Discovery of potent inhibitors of Schistosoma mansoni NAD+ catabolizing enzyme

Article abstract of DOI:10.1021/acs.jmedchem.5b00203

The blood fluke Schistosoma mansoni is the causative agent of the intestinal form of schistosomiasis (or bilharzia). Emergence of Schistosoma mansoni with reduced sensitivity to praziquantel, the drug currently used to treat this neglected disease, has underlined the need for development of new strategies to control schistosomiasis. Our ability to screen drug libraries for antischistosomal compounds has been hampered by the lack of validated S. mansoni targets. In the present work, we describe a virtual screening approach to identify inhibitors of S. mansoni NAD⁺ catabolizing enzyme (SmNACE), a receptor enzyme suspected to be involved in immune evasion by the parasite at the adult stage. Docking of commercial libraries into a homology model of the enzyme has led to the discovery of two in vitro micromolar inhibitors. Further structure-activity relationship studies have allowed a 3-log gain in potency, accompanied by a largely enhanced selectivity for the parasitic enzyme over the human homologue CD38.

Full text of DOI:10.1021/acs.jmedchem.5b00203

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Article
Discovery of potent inhibitors of Schistosoma
+
mansoni NAD catabolizing enzyme
Sylvain A. Jacques, Isabelle Kuhn, Oleksandr Koniev, Francis Schuber, Frances
E. Lund, Alain Wagner, Hélène Muller-Steffner, and Esther Kellenberger
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00203 • Publication Date (Web): 24 Mar 2015
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Discovery of potent inhibitors of Schistosoma
mansoni NAD+ catabolizing enzyme
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Sylvain A. Jacques1,2, Isabelle Kuhn1, Oleksandr Koniev1, Francis Schuber1, Frances E.
Lund3, Alain Wagner1, Hélène Muller-Steffner*1 and Esther Kellenberger*2
1 Laboratoire des Systèmes Chimiques Fonctionnels, CAMB UMR7199 CNRS/ Université de
Strasbourg, MEDALIS Drug Discovery Center, Faculté de Pharmacie, 67401 Illkirch, France
2 Laboratoire d’Innovation Thérapeutique, LIT UMR7200 CNRS/Université de Strasbourg,
MEDALIS Drug Discovery Center, Faculté de Pharmacie, 67401 Illkirch, France
3 Department of Microbiology, University of Alabama at Birmingham, 276 BBRB Box 11,
1720 2nd Avenue South, Birmingham AL, United States of America
KEYWORDS: SmNACE, CD38, docking, virtual screening, structure-activity relationship,
selectivity
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ABSTRACT. The blood fluke Schistosoma mansoni is the causative agent of the intestinal
form of schistosomiasis (or bilharzia). Emergence of Schistosoma mansoni with reduced
sensitivity to praziquantel, the drug currently used to treat this neglected disease, has
underlined the need for development of new strategies to control schistosomiasis. Our ability
to screen drug libraries for anti-schistosomal compounds has been hampered by the lack of
validated S. mansoni targets. In the present work, we describe a virtual screening approach to
identify inhibitors of S. mansoni NAD+ catabolizing enzyme (SmNACE), a receptor enzyme
suspected to be involved in immune evasion by the parasite at the adult stage. Docking of
commercial libraries into a homology model of the enzyme has led to the discovery of two in
vitro micromolar inhibitors. Further structure-activity relationship studies have allowed a 3-
log gain in potency, accompanied by a largely enhanced selectivity for the parasitic enzyme
over the human homolog CD38.
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INTRODUCTION
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Schistosomiasis is a chronic neglected tropical disease. It affects over 200 million people
and is more debilitating than malaria.1, 2 It is caused by helminth parasites of the genus
Schistosoma. The mostly studied species is Schistosoma mansoni, which causes the intestinal
form of the disease. S. mansoni adult worms live in the portal vasculature and produce eggs,
which damage liver and intestinal tissues. The long-term survival of the parasite in the host
involves a remarkable control of the host immune system, in particular through an
appropriately balanced T helper response.3, 4 Molecular mechanisms of this immune-evasion
are far from being unraveled. A better understanding of the host-parasite relationship is
needed to facilitate vaccine design5 and to identify new drugs effective against the adult
worms, especially those with reduced sensitivity to the prevalent antischistosomal drug
praziquantel.6
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In 2005, we identified a novel enzyme, S. mansoni NAD+ catabolizing enzyme (SmNACE),
which is expressed on the outer surface of the S. mansoni adult worm tegument.7 This
enzyme is a distant homolog of the mammalian CD38, a membrane-associated ecto-enzyme
which has the potential to regulate innate and adaptive immune responses.8 Like CD38,
SmNACE is an efficient NAD+ glycohydrolase. However it lacks the cADPR cyclase activity
which is the hallmark of CD38.9 In human, cADPR produced by CD38 is a calcium-
mobilizing metabolite involved in the chemotactic response of human neutrophils and
monocytes to inflammatory chemokines.8 Accordingly, it is tempting to postulate that
SmNACE acts as an “immune evasion” molecule for S. mansoni by decreasing substrate
availability for CD38 and preventing the CD38 expressing host cells from producing cADPR.
To better characterize SmNACE function, and eventually validate its potential as a
pharmacological target for antischistosomal therapy, we set out to identify inhibitors of its
enzymatic activity. We recently screened a subset of the French National Library (13 760
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molecules) and the Prestwick Chemical Library (880 drugs), and identified two micromolar
inhibitors, cyanidin and delphidin.10 However, these two molecules are poor chemical tools
for studying the in vivo function of SmNACE, due to a general lack of selectivity that is
characteristic of the anthocyanidin family of natural products to which they belong.
In the present study, the virtual screening of commercial libraries (ca. 2 million of
molecules) by high-throughput docking into a SmNACE homology model allowed the
identification of a competitive inhibitor (numbered 3a in Scheme 1) of the enzyme. We report
the synthesis of analogs of 3a and their inhibitory effect on SmNACE catalytic function in
vitro. Furthermore, we show that the best inhibitors in the series have nanomolar potency and
are selective for SmNACE versus the human homolog CD38.
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RESULTS AND DISCUSSION
Virtual screening. The 3D structure of SmNACE was modeled by homology to human and
bovine CD38, Aplysia cyclase and human BST1. Because of the low sequence identity
between SmNACE and the template proteins (from 19.7% to 23.7%), the multiple alignment
was guided by structural data, especially disulfide bridges and conserved secondary
structures. The active site was particularly well defined because it was mainly composed of
conserved secondary structure elements.9
The SmNACE model was used to virtually screen a non-redundant selection of commercial
compounds by high-throughput docking (Figure 1). An initial filtering biased the dataset
towards compounds of small molecular weight in agreement with the small size of the active
site of SmNACE. The molecular weight distribution fitted a Gaussian function centered on
310 with a medium height width of 175. More than 60 000 molecules were docked into the
target. About 3% of the docking poses passed the scoring filters. In 6.9% of these selected
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poses, the predicted inter-molecular interactions partly matched that observed in at least one
crystal structure of CD38 in complex with a substrate, products or molecular analogs
(Tanimoto coefficient computed on interaction fingerprints greater than 0.6). Additional
1.4% of poses were retained based on interaction fingerprints, using a machine learning
approach trained to discriminate inter-molecular interactions in crystal structures of CD38 in
complex with a substrate, products or molecular analogs from “decoy” binding modes
generated by the docking into SmNACE of truly inactive compounds. The automated
selection protocol returned 188 molecules. A visual inspection rejected 90 molecules because
of high similarity between structures.
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Biological validation of hits. The 98 selected compounds were purchased and tested for
their ability to inhibit the catalytic activity of recombinant SmNACE in vitro using 1,N6-
etheno NAD+ as substrate. In the case of molecules with intrinsic fluorescence, the inhibition
of SmNACE was re-evaluated using the direct quantification by HPLC of the reaction
products generated using NAD+ as substrate. We observed that 20 molecules produce a
significant inhibition (≥ 10%) at a concentration of 100 µM. We further tested the
preliminary hits by repeating both fluorimetric and HPLC assays at different inhibitor
concentrations. Only five molecules consistently inhibited more than 40% of SmNACE
activity at a concentration of 100 µM. One of these molecules was taxifolin, which was
already investigated as SmNACE inhibitor because of its similarity to cyanidin.10 As
mentioned in the Introduction, cyanidin and taxifolin are natural products with multiple
biological activities due to promiscuous binding to different protein targets. We therefore
focused our study on the other four molecules. We repeated the in vitro enzymatic
experiments on four new lots of chemicals (purchased from a different supplier or
synthesized if not available commercially), as well as on two or three close chemical analogs
of each of the four molecules. Three out of the four putative inhibitors have been discarded
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because the investigated changes in the structure (e.g., loss of a carboxyl group) had dramatic
effects on the inhibitory effect, or because inhibition was not reproducible due to
fluorescence quenching and/or compound degradation. In the end, only one of the four
potential inhibitors has been validated (Figure 2a). The construction of the dose response
curve revealed a half maximal inhibition concentration (IC50) equal to 88 8 µM (Figure 2b).
Though the inhibition potency is modest, it nevertheless corresponds to a fairly good ligand
efficiency (0.30 kcal/mol/heavy atom)11 because the molecular mass of the inhibitor is small
(260.25 Da). For the sake of comparison, we observed exactly the same ligand efficiency for
the racemic form of taxifolin (IC50: 19.5 1.4 µM).10 Lastly, Dixon plots unambiguously
established that, like taxifolin, the validated hit is a competitive inhibitor of SmNACE (Figure
2c). The docking poses of taxifolin and of the validated inhibitor are given as Supplementary
Material (Figure S1).
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Chemistry. The synthetic route of the target modified hydrazide derivatives obtained by
condensation reactions is outlined on Scheme 1. Commercially available methyl 2-
methylfuran-3-carboxylate (1) was converted into the corresponding hydrazide 2 using
hydrazine monohydrate,12 and the hydrazide was reacted with substituted aldehydes or
ketones to give the corresponding compounds 3a–3p.13 Benzylidene-carbohydrazide
derivatives 3d and 3e were treated with TFA and triethylsilane to afford compounds 4b and
4c.14 Treatment of 3e with methyl iodide gave compound 8. Saponification of 1 with NaOH 15
followed by activation of the acid with ethyl chloroformate and condensation with 2-(2-
methoxyphenyl)ethan-1-amine gave amide 6 (Scheme 2). Finally compounds 6 and 8 were
converted into the corresponding phenols 5 and 7, respectively upon treatment with BBr3 at
low temperature. Bis-amide derivatives 11a-11d were obtained by acylation reactions
between acid chloride and hydrazide derivatives (Scheme 3). Carboxylic acid 9 was
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converted into the corresponding acid chloride by treatment with oxalyl chloride and DMF
and reacted with commercially available hydrazide 10 to give 11a. 16 In a similar manner,
compounds 11b-11d were prepared by reacting hydrazide 2 with the corresponding acid
chlorides derived from commercially available or easily prepared carboxylic acids 13b-13d.
Finally, 1,3,4-oxadiazole derivatives 12a–12c were obtained upon treatment of 11a-11c with
dehydrating reagent 2-chloro-1,3-dimethylimidazolinium chloride (DMC) in the presence of
Et3N.17
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Structure-Activity Relationships. We have assessed the importance of the two hydroxyl
groups in the validated hit (3a). To that end we compared the effect on SmNACE inhibition
in vitro of five different substitution patterns of the phenyl ring (Table 1, compounds 3b-3e,
3o). Methylation of the meta hydroxyl group (3b) did not affect the IC50 value. By contrast, a
4-fold decrease of the apparent affinity was observed for the fully dehydroxylated analog (3c,
with IC50=330µM). Single substitution at position 1 (3d, with IC50=0.13µM) resulted in a 3-
log gain in affinity as compared to 3a. Considering 3d, the introduction of a methoxy group
at position 1 (3e, with IC50=67µM) or of a second hydroxyl group at position 2 (3o, with
IC50=8.7µM) was detrimental to the activity.
Starting from the best inhibitor 3d, we tested other substitutions at positions 2-4 of the
phenyl ring (Table 1, compounds 3f-3n). Considering position 2, substitution with fluorine
(3h, with IC50=0.036µM) resulted in a 4-fold improvement of IC50 as compared to 3d,
whereas substitution with chlorine (3f, with IC50=0.15µM) did not significantly modify the
IC50 value, and introduction of an alkyl group (3m and 3n, with respectively IC50=59µM and
IC50=105µM) negatively impacted inhibitory potency. Introduction of chlorine or methoxy
group(s) at positions 3 (3k), 4 (3g and 3j) and 3,5 (3i) also decreased IC50 values (1.5 to 4
fold, IC50=0.032-0.077µM). This round of modification allowed a significant improvement of
ligand efficiency, which reached 0.54 kcal/mol/heavy atom for 3g and 3h.
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We then investigated whether the nature of the linker between the phenyl and furan rings
impacts activity on SmNACE. Increasing the linker flexibility in 3d by reduction of the C=N
double bond (4b) or its replacement by C-C single bond (5) dramatically increased IC50
values (IC50=12.3µM and IC50=131µM, respectively). The same modifications made on the
O-methylated inhibitor 3e (4c, 6) also decreased the inhibitor potency (IC50=11.6µM and 0%
inhibition at 100µM concentration, respectively). The introduction of a methyl group on the
amide nitrogen atom of 3e (8) also produced a substantial drop of inhibition (IC50=513µM),
suggesting that the hydrogen atom of the amide may be involved in an important
intramolecular or intermolecular H-bond. We further modified the H-bonding properties of
three compounds with the introduction of a bis-amide linker, yet no significant changes in
activity were observed (11a–11c vs parent compounds 3d, 3g and 3k). Lastly, a rigid 1,2,4-
oxadiazole linker completely abolished the ability to inhibit SmNACE (12a– 12c, with 0%
inhibition at 100µM concentration).
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Overall, the study of structure-activity relationships has identified eleven potent inhibitors
of SmNACE with IC50 in the 0.032- 0.25µM range (3d, 3f, 3g, 3h, 3i, 3j, 3k, 3l, 11a, 11b and
11c). Dixon plot analysis of inhibition of compounds 3d, 3h, 11b and 11c confirmed the
competitive mode of inhibition in the chemical series developed from the validated hit (3a)
(Figure S2).
Of note, the compounds 3a-3o contained a 2-hydroxyl-phenyl-hydrazone group, found in
Pan Assay Interference Compounds because of possible interference in biological assays. Our
experimental validation however ruled out non-specific inhibition due to formation of
aggregates, spectroscopic properties, metal chelation or high reactivity (more details are
given in the Supplementary Information, Text S2).
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Selectivity for SmNACE over human CD38. Compounds with IC50 equal to or lower than
100 µM were tested in vitro on the human homolog of SmNACE, namely CD38. The
validated hit 3a was not selective, nor did the micromolar inhibitors 3b and 3c. However, all
derivatives with increased potency against SmNACE were highly selective for the parasitic
enzyme (3d – 3l, 3o, 4b – 4c, 11a – 11b). Importantly, the IC50(CD38)/IC50(SmNACE) was
greater than 1000 for seven out of eleven potent inhibitors of SmNACE with IC50 in the
0.032-0.25µM range (3d, 3g, 3i, 3j, 3k, 11b and 11c). Docking models of 3d into SmNACE
and human CD38 suggested that the His / Trp sequence variation in the catalytic site, which
is otherwise highly conserved, is the key structural determinant of the ligand selectivity
(Figure S1).
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CONCLUSION
Our approach to the discovery of new SmNACE inhibitors combined docking using a
homology model of SmNACE and scoring using machine learning and interaction
fingerprinting. We selected and experimentally tested 98 molecules on the recombinant
enzyme, yielding a primary hit rate of about 10%. Only two compounds were validated: the
natural product taxifolin and a new synthetic inhibitor. The two inhibitors are competitive and
have ligand efficiency in the range of 0.30 kcal/mol/non-hydrogen atom. The synthesis of
derivatives of the synthetic inhibitor allowed to improve by 3-log the inhibitor potency, while
dramatically enhancing the selectivity for the parasitic enzyme vs the human homolog. The
six best SmNACE inhibitors (3g, 3i, 3j, 3k, 11b and 11c) have IC50 in the 0.032- 0.077µM
range, corresponding to ligand efficiency of 0.46-0.54 kcal/mol/heavy atom. The compounds
do not inhibit human CD38 at 100 µM concentration. These compounds are promising tools
for the study of the in vivo function of SmNACE, and its validation as a pharmacological
target for antischistosomal therapy.
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MATERIAL AND METHODS
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Homology modeling. The three-dimensional structure of SmNACE (UNIPROT:
Q32TF5_SCHMA) was modeled by homology to human CD38 (PDB: 2pgj, chain A), bovine
CD38 (PDB: 3gc6) and Aplysia cyclase (PDB: 1r15) and human BST1 (PDB: 1isj). The
sequence alignment and the building of segments matching the ten structurally conserved
regions (which include the full active site) were performed using the COMPOSER module of
Sybyl (Tripos, Inc.). Loop construction was performed using COMPOSER if SmNACE
sequence was highly similar to the sequence of at least one of the templates otherwise using
the automodel routine of Modeller (default setting). Six disulfide bridges were constrained
during the modeling: C34-C48, C68-C144, C114-C120, C138-C152, C231-C252, C264-
C273. SmNACE model was energy minimized using Amber (University of California, San
Francisco, CA, USA). The best model was chosen according to Procheck quality scores.
Virtual screening. The chemical library for virtual screening was obtained by merging
commercial screening collections (from Asinex, Bionet, Chembridge ChemicalDivision,
ChemStar, Enamine, InterBioScreen, lifeChemicals, MayBridge, OTAVA, Peakdale,
Pharmeks, TimTec, Princeton and Vitas-M) then by the filtering of duplicates, reactive or non
drug-like compounds, using Pipeline Pilot (Accelrys Software Inc, San Diego). The most
probable ionization state of each molecule of the filtered library was predicted using Filter
(OpenEye Scientific Software, Santa Fe). The 2D structures were converted into three-
dimensional structures using Corina (Molecular Networks GmbH, Erlangen). The high-
throughput docking of the chemical library was performed using Surflex v2.11 The docking
site of SmNACE was defined as the 41 residues surrounding the largest protein cavity in the
model (Q100-M107, I110, R111, S122-E124, G129, F132- L135, W137, N159, V161-Q166,
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A168-A170, Y172, A173, V182, N193, N195-E202) and represented by the protomol
automatically generated from the coordinates of these residues using Surflex (setting on the
adap_proto option). Each structure of the chemical library was docked into the protomol after
an initial minimization of it coordinates (+premin). The conformational search involves five
additional starting conformations (+multistart) and a maximum of 100 conformations per
fragment (-maxconfs). The ten best poses were retained (-ndock_final) and refined by energy
minimization (-remin). The minimal RMS difference between final poses was set to 0.5 (-
divi_rms). The poses were selected according to docking scores (pKi ≥7.0, crash >-2 and
polar ≥4.0), then filtered depending on their binding modes using a Naïve Bayes model
generated with Weka 3.5.5 from a training set of 42 positive and 1540 negative reference
binding modes described with residues-based interaction fingerprints.18 The positive binding
modes were extracted from the X-ray structures of complexes between human or bovine
CD38 and a ligand (PDB: 2htc, 2i65, 2i66, 2i67, 2pqj, 2pgl, 3p5s and 3kou) after addition of
hydrogen atoms using Sybyl (Tripos, Inc) and fragmentation of the ligand into
phosphoribose, nucleoside and nucleotides monophosphate (or their fluorinated analogs). The
negative binding modes were extracted from poses obtained by docking into SmNACE (see
above for detailed settings) of 89 molecules that were tested during a previous screening
campaign and did not inhibit the enzyme at micromolar concentration.10 Alternatively, a
docked pose was retained if the direct comparison with any of the positive binding modes
returned a Tanimoto coefficient greater or equal to 0.6. The chemical library was searched for
close analogs of potential hits. Selected analogs had a Tanimoto coefficient computed from
FCFP_4 fingerprint greater than 0.6 (Accelrys Software Inc, San Diego) and the same
chemical scaffold as the parent molecule.
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Enzyme assays. SmNACE activity was determined by a sensitive continuous fluorometric
assay using 1,N6-etheno NAD+ (ε-NAD+, Sigma) as substrate.7,19 This assay consists in
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measuring the appearance of the reaction product ε-ADP-ribose by the increase of
fluorescence at λem = 410 nm (λexc = 310 nm) at 37 °C in 10 mM potassium phosphate buffer,
pH 7.4, containing 0.05 % (w/v) emulphogen (100µl final volume) in a Spectramax Gemini
XPS fluorescence plate reader (Molecular Devices). The effect of the inhibitors on the initial
rates of the transformation of ε-NAD+ was determined and IC50 values were calculated from
the plot of residual activity against the log of inhibitor concentration (6 data points) using a
non-linear regression program (GraphPad, Prism). The type of inhibition was determined
under the same experimental conditions using three different fixed inhibition concentrations
and varying ε-NAD+ concentrations. Alternatively, the enzyme activity was measured at 20
µM NAD+ as described previously7 by following the product formation by HPLC on a 300 x
3.9 mm µBondapack C18 column (Waters). The isocratic elution was performed at a flow
rate of 1 mL/min (10 mM ammonium phosphate buffer, pH 5.5, with 1.2% (v/v) acetonitrile).
In some cases [adenosine-U-14C] NAD+ (2x105 dpm) was added to the unlabeled substrate.
The eluted compounds were detected by absorbance recordings at 260 nm and by
radiodetection (Flo-one, Packard Radiometric Instruments) when using [14C] NAD+.7
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Chemistry
General experimental procedures: unless otherwise indicated, reactions were carried out
under an atmosphere of argon in flame-dried glassware with magnetic stirring. Air and/or
moisture-sensitive liquids were transferred via syringe. When required, solutions were
degassed by bubbling argon through a needle. Organic solutions were concentrated by rotary
evaporation at 25-60 °C at 15-30 torr. Analytical thin layer chromatography (TLC) was
performed using plates cut from glass sheets (silica gel 60F-254 from Merck). Visualization
was achieved under a 254 or 365 nm UV light and by immersion in an ethanolic solution of
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cerium sulfate, followed by treatment with a heat gun. Column chromatography was carried
out as “Flash Chromatography” using silica gel G-25 (40-63 ꢀM) from Macherey-Nagel.
Materials: All reagents were obtained from commercial sources and used without further
purifications. Dry MeOH and DMF were obtained from Aldrich.
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Instrumentation: UV-Vis spectra and kinetic were recorded on Shimadzu UV-1800
spectrophotometer. IR spectra were recorded on a Nicolet 380 FT-IR spectrometer from
1
Thermo Electron Corporation as a CH2Cl2 solution or solid on a diamond plate. H and 13C
NMR spectra were recorded at 25 °C on Bruker 400 and 500 spectrometers. Recorded shifts
(δ) are reported in parts per million (ppm) and calibrated using residual undeuterated solvent.
Data are represented as follows: Chemical shift, mutiplicity (s = singlet, d = doublet, t =
triplet, q = quartet, quint = quintet, m = multiplet, br = broad), coupling constant (J, Hz) and
integration. High-resolution mass spectra (HRMS) were obtained using an Agilent Q-TOF
(time of flight) 6520 and low resolution mass spectra using an Agilent MSD 1200 SL
(ESI/APCI) with a Agilent HPLC1200 SL. The semi-preparative HPLC system consisted of a
Waters 600 pump, a 2487 detector (Waters), a 5 mL sample loop and a Sunfire C18 column
(150 mm × 19 mm i.d., 5 ꢀm, Waters) with a 40 minutes gradient from 5% to 95%
acetonitrile.
2-Methylfuran-3-carbohydrazide 2
To a solution of methyl 2-methyl-3-furoate 1 (750 mg, 5.35 mmol, 1.0 eq) in MeOH (21
mL) was added hydrazine hydrate (2.68 g, 53.5 mmol, 10.0 eq.) and the resulting mixture
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was heated to reflux for 16 h. The reaction mixture was concentrated under reduced pressure
and the crude solid was purified by silica gel column chromatography (cHex/EtOAc 100% to
0% over 20 min) to afford 2-methylfuran-3-carbohydrazide 2 as a white solid (695 mg, 4.96
mmol, 93%).
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1H NMR (400 MHz, MeOD-d4) δ 7.25 (d, J = 2.01 Hz, 1H), 6.54 (d, J = 1.76 Hz, 1H),
2.42 (s, 3H), NH and NH2 signals missing.
13C NMR (100 MHz, MeOD-d4) δ 166.2, 157.8, 141.9, 115.2, 109.6, 13.4.
IR (film) 3272, 2330, 2341, 1635, 1512, 1337, 1216, 1048 cm-1.
MS (ES+APCI) m/z 141 [M + H]+.
General procedure for the synthesis of analogs 3a–3n:
To a solution of 2 (1.0 eq) in EtOH was added aldehyde/ketone (1.0 eq) and the resulting
mixture was micro-irradiated for 5 to 10 min at 150 °C. The crude mixture was allowed to
cool down to room temperature. Water was added; the precipitate was collected, washed with
water and Et2O and dried under reduced pressure.
(Z)-N'-(3,4-Dihydroxybenzylidene)-2-methylfuran-3-carbohydrazide 3a
86%
1H (400 MHz, MeOD-d4) δ 8.12 (s, 1H), 7.43 (s, 1H), 7.31 (s, 1H), 7.37 (s, 1H), 7.05 (d, J
= 8.03 Hz, 1H), 6.89 – 6.70 (m, 2H), 2.62 (s, 3H), 2 OH and 1 NH signals missing.
13C (100 MHz, MeOD-d4) δ 163.0, 159.6, 150.1, 149.5, 146.9, 142.1, 127.6, 122.6, 116.3,
115.2, 114.2, 109.5, 13.7.
IR (film) 3295, 1607, 1495, 1372, 1298, 1281, 1190, 897, 724 cm-1.
MS (ES+APCI) m/z 261 [M + H]+.
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(Z)-N'-(2-Hydroxybenzylidene)-2-methylfuran-3-carbohydrazide 3d
7
8
9
10
11
12
13
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92%
1H (400 MHz, DMSO-d6) δ 11.64 (s, 1H, NH), 11.28 (s, 1H, OH), 8.57 (s, 1H, H-3), 7.61
(d, J = 2.0 Hz, 1H, H-1), 7.52 (d, J = 7.2 Hz, 1H, HAr), 7.29 (t, J = 7.2 Hz, 1H, HAr), 6.96
(d, J = 1.6 Hz, 1H, H-2), 6.95 – 6.91 (m, 2H, HAr), 2.56 (s, 3H, Me).
13C (100 MHz, DMSO-d6) δ 159.1, 157.6, 157.4, 147.4, 141.1, 131.2, 129.5, 119.3, 118.7,
116.4, 113.9, 108.8, 13.3.
IR (film) 3523 (NH), 3296 (OH), 2359, 2337, 1627 (C=O), 1608 (C=C), 1495 (C=N),
1368, 1298, 1231, 1198, 897, 724 cm-1.
MS (ES+APCI) m/z 245 [M + H]+.
(Z)-N'-(2-Methoxybenzylidene)-2-methylfuran-3-carbohydrazide 3e
52%
1H (400 MHz, MeOD-d4) δ 8.71 (s, 1H), 8.09 (dd, J = 7.6, 1.2 Hz, 1H), 7.42 – 7.38 (m,
2H), 7.04 (d, J = 8.4 Hz, 1H), 6.99 (t, J = 7.6 Hz, 1H), 6.85 (d, J = 1.6 Hz, 1H), 3.89 (s, 3H),
2.60 (s, 3H), 1 NH signal missing.
13C (100 MHz, MeOD-d4) δ 163.1, 159.9, 159.8, 145.5, 142.0, 132.9, 127.6, 123.8, 121.8,
115.1, 112.3, 109.5, 56.2, 13.7.
IR (film) 2350, 1633, 1601, 1428, 1350, 1232, 1014, 749, 732 cm-1.
MS (ES+APCI) m/z 259 [M + H]+.
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(Z)-N'-(3-Chloro-2-hydroxybenzylidene)-2-methylfuran-3-carbohydrazide 3f
7
8
9
10
11
12
13
14
15
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60
59%
1H (400 MHz, MeOD-d4) δ 8.44 (s, 1H), 7.45 (d, J = 1.6 Hz, 1H), 7.40 (dd, J = 8.0, 1.2 Hz,
1H), 7.33 (dd, J = 8.0, 1.6 Hz, 1H), 3.91 (t, J = 8.0 Hz, 1H), 6.84 (d, J = 1.2 Hz, 1H), 2.61 (s,
3H), 1 NH and 1 OH signals missing.
13C (100 MHz, MeOD-d4) δ 160.4 (2C), 155.2, 149.9, 142.2, 132.7, 130.4, 122.6 (2C),
120.9, 114.7, 109.3, 13.7.
IR (film) 3439, 3214, 2860, 1640, 1606, 1359, 1312, 1234, 1203, 1186, 1141, 1125, 936,
744 cm-1.
MS (ES+APCI) m/z 279 [M + H]+.
(Z)-N'-(5-Chloro-2-hydroxybenzylidene)-2-methylfuran-3-carbohydrazide 3g
55%
1H (400 MHz, MeOD-d4) δ 8.42 (s, 1H), 7.50 (d, J = 2.8 Hz, 1H), 7.44 (d, J = 2.0 Hz, 1H),
7.25 (dd, J = 8.8, 2.8 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H), 2.60 (s, 3H),
1 NH and 1 OH signals missing.
13C (100 MHz, MeOD-d4) δ 162.5, 160.3, 157.9, 148.7, 142.2, 132.2, 130.2, 125.2, 121.2,
119.2, 114.8, 109.3, 13.7.
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IR (film) 3420, 3202, 3050, 1640, 1606, 1556, 1486, 1353, 1321, 1286, 1238, 1210, 926,
4
5
6
732 cm-1.
7
8
MS (ES+APCI) m/z 279 [M + H]+.
9
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59
60
(Z)-N'-(3-Fluoro-2-hydroxybenzylidene)-2-methylfuran-3-carbohydrazide 3h
52%
1H (400 MHz, MeOD-d4) δ 8.48 (s, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.26 (td, J = 8.0, 1.6 Hz,
1H), 7.14 (ddd, J = 11.1, 8.1, 2.1 Hz, 1H), 6.88 (td, J = 8.0, 4.4 Hz, 1H), 6.84 (d, J = 2.0 Hz,
1H), 2.61 (s, 3H), 1 NH and 1 OH signals missing.
13C (100 MHz, MeOD-d4) δ 162.5, 160.2, 152.8 (d, J = 241.0 Hz), 149.5, 147.3 (d, J =
13.0 Hz), 142.2, 126.3 (d, J = 2.5 Hz), 122.1 (d, J = 2.7 Hz), 120.2 (d, J = 7.0 Hz), 118.6 (d, J
= 18.2 Hz), 114.8, 109.3, 13.7.
19F (376 MHz, MeOD-d4) δ -139.6 (dd, J = 11.3, 5.5 Hz).
IR (film) 3426, 3214, 3030, 1633, 1607, 1473, 1365, 1246, 1235, 1124, 725 cm-1.
MS (ES+APCI) m/z 263 [M + H]+.
(Z)-N'-(2,4-Dichloro-6-hydroxybenzylidene)-2-methylfuran-3-carbohydrazide 3i
33%
1H (400 MHz, MeOD-d4) δ 8.91 (s, 1H), 7.45 (d, J = 2.0 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H),
6.96 (d, J = 2.0 Hz, 1H), 6.85 (d, J = 1.6 Hz, 1H), 2.60 (s, 3H), 1 NH and 1 OH signals
missing.
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13C (100 MHz, MeOD-d4) δ 161.7, 160.7 (2C), 146.8, 142.3, 137.9, 136.2, 121.4, 117.4,
5
6
115.7, 114.6, 109.3, 13.7.
7
8
IR (film) 3467, 3217, 3005, 1644, 1604, 1551, 1405, 1347, 1296, 1219, 1089, 953, 902,
9
830 cm-1.
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MS (ES+APCI) m/z 314 [M + H]+.
(Z)-N'-(2-Hydroxy-5-methoxybenzylidene)-2-methylfuran-3-carbohydrazide 3j
47%
1H (400 MHz, MeOD-d4) δ 8.44 (s, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.05 (d, J = 2.8 Hz, 1H),
6.92 – 6.89 (m, 1H), 6.85 – 6.83 (m, 2H), 3.78 (s, 3H), 2.60 (s, 3H), 1 NH and 1 OH signals
missing.
13C (100 MHz, MeOD-d4) δ 162.4, 160.1, 154.2, 153.3, 150.1, 142.2, 119.8, 119.7, 118.4,
114.9, 114.4, 109.4, 56.3, 13.7.
IR (film) 3300, 3148, 2825, 1674, 1600, 1516, 1488, 1292, 1263, 1188, 1148, 1039, 858,
733 cm-1.
MS (ES+APCI) m/z 275 [M + H]+.
(Z)-N'-(2-Hydroxy-4-methoxybenzylidene)-2-methylfuran-3-carbohydrazide 3k
60%
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1H (400 MHz, MeOD-d4) δ 8.37 (s, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.27 (d, J = 8.8 Hz, 1H),
6.82 (d, J = 2.0 Hz, 1H), 6.53 – 6.48 (m, 2H), 3.81 (s, 3H), 2.59 (s, 3H), 1 NH and 1 OH
signals missing.
9
13C (100 MHz, MeOD-d4) δ 164.4, 162.3, 161.3, 159.9, 151.1, 142.2, 133.0, 114.9, 112.9,
109.3, 107.7, 102.3, 55.9, 13.7.
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60
IR (film) 3430, 3205, 1622, 1601, 1505, 1263, 1227, 1111, 964, 901, 731 cm-1.
MS (ES+APCI) m/z 275 [M + H]+.
(Z)-N'-(2-Hydroxy-4,6-dimethoxybenzylidene)-2-methylfuran-3-carbohydrazide 3l
68%
1H (400 MHz, MeOD-d4) δ 8.75 (s, 1H), 7.42 (d, J = 2.0 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H),
6.12 (d, J = 2.4 Hz, 1H), 6.09 (d, J = 2.4 Hz, 1H), 3.85 (s, 3H), 3.80 (s, 3H), 2.58 (s, 3H), 1
NH and 1 OH signals missing.
13C (100 MHz, MeOD-d4) δ 165.4, 162.6, 162.0, 161.5, 159.7, 147.8, 142.1, 114.9, 109.4,
102.1, 94.8, 91.2, 56.3, 55.9, 13.7.
IR (film) 3018, 1629, 1599, 1347, 1306, 1215, 1145, 1110, 901, 812 cm-1.
MS (ES+APCI) m/z 305 [M + H]+.
(Z)-N'-(2-Hydroxy-3-methylbenzylidene)-2-methylfuran-3-carbohydrazide 3m
55%
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1H (400 MHz, MeOD-d4) δ 8.40 (s, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.18 (d, J = 7.2 Hz, 1H),
7.15 (d, J = 4.6 Hz, 1H), 6.83 (d, J = 2.0 Hz, 1H), 6.83 (t, J = 7.6 Hz, 1H), 2.61 (s, 3H), 2.26
(s, 3H), 1 NH and 1 OH signals missing.
9
13C (100 MHz, MeOD-d4) δ 160.3 (2C), 157.8, 151.8, 142.2, 133.8, 129.9, 126.9, 120.1,
118.5, 114.7, 109.3, 15.7, 13.7.
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60
IR (film) 3449, 3320, 3069, 1639, 1606, 1552, 1404, 1363, 1304, 1261, 1020, 1053, 862,
731 cm-1.
MS (ES+APCI) m/z 259 [M + H]+.
(Z)-N'-(3-(tert-Butyl)-2-hydroxybenzylidene)-2-methylfuran-3-carbohydrazide 3n
18%
1H (400 MHz, MeOD-d4) δ 8.42 (s, 1H), 7.45 (d, J = 1.2 Hz, 1H), 7.33 (d, J = 7.2 Hz, 1H),
7.15 (d, J = 6.8 Hz, 1H), 6.88 – 6.84 (m, 2H), 2.61 (s, 3H), 1.45 (s, 9H), 1 NH and 1 OH
signals missing.
13C (100 MHz, MeOD-d4) δ 160.1 (2C), 158.7, 152.5, 142.2, 138.3, 130.6, 130.0, 119.9,
119.1, 114.8, 109.4, 35.8, 29.9 (3C), 13.7.
IR (film) 3439, 3236, 2955, 1640, 1605, 1433, 1313, 1232, 1203, 1126, 851, 742 cm-1.
MS (ES+APCI) m/z 301 [M + H]+.
(Z)-N'-(2,3-Dihydroxybenzylidene)-2-methylfuran-3-carbohydrazide 3o
52%
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1H (400 MHz, DMSO-d6) δ 11.64 (s, 1H), 11.11 (s, 1H), 9.19 (s, 1H), 8.53 (s, 1H), 7.61 (d,
J = 1.6 Hz, 1H), 6.95 (d, J = 1.2 Hz, 1H), 6.94 (d, J = 6.2 Hz, 1H), 6.85 (dd, J = 7.2, 0.7 Hz,
1H), 6.73 (t, J = 8.0 Hz, 1H), 2.57 (s, 3H).
9
13C (100 MHz, DMSO-d6) δ 158.5, 157.0, 147.6, 145.5, 145.0, 140.5, 119.4, 118.5, 118.2,
116.7, 113.3, 108.2, 12.8.
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60
IR (film) 3265, 3069, 1628, 1609, 1590, 1524, 1407, 1359, 1219, 950, 868 cm-1.
MS (ES+APCI) m/z 261 [M + H]+.
(Z)-N'-(1-(2-Hydroxyphenyl)ethylidene)-2-methylfuran-3-carbohydrazide 3p
43 %
1H (400 MHz, MeOD-d4) δ 7.61 (dd, J = 8.0, 1.6 Hz, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.28
(ddd, J = 8.6, 6.7, 1.5 Hz, 1H), 6.93 – 6.87 (m, 2H), 6.89 (ddd, J = 8.4, 6.9, 1.6 Hz, 1H), 2.59
(s, 3H), 2.46 (s, 3H), 1 NH and 1 OH signals missing.
13C (100 MHz, MeOD-d4) δ 163.1, 160.3, 160.0, 159.5, 142.1, 132.5, 129.4, 120.8, 119.8,
118.5, 115.3, 109.9, 13.8, 13.7.
IR (film) 3211, 2917, 1644, 1605, 1516, 1490, 1296, 1227, 1193, 1113, 829, 749 cm-1.
MS (ES+APCI) m/z 259 [M + H]+.
2-Methylfuran-3-carboxylic acid 9
A solution of methyl 2-methyl-3-furoate (2.0 g, 14.3 mmol, 1.0 eq) in 20% NaOH (8 mL)
was heated to reflux for 2 h and further stirred for 48 h at room temperature. The resulting
mixture was allowed to cool down to room temperature before addition of 1N HCl under
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vigorous stirring. The resulting precipitate was washed with water and dried under reduced
pressure to give the desired carboxylic acid as a slightly yellow solid. The aqueous layer was
extracted with Et2O (3 x 50 mL) and the combined organic layers were dried over Na2SO4
and concentrated under reduced pressure to give 9 as a slightly orange solid (1.54 g, 12.2
mmol, 86%).
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60
1H (400 MHz, CDCl3) δ 7.26 (d, J = 2.0 Hz, 1H), 6.69 (d, J = 1.6 Hz, 1H), 2.61 (s, 3H), 1
COOH signal missing.
13C (100 MHz, CDCl3) δ 169.7, 161.1, 140.8, 113.0, 111.0, 14.0.
IR (film) 3040, 2885, 2578, 1670, 1590, 1436, 1300, 1125, 944, 735 cm-1.
N-(2-Methoxyphenethyl)-2-methylfuran-3-carboxamide 6
O
OMe
NH
O
A mixture of 2-methylfuran-3-carboxylic acid (100 mg, 0.79 mmol, 1.0 eq.) and Et3N (275
µL, 1.98 mmol, 2.5 eq.) was dissolved in CH2Cl2 (3 mL). The resulting mixture was cooled
down to 0 °C and ethyl chloroformate (83.4 µL, 0.87 mmol, 1.1 eq.) was added. The resulting
mixture was stirred for 1 h before 2-methoxyphenethylamine (174.6 µL, 1.18 mmol, 1.5 eq.)
was added. The solution was warmed up to room temperature and stirred for 24 h. H2O was
added and the layers were separated. The aqueous layer was extracted with CH2Cl2 (3 x 5
mL) and the combined organic layers were dried over Na2SO4 and concentrated under
reduced pressure. The crude material was purified by preparative reverse phase HPLC
(SunFire column, acidic gradients with MeCN-H2O containing 0.05 % TFA) to give 6 as a
white solid (200 mg, 0.77 mmol, 97%).
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1H (400 MHz, MeOD-d4) δ 7.33 (d, J = 2.0 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 6.82 – 6.80
(m, 2H), 6.77 – 6.76 (m, 1H), 6.62 (d, J = 2.0 Hz, 1H), 3.51 (t, J = 7.4 Hz, 2H), 3.76 (s, 3H),
2.84 (t, J = 7.4 Hz, 2H), 2.49 (s, 3H), 1 NH signal missing.
9
13C (100 MHz, MeOD-d4) δ 166.5, 161.3, 157.7, 142.2, 141.7, 130.4, 122.2, 116.9, 115.4,
112.9, 109.9, 55.5, 42.0, 36.75, 13.4.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
IR (film) 3355, 2478, 2071, 1626, 1602, 1487, 1463, 1260, 970 cm-1.
MS (ES+APCI) m/z 260 [M + H]+.
N-(2-Hydroxyphenethyl)-2-methylfuran-3-carboxamide 5
To a solution of N-(2-hydroxyphenethyl)-2-methylfuran-3-carboxamide 6 (25 mg, 0.09
mmol, 1.0 eq.) in CH2Cl2 (500 µL) was added at −78 °C BBr3 (10.9 µL, 0.11 mmol, 1.2 eq.)
and the resulting mixture was stirred at −78 °C for 1 h before warming up to room
temperature. H2O was added and the resulting mixture was stirred for 15 min. The layers
were separated and the aqueous layer was extracted with CH2Cl2 (3 x 5 mL). The combined
organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude
material was purified by preparative reverse phase HPLC (SunFire column, acidic gradients
with MeCN-H2O containing 0.05 % TFA) to give 5 as a white solid (13.5 mg, 0.05 mmol,
57%).
1H (400 MHz, MeOD-d4) δ 7.33 (d, J = 1.6 Hz, 1H, H-1), 7.09 (t, J = 7.8 Hz, 1H, HAr),
6.70 (d, J = 8.0 Hz, 1H, HAr), 6.68 (br s, 1H, HAr), 6.64 – 6.62 (m, 1H, HAr), 6.62 (d, J =
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2.0 Hz, 1H, H-2), 3.49 (t, J = 7.6 Hz, 2H, H-3), 2.79 (t, J = 7.4 Hz, 2H, H-4), 2.50 (s, 3H,
Me), 1 NH and 1 OH signals missing.
7
8
9
13C (100 MHz, MeOD-d4) δ 160.4, 158.6, 157.7, 142.1, 141.7, 140.2, 130.5, 121.0, 116.7,
114.2, 109.1, 42.1, 36.7, 13.4.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
IR (film) 3322 (NH), 2200, 1663 (C=O), 1635 (C=C), 1524 (C=N), 1406, 1216, 1140 cm-1.
MS (ES+APCI) m/z 246 [M + H]+.
N'-(2-Hydroxybenzyl)-2-methylfuran-3-carbohydrazide 4b
To a solution of (Z)-N'-(2-hydroxybenzylidene)-2-methylfuran-3-carbohydrazide 3b (25 mg, 0.10
mmol, 1.0 eq.) in TFA (152 µL) at 0 °C was added Et3SiH (33 µL, 0.20 mmol, 2.0 eq.) and the
resulting mixture was stirred at 0 °C for 4 h. The crude mixture was diluted in toluene and
concentrated under reduced pressure. The crude material was purified by preparative reverse phase
HPLC (SunFire column, acidic gradients with MeCN-H2O containing 0.05 % TFA) to give 4b as a
white solid (20 mg, 0.08 mmol, 79%).
1H (400 MHz, MeOD-d4) δ 7.41 (s, 1H, H-1), 7.30 – 7.24 (m, 2H, HAr), 6.90 – 6.85 (m,
2H, HAr), 6.65 (s, 1H, H-2), 4.40 (s, 2H, H-3), 2.54 (s, 3H, Me), 2 NH and 1 OH signals
missing.
13C (100 MHz, MeOD-d4) δ 164.2, 160.2, 158.0, 142.5, 132.9, 132.2, 120.8, 118.6, 116.3,
113.5, 109.3, 52.2, 13.5.
IR (film) 3495 (NH), 3247 (OH), 2353, 1671 (C=O), 1606 (C=C), 1460 (C=N), 1235,
1200, 1138, 754 cm-1.
MS (ES+APCI) m/z 247 [M + H]+.
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N'-(2-Methoxybenzyl)-2-methylfuran-3-carbohydrazide 4c
4
5
6
7
8
9
To a solution of (Z)-N'-(2-methoxybenzylidene)-2-methylfuran-3-carbohydrazide 3c (22 mg, 0.08
mmol, 1.0 eq.) in TFA (126 µL) at 0 °C was added Et3SiH (27 µL, 0.20 mmol, 2.0 eq.) and the
resulting mixture was stirred at 0 °C for 4 h. The crude mixture was diluted in toluene and
concentrated under reduced pressure. The crude material was purified by preparative reverse phase
HPLC (SunFire column, acidic gradients with MeCN-H2O containing 0.05 % TFA) to give 4c as a
white solid (16.6 mg, 0.06 mmol, 75%).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1H (400 MHz, MeOD-d4) δ 7.42 (td, J = 7.6, 1.6 Hz, 1H), 7.41 (d, J = 2.0 Hz, 1H), 7.35
(dd, J = 7.6, 1.6 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.98 (td, J = 7.6, 1.2 Hz, 1H), 6.12 (d, J =
2.0 Hz, 1H), 4.36 (s, 2H), 3.90 (s, 3H), 2.53 (s, 3H), 2 NH signals missing.
13C (100 MHz, MeOD-d4) δ 164.1, 159.9, 159.8, 142.4, 132.9, 132.2, 121.8, 121.1, 113.7,
111.9, 109.2, 56.1, 51.9, 13.5.
IR (film) 3290, 1667, 1604, 1494, 1485, 1438, 1246, 1200, 1133, 1027, 753 cm-1.
MS (ES+APCI) m/z 261 [M + H]+.
(Z)-N'-(2-Methoxybenzylidene)-N,2-dimethylfuran-3-carbohydrazide 8
To a suspension of NaH (13.9 mg, 0.35 mmol, 1.2 eq.) in dry THF (1.0 mL) was added (Z)-
N'-(2-methoxybenzylidene)-2-methylfuran-3-carbohydrazide 3e (75 mg, 0.29 mmol, 1.0 eq.)
and the resulting mixture was heated under reflux for 1 h. CH3I (18.1 µL, 0.29 mmol, 1.0 eq.)
in THF (200 µL) was added and the reaction mixture was further heated under reflux for 1 h.
The crude mixture was cooled down to room temperature and concentrated under reduced
pressure. The crude material was purified by preparative reverse phase HPLC (SunFire
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column, acidic gradients with MeCN-H2O containing 0.05 % TFA) to give 8 as a white solid
(55.4 mg, 0.20 mmol, 70%).
7
8
9
1H (400 MHz, MeOD-d4) δ 8.21 (s, 1H), 7.46 (dd, J = 8.0, 2.0 Hz, 1H), 7.38 (d, J = 2.0 Hz,
1H), 7.36 (ddd, J = 8.4, 7.3, 1.9 Hz, 1H), 7.05 (dd, J = 8.4, 0.8 Hz, 1H), 6.94 (t, J = 7.4 Hz,
1H), 6.81 (d, J = 2.0 Hz, 1H), 3.91 (s, 3H), 3.48 (d, J = 0.4 Hz, 3H), 2.45 (s, 3H).
13C (100 MHz, MeOD-d4) δ 168.1, 159.7, 158.8, 140.6, 137.7, 132.2, 126.9, 124.4, 121.8,
116.8, 114.1, 112.5, 56.1, 28.7, 13.9.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
IR (film) 2917, 1647, 1601, 1413, 1333, 1246, 1620, 1017, 732 cm-1.
MS (ES+APCI) m/z 273 [M + H]+.
(Z)-N'-(2-Hydroxybenzylidene)-N,2-dimethylfuran-3-carbohydrazide 7
To a solution of (Z)-N'-(2-Methoxybenzylidene)-N,2-dimethylfuran-3-carbohydrazide 8
(18.9 mg, 0.06 mmol, 1.0 eq.) in CH2Cl2 (500 µL) at −78 °C was added BBr3 (7.8 µL, 0.08
mmol, 1.2 eq.) and the resulting mixture was stirred at −78 °C for 1 h before warming up to
room temperature. H2O was added and the resulting mixture was stirred for 15 min. The
layers were separated and the aqueous layer was extracted with CH2Cl2 (3 x 5 mL). The
combined organic layers were dried over Na2SO4 and concentrated under reduced pressure.
The crude material was purified by preparative reverse phase HPLC (SunFire column, acidic
gradients with MeCN-H2O containing 0.05 % TFA) to give 7 as a white solid (9.8 mg, 0.04
mmol, 55%).
1H (400 MHz, MeOD-d4) δ 8.20 (s, 1H, H-3), 7.46 (dd, J = 7.5, 2.0 Hz, 1H, HAr), 7.44 (d,
J = 2.0 Hz, 1H, H-1), 7.24 (ddd, J = 8.4, 7.5, 1.8 Hz, 1H, HAr), 6.90 (td, J = 7.8, 1.2 Hz, 1H,
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HAr), 6.82 (dd, J = 8.4, 0.8 Hz, 1H, HAr), 6.63 (d, J = 2.0 Hz, 1H, H-2), 3.25 (d, J = 0.4 Hz,
3H, N-Me), 2.39 (s, 3H, Me), 1 OH signal missing.
7
8
9
13C (100 MHz, MeOD-d4) δ 167.9, 158.3, 156.9, 144.6, 141.8, 132.3, 131.7, 120.7, 120.1,
117.4, 116.6, 112.3, 28.7, 13.8.
10
11
12
13
14
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16
17
18
19
20
21
22
23
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32
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34
35
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40
41
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
MS (ES+APCI) m/z 259 [M + H]+.
N'-(2-Hydroxybenzoyl)-2-methylfuran-3-carbohydrazide 11a
To a solution of salicylhydrazide (161.4 mg, 1.06 mmol, 1.0 eq.) in THF (5 mL) at 0 °C
was added Et3N (222 µL, 1.59 mmol, 1.5 eq.) and the resulting mixture was stirred at 0 °C for
15 min. To this mixture was added 2-methylfuran-3-carbonyl chloride (230 mg, 1.59 mmol,
1.0 eq.) in THF (1 mL) and the resulting mixture was stirred for 15 min at 0 °C, allowed to
warm up to room temperature and further stirred for 12 h. The crude mixture was diluted with
water and the aqueous layer was extracted with EtOAc. The combined organic layers were
dried over MgSO4 and concentrated under reduced pressure. The crude material was
triturated with MeOH. The solid was filtered off, washed with cold MeOH and dried under
reduced pressure to give 11a as a white solid (117 mg, 0.45 mmol, 42%).
1H (400 MHz, MeOD-d4) δ 7.88 (dd, J = 7.8, 1.6 Hz, 1H, HAr), 7.43 (ddd, J = 8.2, 7.3, 1.8
Hz, 1H, HAr), 7.42 (d, J = 2.1 Hz, 1H, H-1), 6.96 – 6.92 (m, 2H, HAr), 6.80 (d, J = 2.0 Hz,
1H, H-2), 2.57 (s, 3H, Me), 2 NH and 1 OH signals missing.
13C (100 MHz, MeOD-d4) δ 169.8, 165.4, 160.8, 159.4, 142.1, 135.3, 129.5, 120.4, 118.4,
115.9, 114.7, 109.7, 13.5.
IR (film) 3221 (NH), 3005 (OH), 1650 (C=O), 1632 (C=N), 1594, 1523, 1487, 1308, 1213,
1160, 729 cm-1.
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MS (ES+APCI) m/z 259 [M − H]+.
5
6
7
8
General procedure for the synthesis of analogs 11b–11d:
9
To a solution of 2-methylfuran-3-carbohydrazide 2 (1.0 eq.) in THF (5 mL) at 0 °C was
added Et3N (1.5 eq.) and the resulting mixture was stirred at 0 °C for 15 min. To this mixture
was added acid chloride derivative (1.0 eq.) in THF (1 mL) and the resulting mixture was
stirred for 15 min at 0 °C, allowed to warm up to room temperature and further stirred for 12
h. The crude mixture was diluted with water and the aqueous layer was extracted with
EtOAc. The combined organic layers were dried over MgSO4 and concentrated under
reduced pressure. The crude material was triturated with MeOH. The solid was filtered off,
washed with cold MeOH and dried under reduced pressure.
10
11
12
13
14
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17
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59
60
N'-(5-Chloro-2-hydroxybenzoyl)-2-methylfuran-3-carbohydrazide 11b
25%
1H (400 MHz, MeOD-d4) δ 7.90 (d, J = 2.8 Hz, 1H), 7.43 (t, J = 2.2 Hz, 1H), 7.41 (d, J =
2.8 Hz, 1H), 6.96 (d, J = 8.8 Hz, 1H), 2.0 Hz, 1H), 2.57 (s, 3H), 2 NH and 1 OH signals
missing.
IR (film) 3310, 1667, 1608, 1538, 1481, 1421, 1283, 1234, 1123, 824 cm-1.
MS (ES+APCI) m/z 293 [M − H]+.
N'-(2-Hydroxy-4-methoxybenzoyl)-2-methylfuran-3-carbohydrazide 11c
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16%
10
11
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44
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48
49
50
51
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60
1H (400 MHz, MeOD-d4) δ 7.44 (d, J = 3.2 Hz, 1H), 7.42 (d, J = 2.4 Hz, 1H), 7.05 (dd, J =
8.8, 3.2 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 6.80 (d, J = 1.6 Hz, 1H), 3.79 (s, 3H), 2.57 (s, 3H),
2 NH and 1 OH signals missing.
13C (100 MHz, MeOD-d4) δ 169.3, 165.3, 159.4, 154.6, 154.0, 142.1, 122.7, 119.3, 115.9,
114.7, 112.8, 109.7, 56.3, 13.6.
IR (film) 3218, 3003, 2358, 1745, 1635, 1590, 1300, 763 cm-1.
MS (ES+APCI) m/z 289 [M − H]+.
N'-(2-Hydroxy-3-methylbenzoyl)-2-methylfuran-3-carbohydrazide 11d
25%
1H (400 MHz, MeOD-d4) δ 7.64 (dd, J = 8.0, 0.8 Hz, 1H), 7.42 (d, J = 2.0 Hz, 1H), 7.33 –
7.31 (m, 1H), 6.81 (t, J = 7.6 Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 2.57 (s, 3H), 2.23 (s, 3H), 2
NH and 1 OH signals missing.
13C (100 MHz, MeOD-d4) δ 171.8, 165.7, 160.8, 159.4, 142.1, 136.3, 128.0, 125.6, 119.4,
114.7, 113.7, 109.7, 15.8, 13.6
IR (film) 3212, 3011, 2358, 1743, 1626, 1607, 1505, 1298, 1099, 899 cm-1.
MS (ES+APCI) m/z 293 [M − H]+.
General procedure for the synthesis of analogs 12a–12d:
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