5-Membered cyclic hydroxamic acids as HDAC inhibitors

Article abstract of DOI:10.3109/14756366.2014.912214

The new histone deacylases inhibitors (HDACi) were synthesized in the class of 5-membered cyclic hydroxamic acids (5-CHA), showing medium size CHA as a new Zn-binding group. New reaction sequence was proposed for the synthesis of 5-membered alkylidene-cyc

Full text of DOI:10.3109/14756366.2014.912214

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–8
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.912214
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RESEARCH ARTICLE
5-Membered cyclic hydroxamic acids as HDAC inhibitors
Ilze Mutule, Diana Borovika, Elina Rozenberga, Nadezhda Romanchikova, Raivis Zalubovskis, Irina Shestakova, and
Peteris Trapencieris
Department of Organic Chemistry, Latvian Institute of Organic Synthesis, Riga, Latvia
Abstract
Keywords
The new histone deacylases inhibitors (HDACi) were synthesized in the class of 5-membered
cyclic hydroxamic acids (5-CHA), showing medium size CHA as a new Zn-binding group. New
reaction sequence was proposed for the synthesis of 5-membered alkylidene-cyclic-hydroxamic
acids starting from butyrolactone. Compound 10c showed low mM activity on HeLa cell
extracts. From these results, cyclic hydroxamic acids will be further investigated to find more
potent compounds.
Cyclic hydroxamic acids, histone deacylases,
inhibitors, N-hydroxypyrrolidones
History
Received 13 January 2014
Revised 28 March 2014
Accepted 29 March 2014
Published online 17 June 2014
Introduction
The play for medicinal chemists in the development of new
inhibitors for Zn-dependant enzymes is around the existing ZBGs.
In spite of toxic side effects the main part of HDACi have a
hydroxamic acid moiety as the binding group to Zn2+ ion in the
active center of enzyme.
One of research interest in ‘‘epi-drugs’’ is restoration of
epigenetic regulation balance in histone acetylation/deacetylation
processes. The acetylation state of lysine residues is controlled by
two counteracting responsible enzymes – histone acetyl transfer-
ases (HATs) and histone deacylases (HDACs). HDACs catalyse
the removal of acetyl groups from the "-amino groups of the
lysine side chains1,2, leading to differentiation, growth arrest
and apoptosis of malignant cells, representing a new strategy in
cancer therapy. Moreover, HDAC inhibitors (HDACi) have
anti-angiogenic, anti-invasive and immuno-modulatory activities
in vitro and in vivo3. Clinical trial data demonstrated that HDACi
are efficacious as single agents and will be most broadly used
in the clinics in combination with other anticancer agents3,4. In
recent years many synthetic hydroxamic acids as HDACi were
developed for anticancer therapy. Thus, Vorinostat (SAHA) was
the first unselective HDACi licenced by the FDA in October 2006
for the treatment of advanced forms of cutaneous T-cell lymph-
oma (CTCL)5 and now continued to test in many other clinical
trials6. There are many clinical trials for HDAC inhibitors and
epigenetic drugs reviewed with the interpretation of data related
to epigenetic measurements7. Due to the short half life and low
oral bioavailability of hydroxamic acids very limited amount of
new active compounds went through the clinical studies.
Only some recent publications were devoted to the develop-
ment of the novel ZBGs for HDAC inhibitors. Among them are
pyridine-2,6-dicarboxylic acids8, cysteines9, thiols10 and hydra-
zides11. Some of the compounds with these new ZBGs bind to
HDACs with nM binding capacity. During the last decade,
Cohen’s group12–15 has investigated several new ZBGs for
Zn-dependant enzymes, particularly matrix metalloproteinases.
Among a wide variety of cyclic ketones and lactones they
proposed some cyclic hydroxamic and thiohydroxamic acids13–15
.
In the course of our interest in new ZBGs for HDACi and CAi,
we were aimed to find new ZBGs and use the closest structural
analog of the most exploited hydroxamic acids – medium size
cyclic hydroxamic acids. These new structural units for HDACi
will be broadened by enlarging the ring size and functionalizing
the molecules. With this purpose we have analyzed all the
synthetic routes to small and medium size16 and mono-
macrocyclic17 hydroxamic acids. Current investigation is devoted
to 5-membered cyclic hydroxamic acids (5-CHA), synthesis of
the simplest representatives and evaluation of their potential as
HDAC inhibitors.
Almost all of the ꢀ300 Zn-dependant proteases are now
known to be connected with tetrahedral Zn atom in the enzyme
active center2. Despite a large number of publications in this field,
discovery of new successful zinc binding groups (ZBGs) is very
limited6,7. There are only some ZBGs known for HDACi:
bidentate ligands derived from hydroxamic acids, monoacylated
phenylenediamines and monodentate ligands – carboxylic acids.
Materials and methods
Chemistry
Commercial grade reagents and anhydrous solvents were used as
received from suppliers and no attempts were made to purify or to
dry these components further. All non-aqueous reactions were
performed under dry conditions in the atmosphere of argon unless
otherwise specified. Thin layer chromatography (TLC) was
performed using silica gel 60 F254 (Merck, Darmstadt,
Germany) plates. Visualization of TLC plates was made with
Address for correspondence: Peteris Trapencieris, Latvian Institute of
Organic Synthesis, Aizkraukles 21, Riga, LV-1006, Latvia. Tel: +371
67014936. Fax: +371 67550338. E-mail: peteris@osi.lv

2
I. Mutule et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
short wave (254 nm) UV light. Flash column chromatography was 5.0 mmol) in dry THF (2 mL) was stirred at 60 ꢂC for 30 h.
1
carried out using silica gel 0.035–0.070 mm (Merck). H and 13C After cooling to room temperature, the solid was filtered off,
NMR spectra were obtained on NMR Spectrometers – Varian 200 washed with THF and dried to afford 645 mg (60%) of the Wittig
Mercury (200 MHz) (Ex Varian, Agilent Technologies, Santa salt (7a). 1H-NMR (DMSO-D6, 200 MHz, d, ppm): 2.10–2.42 (m,
Clara, CA) and Varian Mercury plus 400 (400 MHz) (Ex Varian, 2H), 3.15–3.40 (m, 1H, overlapped with H2O), 3.42–3.62 (m, 1H),
Agilent Technologies) and are reported in ppm d values, using 4.62 (d, J ¼ 2.6 Hz, 2H), 5.50–5.74 (m, 1H), 7.30–7.40 (m, 5H),
tetramethylsilane as an internal reference. Microanalyses were 7.70–7.95 (m, 15H). MS (ESI), m/z: 452 (M+), calcd for C28H28
performed using a Carlo Erba – 106 (Milan, Italy) elemental PNO2: 452.53.
analyzer. Melting points were determined in capillary tubes using
[(N-Tetrahydro-pyran-2-yloxy)-2-oxopyrrolid-3-yl]-triphenylpho-
sphonium bromide (7b). This compound was obtained from
3-bromo-1-(tetrahydropyran-yloxy)pyrrolidin-2-one (5b) and tri-
phenyl phosphine (6) according to the General Method A as a
white solid. Yield 35%. 1H-NMR (DMSO-D6, 400 MHz, d, ppm):
1.41–1.61 (m, 4H), 1.73–1.78 (m, 2H), 2.21–2.40 (m, 2H), 3.23–
3.40 (m, 1H, overlapped with H2O), 3.41–3.56 (m, 1H), 3.56–3.63
(m, 2H), 3.75–3.93 (m, 1H), 5.49–5.64 (m, 1H), 7.72–7.94 (m,
15H). MS (ESI), m/z: 446.5 (M+), calcd for C27H29PNO3: 446.52.
a Opti Melt automated melting point system (Stanford Research
Systems, Sunnyvale, CA). IR spectra were recorded on Shimadzu
IR Prestige 21 (Kyoto, Japan) IR spectrometer.
Trans-cinnamaldehyde, 2-quinolinecarbaldehyde and 3-formyl
indole are commercialy available. General procedure for alde-
hyde chain elongation18 was employed. 9H-Carbazole-2-carbal-
dehyde19,
9-methyl-9H-carbazole-2-carbaldehyde20
(8h),
3-formyl-indole-1-carboxylic acid tert-butyl ester21 (8a),
3-formyl-N-phenyl-benzenesulfonamide22 (8i), were synthesized
according to literature procedures.
General Method B
(2E,4E)-5-Phenyl-penta-2,4-dienal (8c). Solution of dioxolane
(14a) (541 mg, 2.67 mmol) in a mixture of THF (7 mL) and 10%
HCl in water (7 mL) was stirred at room temperature 2 h and
analyzed by TLC (solvent system 5% EtOAc in PE). The mixture
was diluted with water (50 mL), and extracted with ether
(3 ꢃ 30 mL). The organic layer was washed with brine, dried
over Na2SO4, and evaporated. The residual oil was purified by
flash chromatography on silica gel (eluent 5% EtOAc in PE)
to afford the title compound as yellow oil. Yield 420 mg (100%).
1H-NMR (CDCl3, 400 MHz, d, ppm): 6.25 (dd, J ¼ 15.1 and
8.0 Hz, 1H), 6.98–7.01 (m, 1H), 7.00 (s, 1H), 7.22–7.29
(m, overlapped with CHCl3, 1H), 7.32–7.39 (m, 3H), 7.47–7.50
(m, 2H). GC/MS: 158.1 (M+), calcd for C11H10O: 158.20.
1-Benzyloxy-3-bromopyrrolidin-2-one (5a)
A
solution of 2,4-dibromobutyroyl chloride23 (2; 1.32 g;
4.95 mmol) in dry DCM (30 mL) was added at ꢁ20 ꢂC to a
solution of O-benzylhydroxylamine (3a; 0.58 mL; 5.0 mmol) and
triethylamine (0.70 mL; 5.0 mmol) in dry DCM (20 mL). After
30 min, the reaction mixture was extracted with water (20 mL)
and the organic phase was dried over Na2SO4. Evaporation of the
solvent afforded a colorless oil. Amberlite IRA-400 (in OHꢁ
form) was added to a vigorously stirred mixture of this oil in
DCM (20 mL) and 1.5 g of 50% NaOH solution. After 4 h at room
temperature the mixture was filtered, filtrate washed with water
(2 ꢃ 5 mL) and the organic phase dried over Na2SO4. Evaporation
of the solvent gave colorless oil, which was purified by flash
chromatography on silica gel (eluent 20% EtOAc in PE) to
afford the title compound as a white solid. Yield 800 mg (60%).
1H-NMR (CDCl3, 200 MHz, d, ppm): 2.1–2.3 (m, 1H), 2.41–2.63
(m, 1H), 3.13–3.30 (m, 1H), 3.30–3.45 (m, 1H), 4.30–4.40 (m,
1H), 5.03 (s, 2H), 7.18–7.25 (m, 5H). MS (ESI), m/z: 270/272
(M + H)+, calcd for C11H12BrNO2: 270.13.
(2E,4E,6E)-7-Phenyl-hepta-2,4,6-trienal (8d). This compound
was obtained from dioxolane (14b) according to the General
1
Method B as an orange substance. Yield 91%; H-NMR (CDCl3,
400 MHz, d, ppm): 6.20 (dd, J ¼ 15.2 and 7.8 Hz, 1H), 6.57 (dd,
J ¼ 14.0 and 11.1 Hz, 1H), 6.78–6.95 (m, 3H), 7.19 (dd, J ¼ 15.2
and 11.1 Hz, 1H), 7.27–7.32 (m, 1H), 7.33–7.38 (m, 2H), 7.43–
7.47 (m, 2H), 9.59 (d, J ¼ 7.8 Hz, 1H). GC/MS: 184.1 (M+), calcd
for C13H12O: 184.24.
3-Bromo-1-(tetrahydro-pyran-2-yloxy)pyrrolidin-2-one (5b)
A solution of 2,4-dibromobutanoic acid chloride (2; 2.46 g;
9.3 mmol) in dry DCM (30 mL) at ꢁ20 ꢂC was added to a solution
of tetrahydropyranylhydroxylamine (3b; 1.11 g; 9.5 mmol)24 and
triethylamine (1.33 mL; 9.5 mmol) in dry DCM (30 mL). After
30 min stirring the reaction mixture was extracted with water
(20 mL) and the organic phase was dried over Na2SO4. Evaporation
of the solvent afforded a colorless oil. Amberlite IRA-400 (4.0 g; in
OHꢁ form) was added to a vigorously stirred solution of this oil in a
mixture of DCM (50 mL) and 3.30 g of 50% NaOH solution. After
1 h at room temperature the mixture was filtered, the filtrate
washed with water (2 ꢃ 10 mL), the organic phase separated and
dried over Na2SO4. Evaporation of the solvent gave colorless oil,
which was purified by flash chromatography on silica gel (eluent
20–30% EtOAc in PE) to afford the title compound as a white solid.
(E)-3-Quinolin-3-yl-propenal (8e). This compound was
obtained from dioxolane (14c) according to the General Method
B as a yellowish substance. Yield 75%. 1H-NMR (CDCl3,
400 MHz, d, ppm): 6.94 (dd, J ¼ 16.2 and 7.5 Hz, 1H), 7.60–
7.65 (m, 1H), 7.65 (d, J ¼ 16.2 Hz, 1H), 7.78–7.83 (m, 1H), 7.89
(d, J ¼ 8.4 Hz, 1H), 8.14 (d, J ¼ 8.4 Hz, 1H), 8.32 (d, J ¼ 2.2 Hz,
1H), 9.12 (d, J ¼ 2.2 Hz, 1H), 9.79 (d, J ¼ 7.4 Hz, 1H). GC/MS:
183.1(M+), calcd for C12H9NO: 183.22.
(2E,4E)-5-Quinolin-3-yl-penta-2,4-dienal (8f). This compound
was obtained from dioxolane (14d) according to the General
Method B as a yellowish substance. Yield 78%. 1H-NMR (CDCl3,
400 MHz, d, ppm): 6.36 (dd, J ¼ 15.0 and 7.8 Hz, 1H), 7.19 (dd,
J ¼ 19.1 and 10.1 Hz, 2H), 7.32 (dd, J ¼ 15.0 and 9.9 Hz, 1H),
7.55–7.61 (m, 1H), 7.72 (m, 1H), 7.84 (dd, J ¼ 8.2 and 1.6 Hz,
1H), 8.10 (d, J ¼ 8.6 Hz, 1H), 8.22 (d, J ¼ 2.1 Hz, 1H), 9.07 (d,
J ¼ 2.1 Hz, 1H), 9.67 (d, J ¼ 7.8 Hz, 1H). GC/MS: 209.1 (M+).
MS (ESI), m/z: 210.2 (M + H+), calcd for C14H11NO: 209.25.
1
Yield 1.43 g (58%). H-NMR (CDCl3, 400 MHz, d, ppm): 1.58–
1.72 (m, 3H), 1.75–1.85 (m, 3H), 2.30–2.40 (m, 1H), 3.58–3.72 (m,
2H), 3.75–3.90 (m, 1H), 4.00–4.15 (m, 1H), 4.37–4.43 (m, 1H),
5.17–5.22 (m, 1H). MS (ESI), m/z: 264/266 (M + H)+, calcd for
C9H14NO3: 264.12.
General Method A
(E)-3-(9-Methyl-9H-carbazol-2-yl)-propenal (8g). This com-
(N-Benzyloxy-2-oxopyrrolid-3-yl)-triphenyl phosphonium bro- pound was obtained from dioxolane (14e) according to the
1
mide (7a). A mixture of 1-benzyloxy-3-bromopyrrolidin-one General Method B as a yellow substance. Yield 85%. H-NMR
(5a; 540 mg, 2.0 mmol) and triphenylphosphine (6; 524 mg, (CDCl3, 400 MHz, d, ppm): 6.86 (dd, J ¼ 15.8 and 7.8 Hz, 1H),

DOI: 10.3109/14756366.2014.912214
5-Membered cyclic hydroxamic acids
3
7.28 (d, J ¼ 8.0 Hz, 1H), 7.43 (d, J ¼ 8.4 Hz, 1H), 7.47 6.66 (t, J ¼ 11.8 Hz, 1H), 6.67 (d, J ¼ 15.6 Hz, 1H), 6.88 (dd,
(d, J ¼ 8.2 Hz, 1H), 7.50–7.56 (m, 1H), 7.59 (s, 1H), 7.68 J ¼ 15.6 and 10.6 Hz, 1H), 7.02 (td, J ¼ 11.8 and 2.4 Hz, 1H),
(d, J ¼ 15.8 Hz, 1H), 8.09–8.13 (m, 2H), 9.76 (d, J ¼ 7.8 Hz, 1H). 7.20–7.27 (m, overlapped with CHCl3, 1H), 7.28–7.34 (m, 2H),
GC/MS: 235.1 (M+). MS (ESI), m/z: 236.1 (M + H)+, calcd for 7.38–7.43 (m, 2H). MS (ESI), m/z: 326.6 (M + H)+, calcd for
C16H13NO: 235.29.
C20H23NO3: 325.41.
General Method C
3-[(2E,4E,6E)-7-Phenyl-hepta-2,4,6-trien-(E)-ylidene]-1-(tetra-
hydro-pyran-2-yloxy)-pyrrolidin-2-one (9f). This compound
was obtained from phosphonium salt (7b) and aldehyde (8d)
according to the General Method C as a yellow substance. Yield
3-[1-Benzyloxy-2-oxo-pyrrolidin-(3E)-ylidenemethyl]-indole-1-
carboxylic acid tert-butyl ester (9a). To a solution of phos-
phonium salt (7a) (166 mg, 0.31 mmol) and aldehyde (8a) (84 mg,
0.34 mmol) in abs. EtOH (6 mL) was added triethylamine
(131 mL, 0.93 mmol). Reaction mixture was heated at 85 ꢂC
under argon atmosphere for 4 h with vigorosly stirring, then
evaporated under reduced pressure and residual substance
purified by flash chromatography on silica gel (eluent 20–25%
EtOAc in PE) to afford the title compound as a white substance.
Yield 89 mg (67%). 1H-NMR (CDCl3, 400 MHz, d, ppm): 1.67 (s,
9H), 2.85 (dt, J ¼ 6.4 and 2.7 Hz, 2H), 3.41 (t, J ¼ 6.4 Hz, 2H),
5.10 (s, 2H), 7.28–7.40 (m, 5H), 7.45–7.49 (m, 2H), 7.62
(t, J ¼ 2.8 Hz, 1H), 7.64 (s, 1H), 7.78 (d, J ¼ 2.8 Hz, 1H), 8.08
(d, J ¼ 8.2 Hz, 1H). MS (ESI), m/z: 419.5 (M + H)+, calcd for
C25H26N2O4: 418.50.
1
23%. H-NMR (CDCl3, 400 MHz, d, ppm): 1.58–1.74 (m, 3H),
1.76–1.94 (m, 3H), 2.83 (dd, J ¼ 6.0 and 2.4 Hz, 1H), 2.85 (dd,
J ¼ 7.0 and 2.4 Hz, 1H), 3.61–3.70 (m, 2H), 3.75–3.82 (m, 1H),
4.09–4.17 (m, 1H), 5.21 (t, J ¼ 3.0 Hz, 1H), 6.32 (dd, J ¼ 14.6 and
11.8 Hz, 1H), 6.44 (dd, J ¼ 14.6 and 11.0 Hz, 1H), 6.55 (dd,
J ¼ 14.6 and 10.4 Hz, 1H), 6.61 (dd, J ¼ 14.6 and 11.0 Hz, 1H),
6.64 (d, J ¼ 15.6 Hz, 1H), 6.85 (dd, J ¼ 15.6 and 10.4 Hz, 1H),
7.02 (td, J ¼ 11.8 and 2.6 Hz, 1H), 7.21–7.26 (m, 1H), 7.29–7.35
(m, 2H), 7.39–7.44 (m, 2H). MS (ESI), m/z: 352.6 (M + H)+,
calcd for C22H25NO3: 351.45.
3-[(E)-3-Ouinolin-3-yl-prop-2-en-(E)-ylidene]-1-(tetrahydro-
pyran-2-yloxy)-pyrrolidin-2-one (9g). This compound was
obtained from phosphonium salt (7b) and aldehyde (8e) according
to the General Method C as a yellow substance. Yield 38%.
1H-NMR (DMSO-D6, 400 MHz, d, ppm): 1.50–1.63 (m, 3H),
1.65–1.76 (m, 3H), 2.99 (dt, J ¼ 6.6 and 1.9 Hz, 2H), 3.53–3.59
(m, 1H), 3.62–3.76 (m, 2H), 4.04–4.12 (m, 1H), 5.16 (unresolved
t, J ¼ 2.3 Hz, 1H), 6.97 (td, J ¼ 11.4 and 2.5 Hz, 1H), 7.16
(d, J ¼ 15.6 Hz, 1H), 7.37 (dd, J ¼ 15.6 and 11.4 Hz, 1H), 7.60–
7.63 (m, 1H), 7.70–7.75 (m, 2H), 7.96 (dd, J ¼ 8.5 and 1.2 Hz,
1H), 8.00 (d, J ¼ 8.5 Hz, 1H), 8.49 (d, J ¼ 2.0 Hz, 1H), 9.18
(d, J ¼ 2.0 Hz, 1H). MS (ESI), m/z: 351.1 (M + H)+, calcd for
C21H22N2O3: 350.42.
3-[2-Oxo-1-(tetrahydro-pyran-2-yloxy)-pyrrolidin-(3E)-ylidene-
methyl]-indole-1-carboxylic acid tert-butyl ester (9b). This
compound was obtained from phosphonium salt (7b) and
aldehyde (8a) according to the General Method C as a white
substance. Yield 44%. 1H-NMR (CDCl3, 400 MHz, d, ppm):
1.60–1.69 (m, 3H), 1.70 (s, 9H), 1.81–1.97 (m, 3H), 3.00 (dt,
J ¼ 6.5 and 2.5 Hz, 2H), 3.64–3.71 (m, 1H), 3.77 (q, J ¼ 7.0 Hz,
1H), 3.86–3.93 (m, 1H), 4.13–4.21 (m, 1H), 5.28 (s, 2H), 7.29–
7.41 (m, 2H), 7.63 (t, J ¼ 2.8 Hz, 1H), 7.71 (s, 1H), 7.79
(d, J ¼ 7.5 Hz, 1H), 8.10 (d, J ¼ 8.1 Hz, 1H). MS (ESI), m/z:
413.60 (M + H)+, calcd for C23H28N2O5: 412.49.
3-[(2E,4E)-5-Ouinolin-3-yl-penta-2,4-dien-(E)-ylidene]-1-tetra-
hydro-pyran-2-yloxy)-pyrrolidin-2-one (9h). This compound
was obtained from phosphonium salt (7b) and aldehyde (8f)
according to the General Method C as a yellow substance. Yield
30%. 1H-NMR (DMSO-D6, 400 MHz, d, ppm): 1.49–1.62 (m,
3H), 1.62 (m, 3H), 2.87 (dt, J ¼ 6.4 and 2.1 Hz, 1H), 3.51–3.58
(m, 1H), 3.59–3.72 (m, 2H), 4.00–4.10 (m, 1H), 5.14 (unres
t, J ¼ 2.3 Hz, 1H), 6.63 (dd, J ¼ 14.7 and 11.7 Hz, 1H), 6.84–6.92
(m, 2H), 6.96 (d, J ¼ 15.7 Hz, 1H), 7.41 (dd, J ¼ 15.7 and 11.1 Hz,
1H), 7.51–7.54 (m, 2H), 7.69–7.74 (m, 1H), 7.91–8.00 (m, 2H),
8.40 (d, J ¼ 2.0 Hz, 1H), 9.12 (d, J ¼ 2.0 Hz, 1H). MS (ESI), m/z:
377.1 (M + H)+, calcd for C23H24N2O3: 376.46.
1-Benzyloxy-3[(E)-3-phenyl-prop-2-en-(E)-ylidene]-pyrrolidin-2-
one (9c). This compound was obtained from phosphonium salt
(7a) and cinnamaldehyde (8b) according to the General Method C
as a yellowish substance. Yield 84%. 1H-NMR (CDCl3, 400 MHz,
d, ppm): 2.76 (td, J ¼ 6.7 and 2.6 Hz, 2H), 3.34 (t, J ¼ 6.7 Hz, 2H),
5.06 (s, 2H), 6.77 (dd, J ¼ 15.6 and 10.6 Hz, 1H), 6.89 (d,
J ¼ 15.6 Hz, 1H), 7.10 (dt, J ¼ 10.6 and 2.6 Hz, 1H), 7.25–7.29
(m, 1H), 7.30–7.39 (m, 5H), 7.41–7.47 (m, 4H). MS (ESI), m/z:
306.4 (M + H)+, calcd for C20H19NO2: 305.50.
3-[(E)-3-Phenyl-prop-2-en-(E)-ylidene]-1-(tetrahydro-pyran-2-
yloxy)-pyrrolidin-2-one (9d). This compound was obtained from
phosphonium salt (7b) and cinnamaldehyde (8b) according to the
General Method C as a yellowish substance. Yield 49%. 1H-NMR
(CDCl3, 400 MHz, d, ppm): 1.58–1.73 (m, 3H), 1.78–1.95 (m,
3H), 2.90 (dd, J ¼ 6.1 and 2.6 Hz, 1H), 2.92 (dd, J ¼ 7.3 and
2.6 Hz, 1H), 3.62–3.73 (m, 2H), 3.78–3.85 (m, 1H), 4.10–4.18 (m,
1H), 5.23 (t, J ¼ 3.0 Hz, 1H), 6.84 (dd, J ¼ 21.6 and 15.2 Hz, 1H),
6.85 (s, 1H), 7.09–7.14 (m, 1H), 7.26–7.31 (m, 1H), 7.32–7.37
(m, 2H), 7.44–7.48 (m, 2H). MS (ESI), m/z: 300.5 (M + H)+,
calcd for C18H21NO3: 299.39.
3-[(E)-3-(9-Methyl-9H-carbazol-2-yl)-prop-2-en-(E)-ylidene]-1-
tetrahydro-pyran-2-yloxy)-pyrrolidin-2-one (9i). This com-
pound was obtained from phosphonium salt (7b) and aldehyde
(8g) according to the General Method D as a yellowish substance.
1
Yield 24%. H-NMR (DMSO-D6, 400 MHz, d, ppm): 1.50–1.64
(m, 3H), 1.64–1.77 (m, 3H), 2.97 (dd, J ¼ 6.7 and 2.2 Hz, 1H),
2.99 (dd, J ¼ 7.1 and 2.2 Hz, 1H), 3.53–3.60 (m, 1H), 3.62–3.75
(m, 2H), 3.90 (s, 3H), 4.03–4.13 (m, 1H), 5.15 (unres. t,
J ¼ 2.2 Hz, 1H), 6.97 (dt, J ¼ 10.3 and 2.2 Hz, 1H), 7.12–7.25 (m,
3H), 7.43–7.48 (m, 2H), 7.58 (d, J ¼ 8.3 Hz, 1H), 7.82 (s, 1H),
8.12 (d, J ¼ 3.2 Hz, 1H), 8.14 (d, J ¼ 2.4 Hz, 1H). MS (ESI), m/z:
403.2 (M + H)+, calcd for C25H26N2O3: 402.5
3-[(2E,4E)-5-Phenyl-penta-2,4-dien(E)-ylidene]-1-(tetrahydro-
pyran-2-yloxy)-pyrrolidin-2-one (9e). This compound was
obtained from phosphonium salt (7b) and aldehyde (8c) according
to the General Method C as a yellowish substance. Yield 28%. 1H-
NMR (CDCl3, 400 MHz, d, ppm): 1.56–1.71 (m, 3H), 1.76–1.92
(m, 3H), 2.82 (dd, J ¼ 6.3 and 2.4 Hz, 1H), 2.84 (dd, J ¼ 7.0 and
2.4 Hz, 1H), 3.60–3.69 (m, 2H), 3.74–3.81 (m, 1H), 4.07–4.16 (m,
1H), 5.20 (t, J ¼ 3.0 Hz, 1H), 6.37 (dd, J ¼ 14.8 and 11.9 Hz, 1H),
3-[1-(9-Methyl-9H-carbazol-2-yl)-meth-(E)-ylidene]-1-tetrahy-
dro-pyran-2-yloxy)-pyrrolidin-2-one (9j). This compound was
obtained from phosphonium salt (7b) and aldehyde (8h) accord-
ing to the General Method C as a white substance. Yield 45%.

4
I. Mutule et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
1H-NMR (DMSO-D6, 400 MHz, d, ppm): 1.52–1.64 (m, 3H), (M + H)+, calcd for C15H15NO2: 241.29. Elemental analysis.
1.69–1.79 (m, 3H), 3.22 (dd, J ¼ 7.2 and 2.4 Hz, 1H), 3.24 (dd, Found, %: C 73.44, H 6.00, N 5.66. Calcd for C15H15NO2: C
J ¼ 6.3 and 2.4 Hz, 1H), 3.55–3.61 (m, 1H), 3.66–3.80 (m, 2H), 73.83, H 6.28, N 5.66.
4.07–4.15 (m, 1H), 5.20 (unres. t, J ¼ 2.5 Hz, 1H), 7.19–7.24 (m,
1H), 7.38–7.41 (m, 1H), 7.60 (d, J ¼ 8.2 Hz, 1H), 7.76 (s, 1H),
1-Hydroxy-3-[(2E,4E,6E)-7-phenyl-hepta-2,4,6-trien-(E)-yli-
8.17 (d, J ¼ 7.8 Hz, 1H), 8.19 (d, J ¼ 8.2 Hz, 1H). MS (ESI), m/z:
dene]-pyrrolidin-2-one (10d). This compound was obtained
377.1 (M + H)+, calcd for C23H24N2O3: 376.5
from protected hydroxamic acid (9f) according to the General
1
Method D. Yield 69%; m.p.: 209–214 ꢂC. H-NMR (DMSO-D6,
3-[2-Oxo-1-(tetrahydro-pyran-2-yloxy)-pyrrolidin-(3E)-ylidene-
400 MHz, d, ppm): 2.80 (dt, J ¼ 6.5 and 2.3 Hz, 2H); 3.52 (t,
methyl]-N-phenyl-benzenesulfonamide (9k). This compound J ¼ 6.5 Hz, 2H); 6.45 (dd, J ¼ 14.4 and 12.0 Hz, 1H); 6.51–6.62
was obtained from phosphonium salt (7b) and aldehyde (8i) (m, 2H); 6.63–6.78 (m, 3H); 7.02 (dd, J ¼ 15.5 and 9.8 Hz, 1H);
according to the General Method C as a white substance. Yield 7.24 (t, J ¼ 7.2 Hz, 1H); 7.34 (t, J ¼ 7.7 Hz, 2H); 7.49 (d,
32%. 1H-NMR (DMSO-D6, 400 MHz, d, ppm): 1.51–1.64 (m, J ¼ 7.4 Hz, 2H), 9.94 (br s, 1H). MS (ESI), m/z: 268 (M + H)+,
3H), 1.65–1.77 (m, 3H), 2.90–2.96 (m, 2H), 3.52–3.59 (m, 1H), calcd for C17H17NO2: 267.33. Elemental analysis. Found, %: C
3.60–3.68 (m, 1H), 3.69–3.76 (m, 1H), 4.04–4.12 (m, 1H), 5.18 70.87, H 6.41, N 4.80. Calcd for C17H17NO2: C 70.82, H 5.94,
(s, 1H), 7.02 (t, J ¼ 7.2 Hz, 1H), 7.10 (dd, J ¼ 8.6 and 1.1 Hz, 2H), N 4.86.
7.15 (t, J ¼ 2.7 Hz, 1H), 7.23 (t, J ¼ 7.8 Hz, 2H), 7.60
(t, J ¼ 7.8 Hz, 1H), 7.71 (d, J ¼ 8.1 Hz 1H), 7.75 (d, J ¼ 7.8 Hz,
1-Hydroxy-3-[(E)-3-quinolin-3-yl-prop-2-en-(E)-ylidene]-pyrroli-
din-2-one (10e). This compound was obtained from protected
hydroxamic acid (9g) according to the General Method D. Yield
(69%); m.p.: 234–237 ꢂC. 1H-NMR (DMSO-D6, 400 MHz, d,
ppm): 2.94 (dt, J ¼ 6.2 and 2.3 Hz, 2H), 3.55 (t, J ¼ 6.2 Hz, 2H),
1H), 7.81 (s, 1H), 10.32 (s, 1H). MS (ESI), m/z: 429.2 (M + H)+,
calcd for C22H24N2O5S: 428.5.
1-Hydroxy-3-[1-(1H-indol-3-yl)-meth-(E)-ylidene]-pyrrolidin-2-
one (10a). To a solution of the protected cyclic hydroxamic acid 6.84–6.90 (m, 1H), 7.08 (d, J ¼ 15.7 Hz, 1H), 7.33 (dd, J ¼ 15.7
(9a) (56 mg, 0.125 mmol) in 5 mL dry DCM under argon and 11.3 Hz, 1H), 7.55–7.60 (m, 1H), 7.68–7.73 (m, 1H), 7.91–
atmosphere at 0 ꢂC was added to 2.5 mL trifluoracetic acid. The 7.99 (m, 2H), 8.45 (d, J ¼ 1.8 Hz, 1H), 9.15 (d, J ¼ 2.3 Hz, 1H),
mixture was stirred at room temperature 2 h and analyzed by TLC 9.99 (br s, 1H). MS (ESI), m/z: 267 (M + H)+, calcd for
in solvent system. Then 10% EtOH in DCM (to ascertain that C16H14N2O2: 266.30. Elemental analysis: Found, %: C 70.87, H
reaction had ended) was evaporated under reduced pressure and 4.11, N 10.21. Calcd for C16H14N2O2: C 70.94, H 5.21, N 10.34.
triturated with absolute ether. The precipitate formed was filtered
off and dried in vacuo. The title compound was obtained as a
slightly brown substance. Yield 23.4 mg (82%); m.p.: 245–250 ꢂC.
1-Hydroxy-3-[(2E,4E)-5-quinolin-3-yl-penta-2,4-dien-(E)-yli-
dene]-pyrrolidin-2-one (10f). This compound was obtained
1H-NMR (DMSO-D6, 400 MHz, d, ppm): 2.90 (dt, J ¼ 6.4 and
from protected hydroxamic acid (9h) according to the General
1
Method D. Yield 63%; m.p.: 244–245 ꢂC. H-NMR (DMSO-D6,
2.4 Hz, 2H), 3.59 (t, J ¼ 6.4 Hz, 2H), 7.11 (t, J ¼ 7.5 Hz, 1H), 7.17
(t, J ¼ 7.5 Hz, 1H), 7.40 (unres. t, J ¼ 2.4 Hz, 1H), 7.43 (d,
J ¼ 8.0 Hz, 1H), 7.59 (d, J ¼ 2.4 Hz, 1H), 7.75 (d, J ¼ 7.8 Hz, 1H),
9.80 (s, 1H), 11.66 (s, 1H). MS (ESI), m/z: 229 (M + H)+, calcd
for C13H12N2O2: 228.25. Elemental analysis: Found, %: C 61.86,
H 4.76, N 10.14. Calcd for C13 H12 N2O2: C 62.03, H 6.27, N 9.91
400 MHz, d, ppm): 2.82 (dt, J ¼ 5.7 and 2.3 Hz, 2H), 3.52
(t, J ¼ 6.3 Hz, 2H), 6.53–6.64 (m, 1H), 6.75–6.95 (m, 1H), 7.38
(dd, J ¼ 15.7 and 10.7 Hz, 1H), 7.53–7.60 (m, 1H), 7.65–7.72 (m,
1H), 7.86–7.98 (m, 2H), 8.36 (d, J ¼ 1.7 Hz, 1H), 9.09 (d,
J ¼ 2.2 Hz, 1H), 9.96 (br s, 1H). MS (ESI), m/z: 293 (M + H)+,
calcd for C18H16N2O2: 292.34. Elemental analysis. Found, %: C
72.86, H 4.45, N 9.27. Calcd for C18H16N2O2: C 72.86, H 4.43,
N 9.44.
General Method E
1-Hydroxy-3-[(E)-3-phenyl-prop-2-en-(E)-ylidene]-pyrro-lidin-2-
one (10b). Protected hydroxamic acid (9d) (89 mg, 0.297 mmol)
was dissolved in a mixture of 2.0 mL THF, 4.0 mL AcOH and
1.0 mL water and heated at 60 ꢂC for 5 h. The process was
monitored by TLC in solvent system 5% EtOH in DCM. Reaction
mixture was evaporated under reduced pressure, and the residual
product purified by flash chromatography on silica gel (eluent 5%
EtOH in DCM), to afford the title substance. Yield 50 mg (78%);
m.p.: 190–195 ꢂC. 1H-NMR (DMSO-D6, 400 MHz, d, ppm): 2.90
(dt, J ¼ 6.2 and 2.2 Hz, 2H); 3.55 (t, J ¼ 6.2 Hz, 2H); 6.82 (dt,
J ¼ 11.2 and 2.3 Hz, 1H); 6.91 (d, J ¼ 15.6 Hz, 1H); 7.03 (dd,
J ¼ 15.6 and 11.2 Hz, 1H); 7.28 (t, J ¼ 7.2 Hz, 1H); 7.36
(t, J ¼ 7.4 Hz, 2H); 7.56 (d, J ¼ 7.4 Hz, 2H), 9.95 (s, 1H). MS
(ESI), m/z: 216 (M + H)+, calcd for C13H13NO2: 215.25.
Elemental analysis. Found, %: C 70.12, H 6.32, N 6.14. Calcd
for C13H13NO2: C 70.25, H 5.90, N 6.30.
1-Hydroxy-3-[(E)-3-(9-methyl-9H-carbazol-2-yl)- prop-2-en-(E)-
ylidene]-pyrrolidin-2-one (10g). This compound was obtained
from protected hydroxamic acid (9i) according to the General
1
Method D. Yield 92%; m.p.: 268–270 ꢂC. H-NMR (DMSO-D6,
400 MHz, d, ppm): 2.89–2.99 (m, 2H), 3.55 (t, J ¼ 6.3 Hz, 2H),
3.87 (s, 1H), 6.86 (dt, J ¼ 10.9 and 2.6 Hz, 1H), 7.03–7.21 (m,
3H), 7.43 (t, J ¼ 8.3 Hz, 2H), 7.55 (d, J ¼ 8.3 Hz, 1H), 7.77 (s,
1H), 8.07–8.12 (m, 2H), 9.92 (s, 1H). MS (ESI), m/z: 319
(M + H)+, calcd for C20H18N2O2: 318.38. Elemental analysis.
Found, %: C 74.95, H 4.41, N 8.56. Calcd for C20H18N2O2: C
74.98, H 5.66, N 8.74.
1-Hydroxy-3-[(E)-3-(9-methyl-9H-carbazol-2-yl)-meth-(E)-yli-
dene]-pyrrolidin-2-one (10h). This compound was obtained
from protected hydroxamic acid (9j) according to the General
1
Method E. Yield 71%; m.p.: 258–262 ꢂC. H-NMR (DMSO-D6,
1-Hydroxy-3-[(2E,4E)-5-phenyl-penta-2,4-dien-(E)-ylidene]-pyr-
rolidin-2-one (10c). This compound was obtained from pro-
tected hydroxamic acid (9e) according to the General Method D.
400 MHz, d, ppm): 3.19 (dt, J ¼ 6.3 and 2.6 Hz, 2H), 3.60
(t, J ¼ 6.3 Hz, 2H), 3.87 (s, 3H), 7.18 (t, J ¼ 7.8 Hz, 1H), 7.30
(t, J ¼ 2.7 Hz, 1H), 7.35 (unres. dd, J ¼ 8.2 and 1.0 Hz, 1H), 7.43–
7.48 (m, 1H), 7.57 (d, J ¼ 8.2 Hz, 1H), 7.71 (s, 1H), 8.12–8.17
(m, 2H), 9.97 (br s, 1H). MS (ESI), m/z: 293 (M + H)+, calcd for
C18H16N2O2: 292.34. Elemental analysis. Found, %: C 73.44, H
4.40, N 9.35. Calcd for C18H16N2O2: C 73.40, H 5.47, N 9.51.
1
Yield (82%); m.p.: 222–226 ꢂC. H-NMR (DMSO-D6, 400 MHz,
d, ppm): 2.81 (dt, J ¼ 6.5 and 2.4 Hz, 2H), 3.53 (t, J ¼ 6.5 Hz, 2H),
6.53 (dd, J ¼ 14.5 and 11.8 Hz, 1H), 6.70–6.80 (m, 3H), 7.09 (dd,
J ¼ 15.6 and 10.8 Hz, 1H), 7.22–7.28 (m, 1H), 7.34 (t, J ¼ 7.6 Hz,
2H), 7.50 (d, J ¼ 7.6 Hz, 2H), 9.95 (br s, 1H). MS (ESI), m/z: 242

DOI: 10.3109/14756366.2014.912214
5-Membered cyclic hydroxamic acids
5
3-[1-Hydroxy-2-oxo-pyrrolidin-(3E)-ylidenemethyl]-N-phenyl-
(dd, J ¼ 15.2 and 10.6 Hz, 1H), 6.62 (d, J ¼ 15.6 Hz, 1H), 7.20–
benzenesulfonamide (10i). This compound was obtained from 7.26 (m, 1H, overlapped with CHCl3), 7.27–7.34 (m, 2H), 7.36–
protected hydroxamic acid (9k) according to the General Method 7.41 (m, 2H). GC/MS: 202.1 (M+), calcd for C13 H14O2: 202.26.
E. Yield 67%; m.p. 192–195 ꢂC. 1H-NMR (DMSO-D6, 400 MHz,
d, ppm): 2.90 (dt, J ¼ 6.0 and 2.1 Hz, 2H), 3.57 (t, J ¼ 6.0 Hz, 2H),
[(1E,3E,5E)-6-Phenyl-hexa-1,3,5-trienyl]-1,3-dioxolane
7.00–7.05 (m, 1H), 6.51–6.62 (m, 1H), 7.06–7.12 (m, 3H), 7.19–
7.26 (m, 2H), 7.59 (t, J ¼ 7.8 Hz, 1H), 7.67–7.76 (m, 2H), 7.79 (s,
1H), 10.11 (br s, 1H), 10.32 (br s, 1H). MS (ESI), m/z: 345
(M + H)+, 386 (M + MeCN)+, calcd for C17H16N2O2S: 344.39.
Elemental analysis. Found, %: C 58.65, H 4.80, N 7.76. Calcd for
C17H16N2O2S: C 58.78, H 4.64, N 8.06.
(14b). This compound was obtained from aldehyde (8c) accord-
ing to the General Method B as a yellowish substance. Yield 29%.
1H-NMR (CDCl3, 400 MHz, d, ppm): 3.86–4.06 (m, 4H), 5.32
(d, J ¼ 6.1 Hz, 1H), 5.68 (dd, J ¼ 15.1 and 6.1 Hz, 1H), 6.34
(dd, J ¼ 14.5 and 10.3 Hz, 1H), 6.40–6.52 (m, 2H), 6.58
(d, J ¼ 15.8 Hz, 1H), 6.80 (dd, J ¼ 15.8 and 10.6 Hz, 1H), 7.21
(t, J ¼ 7.1 Hz, 1H), 7.27–7.33 (m, 2H), 7.35–7.41 (m, 2H),
GC/MS: 228 (M+), calcd For C15H16O: 228.3.
3-(2,3-Dihydro-1H-indol-3-ylmethyl)-1-hydroxy-pyrrolidin-2-one
(11a). To a solution of the protected hydroxamic acid (9a)
(130 mg, 0.31 mmol) in 50 mL of EtOH was added to 300 mg 10%
Pd/C and mixture stirred at room temperature with hydrogen
bubbling. Reaction mixture was analyzed in solvent system 10%
EtOAc in DCM. After the reaction was complete, the mixture was
filtered through celite and the filtrate was evaporated. Residual oil
was dissolved in dry DCM (5 mL) at 0 ꢂC and 2.5 mL of
trifluoroacetic acid added. Reaction was monitored by TLC,
solvent system 5% EtOH in DCM. After disappearance of the
starting substance spot, the mixture was evaporated to dryness and
the residual product purified by flash chromatography (eluent 5–
10% EtOH in DCM) to afford the title compound as the TFA salt.
Treating with anionite IRA-400 in Cl form the substance was
turned to hydrogen chloride. Yield of a hygroscopic foam was
3-[(E)-2-1,3-Dioxolan-2-yl-vinyl]-quinoline (14c). This com-
pound was obtained from 2-quinolinealdehyde according to the
1
General Method B as a white substance. Yield 29%. H-NMR
(CDCl3, 400 MHz, d, ppm): 3.94–4.04 (m, 2H), 4.04–4.11 (m,
2H), 5.49 (d, J ¼ 5.8 Hz, 1H), 6.39 (dd, J ¼ 16.2 and 5.8 Hz, 1H),
6.93 (d, J ¼ 16.2 Hz, 1H), 7.50–7.55 (m, 1H), 7.65–7.70 (m, 1H),
7.79 (dd, J ¼ 8.2 and 1.2 Hz, 1H), 8.06 (d, J ¼ 8.7 Hz, 1H), 8.08
(d, J ¼ 2.3 Hz, 1H), 9.00 (d, J ¼ 2.3 Hz, 1H). GC/MS: 227.1 (M+),
calcd for C14H13NO2: 227.3.
3-[(1E,3E)-4-1,3-Dioxolan-2-yl-buta-1,3-dienyl]-quinoline
(14d). This compound was obtained from aldehyde (8e) as a
yellowish substance according to the General Method B. Yield
1
1
33.7 mg (40%). H-NMR (DMSO-D6, 400 MHz, d, ppm): 1.43–
34%. H-NMR (CDCl3, 400 MHz, d, ppm): 3.91–4.00 (m, 2H),
1.87 (m, 3H), 2.09–2.24 (m, 1H), 2.30–2.46 (m, 1H), 3.15–3.24
(m, 1H), 3.30–3.48 (m, 3H), 3.61–3.70 (m, 1H), 6.78–6.87
(m, 2H), 7.04–7.11 (m, 1H), 7.15–7.21 (m, 1H), 9.56 (br s, 1H).
MS (ESI), m/z: 233.1 (M + H)+ calcd for C13H16N2O2: 232.3.
Elemental analysis. Found, %: C 56.90, H 6.55, N 9.36. Calcd for
C13H16N2O2.HCl: C 56.98, H 6.25, N 10.22.
4.00–4.10 (m, 2H), 5.39 (d, J ¼ 5.9 Hz, 1H), 5.88 (dd, J ¼ 15.3
and 5.9 Hz, 1H), 6.63 (dd, J ¼ 15.3 and 10.6 Hz, 1H), 7.51–7.56
(m, 1H), 7.65–7.70 (m, 1H), 7.79 (dd, J ¼ 8.2 and 1.1 Hz, 1H),
8.05–8.09 (m, 2H), 9.00 (d, J ¼ 2.1 Hz, 1H). GC/MS: 253.2(M+),
calcd for C16H15NO2: 253.3.
2-[(E)-2-1,3-Dioxolan-2-yl-vinyl]-9-methyl-9H-carbazole
(14e). This compound was obtained from 9-methyl-9H-carba-
zole-2-carbaldehyde (8h) according to the General Method B as
a white substance. Yield 39%. 1H-NMR (CDCl3, 400 MHz, d,
ppm): 3.85 (s, 3H), 3.97–4.05 (m, 2H), 4.05–4.13 (m, 2H), 5.50
(d, J ¼ 6.0 Hz, 1H), 6.29 (dd, J ¼ 16.1 and 6.3 Hz, 1H), 6.99 (d,
J ¼ 16.1 Hz, 1H), 7.20–7.25 (m, 1H), 7.33 (dd, J ¼ 8.1 and 1.5 Hz,
1H), 7.39 (d, J ¼ 8.1 Hz, 1H), 7.44 (s, 1H), 7.45–7.50 (m, 1H),
8.03 (d, J ¼ 8.1 Hz, 1H), 8.06 (dt, J ¼ 7.7 and 1.0 Hz, 1H). GC/
MS: 279.1(M+). MS (ESI), m/z: 280.2 (M + H)+, calcd for
C18H17NO2: 279.3.
1-Hydroxy-3-phenethyl-pyrrolidin-2-one (11b). To a solution of
protected hydroxamic acid (9c) (45.7 mg, 0.15 mmol) in 40 mL of
EtOH was added 200 mg 10% Pd/C and the mixture stirred with
hydrogen bubbling. Reaction mixture was analyzed in solvent
systems 10% EtOAc in DCM and 3% MeOH in DCM. After
disappearance of the starting spot the mixture was filtered through
celite, filtrate evaporated to dryness and the was residue dried
in vacuo. Yield 20.0 mg (60%) of an off-white powder; m.p. 113–
115 ꢂC. 1H-NMR (CDCl3, 400 MHz, d, ppm): 1.35–1.47 (m, 1H),
1.59–1.72 (m, 3H), 1.78–1.88 (m, 1H), 2.11–2.21 (m, 1H), 2.38–
2.48 (m, 1H), 2.55–2.69 (m, 2H), 3.51–3.60 (m, 2H), 7.13–7.21
(m, 3H), 7.24–7.30 (m, overlapped with CHCl3, 2H), 9.0–10.5 (br
s, 1H). MS (ESI), m/z: 220 (M + H)+, calcd for C13H17NO2:
219.3. Elemental analysis. Found, %: C 70.03, H 7.79, N 6.12.
Calcd for C13H17NO2: C 70.24, H 7.71, N 6.30.
HDAC activity evaluation
HDAC activity in HeLa nuclear extracts
5-Membered cyclic hydroxamic acids 10a–d and 11a were
assayed for the inhibitory activities against HDAC using
BioVision ‘‘HDAC inhibitor drug screening kit’’ according to
General Method B
[(1E,3E)-4-Phenyl-buta-1,3-dienyl]-1,3-dioxolane (14a). To a the manufacturer’s protocol. Briefly, chemical compounds were
mixture of cinnamaldehyde (1.03 g, 8.16 mmol) and (1,3-dioxo- dissolved into DMSO in concentration 10 mM and diluted to 2ꢃ
lan-yl)-tributyl-phosphonium bromide18,25 (13) in dry DMF the desired test concentration with double distilled water (ddH2O).
(20 mL) at 90 ꢂC under argon atmosphere 1M NaOEt solution 50 mL of diluted compounds as well as positive control without
in abs EtOH was added (12.24 mL, 12.24 mmol). Reaction inhibitors, were added to the wells of 96-well plate. For negative
mixture was heated for 16 h at the same temperature, then control, 48 mL of ddH2O and 2 mL of Trichostatin A (TSA) were
cooled and diluted with water (200 mL), and extracted with mixed in the well. For each well 50 mL of reaction mixture
EtOAc (3 ꢃ 50 mL). The organic layer was washed with brine, containing 10 mL 10X HDAC Assay Buffer was also added, 2 mL
dried over Na2SO4 and evaporated. Residual substance was HeLa nuclear extract, 5 mL HDAC fluorescent substrate Boc-
purified by flash chromatography on silica gel (eluent 5–10% Lys(Ac)-AMC and 33 mL ddH2O. Plate was incubated for 30 min
EtOAc in PE) to afford 14a as a yellow substance. Yield 555 mg at 37 ꢂC. The reaction was stopped by adding 10 mL of Lysine
1
(34%). H-NMR (CDCl3, 400 MHz, d, ppm): 3.87–4.07 (m, 4H), Developer and mixed well. Plate was incubated for 30 min at
5.34 (d, J ¼ 6.0 Hz, 1H), 5.75 (dd, J ¼ 15.2 and 6.0 Hz, 1H), 6.55 37 ꢂC. The relative fluorescent units (RFU) were measured in

6
I. Mutule et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
fluorimeter Tecan (Infinite M1000) with Ex./Em. ¼ 350 nm/ Side chain elongation was performed from appropriate aldehydes
460 nm. Set of the RFUs and positive control with 100% HDAC (12) and tributyl phosphonium bromide (13) prepared after
activity, was used to calculate the relative HDAC inhibitory literature precedent depicted in Scheme 218.
activity of chemical compounds as follow: activity remain
Acetals 14a–e prepared by this methodology were deprotected
with compounds ¼ (RFU of compound/RFU of Positive to give the target aldehydes 8c–g. Further reaction of them with
Control) ꢃ 100%.
earlier prepared phosphonium bromides 7a–b afforded protected
cyclic hydroxamic acids 9a–k. Deprotection of the O-THP
protecting group with AcOH in THF-H2O mixture yielded title
compounds 10a–i. O-Benzyl group in compounds 9 was cleaved
in Pd catalyzed reduction conditions and gave cyclic hydroxamic
acids 11a–b.
IC50 calculation
Detection of the IC50 (HDACi) values was revealed after
measurement of RFU for at least five concentrations of
compounds in a range from 1 to 40 mM. Calculation of IC50
was done with GraphPad Prism 5.03 software (GraphPad
Software, La Jolla, CA) on the basis of detected RFUs. All data
are the mean of three independent experiments.
O
Results and discussion
+
O
−
Br
Chemistry
O
10% HCl
13
THF, rt, 2h
R
Synthesis of compounds 10a–d and 11a is described in Scheme 1.
Starting materials - N-protected 3-bromopyrrolidin-2-ones24 were
prepared according to the literature procedure.
R
R
O
n
1
O
1
O
1
n
14a-e
12a-c
8c-g
2,4-Dibromobutyryl chloride (2) was prepared from butyr-
olactone (1) in a two-step process and coupled with O-protected
hydroxylamines 3a–b to give O-protected derivatives 4a–b. They
were cyclized to cyclic hydroxamic acid (5a–b) with NaOH in the
presence of strong anionite IRA-400 in OHꢁ form. Introduction of
the side chain was started in the two-step process from bromides
5a–b. Exchange of bromide functionality was performed with
triphenylphosphine (6) affording phosphonium bromides 7a–b.
8c R =Ph, n=2; 8d R =Ph, n=1; 8e R =3-Quinolinyl, n=1;
1
1
1
8f R =3-Quinolinyl, n=2; 8g R =9-Me-Carbazol-2-yl, n=1;
1
1
12a R =Ph; 12b R =3-Quinolinyl; 12c R =9-Me-Carbazol-2-yl;
1
1
1
14a R =Ph, n=2; 14b R =Ph, n=3; 14c R =3-Quinolinyl, n=1;
1
1
1
14d R =3-Quinolinyl, n=2; 14e R =9-Me-Carbazol-2-yl, n=1
1
1
Scheme 2. Unsaturated aldehyde 8c–g synthesis.
O
Br
Br
Scheme 1. Synthesis of 5-CHA 10 and 11.
1. Br2, PBr3
2. SOCl2
H2N
R
3a-b
H
N
O
R
Cl
Br
O
Br
O
1
2
4a-b
O
O
NaOH
IR-400
−
Br
O
O
N
O
+
R
Ph P
O
1
Br
2
R
n
PPh3
1
n
8a-i
6
N
OR
OR
N
OR
7a-b
9a-k
5a-b
A. (R=THP)
AcOH, THF, H2O
B. (R=Bn)
H2, Pd/C, EtOAc
O
O
R
R
1
1
n
n
N
OH
N
OH
10a-i
11a-b
4a R=Bn; 4b R=THP; 5a R=Bn; 5b R=THP; 7a R=Bn; 7b R=THP;
8a R =3-Indolyl, n=0; 8b R =Ph, n=1; 8c R =Ph, n=2; 8d R =Ph, n=1;
1
1
1
1
8e R =3-Quinolinyl, n=1; 8f R =3-Quinolinyl, n=2; 8g R =9-Me-Carbazol-2-yl, n=1;
1
1
1
8h R =9-Me-Carbazol-2-yl, n=0; 8i R =3-(Phenylaminosulphonyl)-Phenyl, n=0;
1
1
9a R=Bn, R =3-Indolyl, n=0; 9b R=THP, R =3-Indolyl, n=0; 9c R=Bn, R =Ph, n=1;
1
1
1
9d R=THP, R =Ph, n=1; 9e R=THP, R =Ph, n=2; 9f R=THP, R =Ph, n=3;
1
1
1
9g R=THP, R =3-Quinolinyl, n=1; 9h R=THP, R =3-Quinolinyl, n=2;
1
1
9i R=THP, R =9-Me-Carbazol-2-yl, n=1; 9j R=THP R =9-Me-Carbazol-2-yl, n=0;
1
1
9k R=THP, R =3-(Phenylaminosulphonyl)-Phenyl, n=0;
1
10a R =3-Indolyl, n=0; 10b R =Ph, n=1; 10c R =Ph, n=2; 10d R =Ph, n=3;
1
1
1
1
10e R =3-Quinolinyl, n=1; 10f R =3-Quinolinyl, n=2; 10g R =9-Me-Carbazol-2-yl, n=1;
1
1
1
10h R =9-Me-Carbazol-2-yl, n=0; 10i R =3-(Phenylaminosulphonyl)-Phenyl, n=0;
1
1
11a R =3-Indolyl, n=0; 11b R =Ph, n=0
1
1

DOI: 10.3109/14756366.2014.912214
5-Membered cyclic hydroxamic acids
7
Table 1. IC50 values (mM) for the HDAC inhibitory activity of 5-CHA.
hydroxamic acid function and aromatic phenyl ring is important
for the development of new zinc binding groups (ZBGs) for
histone deacetylases inhibition.
#
Structure
IC50 (mM)
O
10a
Nd*
Conclusions
OH
OH
N
N
H
In conclusion, the present work reports on the search of new ZBG
for the development of HDAC inhibitors. We have designed and
synthesized series of 5-CHA as HDAC inhibitors. The new
derivatives have been tested in nuclear extracts from HeLa cell
cultures using tert-butoxycarbonyl-lysine (acetylated)-4-amino-7-
methylcoumarin (Boc-Lys(Ac)-AMC) as the substrate. Some of
the reported derivatives showed low mM activity and they are
good examples for starting fragment-based structure design.
Thus, we have developed – cyclic hydroxamic acids (CHA) as
a new ZBG for HDAC inhibition. Although synthesized com-
pounds are not as effective as known hydroxamic acids, their
some structure-activity profile shows some kind of selectivity, and
make these derivatives interesting for more detailed SAR studies.
Further investigations on design and synthesis of other CHAs
and their development as metallo-enzyme inhibitors are under
the way.
O
10b
33.5
N
O
10c
10d
11a
7.4
40.0
OH
N
O
OH
N
O
Nd**
OH
N
N
H
*HDAC inhibitory activity was not detectable.
**HDAC inhibitory activity was very weak.
Declaration of interest
The authors declare that there are no conflicts of interest.
This study was supported by the Latvian National Research
Programme 2010–2013. ‘‘BIOMEDICINE’’ and COST Action TD0905
‘‘Epigenetics: from Bench to Bedside’’.
Biological activity
HDAC activity with cyclic hydroxamic acids
Cyclic hydroxamic acids 10a–d and 11a were tested on BioVision References
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