Design and synthesis of novel and highly-active pan-histone deacetylase (pan-HDAC) inhibitors

Accepted Manuscript

Design and Synthesis of Novel and Highly-Active Pan-Histone Deacetylase

(pan-HDAC) Inhibitors

Toshihiko Tashima, Hiroaki Murata, Hidehiko Kodama

PII:

S0968-0896(14)00341-1

DOI:

http://dx.doi.org/10.1016/j.bmc.2014.05.001

BMC 11562

Reference:

Bioorganic & Medicinal Chemistry

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2 May 2014

3 May 2014

Please cite this article as: Tashima, T., Murata, H., Kodama, H., Design and Synthesis of Novel and Highly-Active

Pan-Histone Deacetylase (pan-HDAC) Inhibitors, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/

10.1016/j.bmc.2014.05.001

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Bioorganic & Medicinal Chemistry

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Design and Synthesis of Novel and Highly-Active Pan-Histone Deacetylase (pan-

HDAC) Inhibitors

Toshihiko Tashima , Hiroaki Murata, Hidehiko Kodama

Nippon Pharmaceutical Chemicals Co., Ltd., 2-8-18 Chodo, Higashi-Osaka, Osaka 577-0056, Japan

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

Histone deacetylase (HDAC) inhibitions are known to elicit anticancer effects. We designed and

synthesized several HDAC inhibitors. Among these compounds, compound 40 exhibited a more

than 10-fold stronger inhibitory activity compared with that of suberoylanilide hydroxamic acid

(SAHA) against each human HDAC isozyme in vitro (IC₅₀values of 40: HDAC1, 0.0038 µM;

HDAC2, 0.0082 µM; HDAC3, 0.015 µM; HDAC8, 0.0060 µM; HDAC4, 0.058 µM; HDAC9,

0.0052 µM; HDAC6, 0.058 µM). The dose of the administered HDAC inhibitors that contain

hydroxamic acid as the zinc-binding group may be reduced by 40. Because the carbostyril

subunit is a time-tested structural component of drugs and biologically active compounds, 40

most likely exhibits good absorption, distribution, metabolism, excretion, and toxicity

(ADMET). Thus, compound 40 is expected to be a promising therapeutic agent or chemical tool

for the investigation of life process.

Available online

Keywords:

epigenetics

histone deacetylase

histone deacetylase inhibitor

cancer

carbostyril

The 18 human HDACs are subdivided into four classes based

on their amino acid sequences. Class I (HDACs 1-3 and 8), class

1. Introduction

2

At the molecular level, histone deacetylases (HDACs)^1,

catalyze the deacetylation of ε-N-acetyl lysine residues not only

on histone proteins but also on non-histone proteins^3-5, such as

tumor suppressor proteins, transcriptional factors, nuclear

receptors, and signal mediators, and therefore play an important

role in epigenetic transcriptional regulation⁶. From the point of

view of cancer, HDACs have much influence on proliferation,

differentiation, cell cycle arrest, and/or apoptosis. The

deacetylation of lysine residues on histone proteins leads to DNA

transcriptional repression because the coilings of chromatin are

too tight for transcription factors to access them. In this

deacetylated state of chromatin, genes such as p21^WAF1/CIP1, Gadd

45, FAS, and caspase-3, which are related to cell cycle arrest and

apoptosis in tumor cells, are transcriptionally inactivated⁷. Thus,

HDAC inhibition on histone proteins is promising strategy to

achieve anticancer effects. However, the association of HDACs

with their non-histone protein substrates has been revealed. The

acetylation of p53 or Runx3, which is a tumor suppressor protein,

increases its stability and its binding affinity to DNA, and the

acetylation of p53 results in an increase in related transcriptional

activation^3-5. Thus, HDAC inhibition on non-histone proteins

may also result in anticancer effects. In addition, HDAC

overexpression has been recognized in many types of human

cancers⁸. Therefore, HDAC inhibitors are potential anticancer

drug candidates.

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

class IV (HDAC 11) are zinc-dependent metallohydrolases,

whereas class III HDACs (sirtuins 1-7) are NAD⁺-dependent.

p53⁹and Runx3¹⁰have been implicated in HDAC1, and Runx3¹⁰

is implicated in HDAC5. It has been suggested that the overex-

pression of HDAC1-3 is associated with breast cancer^{11, 12}and

that HDAC8 is associated with T-cell lymphoma¹³and neuroblas-

toma tumorigenesis¹⁴. As previously described, HDAC isozymes

are intricately interrelated with histone proteins and non-histone

proteins.

Vorinostat (suberoylanilide hydroxamic acid, SAHA) and

romidepsin have been approved for the treatment of cutaneous T-

cell lymphoma (CTCL) by the US Food and Drug Administration

(FDA) (Figure 1). Vorinostat was used for the treatment of

CTCL in 21 countries in 2011. Recently several HDAC inhibitors

have been investigated in clinical trials as potential cancer

treatments⁶. Novel HDAC inhibitors are expected to cure patients

suffering from a variety of cancers. In the present study, we

synthesized novel and highly-active pan-HDAC inhibitors.

2. Chemistry

Compounds 1-43 were designed and synthesized in this study.

Their structures and synthetic routes are outlined in Schemes 1-7

and Tables 1-4. The letters X, Y, Z, Y, and B in Schemes 1-5

correspond to those in Tables 1-3. The analysis of the structural

———

Corresponding author, Tel.: +81-06-6781-0346; fax.: +81-06-6787-0771; e-mail.: t-tashima@nichiri.co.jp

sulted in different activity results. The rank order of activity

strength based on X-Y tended to be CH=CH > C(CH₃)₂-CH₂>

CH₂-CH₂. Thus, in addition to the zinc-binding group, the inter-

action of both the cap group and the connection unit between the

linker and the cap group to HDAC proteins is crucial for the

activity. The linkers with n = 5 were slightly shorter based on the

results of 16 and 23 at 0.1 µM. Compared with 17 and 24, com-

pounds 13 and 20 showed higher activity at 0.1 µM, respectively,

which indicates that a linker length of n = 6 is the best in this

amide connection series. The comparisons of the amide and the

corresponding reverse amide at 0.1 µM, e.g., 12 (66%) and 19

(68%), 13 (51%) and 20 (45%), 14 (58%) and 21 (44%), 15

(49%) and 22 (57%), and 26 (55%) and 29 (50%), which are

shown in Tables 2 and 3 with the percent inhibition in

features shows that the carbostyril subunit, which is often used in

biologically active compounds, as the cap group and hydroxamic

acid as the zinc-binding group are tethered with an alkyl chain.

The synthetic route of compounds 1-11 is described in Scheme 1.

The nucleophilic substitution of the corresponding carbostyril¹⁵

or coumarin phenoxides 44 to bromides yielded the ethers 45.

The hydroxamic acids 1-11 were obtained by the replacement of

esters 45 with NH₂OH. Second, the synthetic route of compounds

12-18, 26, 30, and 32 is described in Scheme 2. Dicarboxylic

acid mono esters and the corresponding carbostyril anilines 46

were condensed with EDCI, and the esters 47 were then replaced

with NH₂OH¹⁶to afford compounds 12-18, 26, 30, and 32. Third,

the synthetic route used to produce 19-25, 27-29, and 31 is

described in Scheme 3. In this route, amines and the

corresponding carbostyril or coumarin carboxylic acids 48 were

condensed with EDCI, and the esters 49 were replaced with

NH₂OH¹⁶to afford compounds 19-25, 27-29, and 31.

parentheses, did not obviously reveal which type of amide is

better in this assay using rat liver HDACs. The case of A =

NHCO in Table 2 is preferable in drug design because toxic

aniline metabolites cannot be formed.

Compounds 33-35 were then synthesized using the route

shown in Scheme 4. N-Boc-bromobenzylamine 51 was obtained

through Gabriel amine synthesis¹⁷and subsequent protection.

Ethyl acrylate was introduced through a Heck reaction¹⁸in good

yield. Through the coupling of deprotected amines 52 and

carbostyril carboxylic acids with EDCI, the esters were converted

to compounds 33-35 with NH₂OH¹⁶. Amines¹⁹were added to the

carbostyril sulfonyl chlorides 54²⁰, and the esters were converted

with NH₂OH¹⁶to yield compounds 36-38 (Scheme 5). In reverse,

the addition of 6-amino-3,4-dihydrocarbostyril to sulfonic acid

56 derived from 7-bromoheptanoic acid methyl ester 55 provided

39 (Scheme 6).

The assay results with the modified compounds based on these

findings are shown in Table 3. The compounds with a hydro-

phobic cap group, i.e., compounds 26-32, elicited higher activity

than SAHA. The comparisons of the results obtained with the

corresponding less hydrophobic compounds at 0.1 µM, for

example, 26 (55%) and 14 (58%), 27 (63%) and 19 (68%), 28

(51%) and 20 (45%), 29 (50%) and 21 (44%), 30 (52%) and 14

(58%), 31 (70%) and 19 (68%), and 32 (42%) and 14 (58%),

which are shown in Tables 2 and 3 with the percent inhibition in

parentheses, revealed that both cap forms with a hydrophobic

character gave almost the same results in the in vitro enzyme

assay. The amide bonds and the related substituent groups on

positions 1 and 2 of carbostyril did not markedly interact with

HDAC proteins. However, the in vitro and in vivo cell assays

show that the both cap forms may give different results due to

other factors, such as membrane permeability. The compounds

possessing sulfonamide and reverse sulfonamide as the connec-

tion unit, i.e., compounds 36-39, had low activity. Of compounds

with a linker containing an aromatic ring, the para form 34

elicited stronger activity than SAHA.

The compounds with a linker containing an aromatic ring

exhibited low solubility. To improve this low solubility, the

benzene ring was saturated to form a cyclohexane ring in the

molecular design. The assay results of the resulting compounds

are shown in Table 4. The trans forms 40 and 41 elicited stronger

activity than SAHA, but the cis forms 42 and 43 elicited weaker

activity. The lengths between the carbonyl oxygen atom of the

hydroxamic acid group and the carbonyl oxygen atom of the

connection amide group on position 6 of carbostyril were

calculated by MM2 stabilization in a vacuum state using the

Chem3D, software (version 13). Although the approximate rank

order of the linker length was 40 (11.33 Ǻ) > 41 (10.56 Ǻ) > 42

(9.33 Ǻ) > 43 (8.87 Ǻ), showing calculated length in parentheses,

the rank order of the activity strength was 40 > 41 > 43 > 42. The

cyclohexyl linkers were so rigid that the calculated lengths

presumably reflect the actually observed lengths. The linker

length of 40 resulted in the best interaction with HDAC proteins.

The synthetic route used for the production of 40-43 is de-

scribed in Scheme 7. These routes are started from the trans and

cis forms of 1,4-cyclohexanedimethanol 58. After mono-

benzylation, the other hydroxyl group was mesylated and then

replaced with NaN₃. The amines provided by the hydrogenolysis

of azides were coupled to 4,4-dimethyl-3,4-dihydrocarbostyril-6-

carboxylic acid with EDCI to form amides 59. The hydroxyl

groups obtained by the high-presure hydrogenolysis of benzyl

groups were oxidized with Dess-Martin periodinane²¹and then

subject to a Horner-Emmons reaction to generate esters 60. The

replacement of esters with NH₂OH¹⁶gave compounds 40 and 42.

In contrast, through a reduction of the double bond by

hydrogenation, the replacement of esters with NH₂OH¹⁶produced

compounds 41 and 43.

3. Results and discussion

Two types of enzyme assays, using rat liver HDACs and hu-

man HDAC isozymes, were performed in this study. The results

of the enzyme assays performed with SAHA²²and 1-43 using rat

liver HDACs are shown in Tables 1-4. All of the compounds in

which the cap group and the linker were connected by an ether

bond showed low activity at 0.1 µM and high activity at 10 µM

with the exception of compound 4 (Table 1). Although it is well-

known, the linker of HDAC inhibitors has the appropriate

length²³. The weak activity of 4, which possessed a shorter linker

than the other compounds, reflects this fact. The linker lengths of

SAHA and 1-11, with the exception of 4, were similar. It is thus

apparent that the ether bonds in this region of the molecules are

repulsive to HDAC enzymes. Therefore, the ether bonds were

replaced with an amide bond in the next design, and the ether

series compound with the longer linker was not synthesized.

Interestingly, some of the compounds with an amide connect-

ion elicited stronger activity than SAHA (Table 2). This suggests

the existence of a hydrogen bond between this amide connection

and HDAC proteins. The structural features of the cap group re-

The results of enzyme assays with SAHA and several of the

synthesized compounds using human HDAC isozymes are shown

in Table 5. The rank order of the activity strength of the

cyclohexyl series was 40 > 41 = 43 > 42, which is mostly consis-

tent with that obtained in the enzyme assays using rat HDACs.

Surprisingly, compound 40 elicited a more than 10-fold increase

in pan-spectrum inhibitory activity compared with that of SAHA

against human HDAC isozymes, particularly against HDAC8.

Structurally, the active site of HDAC proteins forms a cylindrical

cave, and the zinc is located at the bottom of this cave²⁴. The

analysis of the interaction mode between HDAC proteins and

HADC inhibitors shows that the zinc-binding groups chelate the

zinc atoms at the bottom of the cave and that the cave mouths are

covered with the cap groups. Furthermore, the linkers interact

with the wall surface of the cave. Thus, it is hypothesized that

cyclohexyl linkers such as that found in compound 40 settle in

the cylindrical cave in a thermodynamically more stable state

than straight-chain linkers, such as that found in SAHA, due to

various factors, such as hydrophobic bonds.

5.1.1. 6-(3,4-dihydrocarbostyril-6-yloxy)hexanoic acid

ethyl ester (representative of compound 45) as the general

procedure for the synthesis of the intermediate of 1-11

A solution of 6-hydroxy-3,4-dihydrocarbostyril (corresponding

to 44) (2.00 g, 12.26 mmol) in DMF (10 ml) was added dropwise

to a suspension of NaH (60% in mineral oil, 0.59 g, 14.71 mmol)

in DMF (5 ml) for 14 min at 0°C with a CaCl₂tube. After stirring

for 20 min at 0°C, a solution of 6-bromohexanoic acid ethyl ester

(3.06 g, 13.48 mmol) in DMF (7 ml) was added dropwise to the

reaction mixture for 7 min at 0°C. The reaction mixture was

stirred overnight, during which time the ice was allowed to

gradually melt and was subsequently stirred for 3 h at 110°C.

After cooling, H₂O (100 ml) was added to the mixture, and the

resulting mixture was extracted with AcOEt (100 ml × 3). The

combined organic layer was washed with H₂O (100 ml × 1) and

brine (100 ml × 1), dried over Na₂SO₄(anhyd.), filtered, and

concentrated under reduced pressure. The crude product was

purified through open silica gel column chromatography (n-

hexane:AcOEt = 1:2) to afford a colorless solid (2.56 g, 8.38

mmol, y. 68%). Colorless needles (n-hexane / AcOEt). Mp. 79-

▪ 4. Conclusions

The carbostyril derivatives designed and synthesized in this

study elicited HDAC inhibitory activity. Of these studied struc-

tures, compound 40 exhibited much stronger pan-spectrum

HDAC inhibitory activity than SAHA. Pan-spectrum HDAC

inhibitors and selective-spectrum HDAC inhibitors have their

own advantages. It is true that selective HDAC inhibitors are

very effective for the treatment of chronic diseases due to their

few side effects, but their effect in the treatment of acute diseases

is different. In these cases, pan-spectrum HDAC inhibitors will

exert serious effects even though they might yield some side

effects, depending on the conditions. Many types of cancers are

considered representative examples of acute diseases and have

complicated pathological mechanisms. The analysis of HDACs,

which catalyze the deacetylaion of histone proteins and non-

histone proteins as their substrates, has shown that these proteins

are involved in a variety of processes, including suppression,

onset, progression, and apoptosis of cancer cells, through

complicated mechanisms. If that is true, it is thus possible that

pan-spectrum HDAC inhibitors may be attractive agents for

treatment of cancers. This hypothesis is supported by the fact that

SAHA and romidepsin have been approved for the treatment of

CTCL by FDA. Therefore, the stronger pan-spectrum HDAC

inhibitory activity of compound 40 is one of its advantages. The

dose of the administered HDAC inhibitors that contain

hydroxamic acid as the zinc-binding group may be reduced by

compound 40, which may result in low side effects and toxicities.

Moreover, because the carbostyril subunit is a time-tested

structural component of drugs and biologically active

1

80°C. H-NMR (300 MHz / CDCl₃) δ1.26 (3H, t, J = 7.2 Hz,

CH₂CH₃), 1.47-1.52 (2H, m, CH₂), 1.65-1.81 (4H, m, CH₂× 2),

2.33 (2H, t, J = 7.4 Hz, CH₂), 2.61 (2H, t, J = 7.5 Hz, CH₂), 2.93

(2H, t, J = 7.4Hz, CH₂), 3.92 (2H, t, J = 6.5 Hz, CH₂), 4.13 (2H,

q, J = 7.2 Hz, CH₂CH₃), 6.65-6.69 (2H, m, ArH), 6.72 (1H, s,

ArH), 7.91 (1H, s, NH).

5.1.2. 6-(3,4-dihydrocarbostyril-6-yloxy)hexanoic acid

hydroxyamide (2) as the general procedure for the synthesis of

1-43

A solution of NH₂OH in 50% water (0.40 ml, 6.55 mmol) was

added dropwise to a suspension of 6-(3,4-dihydrocarbostyril-6-

yloxy)hexanoic acid ethyl ester (corresponding to 45) (200 mg,

0.66 mmol) in MeOH (1.2 ml) at 0°C. After stirring for 4 min at

0°C, NaOMe in 25% MeOH (0.75 ml, 3.28 mmol) was added

dropwise to the reaction mixture. After stirring for 1 h at 0^oC,

sat.NH₄Cl (10 ml) was added to the mixture. The precipitate was

gathered, washed with H₂O and then n-hexane, and dried to

afford a colorless solid (173 mg, 0.59 mmol, y. 90%). Colorless

compounds^25-28, 40 may possess good features of absorption,

distribution, metabolism, excretion, and toxicity (ADMET).

Thus, compound 40 is expected to be a good therapeutic agent

for cancers and can be used as a chemical tool to investigate the

process of life phenomena, particularly epigenetics, concerning

HDACs.

1

powder (AcOEt). Mp. 178-179°C. H-NMR (300 MHz / DMSO-

d₆) δ1.31-1.41 (2H, m, CH₂), 1.49-1.59 (2H, m, CH₂), 1.62-1.71

(2H, m, CH₂), 1.96 (2H, t, J = 7.2 Hz, CH₂), 2.39 (2H, t, J = 7.5

Hz, CH₂), 2.82 (2H, t, J = 7.7 Hz, CH₂), 3.87 (2H, t, J = 6.5 Hz,

CH₂), 6.69 (1H, dd, J = 8.6, 2.6 Hz, ArH), 6.75 (1H, d, J = 8.1

Hz, ArH), 6.76 (1H, s, ArH), 8.66 (1H, s, NH), 9.88 (1H, s, NH),

10.33 (1H, bs, NOH). LRMS (ESI+) m/z 293 [M+H]⁺. LRMS

(ESI-) m/z 291 [M-H]^-.

5. Experimental procedures

5.1 Chemistry

5.1.3. 6-(carbostyril-6-yloxy)hexanoic acid hydroxyamide

(1)

The commercial solvents and reagents used in this study were

generally used without further purification. Column chromatog-

raphy was conducted on silica gel 60 (230-400 mesh, Merck).

Colorless powder (AcOEt). Mp. 199-200^oC . ¹H-NMR (300

MHz / DMSO-d₆) δ1.37-1.45 (2H, m, CH₂), 1.52-1.59 (2H, m,

CH₂), 1.70-1.75 (2H, m, CH₂), 1.98 (2H, t, J = 7.2 Hz, CH₂), 3.97

(2H, t, J = 6.5 Hz, CH₂), 6.48 (1H, d, J = 9.3 Hz, ArH), 7.13 (1H,

dd, J = 9.0 Hz, 2.7 Hz, ArH), 7.19 (1H, d, J = 2.4 Hz, ArH), 7.24

(1H, d, J = 9.0 Hz, ArH), 7.83 (1H, d, J = 9.6 Hz, ArH), 8.68 (1H,

s, NH), 10.38 (1H, s, NH), 11.62 (1H, s, NOH). LRMS (ESI+)

m/z 291 [M+H]⁺. LRMS (ESI-) m/z 289 [M-H]^-.

1

The H-NMR spectra were recorded on a Bruker Avance 300

spectrometer. The chemical shifts in the ¹H-NMR spectra are

given in parts per million (ppm, δ) relative to tetramethylsilane

(δ0.00) as the internal standard, and the coupling constants are

reported in hertz. The multiplicities are given as s (singlet), br

(broad), d (doublet), t (triplet), q (quartet), dd (doublet of

doublets), dt (doublet of triplets), and m (multiplet). The LRMS

spectra were recorded on a Shimadzu LCMS-2020 instrument.

The HRMS spectra were recorded on a Shimadzu AXIMA

Confidence instrument. The melting points were measured using

Yanako micro metling point apparatus and were uncorrected.

5.1.4. 6-(4,4-dimethyl-3,4-dihydrocarbostyril-6-yloxy)hex-

anoic acid hydroxyamide (3)

Colorless powder (n-hexane / AcOEt). Mp. 99-102°C. ¹H-

NMR (300 MHz / DMSO-d₆) δ1.20 (6H, m, CH₃× 2), 1.38-1.40

(2H, m, CH₂), 1.52-1.55 (2H, m, CH₂), 1.65-1.70 (2H, m, CH₂),

1.97 (2H, t, J = 7.2 Hz, CH₂), 2.29 (2H, s, CH₂), 3.89 (2H, t, J =

6.5 Hz, CH₂), 6.71 (1H, dd, J = 8.7, 2.4 Hz, ArH), 6.78 (1H, d, J

= 8.4 Hz, ArH), 6.81 (1H, d, J = 2.4 Hz, ArH), 8.66 (1H, s, NH),

9.95 (1H, s, NH), 10.34 (1H, s, NOH). LRMS (ESI-) m/z 319

[M-H]^-.

= 8.1 Hz, ArH), 7.06 (1H, t, J = 8.1 Hz, ArH), 8.66 (1H, s, NH),

10.00 (1H, s, NH), 10.33 (1H, s, NOH). LRMS (ESI+) m/z 293

[M+H]⁺. LRMS (ESI-) m/z 291 [M-H]^-.

5.1.11. 6-(carbostyril-8-yloxy)hexanoic acid hydroxyamide

(10)

5.1.5. 4-(3,4-dihydrocarbostyril-6-yloxy)butanoic acid

hydroxyamide (4)

Colorless powder (AcOEt). Mp. 122-124°C. ¹H-NMR (300

MHz / DMSO-d₆) δ1.43-1.50 (2H, m, CH₂), 1.53-1.63 (2H, m,

CH₂), 1.78-1.87 (2H, m, CH₂), 2.00 (2H, t, J = 7.1 Hz, CH₂), 4.08

(2H, t, J = 6.3 Hz, CH₂), 6.52 (1H, d, J = 9.6 Hz, ArH), 7.07-7.14

(2H, m, ArH), 7.22 (1H, dd, J = 6.0, 3.0 Hz, ArH), 7.88 (1H, d, J

= 9.6 Hz, ArH), 8.68 (1H, s, NH), 10.37 (1H, s, NH), 10.72 (1H,

s, NOH). LRMS (ESI+) m/z 291 [M+H]⁺. LRMS (ESI-) m/z 289

[M-H]^-.

Colorless powder (AcOEt). Mp. 190-191°C. ¹H-NMR (300

MHz / DMSO-d₆) δ1.85-1.94 (2H, m, CH₂), 2.11 (2H, t, J = 7.4

Hz, CH₂), 2.39 (2H, t, J = 7.7 Hz, CH₂), 2.82 (2H, t, J = 7.5 Hz,

CH₂), 3.88 (2H, t, J = 6.3 Hz, CH₂), 6.70 (1H, dd, J = 8.7, 2.7 Hz,

ArH), 6.75 (1H, d, J = 7.8 Hz, ArH), 6.76 (1H, d, J = 2.7 Hz,

ArH), 8.69 (1H, s, NH), 9.89 (1H, s, NH), 10.40 (1H, bs, NOH).

LRMS (ESI+) m/z 265 [M+H]⁺. LRMS (ESI-) m/z 263 [M-H]^-.

5.1.6. 6-(coumarin-6-yloxy)hexanoic acid hydroxyamide (5)

5.1.12. 6-(3,4-dihydrocarbostyril-8-yloxy)hexanoic acid

hydroxyamide (11)

Colorless powder (n-hexane / AcOEt). Mp. 124-126°C. ¹H-

NMR (300 MHz / DMSO-d₆) δ1.37-1.45 (2H, m, CH₂), 1.51-1.61

(2H, m, CH₂), 1.68-2.00 (2H, m, CH₂), 1.98 (2H, t, J = 7.2 Hz,

CH₂), 4.00 (2H, t, J = 7.2 Hz, CH₂), 6.48 (1H, d, J = 9.6 Hz,

ArH), 7.19 (1H, dd, J = 9.0, 3.0 Hz, ArH), 7.28 (1H, d, J = 2.7

Hz, ArH), 7.33 (1H, d, J = 9.0 Hz, ArH), 8.00 (1H, d, J = 9.6 Hz,

ArH), 8.67 (1H, d, J = 1.2 Hz, NH), 10.35 (1H, s, NOH). LRMS

(ESI+) m/z 292 [M+H]⁺. LRMS (ESI-) m/z 290 [M-H]^-.

Colorless oil. ¹H-NMR (300 MHz / CDCl₃) δ1.49-1.54 (2H, m,

CH₂), 1.70-1.82 (4H, m, CH₂× 2), 2.23 (2H, t, J = 6.9 Hz, CH₂),

2.61 (2H, t, J = 7.5 Hz, CH₂), 2.94 (2H, t, J = 7.5 Hz, CH₂), 3.99

(2H, t, J = 6.0 Hz, CH₂), 6.71-6.75 (2H, m, ArH), 6.91 (1H, t, J =

8.0 Hz, ArH), 8.24 (1H, s, NH), 9.84 (1H, bs, NOH). LRMS

(ESI+) m/z 293 [M+H]⁺. LRMS (ESI-) m/z 291 [M-H]^-.

5.1.13. 7-(3,4-dihydrocarbostyril-6-ylcarbamoyl)heptanoic

acid benzyl ester (representative of 47) as the general procedure

for the synthesis of the intermediate of 12-18, 26, 30, and 32

5.1.7. 6-(carbostyril-7-yloxy)hexanoic acid hydroxyamide

(6)

Colorless powder (AcOEt). Mp. 174-175°C. ¹H-NMR (300

MHz / DMSO-d₆) δ1.35-1.45 (2H, m, CH₂), 1.51-1.61 (2H, m,

CH₂), 1.69-1.78 (2H, m, CH₂), 1.98 (2H, t, J = 7.2 Hz, CH₂), 3.99

(2H, t, J = 6.3 Hz, CH₂), 6.29 (1H, d, J = 9.6 Hz, ArH), 6.76-6.79

(2H, m, ArH), 7.54 (1H, d, J = 9.3 Hz, ArH), 7.79 (1H, d, J = 9.3

Hz, ArH), 8.67 (1H, s, NH), 10.35 (1H, s, NH), 11.55 (1H, s,

NOH). LRMS (ESI+) m/z 291 [M+H]⁺. LRMS (ESI-) m/z 289

[M-H]^-.

The reaction mixture of 6-amino-3,4-dihydrocarbostyril (corre-

sponding to 46) (78 mg, 0.48 mmol), suberic acid mono-benzyl

ester (128 mg, 0.48 mmol), HOBt (65 mg, 0.48 mmol), Et₃N (49

mg, 0.48 mmol), and EDCIꢀHCl (93 mg, 0.48 mmol) in DMF (2

ml) was stirred overnight. Sat.NaHCO₃(30 ml) was added to the

mixture, and the resulting mixture was extracted with AcOEt (30

ml × 3). The combined organic layer was washed with 2 N HCl

(30 ml × 1) and then brine (30 ml × 1), dried over Na₂SO₄

(anhyd.), filtered, and concentrated under reduced pressure. The

crude product was purified through open silica gel column

chromatography (AcOEt) to afford a pale orange solid (101 mg,

0.25 mmol, y. 51%). Colorless powder (n-hexane / AcOEt). Mp.

5.1.8. 6-(3,4-dihydrocarbostyril-7-yloxy)hexanoic acid

hydroxyamide (7)

Colorless powder (AcOEt). Mp. 130-132°C. ¹H-NMR (300

MHz / DMSO-d₆) δ1.33-1.38 (2H, m, CH₂), 1.52-1.57 (2H, m,

CH₂), 1.65-1.70 (2H, m, CH₂), 1.97 (2H, t, J = 7.5 Hz, CH₂), 2.41

(2H, t, J = 7.5 Hz, CH₂), 2.78 (2H, t, J = 7.4 Hz, CH₂), 3.87 (2H,

t, J = 6.5 Hz, CH₂), 6.42 (1H, d, J = 2.4 Hz, ArH), 6.47 (1H, dd, J

= 8.3 Hz, 2.6 Hz, ArH), 7.03 (1H, d, J = 8.1 Hz, ArH), 8.66 (1H,

s, NH), 9.96 (1H, s, NH), 10.35 (1H, s, NOH). LRMS (ESI+) m/z

293 [M+H]⁺. LRMS (ESI-) m/z 291 [M-H]^-.

1

87-88°C. H-NMR (300 MHz / CDCl₃) δ1.36-1.38 (4H, m, CH₂

× 2), 1.63-1.74 (4H, m, CH₂× 2), 2.29-2.39 (4H, m, CH₂× 2),

2.61 (2H, t, J = 7.5 Hz, CH₂), 2.94 (2H, t, J = 7.5 Hz, CH₂), 5.11

(2H, s, CH₂), 6.71 (1H, d, J = 8.4 Hz, ArH), 7.17 (1H, dd, J = 8.4

Hz, 2.1 Hz, ArH), 7.23 (1H, s, ArH), 7.34-7.37 (5H, m, ArH),

7.50 (1H, s, NH), 8.13 (1H, s, NH). LRMS (ESI+) m/z 409

[M+H]⁺. LRMS (ESI-) m/z 407 [M-H]^-.

5.1.14. 7-(carbostyril-6-ylcarbamoyl)heptanoic acid hy-

droxyamide (12)

5.1.9. 6-(4,4-dimethyl-3,4-dihydrocarbostyril-7-yloxy)-

hexanoic acid hydroxyamide (8)

Light green powder (CHCl₃/ MeOH). Mp. 210-211°C. ¹H-

NMR (300 MHz / DMSO-d₆) δ1.27-1.29 (4H, m, CH₂× 2), 1.44-

1.51 (4H, m, CH₂× 2), 1.94 (2H, t, J = 7.2 Hz, CH₂), 2.27-2.31

(2H, m, NCH₂), 6.47 (1H, d, J = 9.6 Hz, CH=), 7.23 (1H, d, J =

9.0 Hz, ArH), 7.56 (1H, dd, J = 8.7, 2.1 Hz, ArH), 7.85 (1H, d, J

= 9.6 Hz, CH=), 7.99 (1H, d, J = 1.8 Hz, ArH), 8.66 (1H, br, NH),

9.95 (1H, br, NH), 10.34 (1H, br, NH), 11.69 (1H, br, NOH).

LRMS (ESI+) m/z 332 [M+H]⁺. LRMS (ESI-) m/z 330 [M-H]^-.

Colorless powder (n-hexane / AcOEt). Mp. 62-63°C. ¹H-NMR

(300 MHz / DMSO-d₆) δ1.18 (6H, s, CH₃× 2), 1.34-1.41 (2H, m,

CH₂), 1.49-1.57 (2H, m, CH₂), 1.63-1.70 (2H, m, CH₂), 1.97 (2H,

t, J = 7.4 Hz, CH₂), 2.30 (2H, s, CH₂), 3.87 (2H, t, J = 6.5 Hz,

CH₂), 6.43 (1H, d, J = 2.7 Hz, ArH), 6.52 (1H, dd, J = 8.4, 2.4Hz,

ArH), 7.15 (1H, d, J = 8.4 Hz, ArH), 8.66 (1H, s, NH), 10.01 (1H,

s, NH), 10.33 (1H, bs, NOH). LRMS (ESI+) m/z 321 [M+H]⁺.

LRMS (ESI-) m/z 319 [M-H]^-.

5.1.15. 7-(3,4-dihydrocarbostyril-6-ylcarbamoyl)heptanoic

acid hydroxyamide (13)

5.1.10. 6-(3,4-dihydrocarbostyril-5-yloxy)hexanoic acid

hydroxyamide (9)

Colorless powder (AcOEt). Mp. 196-197°C. ¹H-NMR (300

MHz / DMSO-d₆) δ1.26-1.28 (4H, m, CH₂× 2), 1.46-1.56 (4H,

m, CH₂× 2), 1.94 (2H, t, J = 7.4 Hz, CH₂), 2.25 (2H, t, J = 7.2

Hz, CH₂), 2.41 (2H, t, J = 7.5 Hz, CH₂), 2.82 (2H, t, J = 7.5 Hz,

ArH), 6.76 (1H, d, J = 8.4 Hz, ArH), 7.29 (1H, dd, J = 8.6, 2.3

Hz, ArH), 7.43 (1H, d, J = 1.5 Hz, ArH), 8.65 (1H, s, NH), 9.73

Colorless powder (AcOEt). Mp. 159-160°C. ¹H-NMR (300

MHz / DMSO-d₆) δ1.37-1.45 (2H, m, CH₂), 1.51-1.60 (2H, m,

CH₂), 1.67-1.76 (2H, m, CH₂), 1.97 (2H, t, J = 7.2 Hz, CH₂), 2.40

(2H, t, J = 7.8 Hz, CH₂), 2.79 (2H, t, J = 7.7 Hz, CH₂), 3.94 (2H,

t, J = 6.3 Hz, CH₂), 6.47 (1H, d, J = 7.8 Hz, ArH), 6.58 (1H, d, J

(1H, s, NH), 9.98 (1H, s, NH), 10.33 (1H, s, NOH). LRMS

(ESI+) m/z 334 [M+H]⁺. LRMS (ESI-) m/z 332 [M-H]^-.

5.1.22. 7-(3,4-dihydro-4,4-dimethyl-2-methoxyquinolin-6-

ylcarbamoyl)heptanoic acid hydroxyamide (30)

5.1.16. 7-(4,4-dimethyl-3,4-dihydrocarbostyril-6-yl-

carbamoyl)heptanoic acid hydroxyamide (14)

Colorless powder (MeOH). Mp. 66-71°C. ¹H-NMR (300 MHz

/ MeOH-d₄) δ1.23 (6H, s, CH₃× 2), 1.38-1.40 (4H, m, CH₂× 2),

1.61-1.73 (4H, m, CH₂× 2), 2.09 (2H, t, J = 7.4 Hz, CH₂), 2.31

(2H, s, CH₂), 2.36 (2H, t, J = 7.6 Hz, CH₂), 3.87 (3H, s, OCH₃),

7.08 (1H, d, J = 8.4 Hz, ArH), 7.34 (1H, dd, J = 8.4, 2.4 Hz,

ArH), 7.56 (1H, d, J = 2.4 Hz, ArH). LRMS (ESI+) m/z 376

[M+H]⁺. LRMS (ESI-) m/z 374 [M-H]^-.

Colorless powder (AcOEt). Mp. 167-168°C. ¹H-NMR (300

MHz / DMSO-d₆) δ1.20 (6H, s, CH₃× 2), 1.26-1.28 (4H, m, CH₂

× 2), 1.46-1.56 (4H, m, CH₂× 2), 1.94 (2H, t, J = 7.4 Hz, CH₂),

2.25 (2H, t, J = 7.7 Hz, CH₂), 2.31 (2H, s, CH₂), 6.77 (1H, d, J =

8.7 Hz, ArH), 7.39 (1H, dd, J = 8.4, 2.1 Hz, ArH), 7.51 (1H, d, J

= 2.1 Hz, ArH), 8.64 (1H, s, NH), 9.74 (1H, s, NH), 10.04 (1H, s,

NH), 10.32 (1H, bs, NOH). LRMS (ESI-) m/z 360 [M-H]^-.

5.1.23. 7-(3,4-dihydro-4,4-dimethyl-2-ethoxyquinolin-6-yl-

carbamoyl)heptanoic acid hydroxyamide (32)

5.1.17. 7-(8-fluoro-4,4-dimethyl-3,4-dihydrocarbostyril-6-

ylcarbamoyl)heptanoic acid hydroxyamide (15)

Colorless powder (n-hexane / AcOEt). Mp.162-164°C. ¹H-

NMR (300 MHz / DMSO-d₆) δ1.23 (6H, s, CH₃× 2), 1.25-1.31

(7H, m, CH₂× 2 and OCH₂CH₃), 1.46-1.58 (4H, m, CH₂× 2),

1.94 (2H, t, J = 7.2 Hz, CH₂), 2.23-2.29 (4H, m, CH₂and CH₂),

4.27 (2H, q, J = 7.2 Hz, OCH₂CH₃), 6.95 (1H, d, J = 8.4 Hz,

ArH), 7.32 (1H, br, NH), 7.43 (1H, dd, J = 8.4, 2.4 Hz, ArH),

7.52 (1H, d, J = 2.4 Hz, ArH), 9.78 (1H, br, NH), 10.32, (1H, br,

OH). LRMS (ESI+) m/z 390 [M+H]⁺. LRMS (ESI-) m/z 388 [M-

H]^-.

Colorless powder (CHCl₃/ DMSO). Mp. 173-175°C. ¹H-NMR

(300 MHz / DMSO-d₆) δ1.21 (6H, s, CH₃× 2), 1.25-1.27 (4H, m,

CH₂× 2), 1.46-1.58 (4H, m, CH₂× 2), 1.93 (2H, t, J = 7.2 Hz,

CH₂), 2.26 (2H, t, J = 7.4 Hz, CH₂), 2.37 (2H, s, CH₂), 7.21 (1H,

s, ArH), 7.54 (1H, dd, J = 13.0, 2.1 Hz, ArH), 8.66 (1H, br, NH),

9.95 (1H, br, NH), 10.06 (1H, br, NH), 10.39 (1H, br, NOH).

LRMS (ESI+) m/z 380 [M+H]⁺. LRMS (ESI-) m/z 378 [M-H]^-.

5.1.24. 7-[(4,4-dimethyl-3,4-dihydrocarbostyril-6-

carbonyl)amino]heptanoic acid benzyl ester (representative of

49) as the general procedure for the synthesis of the intermediate

of 19-25, 27-29, and 31

5.1.18. 6-(3,4-dihydrocarbostyril-6-ylcarbamoyl)hexanoic

acid hydroxyamide (16)

Colorless powder (CHCl₃/ DMSO). Mp. 186-188°C. ¹H-NMR

(300 MHz / DMSO-d₆) δ1.17-1.30 (2H, m, CH₂), 1.45-1.61 (4H,

m, CH₂× 2), 1.94 (2H, t, J = 7.4 Hz, CH₂), 2.43 (2H, t, J = 7.2

Hz, CH₂), 2.39-2.43 (2H, m, CH₂), 2.79-2.84 (2H, m, CH₂), 6.75

(1H, d, J = 8.4 Hz, ArH), 7.28 (1H, dd, J = 8.4, 2.1 Hz ArH),

7.43 (1H, d, J = 1.5 Hz ArH), 8.67 (1H, br, NH), 10.14 (1H, br,

NH), 10.33 (1H, br, NOH). LRMS (ESI+) m/z 320 [M+H]⁺.

LRMS (ESI-) m/z 318 [M-H]^-.

The reaction mixture of 4,4-dimethyl-3,4-dihydrocarbostyril-6-

carboxylic acid (corresponding to 48) (251 mg, 1.25 mmol), 7-

aminoheptanoic acid benzyl ester (251 mg, 1.31 mmol), HOBt

(178 mg, 1.31 mmol), Et₃N (133 mg, 1.31 mmol), and EDCIꢀHCl

(252 mg, 1.31 mmol) in DMF (6 ml) was stirred overnight at

room temperature. Then, sat.NaHCO₃(50 ml) was added to the

mixture, and the resulting mixture was extracted with AcOEt (50

ml × 3). The combined organic layer was washed with H₂O (50

ml × 1), 1 N HCl (50 ml × 1) and then brine (50 ml × 1), dried

over Na₂SO₄(anhyd.), filtered, and concentrated under reduced

pressure. The crude product was purified through open silica gel

column chromatography (n-hexane:AcOEt = 1:2) to afford a

colorless oil (117 mg, 0.29 mmol, y.22%). ¹H-NMR (300 MHz /

CDCl₃) δ1.26 (6H, s, CH₃× 2), 1.39-1.43 (4H, m, CH₂× 2), 1.62-

1.69 (4H, m, CH₂× 2), 2.28-2.39 (2H, m, CH₂), 2.51 (2H, s,

CH₂), 3.40-3.46 (2H, m, CH₂), 5.11 (2H, s, CH₂), 6.07 (1H, t, J =

5.4 Hz, NH), 6.79 (1H, d, J = 8.1 Hz, ArH), 7.34-7.36 (5H, m,

ArH), 7.52 (1H, dd, J = 8.1, 1.8 Hz, ArH), 7.80 (1H, d, J = 1.5

Hz, ArH), 8.18 (1H, s, NH).

5.1.19. 8-(3,4-dihydrocarbostyril-6-ylcarbamoyl)octanoic

acid hydroxyamide (17)

Colorless powder (MeOH). Mp. 182-183°C. ¹H-NMR (300

MHz / DMSO-d₆) δ1.22-1.32(6H, m, CH₂× 3), 1.45-1.60 (4H, m,

CH₂× 2), 1.93 (2H, t, J = 7.2 Hz, CH₂), 2.24 (2H, t, J = 7.2 Hz,

CH₂), 2.41 (2H, t, J = 7.5 Hz, CH₂), 2.82 (2H, t, J = 7.4 Hz, CH₂),

6.75 (1H, d, J = 8.7 Hz, ArH), 7.28 (1H, d, J = 8.7 Hz, ArH),

7.43 (1H, s, ArH), 8.66 (1H, br, NH), 9.72 (1H, br, NH), 9.99

(1H, br, NH), 10.33 (1H, br, NOH). LRMS (ESI+) m/z 348

[M+H]⁺. LRMS (ESI-) m/z 346 [M-H]^-.

5.1.20. 7-(coumarin-6-ylcarbamoyl)heptanoic acid

hydroxyamide (18)

5.1.25. 7-[(carbostyril-6-carbonyl)amino]heptanoic acid

hydroxyamide (19)

Colorless powder (CHCl₃/ DMSO). Mp. 187-188°C. ¹H-

NMR (300 MHz / DMSO-d₆) δ1.26-1.32 (4H, m, CH₂× 2), 1.44-

1.64 (4H, m, CH₂× 2), 1.94 (2H, t, J = 7.2 Hz, CH₂), 2.31 (2H, t,

J = 7.4 Hz, CH₂), 6.48 (1H, d, J = 9.6 Hz, CH=), 7.36 (1H, d, J =

9.0 Hz ArH), 7.65 (1H, dd, J = 9.0, 2.7 Hz ArH), 8.05-8.09 (2H,

m, ArH, CH=), 8.66 (1H, br, NH), 10.10 (1H, br, NH), 10.33 (1H,

br, NOH). LRMS (ESI+) m/z 333 [M+H]⁺. LRMS (ESI-) m/z

331 [M-H]^-.

Light beige powder (MeOH). Mp. unmeasurable (decomposed

at 263°C). ¹H-NMR (300 MHz / DMSO-d₆) δ1.26-1.33 (4H, m,

CH₂× 2), 1.46-1.55 (4H, m, CH₂× 2), 1.94 (2H, t, J = 7.5 Hz,

CH₂), 3.21-3.29 (2H, m, NCH₂), 6.55 (1H, d, J = 9.6 Hz, CH=),

7.31 (1H, d, J = 8.7 Hz, ArH), 7.93-7.97 (2H, m, ArH, CH=),

8.16 (1H, d, J = 1.8 Hz, ArH), 8.44 (1H, br, NH), 8.66 (1H, br,

NH), 10.37 (1H, br, NH) , 11.92 (1H, br, NOH). LRMS (ESI+)

m/z 332 [M+H]⁺. LRMS (ESI-) m/z 330 [M-H]^-.

5.1.21. 7-(1,4,4-trimethyl-3,4-dihydrocarbostyril-6-yl-

carbamoyl)heptanoic acid hydroxyamide (26)

5.1.26. 7-[(3,4-dihydrocarbostyril-6-carbonyl)amino]hep-

tanoic acid hydroxyamide (20)

Colorless powder (CHCl₃/ AcOEt). Mp. 118-121°C. ¹H-NMR

(300 MHz / MeOH-d₄) δ1.28 (6H, s, CH₃× 2), 1.38-1.40 (4H, m,

CH₂× 2), 1.61-1.68 (4H, m, CH₂× 2), 2.10 (2H, t, J = 7.4 Hz,

CH₂), 2.37 (2H, t, J = 7.4 Hz, CH₂), 2.48 (2H, s, CH₂), 3.34 (3H,

s, NCH₃), 7.10 (1H, d, J = 8.7 Hz, ArH), 7.50 (1H, d, J = 8.7 Hz,

ArH), 7.60 (1H, s, ArH). LRMS (ESI+) m/z 376 [M+H]⁺. LRMS

(ESI-) m/z 374 [M-H]^-.

Colorless powder (AcOEt). Mp. 189-190°C. ¹H-NMR (300

MHz / DMSO-d₆) δ1.27-1.28 (4H, m, CH₂× 2), 1.46-1.51 (4H,

m, CH₂× 2), 1.94 (2H, t, J = 7.4 Hz, CH₂), 2.47 (2H, t, J = 7.2

Hz, CH₂), 2.91 (2H, t, J = 7.5 Hz, CH₂), 3.18-3.24 (2H, m, CH₂),

6.86 (1H, d, J = 8.1 Hz, ArH), 7.63 (1H, d, J = 8.4 Hz, ArH),

7.67 (1H, s, ArH), 8.25 (1H, t, J = 5.4 Hz, NH), 8.64 (1H, s, NH),

10.26 (1H, s, NH), 10.32 (1H, s, NOH). LRMS (ESI+) m/z 334

[M+H]⁺. LRMS (ESI-) m/z 332 [M-H]^-.

ArH), 8.56 (1H, br, NH), 8.67 (1H, br, NH), 10.37 (1H, br, OH).

LRMS (ESI+) m/z 346 [M+H]⁺. LRMS (ESI-) m/z 344 [M-H]^-.

5.1.27. 7-[(4,4-dimethyl-3,4-dihydrocarbostyril-6-

carbonyl)amino]heptanoic acid hydroxyamide (21)

5.1.33. 7-[(1-methyl-3,4-dihydrocarbostyril-6-carbonyl)-

amino]heptanoic acid hydroxyamide (28)

Colorless powder (AcOEt). Mp. 147-149°C. ¹H-NMR (300

MHz / DMSO-d₆) δ1.26 (6H, s, CH₃× 2), 1.29 (4H, m, CH₂× 2),

1.50 (4H, m, CH₂× 2), 1.94 (2H, t, J = 7.2 Hz, CH₂), 2.38 (2H, s,

CH₂), 3.20-3.26 (2H, m, CH₂), 6.89 (1H, d, J = 8.4 Hz, ArH),

7.65 (1H, dd, J = 8.6, 1.7 Hz, ArH), 7.78 (1H, d, J = 1.5 Hz,

ArH), 8.32 (1H, t, J = 5.9 Hz, NH), 8.66 (1H, s, NH), 10.33 (2H,

s, NOH and NH). LRMS (ESI+) m/z 362 [M+H]⁺. LRMS (ESI-)

m/z 360 [M-H]^-.

Colorless powder (H₂O). Mp.168-169°C. ¹H-NMR (300 MHz /

DMSO-d₆) δ1.24-1.32 (4H, m, CH₂× 2), 1.45-1.54 (4H, m, CH₂

× 2), 1.94 (2H, t, J = 7.2 Hz, CH₂), 2.54-2.59 (2H, m, CH₂), 2.88-

2.93 (2H, m, CH₂), 3.23 (2H, q, J = 6.6 Hz, NCH₂), 3.27 (3H, s,

CH₃), 7.14 (1H, d, J = 8.4 Hz, ArH), 7.71-7.77 (2H, m, ArH),

8.34 (1H, br, NH), 8.65 (1H, br, NH), 10.33 (1H, br, OH). LRMS

(ESI+) m/z 348 [M+H]⁺. LRMS (ESI-) m/z 346 [M-H]^-.

5.1.34. 7-[(1,4,4-trimethyl-3,4-dihydrocarbostyril-6-

carbonyl)amino]heptanoic acid hydroxyamide (29)

5.1.28. 7-[(8-fluoro-4,4-dimethyl-3,4-dihydrocarbostyril-6-

carbonyl)amino]heptanoic acid hydroxyamide (22)

Pale cream powder (H₂O). Mp. 173-174°C. ¹H-NMR (300

MHz / MeOH-d₄) δ1.32 (6H, s, CH₃× 2), 1.38-1.42 (4H, m, CH₂

× 2), 1.59-1.69 (4H, m, CH₂× 2), 2.10 (2H, t, J = 7.4 Hz, CH₂),

2.53 (2H, s, CH₂), 3.37 (2H, t, J = 7.2 Hz, NCH₂), 3.40 (3H, s,

CH₃), 7.22 (1H, d, J = 9.6 Hz, ArH), 7.77 (1H, dd, J = 8.4 Hz,

2.1 Hz, ArH), 7.85 (1H, d, J = 2.1 Hz, ArH). LRMS (ESI+) m/z

376 [M+H]⁺. LRMS (ESI-) m/z 374 [M-H]^-.

Colorless powder (n-hexane / CHCl₃). Mp. 168-169°C. ¹H-

NMR (300 MHz / DMSO-d₆) δ1.27 (6H, s), 1.27-1.32 (4H, m,

CH₂× 2), 1.44-1.54 (4H, m, CH₂× 2), 1.94 (2H, t, J = 7.4 Hz,

CH₂), 2.44 (2H, s, CH₂), 3.20-3.26 (2H, m, CH₂), 7.58 (1H, dd, J

= 11.0, 1.5 Hz, ArH), 7.65 (1H, br, NH), 8.43 (1H, br, NH),

10.33 (1H, br, NH) , 10.36 (1H, br, NOH). LRMS (ESI+) m/z

380 [M+H]⁺. LRMS (ESI-) m/z 378 [M-H]^-.

5.1.35. 7-[(2-methoxyquinolin-6-carbonyl)amino]heptanoic

acid hydroxyamide (31)

5.1.29. 6-[(3,4-dihydrocarbostyril-6-carbonyl)amino]hex-

anoic acid hydroxyamide (23)

Colorless powder (AcOEt). Mp.158-160°C. ¹H-NMR (300

MHz / DMSO-d₆) δ1.27-1.32 (4H, m, CH₂× 2), 1.48-1.56 (4H,

m, CH₂× 2), 1.95 (2H, t, J = 7.2 Hz, CH₂), 3.23-3.27 (2H, m,

NCH₂), 4.00 (3H, s, OCH₃), 7.08 (1H, d, J = 8.7 Hz, CH=), 7.82

(1H, d, J = 8.4 Hz, ArH), 8.09 (1H, dd, J = 8.7, 2.1 Hz, ArH),

8.32 (1H, d, J = 8.7 Hz, CH=), 8.39 (1H, d, J = 2.1 Hz, ArH),

8.58 (1H, br, NH), 10.34, (1H, br, NH). LRMS (ESI+) m/z 346

[M+H]⁺. LRMS (ESI-) m/z 344 [M-H]^-.

Colorless powder (MeOH). Mp. 194-197°C. ¹H-NMR (300

MHz / DMSO-d₆) δ1.22-1.32 (2H, m, CH₂), 1.43-1.55 (4H, m,

CH₂×2), 1.94 (2H, t, J = 7.4 Hz, CH₂), 2.45-2.49 (2H, m, CH₂),

2.91 (2H, t, J = 7.5 Hz, CH₂), 3.18-3.24 (2H, m, NCH₂), 6.86 (1H,

d, J = 8.1 Hz, ArH), 7.11 (1H, br, NH), 7.62-7.68 (2H, m, ArH),

8.26 (1H, br, NH), 8.66 (1H, br, NH), 10.27 (1H, br, NH), 10.33

(1H, br, NOH). LRMS (ESI+) m/z 320 [M+H]⁺. LRMS (ESI-)

m/z 318 [M-H]^-.

5.1.36. (4-bromobenzyl)carbamic acid tert-butyl ester (rep-

resentative of 51) as the general procedure for the synthesis of 51

5.1.30. 8-[(3,4-dihydrocarbostyril-6-carbonyl)amino]oc-

tanoic acid hydroxyamide (24)

(a) The reaction mixture of p-bromobenzyl bromide (corre-

sponding to 50) (4.00 g, 16.004 mmol) and phthalimide

potassium salt (3.26 g, 17.61 mmol) in DMF (10 ml) was stirred

for 6 h at 100°C with a CaCl₂tube. After cooling, H₂O (100 ml)

was added, and the resulting mixture was extracted with AcOEt

(100 ml × 3). The combined organic layer was washed with H₂O

(100 ml × 1) and then brine (100 ml × 1), dried over Na₂SO₄

(anhyd.), filtered, and concentrated under reduced pressure to

afford 2-(4-bromobenzyl)isoindole-1,3-dione (5.28 g, quant. y.)

as a colorless solid. Colorless cotton-like crystal (n-hexane /

Colorless powder (CHCl₃/ MeOH). Mp. 193-194°C. ¹H-NMR

(300 MHz / DMSO-d₆) δ1.22-1.31 (6H, m, CH₂× 3), 1.44-1.53

(4H, m, CH₂× 2), 1.93 (2H, t, J = 7.4 Hz, CH₂), 2.47 (2H, t, J =

7.2 Hz, CH₂), 2.91 (2H, t, J = 7.7 Hz, CH₂), 3.16-3.24 (2H, m,

NCH₂), 6.86 (1H, d, J = 8.1 Hz, ArH), 7.61-7.67 (2H, m, ArH),

8.26 (1H, br, NH), 8.65 (1H, br, NH), 10.27 (1H, br, NH), 10.32

(1H, br, NOH). LRMS (ESI+) m/z 348 [M+H]⁺. LRMS (ESI-)

m/z 346 [M-H]^-.

5.1.31. 7-[(4,4-dimethyl-3,4-dihydrocarbostyril-7-carbon-

yl)amino]heptanoic acid hydroxyamide (25)

1

AcOEt). Mp. 126-129°C. H-NMR (300 MHz / CDCl₃/ TMS)

δ4.80 (2H, s, CH₂), 7.32 (2H, d, J = 7.8 Hz, ArH), 7.44 (2H, d, J

Colorless powder (AcOEt / MeOH). Mp. 193-198°C. ¹H-NMR

(300 MHz / DMSO-d₆) δ1.23 (6H, s, CH₃× 2), 1.26-1.27 (4H, m,

CH₂× 2), 1.46-1.50 (4H, m, CH₂× 2), 1.93 (2H, t, J = 7.2 Hz,

CH₂), 2.36 (2H, s, CH₂), 3.16-3.24 (2H, m, NCH₂), 7.31-7.40 (3H,

m, ArH), 8.37 (1H, t, J = 5.4 Hz, NH), 8.68 (1H, br, NH), 10.25

(1H, br, NH), 10.36 (1H, br, NOH). LRMS (ESI+) m/z 362

[M+H]⁺. LRMS (ESI-) m/z 360 [M-H]^-.

= 8.1 Hz, ArH), 7.71-7.73 (2H, m, ArH), 7.84-7.85 (2H, m, ArH).

(b) The reaction mixture of 2-(4-bromobenzyl)isoindole-1,3-

dione (5.01 g, 15.83 mmol) and H₂NNH₂ꢀH₂O (100%) (1.59 g,

31.67 mmol) in MeOH (50 ml) was stirred for 1 h at reflux. After

cooling, MeOH was removed under reduced pressure.

Concentrated HCl (20 ml) was then added and the resulting

mixture was stirred for 1.5 h at reflux. After cooling, 2 N NaOH

(200 ml) was added to the mixture, and the insoluble matters

were removed by filtration. The filtrate was extracted with

AcOEt (100 ml × 3). The combined organic layer was washed

with H₂O (100 ml × 1) and then brine (100 ml × 1), dried over

Na₂SO₄(anhyd.), filtered, and concentrated under reduced

pressure to afford 4-bromobenzylamine (2.73 g, 14.65 mmol, y.

93%) as a yellow oil. ¹H-NMR (300 MHz / CDCl₃) δ3.83 (2H, s,

CH₂), 7.20 (2H, d, J = 8.4 Hz, ArH), 7.45 (2H, d, J = 8.4 Hz,

ArH).

5.1.32. 7-[(1-methylcarbostyril-6-carbonyl)amino]hep-

tanoic acid hydroxyamide (27)

Light green powder (CHCl₃/ MeOH). Mp. 158-161°C. ¹H-

NMR (300 MHz / DMSO-d₆) δ1.26-1.34 (4H, m, CH₂× 2), 1.44-

1.56 (4H, m, CH₂× 2), 1.94 (2H, t, J = 7.2 Hz, CH₂), 3.25 (2H, q,

J = 6.5 Hz, NCH₂), 3.64 (3H, s, NCH₃), 6.67 (1H, d, J = 9.6 Hz,

CH=), 7.59 (1H, d, J = 9.0 Hz, ArH), 7.97 (1H, d, J = 9.6 Hz,

CH=), 8.08 (1H, dd, J = 8.7, 2.1 Hz, ArH), 8.23 (1H, J = 2.1 Hz,

(c) A solution of Boc₂O (1.29 g, 5.91 mmol) in CH₂Cl₂(10 ml)

was added to the suspension of 4-bromobenzylamine (1.00 g,

5.38 mmol) and Et₃N (0.60 g, 5.91 mmol) in CH₂Cl₂(10 ml) for

5 min at 0°C with a CaCl₂tube. The reaction mixture was stirred

overnight at room temperature. Then, H₂O (50 ml) was added to

the mixture, and the organic layer was separated. The aqueous

layer was extracted with CHCl₃(50 ml × 3). The combined

organic layer was washed with H₂O (50 ml × 1) and then brine

and then brine (30 ml × 1), dried over Na₂SO₄(anhyd.), filtered,

and concentrated under reduced pressure. The crude product was

washed with AcOEt (2 ml), and the insoluble matters were

gathered, washed with the solvent (n-hexane:AcOEt = 1:1) and

then n-hexane, and dried to afford (E)-3-[4-(4,4-dimethyl-3,4-

dihydrocarbostyril-6-carbonyl)aminomethylphenyl]acrylic acid

ethyl ester (298 mg, 0.73 mmol, y. 75%) as a colorless solid.

1

Colorless powder (n-hexane / AcOEt). Mp. 215-217°C. H-NMR

(50 ml

×

1), dried over Na₂SO₄(anhyd.), filtered, and

(300 MHz / DMSO-d₆) δ1.26 (3H, t, J = 7.1 Hz, CH₂CH₃), 1.26

(6H, s, CH₃× 2), 2.39 (2H, s, CH₂), 4.18 (2H, q, J = 7.1 Hz,

CH₂CH₃), 4.49 (2H, d, J = 5.7 Hz, CH₂), 6.59 (1H, d, J = 16.2 Hz,

CH=), 6.91 (1H, d, J = 8.1 Hz, ArH), 7.35 (2H, d, J = 8.1 Hz,

ArH), 7.63 (1H, d, J = 16.2 Hz, CH=), 7.68 (2H, d, J = 8.1 Hz,

ArH), 7.73 (1H, dd, J = 8.3, 2.0 Hz, ArH), 7.85 (1H, d, J = 1.8

Hz, ArH), 8.97 (1H, t, J = 5.9 Hz, NH), 10.38 (1H, s, NH).

concentrated under reduced pressure to afford (4-

bromobenzyl)carbamic acid tert-butyl ester (1.63 g, quant. y.) as

a colorless solid. Colorless powder (n-hexane). Mp. 73-74°C. ¹H-

NMR (300 MHz / CDCl₃) δ1.46 (9H, s, t-Bu), 4.26 (2H, d, J =

6.0 Hz, CH₂), 4.84 (1H, bs, NH), 7.16 (2H, d, J = 8.4 Hz, ArH),

7.45 (2H, d, J = 8.4 Hz, ArH).

5.1.37. (E)-3-(4-aminomethylphenyl)acrylic acid ethyl ester

(corresponding to 52) as the general procedure for the synthesis

of 52

(g) This step was performed following the same procedure

described for compound 2. Compound 34: Colorless powder

(AcOEt / MeOH). Mp. 161-165°C. ¹H-NMR (300 MHz / DMSO-

d₆) δ1.26 (6H, s, CH₃× 2), 2.39 (2H, s, CH₂), 4.61 (2H, d, J = 5.7

Hz, CH₂), 6.42 (1H, d, J = 15.9 Hz, CH=), 6.91 (1H, d, J = 8.4

Hz, ArH), 7.34 (2H, d, J = 8.1 Hz, ArH), 7.43 (1H, d, J = 15.9 Hz,

CH=), 7.52 (2H, d, J = 8.1 Hz, ArH), 7.73 (1H, dd, J = 8.4, 1.8

Hz, ArH), 7.85 (1H, d, J = 1.5 Hz, ArH), 8.96 (1H, t, J = 5.7 Hz,

NH), 9.02 (1H, s, NH), 10.38 (1H, s, NH), 10.74 (1H, s, NOH).

LRMS (ESI+) m/z 394 [M+H]⁺. LRMS (ESI-) m/z 392 [M-H]^-.

(d) The reaction mixture of (4-bromobenzyl)carbamic acid

tert-butyl ester (corresponding to 51) (1.63 g, 5.69 mmol), ethyl

acrylate (0.63 g, 6.26 mmol), P(o-tolyl)₃(0.35 g, 1.14 mmol),

DIEA (2.12 ml, 12.17 mmol), and Pd(OAc)₂(128 mg, 0.57

mmol) in DMF (6 ml) and EtCN (25 ml) was stirred for 10 min at

room temperature under N₂and then stirred for 6.5 h at 90°C

under N₂. After cooling, H₂O (100 ml) was added, and the

mixture was extracted with AcOEt (100 ml × 3). The combined

organic layer was washed with H₂O (100 ml × 1) and then brine

(100 ml × 1), dried over Na₂SO₄(anhyd.), filtered, and

concentrated under reduced pressure. The crude product was

purified through open silica gel column chromatography (n-

hexane:AcOEt = 3:1) to afford (E)-3-[4-(tert-butoxycarbonyl-

aminomethyl)phenyl]acrylic acid ethyl ester (1.33 g, 4.36 mmol,

y. 77%) as a yellow solid. Colorless powder (n-hexane). Mp .64-

5.1.39. (4,4-dimethyl-3,4-dihydrocarbostyril)-6-carboxylic

acid (E)-3-[2-(hydroxycarbamoyl)vinyl]benzylamide (33)

Colorless powder (AcOEt / MeOH). Mp. 159-163°C. ¹H-NMR

(300 MHz / DMSO-d₆) δ1.26 (6H, s, CH₃× 2), 2.39 (2H, s, CH₂),

4.49 (2H, d, J = 5.1 Hz, CH₂), 6.46 (1H, d, J = 15.9 Hz, CH=),

6.92 (1H, d, J = 9.0 Hz, ArH), 7.33-7.60 (5H, m, ArH and CH=),

7.74 (1H, d, J = 9.0 Hz, ArH), 7.86 (1H, s, ArH), 9.01 (1H, s,

NH), 10.39 (1H, s, NH). LRMS (ESI+) m/z 394 [M+H]⁺. LRMS

(ESI-) m/z 392 [M-H]^-.

1

65°C. H-NMR (300 MHz / CDCl₃) δ1.34 (3H, t, J = 7.1 Hz,

CH₂CH₃), 1.46 (9H, s, t-Bu), 4.26 (2H, q, J = 7.1 Hz, CH₂CH₃),

4.33 (2H, d, J = 5.1 Hz, CH₂), 4.89 (1H, bs, NH), 6.42 (1H, d, J =

15.9 Hz, CH=), 7.30 (2H, d, J = 8.1 Hz, ArH), 7.49 (2H, d, J =

8.1 Hz, ArH), 7.67 (1H, d, J = 15.9 Hz, CH=).

5.1.40. (4,4-dimethyl-3,4-dihydrocarbostyril)-7-carboxylic

acid (E)-3-[2-(hydroxycarbamoyl)vinyl]benzylamide (35)

Colorless powder (AcOEt / MeOH). Mp. 196-200°C. ¹H-NMR

(300 MHz / DMSO-d₆) δ1.24 (6H, s, CH₃× 2), 2.37 (2H, s, CH₂),

4.48 (2H, d, J = 5.7 Hz, CH₂), 6.44 (1H, d, J = 15.6 Hz, CH=),

7.29-7.55 (8H, m, ArH and CH=), 9.00 (1H, t, J = 5.9 Hz, NH),

10.27 (1H, s, NH), 10.78 (1H, s, NOH). LRMS (ESI+) m/z 394

[M+H]⁺. LRMS (ESI-) m/z 392 [M-H]^-.

(e) The reaction mixture of (E)-3-[4-(tert-butoxycarbonyl-

aminomethyl)phenyl]acrylic acid ethyl ester (1.27 g, 4.17 mmol)

in TFA (3 ml) and CH₂Cl₂(9 ml) was stirred for 1 h at room

temperature. After removing TFA and CH₂Cl₂under reduced

pressure, sat.NaHCO₃(50 ml) was added to the mixture, and the

mixture was extracted with AcOEt (50 ml × 3). The combined

organic layer was washed with brine (50 ml × 1), dried over

Na₂SO₄(anhyd.), filtered, and concentrated under reduced

pressure to afford (E)-3-(4-aminomethylphenyl)acrylic acid ethyl

5.1.41. carbostyril-6-sulfonyl chloride (representative of 54)

as the general procedure for the synthesis of 54

The reaction mixture of 2-quinolinol (corresponding to 53)

(1.0 g, 6.8 mmol) and chlorosulfonic acid (3.0 ml, 45.0 mmol)

was stirred for 3 h at 65°C under N₂. The mixture was poured

into iced water. The precipitate was gathered, washed with H₂O,

and dried to afford a colorless solid (1.1 g, 4.6 mmol, y. 68%).

¹H-NMR (300 MHz / DMSO-d₆) δ6.51 (1H, dd, J = 9.6, 2.1 Hz,

CH=), 7.25 (1H, dd, J = 8.4 Hz, 2.7 Hz ArH), 7.21 (1H, d, J =

8.4 Hz ArH), 7.91 (1H, s, ArH), 7.98 (1H, d, J = 9.6 Hz, CH=),

12.14 (1H, br, NH).

1

ester (0.82 g, 3.98 mmol, y. 95%) as a yellow oil. H-NMR (300

MHz / CDCl₃) δ1.34 (3H, t, J = 7.2 Hz, CH₂CH₃), 2.52 (2H, bs,

NH₂), 3.90 (2H, s, CH₂), 4.26 (2H, q, J = 7.1 Hz, CH₂CH₃), 6.42

(1H, d, J = 15.9 Hz, CH=), 7.35 (2H, d, J = 8.1 Hz, ArH), 7.50

(2H, d, J = 8.1 Hz, ArH), 7.67 (1H, d, J = 16.2 Hz, CH=).

5.1.38. (4,4-dimethyl-3,4-dihydrocarbostyril)-6-carboxylic

acid (E)-4-[2-(hydroxycarbamoyl)vinyl]benzylamide (34) as

the general procedure for the synthesis of 33-35

5.1.42. 7-(carbostyril-6-sulfonylamino)heptanoic acid

hydroxyamide (36) as the general procedure for the synthesis of

36-38

(f) The reaction mixture of (E)-3-(4-aminomethylphenyl)-

acrylic acid ethyl ester (corresponding to 52) (200 mg, 0.97

mmol), 4,4-dimethyl-3,4-dihydrocarbostyril-6-carboxylic acid

(214 mg, 0.97 mmol), HOBt (132 mg, 0.97 mmol), Et₃N (99 mg,

0.97 mmol), and EDCIꢀHCl (187 mg, 0.97 mmol) in DMF (4 ml)

was stirred overnight. After sat.NaHCO₃(30 ml) was added, the

resulting mixture was extracted with AcOEt (30 ml × 3). The

combined organic layer was washed with 1 N HCl (30 ml × 1)

(b) Carbostyril-6-sulfonyl chloride (corresponding to 54) (138

mg, 0.57 mmol) and then subsequently Et₃N (114 mg, 3.24

mmol) in CH₂Cl₂(0.5 ml) were added to a solution of 7-

aminoheptanoic acid methyl ester hydrochloride (142 mg, 0.73

mmol) in CH₂Cl₂(2 ml) at room temperature. The reaction

mixture was stirred for 16 h at room temperature. H₂O (10 ml),

AcOEt (10 ml), and n-hexane (10 ml) were then sequentially

added to the mixture. The precipitate was gathered, washed with

H₂O, and dried to afford a pink solid (127 mg, 0.35 mmol, y.

ml) and then extracted with AcOEt (15 ml × 1, and then 10 ml ×

3). The combined organic layer was washed with brine (15 ml ×

2), dried over Na₂SO₄(anhyd.), filtered, and concentrated under

reduced pressure. The crude product was purified through open

silica gel column chromatography (AcOEt:MeOH = 7:1) to

1

61%). H-NMR (300 MHz / DMSO-d₆) δ1.15-1.20 (4H, m, CH₂

1

× 2), 1.29-1.44 (4H, m, CH₂× 2), 2.22 (2H, t, J = 7.2 Hz, CH₂),

2.72 (2H, q, J = 6.0 Hz, NCH₂), 3.56 (3H, s, OCH₃) , 6.61 (1H, d,

J = 9.6 Hz, CH=), 7.43 (1H, d, J = 8.7 Hz ArH), 7.53 (1H, t, J =

5.9 Hz, NH), 7.84 (1H, dd, J = 8.7, 2.1 Hz ArH), 8.07 (1H, d, J =

9.6 Hz, CH=), 8.13 (1H, d, J = 2.1 Hz, ArH), 12.01 (1H, br, NH).

afford a pink solid (223 mg, 0.62 mmol, y. 13%). H-NMR (300

MHz / CDCl₃) δ1.30-1.48 (4H, m, CH₂× 2), 1.61-1.67 (2H, m,

CH₂), 1.78-1.89 (2H, m, CH₂), 2.30 (2H, t, J = 7.4 Hz, CH₂),

2.61-2.66 (2H, m, CH₂), 2.95-3.0 (2H, m, CH₂), 3.03-3.08 (2H, m,

NCH₂), 3.67 (3H, s, OCH₃), 6.31 (1H, br, NH), 6.70 (1H, d, J =

8.4 Hz, ArH), 7.02 (1H, dd, J = 8.6, 2.7 Hz, ArH), 7.11 (1H, d, J

= 2.7 Hz, ArH), 7.50 (1H, br, NH).

(c) This step was performed following the same procedure

described for compound 2. Compound 36: Colorless powder

1

(MeOH). Mp. unmeasurable (decomposed at 242°C). H-NMR

5.1.47. 7-(3,4-dihydrocarbostyril-6-ylsulfamoyl)heptanoic

acid hydroxyamide (39)

(300 MHz / DMSO-d₆) δ1.08-1.24 (4H, m, CH₂× 2), 1.32-1.47

(4H, m, CH₂× 2), 1.89 (2H, t, J = 7.4 Hz, CH₂), 2.72-2.74 (2H,

m, NCH₂), 6.61 (1H, d, J = 9.6 Hz, CH=), 7.43 (1H, d, J = 8.7 Hz,

ArH), 7.52 (1H, br, NH), 7.84 (1H, dd, J = 8.7, 1.8 Hz ArH),

8.07 (1H, d, J = 9.6 Hz, CH=), 8.62 (1H, d, J = 1.8 Hz, ArH),

8.96 (1H, br, NH), 10.31 (1H, br, NOH). LRMS (ESI+) m/z 368

[M+H]⁺. LRMS (ESI-) m/z 366 [M-H]^-.

Colorless powder (H₂O). Mp. 133-135°C. ¹H-NMR (300 MHz

/ DMSO-d₆) δ1.16-1.36 (4H, m, CH₂× 2), 1.39-1.49 (2H, m,

CH₂), 1.58-1.68 (2H, m, CH₂), 1.91 (2H, t, J = 7.4 Hz, CH₂),

2.39-2.44 (2H, m, CH₂), 2.83 (2H, t, J = 7.5 Hz, CH₂), 2.95-3.00

(2H, m, NCH₂), 6.80 (1H, d, J = 8.4 Hz, ArH), 6.96-7.00 (2H, m,

ArH), 7.24 (1H, br, NH), 8.67 (1H, br, NH), 10.04 (1H, br, OH),

10.35 (1H, br, NH). LRMS (ESI+) m/z 370 [M+H]⁺. LRMS

(ESI-) m/z 368 [M-H]^-.

5.1.43. 7-(3,4-dihydrocarbostyril-6-sulfonylamino)hep-

tanoic acid hydroxyamide (37)

Colorless powder (CHCl₃/ MeOH). Mp. 181-182°C. ¹H-

NMR (300 MHz / DMSO-d₆) δ1.14-1.24 (4H, m, CH₂× 2), 1.30-

1.47 (4H, m, CH₂× 2), 1.90 (2H, t, J = 7.4 Hz, CH₂), 2.47 (2H, t,

J = 7.2 Hz, CH₂), 2.65-2.72 (2H, m, NCH₂), 2.95 (2H, t, J = 7.5

Hz, CH₂), 6.99 (1H, d, J = 8.1 Hz, ArH), 7.40 (1H, br, NH), 7.53-

7.59 (2H, m, ArH), 8.66 (1H, br, NH), 10.37 (1H, br, NH) , 10.45

(1H, br, NOH). LRMS (ESI+) m/z 370 [M+H]⁺. LRMS (ESI-)

m/z 368 [M-H]^-.

5.1.48. 4,4-dimethyl-3,4-dihydrocarbostyril-6-carboxylic

acid (trans-4-benzyloxymethylcyclohexylmethyl)amide (repre-

sentative of 59) as the general procedure for the synthesis of 59

(a)

A

solution

of

trans-1,4-cyclohexanedimethanol

(corresponding to 58) (300 mg, 2.08 mmol) in DMF (2 ml) was

added to a suspension of NaH (60% in mineral oil, 42 mg, 1.04

mmol) in DMF (1 ml) for 3 min at 0°C. After stirring for 30 min

at 0°C, a solution of BnBr (178 mg, 1.04 mmol) in DMF (2 ml)

was added to the mixture for 1 min at 0°C. The mixture was

stirred overnight at room temperature, and then for 2 h at 60°C.

After cooling, H₂O (30 ml) was added, and the resulting mixture

was extracted with AcOEt (50 ml × 3). The combined organic

layer was washed with H₂O (30 ml × 1) and then brine (30 ml ×

1), dried over Na₂SO₄(anhyd.), filtered, and concentrated under

reduced pressure. The crude product was purified through open

silica gel column chromatography (n-hexane:AcOEt = 2:1) to

afford (trans-4-benzyloxymethylcyclohexyl)methanol (177 mg,

0.75 mmol, y. 36%) as a colorless oil. ¹H-NMR (300 MHz /

CDCl₃) δ0.94-1.01 (4H, m, CH₂× 2), 1.26 (1H, m, CH), 1.45 (1H,

m, CH), 1.82-1.89 (4H, m, CH₂× 2), 3.29 (2H, d, J = 6.6 Hz,

CH₂), 3.46 (2H, d, J = 6.3 Hz, CH₂), 4.50 (2H, s, CH₂), 7.33-7.35

(5H, m, ArH).

5.1.44. 7-(4,4-dimethyl-3,4-dihydrocarbostyril-6-sulfonyl-

amino)heptanoic acid hydroxyamide (38)

Colorless powder (AcOEt). Mp. 125-126°C. ¹H-NMR (300

MHz / MeOH-d₄) δ1.25-1.30 (4H, m, CH₂× 2), 1.34 (6H, s, CH₃

× 2), 1.38-1.60 (4H, m, CH₂× 2), 2.05 (2H, t, J = 7.4 Hz, CH₂),

2.50 (2H, s, CH₂), 2.83 (2H, t, J = 6.9 Hz, CH₂), 7.02 (1H, d, J =

8.4 Hz, ArH), 7.65 (1H, dd, J = 8.3, 2.0 Hz, ArH), 7.78 (1H, d, J

= 2.0 Hz, ArH). LRMS (ESI+) m/z 398 [M+H]⁺. LRMS (ESI-)

m/z 396 [M-H]^-.

5.1.45. 7-(chlorosulfonyl)heptanoic acid methyl ester (56)

The reaction mixture of 7-bromoheptanoic acid methyl ester

(55) (800 mg, 3.6 mmol) and Na₂SO₃(580 mg, 4.6 mmol) in H₂O

(3.6 ml) was stirred for 17 h at reflux. After cooling, the mixture

was washed with Et₂O (10 ml × 3) and concentrated under

reduced pressure to afford a colorless solid (1.4 g, quant. y.). ¹H-

NMR (300 MHz / DMSO-d₆) δ1.21-1.34 (4H, m, CH₂× 2), 1.45-

1.59 (4H, m, CH₂× 2), 2.28 (2H, t, J = 7.5 Hz, CH₂), 2.38 (2H, t,

J = 7.8 Hz, SCH₂), 3.58 (3H, s, OCH₃).

(b) A solution of MsCl 104 mg (0.904 mmol) in CH₂Cl₂(2 ml)

was added to

a

solution of (trans-4-benzyloxymethyl-

cyclohexyl)methanol (177 mg, 0.75 mmol) and Et₃N 229 mg

(2.26 mmol) in CH₂Cl₂(2 ml) for 1 min at 0^oC. The reaction

mixture was stirred overnight at room temperature. Then, H₂O

(30 ml) was added, and the resulting mixture was extracted with

CHCl₃(30 ml × 3). The combined organic layer was washed with

H₂O (30 ml × 1) and then brine (30 ml × 1), dried over Na₂SO₄

(anhyd.), filtered, and concentrated under reduced pressure. The

crude product was purified through open silica gel column

5.1.46. 7-(3,4-dihydrocarbostyril-6-ylsulfamoyl)heptanoic

acid methyl ester (57)

A solution of Et₃N (530 mg, 5.2 mmol) in acetone (2 ml) was

added to a suspension of 7-(chlorosulfonyl)heptanoic acid methyl

ester (56) (1.0 g, 4.6 mmol) and trichlorotriazine (926 mg, 5.0

mmol) in acetone (8 ml) for 5 min at room temperature. The

mixture was stirred for 20 min at 80°C with a microwave

apparatus (Initiator™ Eight, Biotage). After cooling, the mixture

was filtered with Celite®. Then, 2 N NaOH (3.0 ml), THF (2.5

ml), and 6-amino-3,4-dihydrocarbostyril (800 mg, 4.9 mmol)

were added to the filtrate, and the mixture was stirred for 20 min

at 80°C with a microwave apparatus. After cooling, the mixture

was filtered with Celite®. The filtrate was diluted with H₂O (15

chromatography (n-hexane:AcOEt

= 4:1) to afford trans-

methanesulfonic acid 4-benzyloxymethylcyclohexylmethyl ester

1

(199 mg, 0.64 mmol, y. 85%) as a colorless oil. H-NMR (300

MHz / CDCl₃) δ0.97-1.07 (4H, m, CH₂× 2), 1.61-1.64 (1H, m,

CH), 1.67-1.73 (1H, m, CH), 1.83-1.91 (4H, m, CH₂× 2), 3.00

(3H, s, CH₃), 3.29 (2H, d, J = 6.3 Hz, CH₂), 4.03 (2H, d, J = 6.3

Hz, CH₂), 4.50 (2H, s, CH₂), 7.30-7.35 (5H, m, ArH).

(c) The reaction mixture of trans-methanesulfonic acid 4-

benzyloxymethylcyclohexylmethyl ester (200 mg, 0.64 mmol)

δ0.95-1.08 (4H, m, CH₂× 2), 1.37 (6H, s, CH₃× 2), 1.55 (2H, bs,

CH × 2), 1.85-1.88 (4H, m, CH₂× 2), 2.51 (2H, s, CH₂), 3.33 (2H,

t, J = 6.5 Hz, CH₂), 3.47 (2H, d, J = 6.3 Hz, CH₂), 6.13 (1H, m,

NH), 6.79 (1H, d, J = 8.1 Hz, ArH), 7.52 (1H, dd, J = 8.3, 2.0 Hz,

ArH), 7.81 (1H, d, J = 1.8 Hz, ArH), 8.11 (1H, s, NH).

and NaN₃(42 mg, 0.64 mmol) in DMF (2 ml) was stirred over-

night at room temperature, and then for 1.5 h at 80°C. After

cooling, H₂O (30 ml) was added, and the resulting mixture was

extracted with AcOEt (30 ml × 3). The combined organic layer

was washed with H₂O (30 ml × 1) and then brine (30 ml × 1),

dried over Na₂SO₄(anhyd.), filtered, and concentrated under

reduced pressure. The crude product was purified through open

silica gel column chromatography (n-hexane:AcOEt = 5:1) to

afford (trans-4-azidomethylcyclohexylmethoxymethyl)benzene

(184 mg, quant. y.) as a colorless oil. ¹H-NMR (300 MHz /

CDCl₃) δ0.96-1.03 (4H, m, CH₂× 2), 1.45-1.58 (2H, bs, CH × 2),

1.81-1.89 (4H, m, CH₂× 2), 3.13 (2H, d, J = 6.9 Hz, CH₂), 3.29

(2H, d, J = 6.3 Hz, CH₂), 4.50 (2H, s, CH₂), 7.33-7.34 (5H, m,

ArH).

(g) A suspension of Dess-Martin periodinane (68 mg, 0.16

mmol) in CH₂Cl₂(3 ml) was added to a solution of 4,4-dimethyl-

3,4-dihydrocarbostyril-6-carboxylic

acid

trans-(4-hydroxy-

methylcyclohexylmethyl)amide (50 mg, 0.15 mmol) in CH₂Cl₂(1

ml) for 3 min at room temperature. The reaction mixture was

stirred overnight at room temperature. A solution of slight

amount of Na₂S₂O₃in sat.NaHCO₃(30 ml) was added to the

mixture, and the resulting mixture was extracted with CHCl₃(30

ml × 3). The combined organic layer was washed with brine (30

ml × 1), dried over Na₂SO₄(anhyd.), filtered, and concentrated

under reduced pressure to afford 4,4-dimethyl-3,4-dihydro-

carbostyril-6-carboxylic acid (trans-4-formylcyclohexylmethyl)-

(d) The suspension of (trans-4-azidomethylcyclohexyl-

methoxymethyl)benzene (1.57 g, 6.65 mmol) and 10% Pd-C

(0.16 g) in EtOH (15 ml) in a glass autoclave reactor was stirred

for 7 h at room temperature under H₂atmosphere at 0.18 MPa.

After the pressure was leaked, the reaction mixture was filtered,

washed with MeOH, and concentrated under reduced pressure to

afford (trans-4-benzyloxymethylcyclohexyl)methylamine (1.36 g,

5.807 mmol, y. 96%) as a colorless solid. Colorless powder

(AcOEt / MeOH). Mp. 149-153°C. ¹H-NMR (300 MHz / CDCl₃)

δ0.97-1.03 (4H, m, CH₂× 2), 1.60-1.63 (2H, m, CH × 2), 1.87-

1.90 (4H, m, CH₂× 2), 2.76 (2H, d, J = 6.6 Hz, CH=), 3.26 (2H,

d, J = 6.6 Hz, CH=), 4.47 (2H, s, CH₂), 4.82 (2H, bs, NH₂), 7.32-

7.34 (5H, m, ArH).

1

amide (41 mg, 0.12 mmol, y. 83%) as a colorless oil. H-NMR

(300 MHz / CDCl₃) δ1.05-1.18 (4H, m, CH₂× 2), 1.37 (6H, s,

CH₃× 2), 1.60 (2H, m, CH × 2), 1.93-2.05 (4H, m, CH₂× 2),

2.51 (2H, s, CH₂), 3.35 (2H, t, J = 6.5 Hz, CH₂), 6.15 (1H, t, J =

5.7 Hz, NH), 6.81 (1H, d, J = 8.1 Hz, ArH), 7.52 (1H, dd, J = 8.1,

1.8 Hz, ArH), 7.81 (1H, d, J = 1.5 Hz, ArH), 8.39 (1H, s, NH),

9.63 (1H, d, J = 1.2 Hz, CHO).

(h) A solution of triethyl phosphonoacetate (30 mg, 0.13

mmol) in THF (2 ml) was added to a suspension of NaH (60% in

mineral oil, 5 mg, 0.13 mmol) in THF (5 ml) for 2 min at 0°C

under N₂using a syringe attached to a needle. After the reaction

mixture was stirred for 15 min at 0°C, a solution of 4,4-dimethyl-

3,4-dihydrocarbostyril-6-carboxylic acid (trans-4-formylcyclo-

hexylmethyl)amide (41 mg, 0.120 mmol) in THF (2 ml) was

added to the mixture for 4 min at 0°C under N₂with a syringe.

After stirring for 6 h at 0°C under N₂, THF was removed under

reduced pressure. Then, H₂O (30 ml) was added to the residue,

and the mixture was extracted with AcOEt (30 ml × 3). The

combined organic layer was washed with brine (30 ml × 1), dried

over Na₂SO₄(anhyd.), filtered, and concentrated under reduced

pressure. The crude product was purified through open silica gel

column chromatography (n-hexane:AcOEt = 1:4) to afford (E)-3-

[trans-4-[[(4,4-dimethyl-3,4-dihydrocarbostyril-6-carbonyl)-

amino]methyl]cyclohexyl]acrylic acid ethyl ester (representative

(e) The reaction mixture of (trans-4-benzyloxymethylcyclo-

hexyl)methylamine (106 mg, 0.46 mmol), 4,4-dimethyl-3,4-di-

hydrocarbostyril-6-carboxylic acid (100 mg, 0.46 mmol), HOBt

(62 mg, 0.46 mmol), Et₃N (46 mg, 0.46 mmol), and EDCIꢀHCl

(87 mg, 0.46 mmol) in DMF (2 ml) was stirred overnight at room

temperature. Then, sat.NaHCO₃(30 ml) was added, and the

resulting mixture was extracted with AcOEt (30 ml × 3). The

combined organic layer was washed with 1 N HCl (30 ml × 1)

and then brine (30 ml × 1), dried over Na₂SO₄(anhyd.), filtered,

and concentrated under reduced pressure. The crude product was

purified through open silica gel column chromatography (n-

hexane:AcOEt

= 1:4) to afford 4,4-dimethyl-3,4-dihydro-

carbostyril-6-carboxylic acid (trans-4-benzyloxymethylcyclo-

hexylmethyl)amide (representative of 59) (94 mg, 0.22 mmol, y.

47%) as a colorless solid. Colorless powder (n-hexane/AcOEt).

1

of 60) (33 mg, 0.08 mmol, y. 66%) as a colorless oil. H-NMR

(300 MHz / CDCl₃) δ1.01-1.16 (4H, m, CH₂× 2), 1.29 (3H, t, J =

7.2 Hz, CH₂CH₃), 1.37 (6H, s, CH₃× 2), 1.72 (2H, bs, CH × 2),

1.84-1.91 (4H, m, CH₂× 2), 2.51 (2H, s, CH₂), 3.33 (2H, t, J =

6.5 Hz, CH₂), 4.18 (2H, q, J = 7.1 Hz, CH₂CH₃), 5.77 (1H, dd, J

= 15.8, 1.4 Hz, CH=), 6.14 (1H, t, J = 5.7 Hz, NH), 6.80 (1H, d, J

= 8.1 Hz, ArH), 6.90 (1H, dd, J = 15.8 Hz, 6.8 Hz, CH=), 7.52

(1H, dd, J = 8.1, 1.8 Hz, ArH), 7.81 (1H, d, J = 1.5 Hz, ArH),

8.23 (1H, s, NH).

1

Mp. 87-88°C. H-NMR (300 MHz / CDCl₃) δ0.96-1.07 (4H, m,

CH₂× 2), 1.36 (6H, s, CH₃× 2), 1.69 (2H, bs, CH × 2), 1.83-1.90

(4H, m, CH₂× 2), 2.51 (2H, s, CH₂), 3.29 (2H, d, J = 6.6 Hz,

CH₂), 3.31 (2H, t, J = 6.3 Hz, CH₂), 4.49 (2H, s, CH₂), 6.12 (1H,

t, J = 5.4 Hz, NH), 6.80 (1H, d, J = 8.1 Hz, ArH), 7.33-7.34 (5H,

m, ArH), 7.51 (1H, dd, J = 8.1, 1.8 Hz, ArH), 7.81 (1H, d, J = 1.8

Hz, ArH), 8.29 (1H, s, NH). LRMS (ESI+) m/z 435 [M+H]⁺.

LRMS (ESI-) m/z 433 [M-H]^-.

5.1.50. 4,4-dimethyl-3,4-dihydrocarbostyril-6-carboxylic

acid (E)-[trans-4-(2-hydroxycarbamoylvinyl)cyclohexyl-

methyl]amide (40)

5.1.49. (E)-3-[trans-4-[[(4,4-dimethyl-3,4-dihydro-carbo-

styril-6-carbonyl)amino]methyl]cyclohexyl]acrylic acid ethyl

ester (representative of 60) as the general procedure for the

synthesis of 60

Colorless cotton-like crystal (n-hexane / AcOEt / MeOH). Mp.

184-187°C. ¹H-NMR (300 MHz / DMSO-d₆) δ1.01-1.06 (4H, m,

CH₂× 2), 1.26 (6H, s, CH₃× 2), 1.52 (2H, bs, CH × 2), 1.73-1.80

(4H, m, CH₂× 2), 2.38 (2H, s, CH₂), 3.11 (2H, t, J = 5.9 Hz,

CH₂), 5.70 (1H, d, J = 15.6 Hz, CH=), 6.59 (1H, dd, J = 15.5, 6.8

Hz, CH=), 6.89 (1H, d, J = 8.4 Hz, ArH), 7.67 (1H, d, J = 8.1 Hz,

ArH), 7.79 (1H, s, ArH), 8.35 (1H, t, J = 5.6 Hz, NH), 10.34 (1H,

s, NH). LRMS (ESI+) m/z 400 [M+H]⁺. LRMS (ESI-) m/z 398

[M-H]^-. HRMS (MALDI+) m/z calcd for C₂₂H₃₀N₃O₄⁺[M+H]⁺

400.2231, found 400.2222.

(f) The suspension of 4,4-dimethyl-3,4-dihydrocarbostyril-6-

carboxylic acid (trans-4-benzyloxymethylcyclohexylmethyl)-

amide (corresponding to 59) (81 mg, 0.19 mmol) and 10% Pd-C

(8 mg) in EtOH (3 ml) in a glass autoclave reactor was stirred for

7 h at room temperature under H₂atmosphere at 0.30 MPa. After

the pressure was leaked, the reaction mixture was filtered,

washed with MeOH, and concentrated under reduced pressure to

afford 4,4-dimethyl-3,4-dihydrocarbostyril-6-carboxylic acid

(trans-4-hydroxymethylcyclohexylmethyl)amide (51 mg, 0.15

mmol, y. 80%) as a colorless oil. ¹H-NMR (300 MHz / CDCl₃)

5.1.51. 4,4-dimethyl-3,4-dihydrocarbostyril-6-carboxylic

acid (E)-[cis-4-(2-hydroxycarbamoylvinyl)cyclohexyl-

methyl]amide (42)

5.2 Biology

The enzyme assays using rat liver HDACs were conducted

according to the literature^{29, 30}. Rat liver HDACs were purchased

from Merck KGaA (Germany). The enzyme assays using human

HDAC isozymes were performed based on the protocol³¹

generated by Promega. The assay was performed using HDAC-

Glo^TMI/II assay kit and the G6420 screening system (Promega,

USA). The human HDAC isozymes were purchased from Singal

Chem (Canada).

Colorless powder (AcOEt / MeOH). Mp. 210-213°C. ¹H-NMR

(300 MHz / DMSO-d₆) δ1.26 (6H, s, CH₃× 2), 1.53 (8H, m, CH₂

× 4), 1.73-1.77 (2H, m, CH × 2), 2.38 (2H, s, CH₂), 3.20 (2H, t, J

= 6.5 Hz, CH₂), 5.74 (1H, d, J = 15.6 Hz, CH=), 6.70 (1H, dd, J =

15.6, 6.0 Hz, CH=), 6.89 (1H, d, J = 8.4 Hz, ArH), 7.66 (1H, dd,

J = 8.4, 1.8 Hz, ArH), 7.78 (1H, d, J = 1.5 Hz, ArH), 8.33 (1H, t,

J = 5.4 Hz, NH), 8.85 (1H, s, NH), 10.34 (1H, s, NH), 10.55 (1H,

s, NOH). LRMS (ESI+) m/z 400 [M+H]⁺. LRMS (ESI-) m/z 398

[M-H]^-. HRMS (MALDI+) m/z calcd for C₂₂H₃₀N₃O₄⁺[M+H]⁺

400.2231, found 400.2253.

5.2.1. Enzyme assay using rat liver HDACs

The incubation buffer composed of 25 mM Tris-HCl, 137 mM

NaCl, 2.7 mM KCl, and 1 mM MgCl₂(pH 8.0) was used in this

assay. The reagents included 1 µM of the test compound (1%

DMSO), 50 µM of the rat liver HDACs (32 µg/ml), and 50 µl of

Boc-Lys(Ac)-AMC (250 µM); the final assay concentrations are

shown in parentheses. Because the activity of an enzyme depends

on the lot number, the concentration used was adjusted when

necessary. Immediately after the test compound and the enzyme

were mixed, the mixture was read on a fluorometer to determine

the background. After preincubation for 15 min at 37°C, pre-

warmed Boc-Lys(Ac)-AMC was added to initiate the reaction.

The reaction mixture was sealed and incubated for 30 min at

37°C, and then 10 µl of diluted Developer Concentrate was

added to quench the reaction. After incubation for 1 h at 37°C,

the plate was read on a fluorometer (excitation: 360 nm,

emission: 465 nm).

5.1.52. 4,4-dimethyl-3,4-dihydrocarbostyril-6-carboxylic

acid [trans-4-(2-hydroxycarbamoylethyl)cyclohexylmethyl]-

amide (41) as the general procedure for the synthesis of 41 and

43

(j) The suspension of (E)-3-[trans-4-[[(4,4-dimethyl-3,4-

dihydrocarbostyril-6-carbonyl)amino]methyl]cyclohexyl]acrylic

acid ethyl ester (representative of 60) (107 mg, 0.26 mmol) and

10% Pd-C (11 mg) in EtOH (4 ml) in a glass autoclave reactor

was stirred for three days at room temperature under H₂

atmosphere at 0.16 MPa. After the pressure was leaked, the

reaction mixture was filtered and washed with MeOH. The

filtrate was concentrated under reduced pressure. The crude

product was purified through open silica gel column

chromatography (n-hexane:AcOEt = 1:4) to afford 3-[trans-4-

[[(4,4-dimethyl-3,4-dihydrocarbostyril-6-carbonyl)amino]-

methyl]cyclohexyl]propionic acid ethyl ester (78 mg, 0.19 mmol,

y. 72%) as a colorless oil. H-NMR (300 MHz / CDCl₃) δ0.95-

5.2.2. Enzyme assay using human HDAC isozymes

1

The fluorometric activity assay kit used to determine HDAC

activity was the HDAC-Glo^TMI/II assay and screening system

(G6420, Promega). Arbitrary human HDAC isozymes were

incubated with the test compounds for 30 min at room temper-

ature, and the reaction was initiated by the addition of HDAC-

Glo^TMI/II substrate and the Developer Reagent. The reaction

mixtures were incubated for 30 min at room temperature. The

plate was read on a fluorometer (excitation: 360 nm, emission:

460 nm). The activity was calculated as a percentage of the

activity of the control. The 50% inhibition concentration (IC₅₀)

values for the test compounds were calculated using the

SigmaPlot software.

1.04 (4H, m, CH₂× 2), 1.25 (3H, t, J = 7.1 Hz, CH₂CH₃), 1.37

(6H, s, CH₃× 2), 1.50-1.58 (2H, m, CH₂), 1.66 (2H, bs, CH × 2),

1.77-1.85 (4H, m, CH₂× 2), 2.31 (2H, t, J = 7.8 Hz, CH₂), 2.51

(2H, s, CH₂), 3.31 (2H, t, J = 6.3 Hz, NCH₂), 4.12 (2H, q, J = 7.2

Hz, CH₂CH₃), 6.18 (1H, t, J = 5.7 Hz, NH), 6.80 (1H, d, J = 8.1

Hz, ArH), 7.51 (1H, dd, J = 8.3 Hz, 1.8 Hz, ArH), 7.81 (1H, d, J

= 1.8 Hz, ArH), 8.29 (1H, s, NH).

(k) This step was performed following the same procedure

described for compound 2. 41: Colorless powder (AcOEt /

MeOH). Mp. 195-197°C. ¹H-NMR (300 MHz / DMSO-d₆)

δ0.81-0.88 (4H, m, CH₂× 2), 1.26 (6H, s, CH₃× 2), 1.37-1.42

(2H, m, CH₂), 1.47 (2H, bs, CH × 2), 1.71-1.73 (4H, m, CH₂× 2),

1.95 (2H, t, J = 7.7 Hz, CH₂), 2.38 (2H, s, CH₂), 3.08 (2H, t, J =

6.3 Hz, NCH₂), 6.88 (1H, d, J = 8.4 Hz, ArH), 7.66 (1H, dd, J =

8.4, 1.8 Hz, ArH), 7.68 (1H, d, J = 1.8 Hz, ArH), 8.32 (1H, t, J =

5.7 Hz, ArH), 8.65 (1H, s, NH), 10.34 (2H, s, NOH and NH).

LRMS (ESI+) m/z 402 [M+H]⁺. LRMS (ESI-) m/z 400 [M-H]^-.

HRMS (MALDI+) m/z calcd for C₂₂H₃₁N₃O₄Na⁺[M+Na]⁺

424.2207, found 424.2238.

Abbreviations

DNA, deoxyribonucleic acid; Gadd 45, growth arrest and DNA

damage 45; DMF, N,N-dimethylformaldehyde; Et, ethyl; rt, room

temperature; quant., quantitative; y., yield; Me, methyl; EDCI, 1-

ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBt,

hydroxybenzotriazole; Bn, benzyl; Boc, tert-butoxycarbonyl; Ac,

acetyl; DIEA, N,N-diisopropylethylamine; TFA, trifluoroacetic

acid; MW, microwave; Ms, mesyl; Pa, pascal; compnd,

compound; NMR, nuclear magnetic resonance; Mp, melting point;

LRMS, low-resolution mass spectrometry; HRMS, high-resolution

mass spectrometry; anhyd., anhydrous; Hz, hertz; DMSO,

dimethylsulfoxide; Bu, butyl; ESI, electrospray ionization;

MALDI, matrix assisted laser desorption / ionization; calcd,

calculated; Lys, lysine; AMC, 7-amido-4-methylcoumarin

5.1.53. 4,4-dimethyl-3,4-dihydrocarbostyril-6-carboxylic

acid [cis-4-(2-hydroxycarbamoylethyl)cyclohexylmethyl]-

amide (43)

Colorless powder (AcOEt). Mp. 129-132°C. ¹H-NMR (300

MHz / DMSO-d₆) δ1.25 (6H, s, CH₃× 2), 1.30-1.52 (10H, m,

CH₂× 5), 1.70-1.76 (2H, m, CH × 2), 1.95 (2H, t, J = 7.5 Hz,

C(O)CH₂), 2.38 (2H, s, CH₂), 3.20 (1.7H, t, J = 6.6 Hz, NCH₂),

6.88 (1H, d, J = 8.1 Hz, ArH), 7.66 (1H, dd, J = 8.1, 1.5 Hz,

ArH), 7.80 (1H, d, J = 1.5 Hz, ArH), 8.31 (1H, br, NH), 8.65 (1H,

br, NH), 10.33 (2H, br, NOH and NH). LRMS (ESI+) m/z 402

[M+H]⁺. LRMS (ESI-) m/z 400 [M-H]^-. HRMS (MALDI+) m/z

calcd for C₂₂H₃₁N₃O₄Na⁺[M+Na]⁺424.2207, found 424.2212.

References and notes

1. Paris, M.; Porcelloni, M,; Binaschi, M.; Fattori, D. J. Med. Chem.

2008, 51, 1505-1529

2. Haberland, M.; Montgomery, R. L.; Olson, E. N. Nat. Rev. Genet.

2009, 10, 32–42.

3. Dokmanovic, M.; Clarke, C.; Marks, P. A. Mol. Cancer Res. 2007,

5, 981-989.

4. Witt, O.; Deubzer, H. E.; Milde, T.; Oehme, I. Cancer Lett. 2009,

277, 8−21

19. Pinto, A.; Wang, M.; Horsman, M.; Boddy, C. N. Org. Lett., 2012,

14, 2278-2281.

5. Kim. H.-J.; Bae, S.-C. Am. J. Transl. Res. 2011, 3, 166-179.

6. Arrowsmith, C. H.; Bountra, C.; Fish, P. V.; Lee, K.; Schapira, M.

Nat. Rev. Drug Discov. 2012, 11, 384-400.

20. Walker, S. K.; Lazarus, M. B.; Gross, B. J. PCT Int. Appl.,

2010141074.

21. Dess, D. B.; Martin, J. C. J. Am. Chem. Soc., 1991, 113, 7277-

7. Jenuwein, T.; Allis, C. D. Science 2001, 293, 1074-1080.

8. Weichert, W. Cancer Lett. 2009, 280, 168−176.

9. Ito A,; Kawaguchi Y.; Lai, C.-H.; Kovacs, J. J.; Higashimoto, Y.;

Appella, E.; Yao, T.-P. EMBO J. 2002, 21, 6236-6245.

10. Jin, Y.-H.; Jeon, E.-J.; Li, Q.-L.; Lee, Y. H.; Choi, J.-K.; Kim,

W.-J.; Lee, K.-Y.; Bae, S.-C. J. Bio. Chem. 2004, 279, 29409-

29417

11. Kawai, H.; Li, H.; Avraham, S.; Jiang, S.; Avraham, H. K. Int. J.

Cancer 2003, 107, 353–358

12. Müller, B. M.; Jana, L.; Kasajima, A.; Lehmann, A.; Prinzler, J.;

Budczies, J.; Winzer, K.-J.; Dietel, M.; Weichert W.; Denkert, C.

BMC Cancer 2013, 13, 215-222

13. Balasubramanian, S.; Ramos, J.; Luo, W.; Sirisawad, M.; Verner,

E.; Buggy, J. J. Leukemia 2008, 22, 1026–1034.

14. Oehme, I.; Deubzer, H. E.; Wegener, D.; Pickert, D.; Linke, J.-P. ;

Hero, B.; Kopp-Schneider, A.; Westermann, F.; Ulrich, S. M.;

Deimling, A.v.; Fischer, M.; Witt, O. Clin. Cancer Res. 2009, 15,

91-99.

7287.

22. The SAHA used for the assays performed in this study was

synthesized using the same method that was used to synthesize

HDAC inhibitors 12-14, with the exception that the carbostyril

skeleton was replaced with benzene ring. SAHA: Colorless

powder (n-hexane / AcOEt / MeOH). Mp. 158-159°C. ¹H-NMR

(300 MHz / DMSO-d₆) δ1.26-1.29 (4H, m, CH₂× 2), 1.44-1.51

(2H, m, CH₂), 1.55-1.60 (2H, m, CH₂), 1.94 (2H, t, J = 7.4 Hz,

CH₂), 2.29 (2H, t, J = 7.4 Hz, CH₂), 7.01 (1H, t, J = 7.4 Hz, ArH),

7.27 (2H, t, J = 7.8 Hz, ArH), 7.59 (2H, d, J = 7.5 Hz, ArH), 8.66

(1H, s, NH), 9.88 (1H, s, NH), 10.35 (1H, s, NOH). LRMS (ESI+)

m/z 265 ([M+H]⁺). LRMS (ESI-) m/z 263 ([M-H]^-).

23. Suzuki, T.; Nagano, Y.; Kouketsu, A.; Matsuura, A.; Maruyama,

S.; Kurotaki, M.; Nakagawa, H.; Miyata, N. J. Med. Chem. 2005,

48, 1019-1032.

24. Salmi-Smail, C.; Fabre, A.; Dequiedt, F.; Restouin, A.; Castellano,

R.; Garbit, S.; Roche, P.; Morelli, X.; Brunel J. M.; Collette, Y. J.

Med. Chem. 2010, 53, 3038–3047.

15. Our company, which was founded in 1919, is a carbostyril

supplier. Thus, the carbostyrils used for the starting material were

products produced by our company. These can be obtained from

the following website: http://www.nichiri.co.jp/.

16. Cho, Y. S.; Whitehead, L.; Li, J.; Chen, C. H.-T.; Jiang, L.;

Vogtle, M.; Francotte, E.; Richert, P.; Wagner, T,; Traebert, M.;

Lu, Q,; Cao, X.; Dumotier, B.; Fejzo, J.; Rajan, S.; Wang, P.; Yan-

Neale, Y.; Shao, W.; Atadja P.; Shultz, M. J. Med. Chem., 2010,

53, 2952-2963.

17. Bradsher, C. K.; Hunt, D. A. J. Org. Chem., 1981, 46, 327-330.

18. Seefeld, M. A.; Miller, W. H.; Newlander, K. A.; Burgess, W. J.;

DeWolf Jr., W. E.; Elkins, P. A.; Head, M. S.; Jakas, D. R.;

Janson, C. A.; Keller, P. M.; Manley, P. J.; Moore, T. D.; Payne,

D. J.; Pearson, S.; Polizzi, B. J.; Qiu, X.; Rittenhouse, S. F.;

Uzinskas, I. N.; Wallis, N. G.; Huffman, W. F. J. Med. Chem.,

2003, 46, 1627-1635.

25. Berrino, R.; Cacchi, S.; Fabrizi, G.; Goggiamani, A. J. Org. Chem.

2012, 77, 2537-2542.

26. Oshiro, Y.; Sato, S.; Kurahashi, N.; Tanaka, T.; Kikuchi, T.;

Tottori, K.; Uwahodo, Y.; Nishi, T. J. Med. Chem. 1998, 41, 658-

667.

27. Tamura, Y.; Terashima, M.; Higuchi, Y.; Ozaki, K. Chemistry &

Industry 1970, 45, 1435.

28. Nakagawa, K.; Murakami, N.; Yoshizaki, S.; Tominaga, M.; Mori,

H.; Yabuuchi, Y.; Shintani, S. J. Med. Chem. 1974, 17, 529-533

29. Hoffmann, K.; Brosch, G.; Loidl, P.; Jung, M. Nucleic Acids Res.

1999, 27, 2057-2058

30. Yoshida, M.; Furumai, R.; Nishiyama, M.; Komatsu, Y.; Nishino,

N.; Horinouchi, S. Cancer Chemother Pharmacol. 2001, 48

(Suppl 1), 20-26.

31. Promega Corporation, www.promega.com

Table(s)

Table 2. In vitro inhibitory activity (%) of SAHA and compounds 12-25

against rat liver HDACs (n = 2)

Table 1. In vitro inhibitory activity (%) of SAHA and compounds 1-11

O

6

against rat liver HDACs (n = 2)

X

5

O

Y

HO

A

6

X

Z

-

7

N

Y

HO

N

O

n

H

-

7

Z

1

O

n

8

H

2

O

B

Z

8

% inhibition

position

n

0.1 M

compnd

A

B

X-Y

Z

n

0.1 M

10 M

96

compnd

X-Y

10 M

-

SAHA

-

SAHA

12

-

34

66

51

-

H

96

94

34

19

99

98

CH=CH

CH₂-CH₂

NH

6

1

2

3

4

6

5

3

CONH

CH=CH

NH

CH₂-CH₂

13

91

88

65

13

11

9

58

49

C(CH₃)₂-CH₂

CH₂-CH₂

CH=CH

C(CH₃)₂-CH₂

H

F

14

CH₂-CH₂

6

99

CONH

NH

15

CONH

NHCO

H

NH

O

CH=CH

CH₂-CH₂

O

16

17

18

19

20

6

5

7

6

95

97

24

30

46

68

45

5

6

7

8

6

7

5

91

93

91

12

21

13

11

NH

98

C(CH₃)₂-CH₂

CH₂-CH₂

CH=CH

NH

100

98

CH₂-CH₂

9

5

8

5

86

80

7

4

C(CH₃)₂-CH₂

CH₂-CH₂

NHCO

H

F

NH

21

22

6

98

99

44

57

CH=CH

10

11

CH₂-CH₂

H

23

24

25

6

7

5

7

6

94

98

99

21

40

63

CH₂-CH₂

NHCO

H

NH

C(CH₃)₂-CH₂

Table 3. In vitro inhibitory activity (%) of SAHA and compounds 26-39

Table 5. In vitro inhibitory activity (IC₅₀) of SAHA and various synthesized

against rat liver HDACs (n = 2)

compounds against human HDAC isozymes (n > 2)

O

inhibition (IC₅₀; M)

6

X

Y

HO

B

Class IIa

Class IIb

Class I

-

N

H

A

W

2

compnds HDAC1 HDAC2 HDAC3 HDAC8 HDAC4 HDAC9 HDAC6

Z

7

1

SAHA

12

0.030

0.035

0.014

0.028

0.045

0.018

0.11

0.054

0.091

0.11

0.048

0.094

0.15

0.063

0.12

0.038

0.058

0.025

0.40

0.066

0.63

% inhibition

position

-

B

10 M 0.1 M

13

compnd

SAHA

A

X-Y

Z-W

14

19

0.0091

0.024

0.010

0.025

0.089

0.18

0.033

0.073

0.047

0.28

0.045

0.046

0.55

0.28

-

96

34

55

63

51

(CH₂)₆

C(CH₃)₂-CH₂

CH=CH

CONH

NHCO

NMe-C=O

26

27

28

6

98

99

20

21

27

0.024

0.030

0.089

0.020

0.093

0.090

0.085

0.069

0.098

0.053

0.055

0.072

0.12

0.063

0.086

0.035

0.039

0.041

0.43

0.092

0.086

CH₂-CH₂

29

31

33

34

40

0.043

0.052

0.67

0.10

0.17

0.028

0.21

0.059

0.080

0.20

0.28

0.078

2.0

0.12

0.034

0.57

0.13

0.040

0.29

(CH₂)₆

C(CH₃)₂-CH₂

NHCO

NMe-C=O

29

30

31

32

6

99

50

(CH₂)₆

CONH C(CH₃)₂-CH₂N=C-OMe

99

100

97

52

70

42

6

0.10

NHCO

N=C-OMe

CH=CH

0.066

0.022

0.065

0.049

0.38

0.037

0.11

0.0038 0.0082 0.015 0.0060 0.058 0.0052 0.058

CONH C(CH₃)₂-CH₂N=C-OEt

41

42

43

0.035

0.19

0.035

0.31

0.089

0.37

0.028

0.021

0.024

0.22

0.75

0.33

0.057

0.091

0.050

0.44

0.72

0.53

95

98

96

22

46

27

NH-CO

6

7



NHCO C(CH₃)₂-CH₂

33

34

35



0.041

0.025

0.087

(CH₂)₆NHSO₂

94

91

18

15

17

11

6

36

37

38

39

CH=CH

CH₂-CH₂

(CH₂)₆NHSO₂

(CH₂)₆SO₂NH

NH-CO

C(CH₃)₂-CH₂

CH₂-CH₂

Table 4. In vitro inhibitory activity (%) of SAHA and compounds 40-43

against rat liver HDACs (n = 2)

% inhibition

compnd

SAHA

10 M 0.1 M

96

98

34

55

O

N

H

N

trans

40

HO

N

H

O

N

H

N

99

43

N

H

41

42

O

N

H

N

cis

92

97

22

27

HO

N

H

O

N

H

N

H

43

Figure(s)

Scheme 3.

O

X

Z

X

Z

RO

X

Y

HO

Y

a

N

n

H

W

O

Figure 1. Approved HDAC inhibitors for cancer treatment

B

49

48

O

6

N

H

b

Y

O

N

O

NH

H

O

HO

N

HO

N

n

W

H

N

O

Z

H

O

7

O

19-25, 27-29, 31

B

NH

Vorinostat (SAHA)

O

S

Reagents and conditions: (a) EDCI^.HCl, HOBt, Et₃N, DMF, RO₂C(CH₂)nNH₂

(R=Bn, Me; n=5-7), rt, 18-86%; (b) NH₂OH, H₂O, MeOH, NaOMe, 0C, 22-

82%.

Romidepsin

Scheme 1.

O

6

X

Z

Scheme 4.

X

Y

a

HO

O

EtO

n

O

Br

7

Br

m, p

Z

O

NHBoc

a, b, c

Br

d, e

44

NH₂

45

EtO

51

50

O

52

6

7

X

Y

b

HO

O

N

H

6

7

O

n

Z

O

f, g

HO

N

1-11

N

H

N

H

O

m, p

33-35

Reagents and conditions: (a) NaH, DMF, 0C, then EtO₂C(CH₂)nBr (n=3, 5),

rt, 25%-quant.y.; (b) NH₂OH, H₂O, MeOH, NaOMe, 0C, 40%-quant.y.

Reagents and conditions: (a) phthalimide potassium salt, DMF, 100^oC, quant.

.

y.; (b) H₂NNH₂H₂O, MeOH, reflux, 93-95%; (c) Boc₂O, Et₃N, CH₂Cl₂, 0^oC

to rt, 95%-quant. y.; (d) ethyl acrylate, Pd(OAc)₂, P(o-tolyl)₃, EtCN, DMF,

DIEA, 90^oC, N₂, 77-96%; (e) TFA, CH₂Cl₂, rt, 95%-quant. y.; (f) EDCI^.HCl,

HOBt, Et₃N, DMF, 4,4-dimethyl-3,4-dihydrocarbostyril-6 (or 7)-carboxylic

acid, rt, 70-75%; (g) NH₂OH, H₂O, MeOH, NaOMe, 0^oC, 11-75%.

Scheme 2.

X

Z

H₂N

X

Z

H

Y

a

Y

R'O

N

n

O

n

O

Scheme 5.

B

46

47

O

X

Z

X

H

b

Y

a

S

X

N

Cl

Y

HO

N

H

O

54

O

N

H

O

53

b,c

B

O

12-18, 26, 30, 32

H

N

S

X

HO

N

Y

6

Reagents and conditions: (a) EDCI^.HCl, HOBt, Et₃N, DMF,

RO₂C(CH₂)nCO₂H (R=Bn, Me; n=5-7), rt, 51-91%; (b) NH₂OH, H₂O,

MeOH, NaOMe, 0C, 31%-quant. y.

H

O

N

H

O

36-38

Reagents and conditions: (a) chlorosulfonic acid, 65C, N₂, 50-89%; (b)

.

MeO₂C(CH₂)₆NH₂HCl, Et₃N, CH₂Cl₂, rt, 58-72%; (c) NH₂OH, H₂O, MeOH,

NaOMe, 0C, 14-67%.

Scheme 6.

H

OH

MeO

N

MeO

a

b

Br

S

6

S

6

O

55

O

N

O

56

H

57

H

N

c

HO

S

6

O

N

H

O

39

Reagents and conditions: (a) Na₂SO₃, H₂O, reflux; (b) trichlorotriazine, Et₃N,

acetone, 80^oC, MW, then 2 N NaOH, 6-amino-3,4-dihydro-carbostyril, 80C,

MW, 13% for two steps; (c) NH₂OH, H₂O, MeOH, NaOMe, 0^oC, 39%.

Scheme 7.

O

OH

a, b, c, d, e

N

f, g , h

H

HO

BnO

N

O

trans, cis

59

H

58

O

N

i

N

H

EtO

N

O

HO

N

H

O

H

60

O

j, k

O

trans: 40, cis: 42

O

N

H

N

HO

N

O

H

trans: 41, cis: 43

O

Reagents and conditions: (a) NaH (0.5eq.), DMF, 0^oC then BnBr (0.5eq.),

0C, 22-36%; (b) MsCl, Et₃N, CH₂Cl₂, 0^oC to rt, 85-100%; (c) NaN₃, DMF,

80C, 86%-quant. y.; (d) H₂, 10%Pd-C, EtOH, rt, 0.18 MPa, 95-96%; (e)

EDCI^.HCl, HOBt, Et₃N, DMF, 4,4-dimethy-3,4-dihydro-carbostyril-6-

carboxylic acid, rt, 47-94%; (f) H₂, 10%Pd-C, EtOH, rt, 0.30 MPa, 80-99%;

(g) Dess-Martin periodinane, CH₂Cl₂, 0C to rt, 41-83%; (h) NaH,

(EtO)₂P(=O)CH₂CO₂Et, THF, 0^oC, 60-66%; (i) NH₂OH, H₂O, MeOH,

NaOMe, 0C, 20-67%; (j) H₂, 10%Pd-C, EtOH, rt, 0.16MPa, 55-80%; (k)

NH₂OH, H₂O, MeOH, NaOMe, 0C, 47-80%.

Graphical Abstract

Design and Synthesis of Novel and Highly-

Active Pan-Histone Deacetylase (pan-HDAC)

Inhibitors

Leave this area blank for abstract info.

Toshihiko Tashima, Hiroaki Murata, Hidehiko Kodama

O

IC₅₀values of 40: HDAC1, 0.0038 M;

HDAC2, 0.0082 M; HDAC3, 0.015 M;

HDAC8, 0.0060 M; HDAC4, 0.058 M;

HDAC9, 0.0052 M; HDAC6, 0.058 M

N

H

trans

N

HO

N

O

H

40

O

Article Doi

Design and synthesis of novel and highly-active pan-histone deacetylase (pan-HDAC) inhibitors

DOI: 10.1016/j.bmc.2014.05.001

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2014.05.001

Full text of DOI:10.1016/j.bmc.2014.05.001

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