A Linker for the Solid-Phase Synthesis of Hydroxamic Acids and Identification of HDAC6 Inhibitors

Article abstract of DOI:10.1021/acscombsci.7b00068

We herein present broadly useful, readily available and nonintegral hydroxylamine linkers for the routine solid-phase synthesis of hydroxamic acids. The developed protocols enable the efficient synthesis and release of a wide range of hydroxamic acids from various resins, relying on high control and flexibility with respect to reagents and synthetic processes. A trityl-based hydroxylamine linker was used to synthesize a library of peptide hydroxamic acids. The inhibitory effects of the compounds were examined for seven HDAC enzyme subtypes using a chemiluminescence-based assay.

Full text of DOI:10.1021/acscombsci.7b00068

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Article
Solid-Phase Synthesis of Hydroxamic Acids and Identification
of HDAC Inhibitors Using a Chemi-luminescence-based Assay
Claus Bang, Jakob Jensen, Emil Cohrt, Lasse Olsen, Saba Siyum, Kim Mortensen, Tine S. Poulsen,
Jens Berthelsen, Liang Yang, Michael Givskov, Katrine Qvortrup, and Thomas Eiland Nielsen
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.7b00068 • Publication Date (Web): 26 Jul 2017
Downloaded from http://pubs.acs.org on August 2, 2017
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A Linker for the Solid-Phase Synthesis of
Hydroxamic Acids and Identification of HDAC6
Inhibitors
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Claus G. Bang,[a] Jakob F. Jensen,[a] Emil O´Hanlon Court,[a] Lasse B. Olsen,[a] Saba G.
Siyum,[a] Kim T. Mortensen,[a] Tine S. Poulsen,[b] Jens Berthelsen,[b] Liang Yang,[c] Michael
Givskov,[b], [c] Katrine Qvortrup*[a] and Thomas E. Nielsen*[ a], [b], [c]
[a]Department of Chemistry, Technical University of Denmark, DK-2800 Kgs. Lyngby,
Denmark, [b]Department of Immunology and Microbiology, University of Copenhagen, DK-2200
Copenhagen N, Denmark, [c]Singapore Centre for Environmental Life Sciences Engineering,
Nanyang Technological University, Singapore 637551, Singapore
KEYWORDS: Solid-phase synthesis, linker, hydroxamic acid, HDAC inhibitor.
ABSTRACT
We herein present broadly useful, readily available and non-integral hydroxylamine linkers for
the routine solid-phase synthesis of hydroxamic acids. The developed protocols enable the
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efficient synthesis and release of a wide range of hydroxamic acids from various resins, relying
on high control and flexibility with respect to reagents and synthetic processes. A trityl-based
hydroxylamine linker was used to synthesize a library of peptide hydroxamic acids. The
inhibitory effects of the compounds were examined for seven HDAC enzyme subtypes using a
chemiluminescence-based assay.
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Introduction
Hydroxamic acids belong to an important class of compounds with a plethora of biological
properties, including antibacterial, antifungal and anticancer activity (Fig. 1).1,2 The ability of
hydroxamates to chelate metal ions, such as Fe3+ and Zn2+, has been widely explored in biology
and medicine, The siderophore desferrioxamene B (Desferal, 1) is used medically to treat iron
overdose, which can arise either from multiple blood transfusions or an underlying genetic blood
disease.3 A large number of recent studies have dealt with hydroxamic acids as potent inhibitors
of Zn2+-containing enzymes, such as matrix metallo-proteinases (MMPs), and histone
deacetylases (HDACs).4 HDAC inhibitors represent a promising class of anticancer agents, with
two hydroxamic acid-based inhibitors now approved for cutaneous and peripheral T-cell
lymphoma, respectively. Recognizing the diverse anti-cancer activities of Vorinostat (SAHA,
3)5 and Belonistat (4),6 huge efforts have been made to explore hydroxamic acids for the
treatment of various cancers. In addition to promising anticancer effects, HDAC inhibitors have
also been suggested for the treatment of a range of other diseases, such as internal fibrosis,
autoimmune, inflammatory diseases, metabolic disorders and many more.7 However, disruption
of multiple pathways by these inhibitors, and the lack of specificity by these inhibitors to a target
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enzyme, may contribute to the cytotoxicity observed in many clinical trials. Therefore, there is
an increasing need to develop more potent and isoform-selective HDAC inhibitors. Following
the observation of clinical benefits of HDAC inhibitors in various diseases, there has been a
substantial refocus on the organic chemistry and biomedical properties of the hydroxamic acid
class of compounds.
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Figure 1. Clinically important hydroxamic acids.
Discussion
Although a high number of potent hydroxamic acids have been identified in drug and probe
discovery efforts,8 only few general methods for the parallel and combinatorial synthesis of
hydroxamic acids have been reported. There have been notable reports describing the solid-phase
synthesis of hydroxamic acids,9 e.g. hydroxylamine derivatives, being either N-tethered to
MBHA,10 and Tentagel resins,11 or O-tethered to Wang,12 Sasrin,13 and trityl resins,14 have served
as the starting point for the synthesis of a range of hydroxamic acids. Alternatively, a reported
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stepwise method relies on the hydrolysis of ester-bound compounds, followed by attachment of
the formed carboxylic acids to a hydroxylamine resin using a peptide coupling agent, before final
cleavage to generate hydroxamic acids.15 The solid-phase synthesis of hydroxamic acids via
direct hydroxyaminolysis of ester-linked substrates has also been reported.16
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The use of non-integral linkers is usually preferred because these provide control and flexibility
for the synthetic process.17 The exact degree of loading and functionalization are also easily
controlled and assessed. Along these lines, any functionalized solid support can be used with
such linkers. When said linker is attached through an amide bond, the quality of functionalized
starting resin is simply controlled by the purity of the linker. When the first building block is
attached to the linker through a more demanding bond, this may advantageously be formed in
solution before attaching the linker construct to the solid support.18 The majority of linkers used
in solid-phase synthesis are consequently of the non-integral type.
Results
As part of ongoing efforts to synthesize hydroxamic acids, we set out to develop a non-integral
hydroxylamine linker for the solid-phase synthesis and release of hydroxamic acids. Important
criteria comprise the applicability to protocols that effectively may lead to a wide variety of
structurally diverse hydroxamic acids, notably with high control and flexibility for the synthetic
process, on any kind of solid support. Initially, the p-methoxybenhydryl-linker 10 was
investigated for the solid-phase synthesis of hydroxamic acids. The p-methoxybenhydryl-linker
system was first reported by Walter19 and Brown,20 for the preparation of primary and secondary
amides, respectively.
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To construct linker 10, phenylmagnesium bromide was added to vanillin, followed by
alkylation with 4-bromobutanoate. Subsequent treatment with AcBr gave the bromide 6 in high
yield. However, much to our surprise, reaction with FmocNHOH in the presence of DIPEA gave
the N-alkylated derivative 7 as the sole product. Chan et al.21 had previously reported a related
synthetic sequence using Fmoc-NR-OH (R = alkyl chain), where O-alkylation was forced. To
synthesize the desired O-alkylated hydroxylamine-linker 10, bromide 6 was treated with N-
hydroxyphthalimide, followed by removal of the phthalimide with hydrazine. Fmoc-protection
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Scheme 1. Synthesis of p-methoxybenhydryl-based linker 10 for the solid-phase synthesis and
release of hydroxamic acids
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followed by treatment with Novozyme-435 led to the desired linker 10. Using standard reagents
for solid-phase peptide synthesis, the linker was immobilized and synthetically elaborated on an
amino-functionalized PEGA resin. Hydroxamic acids were released when the resin was treated
with CH2Cl2/TFA (1:1) for 24 hours. The linker resists 20% TFA (aq) for 1 hour, and is thereby
compatible with removal of highly acid-labile protecting groups. However, fluctuating yields
and purities were generally observed with this linker system, often as a result of hydroxamate
hydrolysis during periods of prolonged acidic treatment.
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In search of a more acid-labile construct, we became interested in trityl-based linker systems
(linker 14). The trityl-group was pioneered in linkers for solid-phase synthesis by Leznoff22 and
Fréchet.23 Cleavage hereby becomes more straightforward, relying on either dilute TFA or
HBr.23 More detailed studies indicated that anhydrous 0.3 M HCl (dioxane) was sufficient for
cleavage.22 Since then, trityl linker constructs have been designed to release a variety of
functionalized molecules from solid support, particularly in integral linker constructs, e.g.
peptides,24 hydroxamic acids,21 amines, acids, thiols,25 but also as non-integral linkers through 4-
carboxy derivatives.26
Starting with 4-hydroxybenzophenone 11, addition of phenylmagnesium bromide27 gave the
trityl alcohol, which was alkylated with ethyl 4-bromobutanoate at the phenolic position to
provide intermediate trityl ester 12 in high yield. Bromination with acetyl bromide in toluene
afforded the trityl bromide, followed by treatment with Fmoc-NHOH in CH2Cl2 in the presence
of DIPEA to generate the Fmoc-protected hydroxylamine ester 13. In this case, only O-
substitution was observed with a sterically demanding alkylating reagent. Exposing the ester to
hydrolysis with Novozyme-435 completed the synthesis of the trityl linker 14.
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Scheme 2. Synthesis of trityl-based linker 14 for the solid-phase synthesis and release of
hydroxamic acids
Using standard reagents for solid-phase peptide synthesis, the linker was easily immobilized and
synthetically elaborated on an amino-functionalized PEGA resin (Table 1). Rewardingly,
hydroxamic acids were cleanly released when cleavage was performed with TFA/CH2Cl2 (1:10)
in the presence of 2% (v/v) TIPS (triisopropylsilane) for 5 min. In comparison, the commercially
available amino-functionalised polystyrene resin functionalized with the hydroxylamine linker
14 was tested under the same conditions. Here, hydroxamic acids were cleanly released after 5
min. of treatment with 1% TFA (CH2Cl2). The reduced reactivity of linker 14 at the PEGA resin
is explained by a reduced proton activity in the polyethylene glycol polyacrylamide-
functionalised resin (PEGA). The PEG based resins serve as proton sponges.
For preliminary biological screening of small-molecule and peptide hydroxamic acids, we
generated a library of potential HDAC inhibitors (21 A–BV; Table 1). In previous studies, we
have identified the thiophene-group as a promising sub-structure in HDAC-inhibitors, generally
leading to inhibitors of good activity and selectivity.28 Based on these results, further
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investigation into thiophene-hydroxamic acids was chosen as the focus of this study. The library
was synthesized using 4 different thiophene spacer-groups 17,29 18, 19, 20 which was used in
combination with various terminating moieties (Scheme 3).
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Scheme 3. Application of a trityl-based linker for the solid-phase synthesis and release of
thiophene hydroxamic acids 21A-BV.
Much to our delight the synthesized hydroxamic acids 21A–BV showed activity against
HDAC subtype enzymes as well as a marked isoform-selectivity, thus indicating the potential of
the compound collection to identify important binding interactions through additional structure–
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activity studies. Specifically, 11 compounds (21N, 21R, 21S, 21AB, 21AD, 21AF, 21AJ, 21AN,
21AX, 21BK, 21Bl) were found to selectively inhibit HDAC6. This enzyme is believed to play
an important role in degradation and clearance of misfolded proteins, as well as cell protection in
response to environmental stress by interaction with stress granules.30 HDAC6 is implicated in
many cellular pathways relevant to tumor cell progression and malignant behavior and has been
proposed as an attractive target for cancer treatment. In this context, the reported HDAC6
isoform-selective inhibitors may be relevant to test for their capability to stimulate immune
recognition of cancer cells.
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Figure 2. Thiophene hydroxamic acids identified as selective HDAC6 inhibitors
Conclusion
In summary, we have developed two broadly useful non-integral hydroxylamine linkers, based
on a trityl and a biphenyl group, respectively. The Fmoc-protected linkers are readily made on
multi-gram scale from simple starting materials and conveniently attached to solid support
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through standard amide bond forming reactions. Optimized protocols enable efficient multi-step
synthesis and formation of a variety of hydroxamic acids from common solid supports. The
linkers show broad applicability to a range of reactions commonly employed in the solid-phase
organic synthesis of molecular libraries, such as amination, acylation, sulfonylation, Cu-
catalyzed click reactions, and Wittig reactions. The flexibility for the synthetic process was
utilized to generate a proof-of-concept collection of 72 thiophene-based hydroxamic acids. The
inhibitory effects of the compounds were examined against 7 HDAC enzyme subtypes using a
Chemiluminescence-based assay, revealing several highly potent, and even subtype-selective,
HDAC inhibitors, including single-digit nM inhibitors of HDAC6.
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Table 1. Chemical and biological data for hydroxamic acids 21A-BV.
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Table 1 continued
EXPERIMENTAL SECTION
General Methods
All reagents and materials used were purchased from ordinary chemical suppliers and were used
without purification. The solvents used were of standard HPLC grade. Solid-phase synthesis
was carried out using plastic-syringe techniques. Flat-bottomed PE-syringes were fitted with PP-
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filters and situated in Teflon valves equipped with Teflon tubing allowing for a moderate
vacuum to be applied to the syringes.
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Yields of solid-phase synthesis protocols are corrected for salt contents and given as percentage
of product mass recovery to the theoretical product loading mass, calculated from the resin
loading (4 mmol/g) as specified by the supplier.
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Thin layer chromatography was performed on aluminum plates, precoated with silica gel (Merck
25, 20 x 20 cm, 60 F254) using the indicated eluent systems. Visualization was carried out by
illumination with a UV-lamp (254 nm) or by staining with a solution of phosphomolybdic acid
(10 g) in absolute ethanol (100 mL).
Flash column chromatography was performed on silica gel (FLUKA 60758 silica gel 60, particle
size 0.035-0.070 mm, 220-440 mesh ASTM) in varying sizes of glass columns, equipped with a
sintered glass filter, a joint pressure inlet and a stop cock, using the indicated eluent systems and
collecting fractions of the indicated volumes.
Products were analyzed on a Waters Alliance reverse-phase HPLC system consisting of a Waters
2695 Separations Module equipped with a Symmetry C18 column (3.5 µm, 4.6 x 75 mm, column
temp 25 °C, flow rate 1 mL/min) and a Waters Photodiode Array Detector (detecting at 215 nm).
Elution was carried out in a linear reversed phase gradient fashion (gradient A: 0% organic for
0.2 min, 0% organic to 100% organic in 10 min, hold for 1 min, 100% organic to 0% organic in
0.3 min, hold for 1.5 min, gradient B: 0% organic for 0.2 min, 0% organic to 40% organic in 10
min, 40% organic to 100% organic in 0.8 min, hold for 1 min, 100% organic to 0% organic in
0.3 min, hold for 1.5 min) combining water and acetonitrile (buffered with 0.1% (v/v) TFA).
Preparative RP-HPLC was carried out on a Waters Alliance reverse-phase HPLC system
consisting of a Waters 2545 Binary Gradient Module equipped with an xBridge TM
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Prep BEH130 C18 column OBDTM (5 µm, 19 x 100 mm, column temp 25 °C, flow rate 20
mL/min), a Waters Photodiode Array Detector (detecting at 210-600 nm), a Waters UV Fraction
Manager and a Waters 2767 Sample Manager. Elution was carried out in a linear reversed phase
gradient fashion combining water and acetonitrile (buffered with 0.2% (v/v) TFA).
1D and 2D NMR spectra were recorded using a Varian Unity Inova-500 MHz, a Varian
Mercury-300 MHz instrument, a Bruker Ascend-400 MHz instrument equipped with a 5 mm
Prodigy cryoprobe or a Bruker Avance-800 MHz instrument, equipped with a 5 mm cryoprobe
TCI, in DMSO-d6 or CDCl3 using the residual DMSO or CHCl3 solvent peaks, respectively, as
the internal standard. All 13C NMR spectra were proton decoupled. DQF-COSY, HSQC, HMBC
and 2D NOESY spectra were acquired using standard pulse sequences.
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LC-DAD-HRMS was performed on an Agilent 1100 LC system equipped with a Agilent
Technologies Diode Array Detector and a Luna C18 column (3µm, 50 mm × 2 mm, column
temp 40°C, flow rate 400 µL/min). Separation was achieved using a linear reversed phase
gradient (20% to 100% organic in 8 min, hold for 2 min, 100% to 20% organic in 1 min, hold for
4 min) again combining water and acetonitrile (buffered with 20 mM HCO2H). The LC was
coupled to a Micromass LCT orthogonal time-of-flight mass spectrometer, equipped with Lock
Mass probe and operated in positive electrospray mode.
Linker synthesis
4-[hydroxy(diphenyl)methyl]phenol (11B)
Magnesium turnings (18.8 g, 0.774 mol, 3.1 equiv.) and a few iodine crystals were added to dry
THF (1 L) in a flame-dried flask under N2 atmosphere. Stirring was applied and the reaction
mixture turned yellow. Bromobenzene (79 mL, 0.755 mol, 3 equiv.) was then added carefully by
syringe, and the reaction was heated to reflux. As the Grignard reaction began a larger amount of
the remaining bromobenzene could be poured into the reaction mixture. The reaction was
refluxed for 6 h and excess Mg was filtered out. Upon cooling, the brownish Grignard product
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was slowly added to an ice-cooled (0 °C) solution of 4-hydroxybenzophenone (50 g, 0.252 mol,
1 equiv.) in THF (1.5 L). Mechanical stirring was applied, and the reaction was heated to reflux
overnight. TLC analysis indicated incomplete conversion of 4-hydroxybenzophenone, and
additional Grignard reagent was therefore added to the reaction following reflux for further 24 h.
The yellow reaction mixture was quenched with saturated NH4Cl (aq) to pH 7 and extracted with
EtOAc (3 x 500 mL). The combined organic phases were washed with H2O (2 x 200 mL) and
brine, and subsequently dried over anhydrous Na2SO4, filtered and concentrated in vacuo to
leave behind a yellow oil. The resulting oil was co-evaporated from toluene (300 mL) to give a
yellow solid which was re-crystallized from EtOAc, toluene and heptane. The title product was
obtained as a yellow solid (43.7 g, 63%). RP-HPLC purity > 92%, Rt = 7.65 min at 254 nm. 1H-
NMR (300 MHz, DMSO-d6) δ 9.33 (s, 1H), 7.15-7.35 (m, 10H), 6.95 (m,2H), 6.65 (m, 2H), 6.24
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(s,1H); C NMR (75 MHz, DMSO-d6) δ 156.0, 148.3, 138.3, 129.0, 127.7, 127.4, 126.4, 114.2,
80.3, IR(neat) ν (cm-1): 3476, 3300, 1612, 1512, 1444, 1173, 1151. UPLC-MS (ESI+): m/z =
259.1 [M+H-H2O]+, found: 259.3, mp. 155-157 °C.
Ethyl 4-(4-hydroxydiphenylmethyl)phenoxy)butanoate (12)
4-(Hydroxydiphenylmethyl)phenol 11B (15.8 g, 57.0 mmol, 1 equiv.) was dissolved in DMF
(250 mL). K2CO3 (11.8, 85.5 mmol, 1.5 equiv.) was added along with ethyl 4-bromobutyrate
(14.7 mL, 102.6 mmol, 1.8 equiv.), and the reaction mixture was stirred for 4 days at room
temperature. TLC analysis showed that the reaction had not reached completion, and further
K2CO3 (3.94 g, 28.5 mmol, 0.5 equiv.) and ethyl 4-bromobutyrate (11.1 mL, 77.7 mmol, 1.4
equiv.) were added. After 72 h, a second batch of K2CO3 (3.94 g, 28.5 mmol, 0.5 equiv.) and
ethyl 4-bromobutyrate (8.2 mL, 57.0 mmol, 1 equiv.) proved necessary in order for the reaction
to run to completion. The reaction mixture was diluted with an H2O-EtOAc mixture (1:1, 1 L).
The two-phase system was separated, and the aqueous phase was extracted with EtOAc (4 x 500
mL). The combined organic phases were dried over Na2SO4, filtered and concentrated in vacuo
followed by co-evaporation from toluene (150 mL). The title product precipitated as a brown
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solid (16.9 g, 76%) upon the sequential addition of toluene (30 mL) and heptane (200 mL) under
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vigorous stirring. RP-HPLC purity > 88%, Rt = 9.54 min at 254 nm. H-NMR (300 MHz,
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DMSO-d6): δ = 7.16-7.33 (m, 10H), 7.07 (m, 2H), 6.83 (m, 2H), 6.34 (bs, 1H), 4.06 (q, J=7,1
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Hz, 2H), 3.95 (t, J=6.7 Hz, 2H), 2.44 (t, J=6.7 Hz, 2H), 1.95 (p, J=6.7 Hz, 2H), 1.17 (t, J=7.1 Hz,
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3H); C NMR (75 MHz, DMSO-d6): δ = 172.6, 157.1, 148.1, 140.0, 129.0, 127.7, 127.5, 126.5,
113.3, 80.2, 66.4, 59.9, 30.2, 24.3, 14.1. IR(neat) ν (cm-1): 3524, 2989, 2936, 1715, 1231. UPLC-
MS (ESI+): m/z = 413.2 [M+Na]+, found: 413.4, mp. 101-103 °C,
Ethyl 4-(4-(((N-Fmoc-amino)oxy)diphenylmethyl)phenoxy)butanoate (13)
The trityl ester 12 (16 g, 41.0 mmol, 1 equiv.) was dissolved in dry dichloromethane (150 mL),
and was hereafter treated with acetylbromide in small portions at room temperature under N2
atmosphere. The dark red reaction mixture was concentrated in vacuo after 1 h of stirring, and
the resulting crude dark red oil was then co-evaporated from dichloromethane twice (2 x 350
mL). The residue oil was re-dispersed in dichloromethane (200 mL) and mixed with N-Fmoc
hydroxylamine (10.5 g, 41.0 mmol, 1 equiv.) along with DIPEA (9.2 mL, 53.2 mmol, 1.3 equiv.)
under the formation of white smoke. The solution was left to react for 72 hours (less would
probably be sufficient) at rt, after which the reaction was at an end. The product was
concentrated in vacuo and then dispersed in EtOAc, upon which DIPEA•HBr precipitated as a
white solid. The precipitate was filtered out and solvent was removed by vacuum to yield a
brown syrup, which was purified by flash chromatography (EtOAc/heptane 3:7). The isolated oil
was dissolved in a small amount of dichloromethane and left to crystallize on an oil pump
overnight to afford the title compound as a yellow solid (19.0 g, 75%). Rf = 0.54
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(EtOAc:Heptane 3:1), RP-HPLC purity > 60%, Rt = 9.31 min at 254 nm. H-NMR (300 MHz,
DMSO-d6) δ 9.98 (bs, 1H), 7.87 (d, J = 7.4 Hz, 2H), 7.56 (d, J = 7.4 Hz, 2H), 7.40 (t, J = 7.4 Hz,
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2H), 7.35 – 7.25 (m, 12H), 7.13 (d, J = 8.8 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 4.10 – 3.88 (m,
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7H), 2.40 (t, J = 7.1 Hz, 2H), 1.92 (p, J = 7.1 Hz, 2H), 1.16 (t, J = 7.1 Hz, 3H).; C NMR (75
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MHz, DMSO-d6): δ = 172.5, 157.7, 157.2, 143.5, 143.0, 140.7, 133.9, 130.9, 128.7, 127.7,
127.6, 127.3, 127.1, 125.2, 120.1, 113.2, 91.6, 66.4, 66.0, 59.9, 46.3, 30.1, 24.2, 14.1. IR(neat) ν
(cm-1): 3269, 3060, 2978, 2945, 1723, 1508, 1447, 1177. HRMS (ESI+): m/z = 650.2519
[M+Na]+, found: 650.2540, mp. 57-58 °C.
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4-(4-(((N-Fmoc-amino)oxy)diphenylmethyl)phenoxy)butanoic acid (14)
N-Fmoc hydroxylamine trityl ester 13 (11.8 g, 18.8 mmol, 1 equiv.) was dissolved in acetone
(125 mL). A Phosphate buffer consisting of 0.1 M KH2PO4 plus 0.10 M NaOH was then slowly
added to the solution, and at the point of incipient precipitation, the addition of Phosphate buffer
was stopped, and a small amount of acetone was used to dissolve the precipitate. Novozym 435
(24 g) was applied to the solution and the reaction was shaken gently at room temperature. The
reaction had run to completion after 7 days, and the suspension was then filtered through celite,
wherein the enzymes were washed with acetone and water. Solvent from the filtrate was
evaporated in vacuo and the product appeared as a yellow solid, which was dissolved in EtOAc
(150 mL) and H2O (150 mL). The phases were separated and the aqueous phase was extracted
with EtOAc (3 x 200 mL). The combined organic phases were washed with brine (100 mL) and
H2O (100 mL), then dried over Na2SO4, filtered and finally concentrated in vacuo to give a
yellow oil. Remaining solvent was removed on an oil pump and the product was obtained as a
yellow crystalline solid (9.0 g, 80%). The linker was used directly in the succeeding solid phase
reactions. RP-HPLC purity > 76%, Rt = 9.91 min at 254 nm. 1H-NMR (300 MHz, DMSO-d6): δ
= 12.18 (bs, 1H), 9.98 (bs, 1H), 7.87 (d, J = 7.4 Hz, 2H), 7.57 (d, J = 7.4 Hz, 2H), 7.40 (t, J = 7.4
Hz, 2H), 7.35 – 7.25 (m, 12H), 7.14 (d, J = 8.8 Hz, 2H), 6.84 (d, J = 8.9 Hz, 2H), 4.07 – 3.87 (m,
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5H), 2.35 (t, J = 7.1 Hz, 2H), 1.91 (t, J = 13.1, 7.1 Hz, 2H); 13C NMR (75 MHz, DMSO-d6): δ =
174.1, 157.8, 157.3, 143.5, 143.0, 140.7, 133.9, 131.0, 128.8, 127.7, 127.6, 127.3, 127.1, 125.2,
120.2, 113.2, 91.6, 66.5, 66.0, 49.3, 30.1, 24.3. IR(neat) ν (cm-1): 3260, 3060, 2948, 1704, 1229,
1178. UPLC-MS (ESI+): m/z = 600.2 [M+H]+, found: 600.6. HRMS (ESI+): m/z = 622.2206
[M+Na]+, found: 622.2181, mp. 67 °C.
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General Solid-Phase Procedures
Prior to usage, the amino functionalized poly(ethylene glycol) dimethyl acrylamide resin (PEGA
800, 0.4 mmol/g) was washed with H2O (x4), EtOH (x4), DMF (x4), MeOH (x4) and CH2Cl2
(x4) and then lyophilized.
Coupling of the first amino acid building block to the resin was carried out as follows. Dry resin
was weighed in a syringe, equipped with a PP-filter. The amino acid (3 equiv) was weighed,
dissolved in DMF (0.02 mL/mg resin) and N-ethylmorpholine (4 equiv) was added using a
microliter
pipette.
N-[(1H-Benzotriazol-1-yl)(dimethylamino)methylene]-N-
methylmethanaminium tetrafluoroborate N-oxide (TBTU, 2.9 equiv) was weighed and likewise
added. The solution was transferred to the resin, the swelled resin stirred gently with a spatula
and allowed to react for 2 h. The resin was filtered, washed with DMF (x6) and CH2Cl2 (x6) and
lyophilized. The Fmoc-group was removed by swelling the resin in a solution of piperidine
(20% v/v) in DMF for 2 min, filtering and then swelling the resin again in a fresh solution of
piperidine (20% v/v) in DMF for 18 min. The resin was washed with DMF (x6) and CH2Cl2 (x6)
and lyophilized.
Specific
Solid-Phase
Procedures
N-hydroxy-5-((2-propylpentanamido)methyl)thiophene-2-carboxamide (21.A)
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1H NMR (300 MHz, DMSO-d6): δ = 11.1 (s, 1H), 9.05 (s, 1H), 8.49 (t, J = 5.7 Hz, 1H), 7.40 (s,
1H), 6.90 (d, J = 3.6 Hz, 1H), 4.38 (d, J = 5.7 Hz, 2H), 2.1 (m, 1H), 0.81 (t, J = 6.8 Hz, 6H),
1.28-1.12 (m, 8H). UPLC-MS (ESI+): m/z = 299.1 [M+H]+, found: 299.4. Purity = 95%, Rt =
5.88 min. Yield = 78%.
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1-benzyl-N-((5-(hydroxycarbamoyl)thiophen-2-yl)methyl)-1H-indole-3-carboxamide (21.B)
1H NMR (300 MHz, DMSO-d6): δ = 11.10 (s, 1H), 8.63 (t, J = 5.9 Hz, 1H), 8.13 (m, 2H), 7.50
(d, J =7.8 Hz, 1H), 7.35-7.07 (m, 8H), 6.98 (d, J = 3.7 Hz, 1H), 5.45 (s, 2H), 4.58 (d, J = 5.8 Hz,
2H). UPLC-MS (ESI+): m/z = 406.1 [M+H]+, found: 406.4. Purity = 84%, Rt = 6.65 min. Yield
= 81%.
5-((3-(1H-benzo[d]imidazol-1-yl)propanamido)methyl)-N-hydroxythiophene-2-
carboxamide (21.C)
1H NMR (300 MHz, DMSO-d6): δ = 11.14 (s, 1H), 9.14 (s, 1H), 8.70 (t, J = 5.8 Hz, 1H), 7.89 (d,
J = 7.8 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.72-7.60 (m, 1H), 7.49-7.45 (’t’, J = 4.4 Hz, 2H), 7.37
(s, 1H), 6.78 (d, J = 3.8 Hz, 1H), 4.64 (t, J = 6.6 Hz, 2H), 4.33 (d, J = 5.8 Hz, 2H), 2.80 (t, J =
6.6 Hz, 2H). UPLC-MS (ESI+): m/z = 345.1 [M+H]+, found: 345.4. Purity = 87%, Rt = 3.48 min.
Yield = 72%.
5-((1H-imidazol-1-yl)methyl)-N-((5-(hydroxycarbamoyl)thiophen-2-yl)methyl)furan-2-
carboxamide (21.D)
1H NMR (300 MHz, DMSO-d6): δ = 11.14 (s, 1H), 9.11 (t, J = 6.0 Hz, 1H), 8.78 (s, 1H), 7.74-
7.59 (m, 2H), 7.46 (s, 1H), 7.41 (s, 1H), 7.13 (d, J = 3.4 Hz, 1H), 6.96 (d, J = 3.6 Hz, 1H), 6.66
(d, J = 3.4 Hz, 1H), 5.47 (s, 2H), 4.53 (d, J = 6.0 Hz, 2H). UPLC-MS (ESI+): m/z = 347.1
[M+H]+, found: 347.3. Purity = 68%, Rt = 3.35 min. Yield = 61%.
N-hydroxy-5-((2-((2-(pyrrolidin-1-yl)ethyl)thio)acetamido)methyl)thiophene-2-
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carboxamide (21.E)
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1H NMR (300 MHz, DMSO-d6): δ = 11.16 (s, 1H), 9.07 (s, 1H), 8.83 (t, J = 5.9 Hz, 1H), 7.42 (s,
1H), 6.95 (d, J = 3.8 Hz, 1H), 4.41 (d, J = 5.9 Hz, 2H), 3.24 (s, 4H), 2.93-2.85 (m, 4H), 1.89 (s,
6H). UPLC-MS (ESI+): m/z = 344.1 [M+H]+, found: 344.3. Purity > 99%, Rt = 3.24 min. Yield
= 84%.
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5-((4-(1H-1,2,4-triazol-1-yl)butanamido)methyl)-N-hydroxythiophene-2-carboxamide
(21.F)
1H NMR (300 MHz, DMSO-d6): δ = 11.12 (s, 1H), 9.06 (s, 1H), 8.53 (t, J = 5.8 Hz, 1H), 8.46 (s,
1H), 7.93 (s, 1H), 7.40 (s, 1H), 6.91 (d, J = 3.7 Hz, 1H), 4.37 (d, J = 5.8 Hz, 2H), 4.16 (t, J = 6.7
Hz, 2H), 2.07 (d, J = 6.8 Hz, 2H) 2.05-1.93 (p, J = 6.9 Hz, 2H). UPLC-MS (ESI+): m/z = 310.1
[M+H]+, found: 310.4. Purity = 87%, Rt = 3.17 min. Yield = 91%.
N-hydroxy-5-((3'-methyl-[1,1'-biphenyl]-3-carboxamido)methyl)thiophene-2-carboxamide
(21.G)
1H NMR (300 MHz, DMSO-d6): δ = 11.13 (s, 1H), 9.32 (t, J = 5.8 Hz, 1H), 9.06 (s, 1H), 8.14 (s,
1H), 7.86 (d, J = 7.9 Hz, 1H), 7.82 (d, J = 7.9 Hz, 1H), 7.59-7.53 (m, 2H), 7.51 (d, J = 7.8 Hz,
1H), 7.45 (s, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.22 (d, J = 7.4 Hz, 1H), 7.03 (d, J = 3.6 Hz, 1H),
4.65 (d, J = 5.8 Hz, 2H), 2.39 (s, 3H). UPLC-MS (ESI+): m/z = 367.1 [M+H]+, found: 367.3.
Purity > 99%, Rt = 6.92 min. Yield = 48%.
N-hydroxy-5-((4'-methyl-[1,1'-biphenyl]-3-carboxamido)methyl)thiophene-2-carboxamide
(21.H)
1H NMR (300 MHz, DMSO-d6): δ = 11.13 (s, 1H), 9.32 (t, J = 5.9 Hz, 1H), 9.06 (s, 1H), 8.12 (s,
1H), 7.82 (t, J = 7.3 Hz, 2H), 7.62 (d, J = 8.0 Hz, 2H), 7.54 (t, J = 7.7 Hz, 1H), 7.44 (s, 1H), 7.29
(d, J = 8.0 Hz, 2H), 7.02 (d, J = 3.7 Hz, 1H), 4.63 (d, J = 5.7 Hz, 2H), 2.34 (s, 3H). UPLC-MS
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(ESI+): m/z = 367.1 [M+H]+, found: 367.3. Purity > 99%, Rt = 6.94 min. Yield = 90%.
5-((3',5'-dimethoxy-[1,1'-biphenyl]-4-carboxamido)methyl)-N-hydroxythiophene-2-
carboxamide (21.I)
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1H NMR (300 MHz, DMSO-d6): δ = 11.12 (s, 1H), 9.26 (t, J = 5.7 Hz, 1H), 9.06 (s, 1H), 7.94 (d,
J = 8.4 Hz, 2H), 7.77 (d, J = 8.4 Hz, 2H), 7.44 (s, 1H), 7.00 (d, J = 3.7 Hz, 1H), 6.83 (d, J = 2.2
Hz, 2H), 6.52 (t, J = 2.2 Hz, 1H), 4.61 (d, J = 5.7 Hz, 2H), 3.79 (s, 6H). UPLC-MS (ESI+): m/z =
413.1 [M+H]+, found: 413.4. Purity = 95%, Rt = 6.53 min. Yield = 45%.
N-hydroxy-5-((4-(pyren-1-yl)benzamido)methyl)thiophene-2-carboxamide (21.J)
1H NMR (300 MHz, DMSO-d6): δ = 11.15 (s, 1H), 9.37 (t, J = 5.5 Hz, 1H), 9.07 (s, 1H), 8.42-
8.00 (m, 9H), 8.11-8.09 (d, J = 7.9 Hz, 2H), 7.74 (d, J = 8.0 Hz, 2H), 7.47 (s, 1H), 7.04 (d, J =3.6
Hz, 1H), 4.68 (d, J = 5.5 Hz, 2H). UPLC-MS (ESI+): m/z = 477.1 [M+H]+, found: 477.3; Purity
= 69%, Rt = 8.56 min.
N-((5-(hydroxycarbamoyl)thiophen-2-yl)methyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-
carboxamide (21.K)
1H NMR (300 MHz, DMSO-d6): δ = 11.11 (s, 1H), 9.02 (t, J = 5.9 Hz, 1H), 7.45-7.37 (s, 2H),
7.36 (d, J = 2.2 Hz, 1H), 6.95 (d, J = 3.7 Hz, 1H), 6.93-6.88 (dd, J = 1.1 Hz, 7.6 Hz, 1H), 4.55
(d, J = 5.8 Hz, 2H), 4.25 (m, 4H). UPLC-MS (ESI+) m/z: 335.1 [M+H]+, found: 335.3. Purity =
90%, Rt = 4.81 min. Yield = 87%.
N-hydroxy-5-((6-methoxy-2-naphthamido)methyl)thiophene-2-carboxamide (21.L)
1H NMR (300 MHz, DMSO-d6): δ = 11.12 (s, 1H), 9.26 (t, J = 5.9 Hz, 1H), 8.39 (s, 1H), 7.94-
7.83 (m, 3H), 7.44 (s, 1H), 7.37 (d, J = 2.4 Hz, 1H), 7.21 (dd, J = 2.5 Hz, 9.0 Hz, 1H), 7.02 (d, J
= 3.7 Hz, 1H), 4.64 (d, J = 5.9 Hz, 2H), 3.88 (s, 3H). UPLC-MS (ESI+): m/z = 357.1 [M+H]+,
found: 357.4. Purity = 95%, Rt = 5.94 min. Yield = 74%.
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6
carboxamide (21.M)
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UPLC-MS (ESI+): m/z = 381.1 [M+H]+, found: 381. 3. Purity = 67%, Rt = 7.18 min.
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N-hydroxy-5-((4-((p-tolyloxy)methyl)benzamido)methyl)thiophene-2-carboxamide (21.N)
1H NMR (300 MHz, DMSO-d6): δ = 11.11 (s, 1H), 9.19 (t, J = 5.8 Hz, 1H), 9.05 (s, 1H), 7.85 (d,
J = 8.2 Hz, 2H), 7.49 (d, J = 8.2 Hz, 2H), 7.42 (s, 1H), 7.05 (d, J =8.6 Hz, 2H), 6.98 (d, J = 3.7
Hz, 1H), 6.86 (d, J = 8.6 Hz, 2H), 5.11 (s, 2H), 4.59 (d, J = 5.8 Hz, 2H), 2.19 (s, 3H). UPLC-MS
(ESI+): m/z = 397.1 [M+H]+, found: 397.4. Purity = 92%, Rt = 7.06 min. Yield = 61%.
5-((9,10-dioxo-9,10-dihydroanthracene-2-carboxamido)methyl)-N-hydroxythiophene-2-
carboxamide (21.O)
1H NMR (300 MHz, DMSO-d6): δ = 11.14 (s, 1H), 9.69 (t, J = 5.9 Hz, 1H), 9.07 (s, 1H), 8.66 (d,
J = 1.7 Hz, 1H), 8.37-8.32 (dd, J = 1.7 Hz, 8.1 Hz, 1H), 8.30 (s, 1H), 8.27-8.18 (m, 2H), 7.98-
7.90 (m, 2H), 7.45 (s, 1H), 7.04 (d, J = 3.8 Hz, 1H), 4.66 (d, J = 5.8 Hz, 2H). UPLC-MS (ESI+):
m/z = 407.1 [M+H]+, found: 407.4. Purity = 86%, Rt = 6.17 min. Yield = 68%.
N-hydroxy-5-((3-(3-(pyridin-4-yl)-1,2,4-oxadiazol-5-yl)propanamido)methyl)thiophene-2-
carboxamide (21.P)
1H NMR (300 MHz, DMSO-d6): δ = 11.12 (s, 1H), 8.84 (d, J = 5.6 Hz, 2H), 8.72 (t, J = 5.8 Hz,
1H), 8.02-7.95 (m, 2H), 7.39 (s, 1H), 6.92 (d, J = 3.7 Hz, 1H), 4.40 (d, J = 5.8 Hz, 2H), 3.29 (m,
2H), 2.75 (t, J = 6.9 Hz, 2H). UPLC-MS (ESI+): m/z = 374.1 [M+H]+, found: 374.4. Purity =
96%, Rt = 3.39 min. Yield = 75%.
N-((5-(hydroxycarbamoyl)thiophen-2-yl)methyl)-4-methyl-2-(4-methylpiperazin-1-
yl)pyrimidine-5-carboxamide (21.Q)
1H NMR (300 MHz, DMSO-d6): δ = 11.15 (s, 1H), 10.33 (s, 1H), 9.03 (t, J = 5.9 Hz, 1H), 8.46
(s, 1H), 7.44 (s, 1H), 6.99 (d, J = 3.8 Hz. 1H), 4.56 (d, J = 5.9 Hz, 2H), 3.36-3.19 (m, 4H), 3.01
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(s, 3H), 2.78 (s, 4H), 2.46 (s, 3H). UPLC-MS (ESI+): m/z = 391.1 [M+H]+, found: 391.5. Purity
= 92%, Rt = 3.56 min. Yield = 70%.
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5-([1,1'-biphenyl]-4-carboxamidomethyl)-N-hydroxythiophene-2-carboxamide (21.R)
1H NMR (300 MHz, DMSO-d6): δ = 11.12 (s, 1H), 9.25 (t, J = 5.8 Hz, 1H), 9.05 (s, 1H), 7.95 (d,
J = 8.2 Hz, 2H), 7.77 (d, J = 8.2 Hz, 2H), 7.71 (d, J = 7.5 Hz, 2H), 7.53-7.34 (m, 4H), 6.99 (d, J
= 3.8 Hz, 1H), 4.61 (d, J = 5.6 Hz, 2H). UPLC-MS (ESI+): m/z = 353.1 [M+H]+, found: 353.3.
Purity = 96%, Rt = 6.45 min. Yield = 77%.
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N-hydroxy-5-((3'-methyl-[1,1'-biphenyl]-4-carboxamido)methyl)thiophene-2-carboxamide
(21.S)
1H NMR (300 MHz, DMSO-d6): δ = 11.13 (s, 1H), 9.25 (t, J = 5.9 Hz, 1H), 7.95 (d, J = 8.4 Hz,
2H), 7.76 (d, J = 8.4 Hz, 2H), 7.56-7.47 (m, 2H), 7.44 (s, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.20 (d, J
= 7.6 Hz, 1H), 7.00 (d, J = 3.7 Hz, 1H), 4.62 (d, J = 5.8 Hz, 2H), 2.37 (s, 3H). UPLC-MS (ESI+):
m/z = 367.1 [M+H]+, found: 367.3. Purity > 99%, Rt = 6.93 min. Yield = 85%.
5-((4'-chloro-[1,1'-biphenyl]-4-carboxamido)methyl)-N-hydroxythiophene-2-carboxamide
(21.T)
1H NMR (300 MHz, DMSO-d6): δ = 11.12 (s, 1H), 9.27 (t, J = 5.8 Hz, 1H), 9.06 (s, 1H), 7.96
(d, J = 8.4 Hz, 2H), 7.77 (t, J = 8.4 Hz, 4H), 7.53 (d, J = 8.5 Hz, 2H), 7.44 (s, 1H), 7.01 (d, J =
3.7 Hz, 1H), 4.62 (d, J = 5.7 Hz, 2H). UPLC-MS (ESI+): m/z = 387.1 [M+H]+, found: 387.3.
Purity = 85%, Rt = 7.11 min. Yield = 56%.
5-((2',4'-dimethoxy-[1,1'-biphenyl]-4-carboxamido)methyl)-N-hydroxythiophene-2-
carboxamide (21.U)
1H NMR (300 MHz, DMSO-d6): δ = 11.12 (s, 1H), 9.19 (t, J = 5.9 Hz, 1H), 7.85 (d, J = 8.4 Hz,
2H), 7.52 (d, J = 8.4 Hz, 2H), 7.43 (s, 1H), 7.25 (d, J = 8.4 Hz, 1H), 6.99 (d, J = 3.7 Hz, 1H),
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6.65 (s, 1H), 6.61 (dd, J = 2.5 Hz, 8.4 Hz, 1H), 4.60 (d, J = 5.8 Hz, 2H), 3.79 (s, 3H), 3.75 (s,
3H). UPLC-MS (ESI+): m/z = 413.1 [M+H]+, found: 413.4. Purity = 93%, Rt = 6.46 min. Yield =
61%.
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N-hydroxy-5-((4'-methyl-[1,1'-biphenyl]-4-carboxamido)methyl)thiophene-2-carboxamide
(21.V)
1H NMR (300 MHz, DMSO-d6): δ = 11.13 (s, 1H), 9.24 (t, J = 5.9 Hz, 1H), 7.96 (d, J = 8.3 Hz,
2H), 7.76 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 7.45 (s, 1H), 7.30 (d, J = 7.9 Hz, 2H),
7.02 (d, J = 3.7 Hz, 1H), 4.63 (d, J = 5.9 Hz, 2H), 2.35 (s, 3H). UPLC-MS (ESI+): m/z = 367.1
[M+H]+, found: 367.3. Purity > 99%, Rt = 6.93 min. Yield = 56%.
N-hydroxy-5-((4-(6-methoxynaphthalen-2-yl)benzamido)methyl)thiophene-2-carboxamide
(21.W)
1H NMR (300 MHz, DMSO-d6): δ = 11.13 (s, 1H), 9.27 (t, J = 5.9 Hz, 1H), 9.06 (s, 1H), 8.23 (s,
1H), 7.98 (d, J = 8.4 Hz, 2H), 7.91-7.88 (d, J = 8.4 Hz, 2H), 7.93-7.90 (d, J = 8.8 Hz, 2H), 7.86
(d, J = 1.4 Hz, 1H), 7.44 (s, 1H), 7.35 (d, J = 2.3 Hz, 1H), 7.19 (dd, J = 2.3 Hz, 8.8 Hz, 1H), 7.00
(d, J = 3.8 Hz, 1H), 4.63 (d, J = 5.7 Hz, 2H), 3.88 (s, 3H). UPLC-MS (ESI+): m/z = 433.1
[M+H]+, found: 433.4. Purity = 75%, Rt = 7.25 min. Yield = 92%.
N-hydroxy-5-((4-(isoquinolin-4-yl)benzamido)methyl)thiophene-2-carboxamide (21.X)
1H NMR (300 MHz, DMSO-d6): δ = 11.15 (s, 1H), 9.36 (t, J = 5.8 Hz, 1H), 9.07 (s, 1H), 8.49 (s,
1H), 8.25 (d, J = 8.01 Hz, 1H), 8.07 (d, J = 8.01 Hz, 2H), 7.88-7.80 (m, 2H), 7.76 (t, J = 7.4 Hz,
1H), 7.68 (d, J = 8.0 Hz, 2H), 7.47 (s, 1H), 7.29 (t, J = 7.5 Hz, 1H), 7.04 (d, J = 3.4 Hz, 1H),
4.68 (d, J = 5.6 Hz, 2H). UPLC-MS (ESI+): m/z = 404.1 [M+H]+, found: 404.4. Purity = 75%,
Rt = 4.32 min. Yield = 73%.
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5-((2-fluoro-[1,1'-biphenyl]-4-carboxamido)methyl)-N-hydroxythiophene-2-carboxamide
(21.Z)
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1H NMR (300 MHz, DMSO-d6): δ = 11.14 (s, 1H), 9.35 (t, J = 5.9 Hz, 1H), 9.07 (s, 1H), 7.85-
7.38 (m, 9H), 7.01 (d, J = 3.7 Hz, 1H), 4.63 (d, J = 5.8 Hz, 2H). UPLC-MS (ESI+): m/z = 371.1
[M+H]+, found: 371.4. Purity = 89%, Rt = 6.70 min. Yield = 79%.
5-((2',6'-dimethyl-[1,1'-biphenyl]-4-carboxamido)methyl)-N-hydroxythiophene-2-
carboxamide (21.AA)
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UPLC-MS (ESI+): m/z = 381.1 [M+H]+, found: 381.4. Purity = 68%, Rt = 7.20 min.
5-((4'-chloro-[1,1'-biphenyl]-3-carboxamido)methyl)-N-hydroxythiophene-2-carboxamide
(21.AB)
UPLC-MS (ESI+): m/z = 387.1 [M+H]+, found: 387.3. Purity = 91%, Rt = 7.09 min. Yield =
77%.
5-((4'-(benzyloxy)-[1,1'-biphenyl]-4-carboxamido)methyl)-N-hydroxythiophene-2-
carboxamide (21.AC)
1H NMR (300 MHz, DMSO-d6): δ = 11.12 (s, 1H), 9.21 (t, J = 5.9 Hz, 1H), 9.05 (s, 1H), 7.91 (d,
J = 8.3 Hz, 2H), 7.72 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.7 Hz, 2H), 7.48-7.30 (m, 6H), 7.10 (d, J
= 8.8 Hz, 2H), 6.99 (d, J = 3.7 Hz, 1H), 5.15 (s, 2H), 4.60 (d, J = 5.6 Hz, 2H). UPLC-MS (ESI+):
m/z = 459.1 [M+H]+, found: 459.5. Purity = 75%, Rt = 7.76 min. Yield = 49%.
N-((5-(hydroxycarbamoyl)thiophen-2-yl)methyl)-1-methyl-1H-imidazole-4-carboxamide
(21.AD)
1H NMR (300 MHz, DMSO-d6): δ = 11.13 (s, 1H), 9.09 (t, J = 5.8 Hz, 1H), 8.36 (s, 1H), 7.88 (s,
1H), 7.42 (s, 1H), 6.97 (d, J = 3.7 Hz, 1H), 4.56 (d, J = 5.8 Hz, 2H), 3.76 (s, 3H). UPLC-MS
(ESI+): m/z = 281.1 [M+H]+, found: 281.3. Purity = 96%, Rt = 2.98 min. Yield = 80%.
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1-(furan-2-ylmethyl)-N-((5-(hydroxycarbamoyl)thiophen-2-yl)methyl)piperidine-3-
carboxamide (21.AE)
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1H NMR (300 MHz, DMSO-d6): δ = 11.14 (s, 1(s, 1H), 8.82 (s, 1H), 7.80 (s, 1H), 7.41 (s, 1H),
6.91 (d, J = 3.7 Hz, 1H), 6.67 (s, 1H), 6.54 (dd, J = 1.8 Hz, 3.2 H), 9.06 Hz, 1H), 4.37 (d, J = 5.8
Hz, 2H), 3.26 (m, 2H), 3.04-2.58 (m, 5H), 1.97-1.56 (m, 4H). UPLC-MS (ESI+): m/z = 364.1
[M+H]+, found: 364.4. Purity > 99%, Rt = 3.67 min. Yield = 96%.
N-hydroxy-5-((3-methyl-1H-indene-2-carboxamido)methyl)thiophene-2-carboxamide
(21.AF)
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1H NMR (300 MHz, DMSO-d6): δ = 11.12 (s, 1H), 8.53 (t, J = 5.9 Hz, 1H), 7.48 (m, 2H), 7.43
(s, 1H), 7.37-7.26 (m, 2H) , 6.98 (d, J = 3.8 Hz, 1H), 4.53 (d, J = 5.9 Hz, 2H), 3.65 (s, 2H), 2.42
(s, 3H). UPLC-MS (ESI+): m/z = 329.1 [M+H]+, found: 329.3. Purity = 90%, Rt = 5.94 min.
Yield = 64%.
N-((5-(hydroxycarbamoyl)thiophen-2-yl)methyl)benzo[c][1,2,5]oxadiazole-5-carboxamide
(21.AG)
1H NMR (300 MHz, DMSO-d6): δ = 9.60 (s, 1H), 8.53 (s, 1H), 8.12 (m, 1H), 8.00-7.90 (m, 1H),
7.25 (s, 1H), 7.08 (s, 1H), 6.91 (s, 1H), 4.65 (d,
J
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5.8 Hz, 2H).
UPLC-MS (ESI+): m/z = 319.1 [M+H]+, found: 319.3. Purity = 71%, Rt = 5.14 min. Yield =
58%.
5-((4-(1-benzyl-1H-pyrazol-4-yl)benzamido)methyl)-N-hydroxythiophene-2-carboxamide
(21.AH)
1H NMR (300 MHz, DMSO-d6): δ = 11.11 (s, 1H), 9.14 (t, J = 5.8 Hz, 1H), 9.05 (s, 1H), 8.38 (s,
1H), 7.99 (s, 1H), 7.84 (d, J = 8.3 Hz, 2H), 7.66 (d, J = 8.2 Hz, 2H), 7.42 (s, 1H), 7.38-7.21 (m,
5H), 6.98 (d, J = 3.6 Hz, 1H), 5.33 (s, 2H), 4.58 (d, J = 5.6 Hz, 2H). Purity = 96%, Rt = 6.20
min; UPLC-MS (ESI+): m/z = 433.1 [M+H]+, Found: 433.4. Yield = 58%.
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1H NMR (300 MHz, DMSO-d6): δ = 11.11 (s, 1H), 9.14 (t, J = 5.8 Hz, 1H), 9.05 (s, 1H), 7.79 (d,
J = 8.2 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H), 7.42 (s, 1H), 6.97 (d, J = 3.8 Hz, 1H), 6.38 (s, 2H),
4.58 (d, J = 5.8 Hz, 2H), 2.12 (s, 1H), 1.93-1.39 (m, 10H). UPLC-MS (ESI+): m/z = 385.1
[M+H]+, found: 385.4; Purity > 99%, Rt = 8.23 min. Yield = 57%.
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5-((4-(furan-3-yl)benzamido)methyl)-N-hydroxythiophene-2-carboxamide (21.AJ)
1H NMR (300 MHz, DMSO-d6): δ = 11.13 (s, 1H), 9.20 (t, J = 5.5 Hz, 1H), 9.05 (s, 1H), 8.30 (s,
1H), 7.90 (d, J = 8.2 Hz, 2H), 7.78 (s, 1H), 7.73 (d, J = 8.3 Hz, 2H), 7.45 (s, 1H), 7.04 (s, 1H),
7.02 (d, J = 3.2 Hz, 1H), 4.63 (d, J = 5.5 Hz, 2H), UPLC-MS (ESI+): m/z = 343.1 [M+H]+,
found: 343.3; Purity = 84%, Rt = 5.81 min. Yield = 55%.
N-hydroxy-5-((4-(thiophen-3-yl)benzamido)methyl)thiophene-2-carboxamide (21.AK)
1H NMR (300 MHz, DMSO-d6): δ = 11.13 (s, 1H), 9.22 (t, J = 5.7 Hz, 1H), 9.06 (s, 1H), 8.03 (s,
1H), 7.93 (d, J = 8.3 Hz, 2H), 7.85 (d, J = 8.3 Hz, 2H), 7.70-7.60 (m, 2H), 7.45 (s, 1H), 7.01 (d, J
= 3.3 Hz, 1H), 4.63 (d, J = 5.6 Hz, 2H). UPLC-MS (ESI+): m/z = 359.1 [M+H]+, found: 359.4;
Purity = 67%, Rt = 6.22 min.
N-((5-(hydroxycarbamoyl)thiophen-2-yl)methyl)-2-methoxynicotinamide (21.AL)
1H NMR (300 MHz, DMSO-d6): δ = 12.54 (s, 1H), 11.12 (s, 1H), 8.92 (t, J = 5.9 Hz, 1H), 8.30
(dd, J = 2.0 Hz, 4.9 Hz, 1H), 8.14 (dd, J = 2.0 Hz, 7.5 Hz, 1H), 7.43 (s, 1H ), 7.12 (dd, J = 4.9
Hz, 7.5 Hz, 1H), 6.98 (d, J = 3.7 Hz, 1H), 4.68-4.57 (m, 2H), 3.95 (s, 3H). UPLC-MS (ESI+):
m/z = 308.1 [M+H]+, found: 308.3. Purity = 80%, Rt = 4.58 min. Yield = 80%.
5-([1,1':4',1''-terphenyl]-4-carboxamidomethyl)-N-hydroxythiophene-2-carboxamide
(21.AM)
1H NMR (300 MHz, DMSO-d6): δ = 11.13 (s, 1H), 9.27 (t, J = 5.9 Hz, 1H), 9.06 (s, 1H), 7.98 (d,
J = 8.4 Hz, 2H), 7.89-7.68 (m, 8H), 7.52-7.33 (m, 4H), 7.01 (d, J = 3.7 Hz, 1H), 4.63 (d, J = 5.7
Hz, 2H). UPLC-MS (ESI+): m/z = 429.1 [M+H]+, found: 429.4. Purity = 66%, Rt = 7.85 min.
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UPLC-MS (ESI+): m/z = 269.1 [M+H]+, Found: 269.3. Purity > 77%, Rt = 5.37 min.
N-hydroxy-5-((4-methoxybenzamido)methyl)thiophene-2-carboxamide (21.AO)
1H NMR (300 MHz, DMSO-d6): δ = 11.12 (s, 1H), 10.30 (s, 1H), 9.17-8.96 (m, 1H), 7.92-7.78
(m, 2H), 7.73-7.48 (m, 2H), 7.54 (t, J = 3.7 Hz, 1H), 6.98 (m, 1H), 4.58 (m, 2H), 3.78 (s, 3H).
UPLC-MS (ESI+): m/z = 307.1 [M+H]+, found: 307.3. Purity = 65%, Rt = 4.84 min.
N-hydroxy-5-((4-methoxybenzamido)methyl)thiophene-2-carboxamide (21.AP)
UPLC-MS (ESI+): m/z = 291.1 [M+H]+, Found: 291.2, HRMS (ESI+): m/z = 291.0798 [M+H]+,
Found: 291.0799. Purity > 81%, Rt = 5.25 min.
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5-((4-fluorobenzamido)methyl)-N-hydroxythiophene-2-carboxamide (21.AQ)
UPLC-MS (ESI+): m/z = 295.1 [M+H]+, Found: 295.2. Purity > 84%, Rt = 4.98 min.
N-((5-(hydroxycarbamoyl)thiophen-2-yl)methyl)-1H-indole-5-carboxamide (21.AR)
UPLC-MS (ESI+): m/z = 316.1 [M+H]+, found: 316.3. Purity = 62%, Rt = 4.60 min.
N-hydroxy-5-((4-(trifluoromethyl)benzamido)methyl)thiophene-2-carboxamide (21.AS)
UPLC-MS: m/z = 345.1 [M+H]+, Found: 345.2. Purity > 82%, Rt = 6.04 min.
N2-([1,1'-biphenyl]-2-yl)-N5-hydroxythiophene-2,5-dicarboxamide (21.AT)
1H NMR (300 MHz, DMSO-d6): δ = 11.36 (s, 1H), 9.98 (s, 1H), 9.24 (s, 1H), 7.53 (d, J = 3.5
Hz, 1H), 7.78-7.57 (m, 3H), 7.48-7.23 (m, 7H). UPLC-MS (ESI+): m/z = 339.1 [M+H]+, found:
339.3; Purity = 78%, Rt = 6.36 min.
N2-(4-fluorobenzyl)-N5-hydroxythiophene-2,5-dicarboxamide (21.AU)
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UPLC-MS (ESI+): m/z = 295.1 [M+H]+, Found: 295.2, HRMS (ESI+): m/z = 295.0547 [M+H]+,
Found: 295.0546. Purity > 74%, Rt = 5.41 min at 254 nm.
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1H NMR (300 MHz, DMSO-d6): δ = 11.35 (s, 1H), 9.23 (s, 1H), 9.16 (t, J = 6.1 Hz, 1H), 7.77-
7.59 (m, 2H), 7.55 (d, J = 3.7 Hz, 1H), 7.37-7.19 (m, 4H), 4.42 (d, J = 6.0 Hz, 2H). UPLC-MS
(ESI+): m/z = 277.1 [M+H]+, found: 277.3; Purity = 72%, Rt = 5.14 min. Yield = 49%.
N2-hydroxy-N5-phenethylthiophene-2,5-dicarboxamide (21.AW)
UPLC-MS (ESI+): m/z = 291.1 [M+H]+, Found: 291.3, HRMS (ESI+): m/z = 291.0798 [M+H]+,
Found: 291.0800. Purity > 73%, Rt = 5.50 min.
N2-cyclohexyl-N5-hydroxythiophene-2,5-dicarboxamide (21.AX)
UPLC-MS (ESI+): m/z = 269.1 [M+H]+, found: 269.3. Purity: > 77%, Rt = 5.37 min.
N2-hydroxy-N5-(4-methylbenzyl)thiophene-2,5-dicarboxamide (21.AZ)
1H-NMR (300 MHz, CDCl3): δ = 11.38 (cs, 1H), 9.15 (t, J = 6.0Hz, 1H), 8.77 (dd, J = 4.4 Hz,
1.2 Hz, 0.5H), 8.54 (dd, J = 8.5 Hz, 1.2 Hz, 0.5H), 7.74 (d, J =3.9 Hz, 1H), 7.56 (d, J = 3.9 Hz,
1H), 7.19 (d, J = 8.1 Hz, 2H), 7.13 (d, J = 8.1 Hz, 2H), 4.40 (d, J = 6.0Hz, 2H), 2.29 (s, 3H).
UPLC-MS (ESI+): m/z = 291.1 [M+H]+, Found: 291.3, HRMS (ESI+): m/z = 291.0798 [M+H]+,
Found: 291.0880. Purity > 77%, Rt = 5.72 min at 254 nm.
N2-hydroxy-N5-(4-methylpyridin-2-yl)thiophene-2,5-dicarboxamide (21.BA)
UPLC-MS (ESI+): m/z = 278.1 [M+H]+, Found: 278.3. Purity = 51%, Rt = 3.56 min. Yield =
58%.
(1-((5-(hydroxycarbamoyl)thiophen-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl
benzoate
(21.BB)
1H NMR (300 MHz, DMSO-d6): δ = 9.96 (s, 1H), 8.35 (s, 1H), 7.96-7.90 (m, 2H), 7.66-7.57 (m,
2H), 7.50 (t, J = 7.9 Hz, 2H), 7.20 (d, J = 3.8 Hz, 1H), 5.88-5.81 (m, 2H), 5.38 (s, 2H). UPLC-
MS (ESI+): m/z = 359.1 [M+H]+, found: 359.3. Purity = 86%, Rt = 5.68 min. Yield = 55%.
ACS Paragon Plus Environment
29