vacuo. Flash chromatography

(petroleum ether/ethyl acetate 9:1) gave 38 as a colorless oil (0.19 g,

46% yield). ¹H NMR (200 MHz, CDCl₃): δ 1.29-1.19 (m, 10 H), 1.5-

1.92 (m, 6H), 3.1-3.4 (m, 2H), 3.68 (d, J = 7.2 Hz, 2H), 3.77 (s, 3H), 4.16

(q, J= 7.2 Hz, 4H), 6.83 (d, J= 7.6 Hz, 2H), 7.11-7.04(m, 1H), 7.38-7.19

(4H), 7.55(d, 7.6Hz, 2H), 9.34 (bs, 1H). MS(ESI) m/z: 498.8 [M þ H]^þ.

Anal. Calcd for C₂₈H₃₈N₂O₆: C, 67.45; H, 7.68; N, 5.62. Found: C, 67.40;

H, 7.64; N, 5.59. [R]_D-22.5 (c 1.0, CHCl₃).

(6S)-6-(Dibenzylamino)-7-oxo-7-(phenylamino)hept-1-

en-3-yl Acetate (36). To a solution of aniline (1.32 mL, 16.35 mmol)

in CH₂Cl₂(57 mL), trimethylaluminum (8.2 mL of a 2 M solution in

toluene, 16.40 mmol) was added at 0 ꢀC, and the resulting mixture was

stirred for 30 min. A solution of 35 (1.75 g, 5.45 mmol) in CH₂Cl₂

(30 mL) was added at 0 ꢀC. After the addition, the reaction mixture was

stirred overnight at room temperature. The mixture was quenched with

H₂O (3 mL), and an amount of 5 g of anhydrous Na₂SO₄was added.

The resulting mixture was stirred for 30 min, filtered through Celite,

washed with ethyl acetate, and concentrated in vacuo. The residue was

purified by flash chromatography (petroleum ether/ethyl acetate 7:3) to

give a mixture of diastereomeric alcohols in 95% yield. To a solution of

this mixture of alcohols (1.72 g, 4.15 mmol) in CH₂Cl₂(10 mL),

triethylamine (2.3 mL, 16.6 mmol), a catalytic amount of DMAP, and

acetic anhydride (0.59 mL, 6.2 mmol) were added at 0 ꢀC. The resulting

mixture was stirred overnight at room temperature. The reaction was

quenched with aqueous NH₄Cl_ssand extracted with CH₂Cl₂. The

organic extracts were dried (anhydrous Na₂SO₄), filtered, and concen-

trated in vacuo. Flash chromatography (petroleum ether/ethyl acetate

8:2) gave 36 as a mixture of two diastereoisomers and as a colorless oil

(92%). ¹H NMR (200 MHz, CDCl₃): δ 2.1-1.6 (m, 4H^Rþβ, 3H,

overlapped), 3.31-3.25 (m, 1H), 3.65-3.54 (m, 3H), 3.83 (d, J = 13.0

(S)-Ethyl 8-((tert-Butoxycarbonyl)(4-methoxybenzyl)ami-

no)-2-(hydroxyimino)-9-oxo-9-(phenylamino)nonanoate (39).

Compound 39 was prepared from the corresponding N-protected deriva-

tive using the procedure described for 3 in 50% yield (the product is a

colorless oil). ¹H NMR (200 MHz, CDCl₃): δ 0.85-2.15 (m, 22H), 3.83

(bs, 3H), 4.49-4.22 (m, 5H), 7.45-6.75 (m, 9H). MS(ESI) m/z: 577.8

[M þ Na]^þ. Anal. Calcd for C₃₀H₄₁N₃O₇: C, 64.85; H, 7.44; N, 7.56.

Found: C, 64.89; H, 7.41; N, 7.53. [R]_D-63.6 (c 1.0, CHCl₃).

(S)-tert-Butyl (8-(Hydroxyimino)-9-(methylamino)-1,9-di-

oxo-1-(phenylamino)nonan-2-yl)(4-methoxybenzyl)carba-

mate (40). Compound 40 was prepared from 39 using the procedure

described for 5 in 70% yield (the product is colorless oil). ¹H NMR (200

MHz, CDCl₃): δ 1.42-1.23 (m, 13H), 1.76 (m, 2H), 2.03 (m, 2H), 2.57

(m, 2H), 2.81 (d, J = 4.8 Hz, 3H), 3.75 (bs, 3H), 4.37-4.88 (m, 3H),

7.26-6.68 (m, 9H), 8.64 (bs, 1H). MS(ESI) m/z: 539.2 [M - H]^-.

Anal. Calcd for C₂₉H₄₀N₄O₆: C, 64.42; H, 7.46; N, 10.36. Found: C,

64.36; H, 7.44; N, 10.34.

(S)-2-(Hydroxyimino)-8-((4-methoxybenzyl)amino)-N¹m-

ethyl-N⁹-phenylnonanediamide (41). To a solution of 40 (0.015

xg, 0.03 mmol) in CH₂Cl₂, TFA (1 mL) was added. The reaction

mixture was stirred for 1 h at room temperature. The solvent was

removed under reduced pressure and the residue purified by flash

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chromatography (dichloromethane/methanol 9:1) to yield 41 as a

colorless oil (82%). H NMR (400 MHz, CDCl₃): δ 1.47-1.23 (m,

6H), 1.66 (m, 1H), 1.81 (m, 1H), 2.55 (m, 2H), 2.80 (bs, 2H), 3.33 (m,

2005 Builder module. Partial charges were assigned by using the CVFF

force field.²⁴The conformational space of compounds was sampled

through 200 cycles of simulated annealing. An initial temperature of

1000 K was applied to the system for 1000 fs with the aim of

surmounting torsional barriers. Successively, temperature was linearly

reduced to 300 K with a decrement of 0.5 K/fs. The resulting structures

were subjected to an energy minimization protocol within the Insight

2005 Discovery module (CVFF force field, conjugate gradient algo-

rithm; maximum rms derivative of <0.001 kcal/Å; ε = 1). In order to

properly analyze the electronic properties, all conformers, obtained from

molecular dynamics and mechanics calculations, were subjected to a full

geometry optimization through semiempirical calculations, using the

quantum mechanical method PM6 in the Mopac2009 package.^25,26The

EF (eigenvector following routine) algorithm of geometry optimization

was used, with a GNORM value set to 0.01. To reach a full geometry

optimization, the criterion for terminating all optimizations was in-

creased by a factor of 100, using the keyword PRECISE. All resulting

PM6 conformers were subsequently ranked by (i) conformational

energy (ΔE < 5 kcal), (ii) interatomic distance between the oxime

1

1H), 3.75 (bs, 5H), 6.68 (bs, 1H), 6.84 (d, J = 8.0 Hz, 2H), 7.08 (m, 1H),

7.23 (m, 2H), 7.30 (m, 3H), 7.57 (d, J = 7.6 Hz, 2H), 9.50 (bs, 1H). ¹³

C

NMR (100 MHz, CDCl₃): δ 23.1, 25.3, 25.6, 26.0, 29.0, 33.1, 52.2, 55.3,

62.7, 114.1 (ꢀ2), 119.6 (ꢀ2), 124.3, 129.0 (ꢀ4), 129.7, 137.6, 154.2,

158.9, 160.9, 172.0. MS(ESI) m/z: 439.2 [M - H]^-. Anal. Calcd for

(C₂₄H₃₂N₄O₄: C, 65.43; H, 7.32; N, 12.72. Found: C, 65.49; H, 7.34; N,

12.68. [R]_D-18.0 (c 0.1, CHCl₃). The enantiomeric integrity the final

product was determined via conversion of 41 to the Mosher amide using

commercially available (S)-(þ)-R-methoxy-R-trifloromethylphenylacetyl

chloride and (R)-(-)-R-methoxy-R-trifloromethylphenylacetyl chloride

under Schotten-Baumann conditions. (S)-amide: ¹⁹F NMR (215 MHz,

CDCl₃) δ -69.5. (R)-amide: ¹⁹F NMR (215 MHz, CDCl₃) δ -71.3.

(S)-Diethyl 2-(6-(Dibenzylamino)-7-oxo-7-(phenylamino)-

heptyl)malonate (42). To a solution of two regioisomers 37a and

37b (0.1 g, 0.18 mmol) in MeOH (5 mL) was added PtO₂(0.05 equiv),

and the mixture was stirred under H₂(1 bar) for 10 min. The catalyst was

filtered off, and the filtrate was concentrated in vacuo. Flash chroma-

tography (petroleum ether/ethyl acetate 85:15) gave pure 42 as color-

less oil (60%). ¹H NMR (200 MHz, CDCl₃): δ 1.5-1.2 (m, 10 H), 2.1-

1.57 (m, 6H), 3.38-3.16 (m, 2H), 3.6 (d, J = 13.2 Hz, 2H), 3.85 (d, J =

13.2 Hz, 2H), 4.2 (q, J = 7.2 Hz, 4H), 7.1 (m, 1H), 7.48-7.2 (m, 13H),

7.55 (m, 1H), 9.0 (bs, 1H). MS(ESI) m/z: 558.8 [M þ H]^þ. Anal. Calcd

for C₃₄H₄₂N₂O₅: C, 73.09; H, 7.58; N, 5.01. Found: C, 73.19; H, 7.55;

N, 4.98. [R]_D-153.6 (c 1.0, CHCl₃).

ꢁ

carbon of ZBG and the Cap centroid (distance: <5 Å, 5-7.5 Å, 7.5-10

ꢁ

Å, >10 Å), (iii) torsional angles (amide bonds conformation (cis and

trans), oxime configuration (E and Z)), and (iv) intramolecular hydro-

gen bonds. Since all inhibitors crystallized into HDAC enzymes show an

extended conformation of the linker, in order to analyze both the

conformational preferences and the ability to coordinate zinc of newly

compounds R-oxime amide ZBG, the most stable stereoisomers of the

main tautomer of 5 with an extended conformation (distance ZBG-CAP

of >7.5 Å) were selected for DFT calculations. Moreover, X-ray

structures of metal oximes complexes (CSD codes BICFUP, CABTUU,

CAYCIP, CAYCOV, CAYDEM, EYOTER, FIDWOF, FIDWUL, GIF-

SAF, GEPMET, IXEBIW, LOVQUI, MEHZUU, WASXEV, ZUDLUF)

and metal-amide complexes (CSD codes BUNWEN, LUHBUL) were

selected and downloaded from the Cambridge Structural Database

(CSD) using the CSDS (Cambridge Structural Database System)

software Conquest 1.12. These complexes were analyzed using Insight

2005 (Accelrys, San Diego, CA).

(S)-2-(Dibenzylamino)-8-(hydroxyimino)-N⁹-methyl-N¹-

phenylnonanediamide (43). Compound 43 was prepared from

appropriate R-oxime ester using the procedure described for 5 in 84%

yield (the product is a colorless oil). ¹H NMR (400 MHz, CDCl₃): δ

1.42 (m, 2H), 1.65-1.54 (m, 5H), 1.95 (m, 1H), 2.62 (m, 2H), 2.83 (d, J

= 5.2 Hz, 3H), 3.23-3.2 (m, 1H), 3.56 (d, J = 13.6 Hz, 2H), 3.79 (d, J =

13.6 Hz, 2H), 6.15 (bs, 1H), 7.07 (m, 1H), 7.35-7.24 (m, 12H), 7.49

(d, J = 8.0 Hz, 2H), 8.2 (bs, 1H), 8.94 (s, 1H). ¹³C NMR (100 MHz,

CDCl₃): δ 23.4, 24.5, 25.8, 26.0, 28.3, 29.9, 54.6 (ꢀ2), 62.8, 119.2 (ꢀ2),

123.9, 127.5 (ꢀ2), 128.7 (ꢀ4), 128.8 (ꢀ4), 129.0 (ꢀ2), 137.9, 138.8

(ꢀ2), 155.7, 163.9, 172.0. MS(ESI) m/z: 523.3 [M þ Na]^þ. Anal. Calcd

for C₃₀H₃₆N₄O₃: C, 71.97; H, 7.25; N, 11.19, Found: C, 72.05; H, 7.23;

N, 11.16. [R]_D-80.5 (c 0.2, CHCl₃).

Molecular Modeling Studies. Molecular modeling calculations

were performed on SGI Origin 200 8XR12000 and E4 Server Twin 2 ꢀ

Dual Xeon 5520, equipped with two nodes. Each node was 2 ꢀ Intel Xeon

QuadCore E5520, 2.26 GHz, 36 GB RAM. The molecular modeling

graphics were carried out on SGI Octane 2 workstations. Experimentally

determined structures of human histone deacetylase HDAC 2 (PDB code

3MAX); HDAC 4 (PDB codes 2VQW, 2VQV, 2VQJ, 2VQM, 2VQO, and

2VQQ); HDAC 7 (PDB codes 3C0Y, 3C0Z, and 3C10) and HDAC 8

(PDB codes 3EW8, 3EWF, 3EZP, 3EZT, 3F06, 3F07, 3F0R, 2V5W, 2V5X,

1W22, 1VKG, 1T69, 1T67, and 1T64) were downloaded from the Protein

Data Bank (PDB, http://www.rcsb.org/pdb/) and analyzed using the

homology module of Insight 2005 (Accelrys, San Diego, CA). Hydrogens

were added to all the PDB structures assuming a pH of 7.2. Sequence

alignments of all human histone deacetylase were performed using PRO-

MALS3D server (http://prodata.swmed.edu/promals3d/promals3d.php).²³

Conformational Analysis. The apparent pK_avalues of newly designed

compounds 2-5, 14, 17, 18, 20, 22-23, 32, 33, 40, 41, and 43, in their

tautomeric forms, were estimated using the pharma algorithms of ACD/

pKa DB, version 12.00, software (Advanced Chemistry Development

Inc., Toronto, Canada). Accordingly, the percentage of neutral/ionized

forms was computed at pH 7.2 (cytoplasm) using the Handerson-

Hasselbalch equation. Compounds 2-5, 14, 17, 18, 20, 22-23, 32, 33,

40, 41, and 43 were built taking into account the prevalent ionic forms of

each tautomer at the considered pH value (i.e., 7.2) using the Insight

Density Functional Calculations. The selected PM6 conformers of 5,

both in their basic conformation and in complex with Zn^2þcation, were

optimized at the density functional level of theory by B3 exchange,²⁷Lee,

Yang, and Parr (LYP) correlation functionals.²⁸6-311þþG** basis set

functions were used for nonmetal atoms,²⁹while LanL2DZ pseudopo-

tential and related basis set were chosen for zinc.³⁰The presence of

diffuse functions allowed an adequate representation of anionic species

molecular orbitals. Any geometrical constraint was imposed during the

optimization process. Vibrational frequency calculations at the same

level of theory were ensured to verify that every structure represents a

minimum on the potential energy surface and zero point correction was

included in all energies. The metal complexes binding energies (BE)

were determined by considering the coordination of zinc to the ZBG

portion of oximes as

products

reactants

BE ¼ ∑ E_i- ∑ E_j

i

j

The BE evaluation was considered in absolute value in order to classify

the coordination compounds according to the ZBG-metal bond strength.

Zn^2þand oximes represent the reactants that give rise to the product as

metal complex. As we were interested in the metal-ligands interaction, we

performed the natural bond orbital (NBO) analysis of every minimum to

elucidate the features of the connection to zinc.³¹The consequences of the

metal retrodonation to ligands, according to the electroneutrality principle,

were highlighted through the atomic charges evaluations, both in oximes

and in zinc complexes. The methodology used to calculate the atomic

charges was the atomic polar tensor (APT),³²which provides values more

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ARTICLE

reliable than Mulliken and natural population analysis approaches especially

for organometallic compounds.³³All calculations were carried out by the

Gaussian 09 suite of programs.³⁴

wide variance of the input structures to be successively minimized, an

energy tolerance value of 10⁶kcal/mol from the previous structure has

been used. After the energy minimization step (conjugate gradient, 2500

iterations, ε = 1), an energy check criterion (energy range of 50 kcal/

mol) and a structure similarity check (rms tolerance of 0.3 kcal/Å) were

applied to select the 20 acceptable structures. Each subsequent structure

was generated from the last accepted structure. All the accepted

complexes resulting from the Monte Carlo/minimization approach

were subjected to a molecular dynamics simulated annealing protocol,

including 5 ps of a dynamic run divided in 50 stages (100 fs each) during

which the temperature of the system was linearly decreased from 500 to

300 K (Verlet velocity integrator; time step of 1.0 fs). In simulated

annealing, the temperature is altered in time increments from an initial

temperature to a final temperature. The temperature is changed by

adjusting the kinetic energy of the structure (by rescaling the velocities of

the atoms). Molecular dynamics calculations were performed using a

constant temperature and constant volume (NVT) statistical ensemble

and the direct velocity scaling as temperature control method

(Temp Window = 10 K). In the first stage, initial velocities were

randomly generated from the Boltzmann distribution, according to

the desired temperature, while during the subsequent stages initial

velocities were generated from Dynamics Restart Data. A temperature

of 500 K was applied with the aim of surmounting torsional barriers, thus

allowing an unconstrained rearrangement of the ligand and the heme

(initial vdW and Coulombic scale factors of 0.1). Temperature was

successively linearly reduced to 300 K in 5 ps, and concurrently the scale

factors have been similarly decreased from their initial values (0.1) to

their final values (1.0). A final round of 10⁴minimization steps

(conjugate gradient, ε = 1) followed the last dynamics steps, and the

minimized structures were saved in a trajectory file. After this procedure,

the resulting docked complexes were ranked by their conformational

energy. In order to choose the structure representing the most probable

binding mode, taking into account all experimentally determined

structures of human HDACs crystallized in complex with inhibitors,

all resulting docked complexes were analyzed and selected by discarding

those presenting the inhibitor with a cis alkyl chain or amide bond

conformation (Homology module of Insight 2005 (Accelrys, San Diego,

CA)). The geometry of π-π interactions was evaluated considering³⁸

(i) the distance between the centroids of the aromatic rings, (ii) the

angle between the planes of the rings, (iii) the offset value, and (iv) the

direction of the dipole vectors.

Docking Procedure. In order to find the bioactive conformation,

docking studies were carried out on 32 and 41 in complex with human

histone deacetylase HDAC 7 (PDB code 3C0Y), using a docking

methodology (Affinity, SA_Docking; Insight2005, Accelrys, San Diego,

CA) which considers all the systems flexible (i.e., ligand and protein).

Atomic potentials were assigned using the esff.frc,³⁵a force field

including zinc parameters, while the atomic partial charges were assigned

using the CVFF force field. Although in the subsequent dynamic

docking protocol all the systems were perturbed by means of Monte

Carlo and simulated annealing procedures, nevertheless the dynamic

docking procedure formally requires a reasonable starting structure. In

order to define the ZBG starting conformation of 32 and 41, all

conformers of 5 in complex with Zn^2þcation, obtained from DFT

calculations, were placed in the active site of experimentally determined

structures of human HDACs and superimposed on both the substrate

and the inhibitors, taking into account the coordination atoms and the

catalytic zinc ion. The conformations that did not show any steric

overlap with catalytic-site amino acids were selected as ZBG starting

conformations for docking calculations. Accordingly, PM6 conformers

of 32 and 41 characterized by a five-membered coordination ring

(N-N) or a six-membered coordination ring (O-O) and by a linker

extended conformation (distance ZBG-CAP > 7.5 Å) were selected. The

ligands were placed in the active site of the enzyme on the basis of

previously reported analysis on ZBG and on the basis of the orientation

of crystallized inhibitors. On the other hand, for the HDAC enzyme, the

crystal structure of catalytic domain of human histone deacetylase

HDAC 7 (PDB code 3C0Y) was selected and the water molecules were

maintained in the crystal structure with the exception of those that

would sterically overlap with the ligands. Flexible docking was achieved

using the Affinity module in the Insight 2005 suite, setting the

SA_Docking procedure³⁶and using the Cell_Multipole method for

nonbond interactions.³⁷A binding domain area was defined as a flexible

subset around the ligand that consisted of all residues and water

molecules having at least one atom within a 10 Å radius from any given

ligand atom. This binding area was enlarged in order to allow the

conformational change at the structural zinc-binding domain. In parti-

cular, water molecules and residues having at least one atom within 10 Å

radius from structural zinc ion were added to the binding area. All atoms

included in the binding domain area were left free to move during the

entire course of docking calculations, whereas a tethering restraint was

applied on zinc and potassium binding domains, in order to avoid

unrealistic results. In particular, the distance between the amino acid

coordination atoms and the ions was constrained within 2.5 Å using a

force constant of 100 (kcal/mol)/Å. Moreover, in order to obtain more

representative results, we increased the variance of docking procedure,

applying for each selected starting complex a tether restraint on ligand

coordination atoms or on only a single coordination atom. The selected

atoms were tethered with a force constant of 30 kcal/Å². A Monte

Carlo/minimization approach for the random generation of a maximum

of 20 acceptable ligand/enzyme complexes, for each compound, was

used. During the first step, starting from the previously obtained roughly

docked structures, the ligand was moved by a random combination of

translation, rotation, and torsional changes (Flexible_Ligand option,

considering all rotatable bonds) to sample both the conformational

space of the ligand and its orientation with respect to the enzyme

(MxRChange = 3 Å; MxAngChange = 180ꢀ). During this step, van der

Waals (vdW) and Coulombic terms were scaled to a factor of 0.1 to

avoid very severe divergences in the Coulombic and vdW energies. If the

energy of a complex structure resulting from random moves of the ligand

was higher by the energy tolerance parameter than the energy of the last

accepted structure, it was not accepted for minimization. To ensure a

’ ASSOCIATED CONTENT

S

Supporting Information. Tables of conditions for Suzuki

b

couplings on the free oxime amide 19, multiple sequences align-

ment of classes I and IIa HDAC enzymes interacting regions,

analysis of all human HDAC X-rays, X-ray structure of 3, and

comparison between HDAC 7 and HDAC 8 loop Gly763-Pro767

(human HDAC 7 numbering). This material is available free of

charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION

Corresponding Author

*For W.C.: phone, þ390691394441; fax, þ390691393638; e-mail,

Walter.Cabri@sigma-tau.it. For C.F.: phone, þ39081678544;

fax, þ39081678552; e-mail, cfattoru@unina.it. For M.R.: phone,

þ39089969254; fax, þ39089969602; e-mail, mrodriquez@unisa.it.

’ ACKNOWLEDGMENT

We thank Dr. Francesco Berrettini (C.I.A.D.S., University of

Siena, Italy) for assistance in X-ray analysis. We thank also Fabio

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Journal of Medicinal Chemistry

ARTICLE

Vaiano and Davide Paoletti (University of Siena) for skillful

technical assistance.

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’ ABBREVIATIONS USED

HDACs, histone deacetylases; HDACIs, histone deacetylase

inhibitors; SAHA, suberoylanilide hydroxamic acid; ZBG, zinc

binding group; CDI, 1,1⁰-carbonyldiimidazole; APHA, aroylpyr-

rolylhydroxyamides; CTCL, cutaneous T-cell lymphoma; DMT-

MM, 4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methylmorpholinium

chloride; NMM, 1-methylmorpholine; PMB, p-methoxybenzyl;

SARs, structure-activity relationships; SA, simulated annealing;

MM, molecular mechanics; DFT, density functional theory;

CSD, Cambridge Structural Database; PDB, Protein Data Bank;

TFMK, triﬂuoromethylketone;EF, eigenvector following; BE, bind-

ing energy; NBO, natural bond orbital; APT, Atomic Polar

Tensor; vdW, van der Waals

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Oxime amides as a novel zinc binding group in histone deacetylase inhibitors: Synthesis, biological activity, and computational evaluation

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