Solid phase synthesis of 1,3,4-oxadiazin-5 (6R)-one and 1,3,4-oxadiazol-2-one scaffolds from acyl hydrazides

Article abstract of DOI:10.1039/c4ob01883d

Solid phase synthesis of 1,3,4-oxadiazin-5(6R)-one and 1,3,4-oxadiazol-2-one scaffolds from resin-bound acyl hydrazides is described. We demonstrate here that the reactions of resin-bound aryl or hetero-aromatic acyl hydrazides with 2-substituted-2-bromoacetic acids and 4-nitrophenyl chloroformate and subsequent treatment with DIEA lead to intramolecular cyclization reactions to produce six-membered 1,3,4-oxadiazin-5(6R)-ones and five-membered 1,3,4-oxadiazol-2-ones, respectively. We also show that acyl hydrazide-derived 1,3,4-oxadiazol-2-ones may be useful serine hydrolase inhibitors. This journal is

Full text of DOI:10.1039/c4ob01883d

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Solid phase synthesis of 1,3,4-oxadiazin-5
(6R)-one and 1,3,4-oxadiazol-2-one scaffolds
from acyl hydrazides†
Cite this: DOI: 10.1039/c4ob01883d
Received 3rd September 2014,
Accepted 22nd October 2014
Bani Kanta Sarma,a Xiaodan Liu,b Hao Wu,b Yu Gaob and Thomas Kodadek*b
DOI: 10.1039/c4ob01883d
www.rsc.org/obc
Solid phase synthesis of 1,3,4-oxadiazin-5(6R)-one and 1,3,4-oxa-
diazol-2-one scaffolds from resin-bound acyl hydrazides is
described. We demonstrate here that the reactions of resin-bound
aryl or hetero-aromatic acyl hydrazides with 2-substituted-2-bromo-
acetic acids and 4-nitrophenyl chloroformate and subsequent
treatment with DIEA lead to intramolecular cyclization reactions to
produce six-membered 1,3,4-oxadiazin-5(6R)-ones and five-mem-
bered 1,3,4-oxadiazol-2-ones, respectively. We also show that acyl
hydrazide-derived 1,3,4-oxadiazol-2-ones may be useful serine
hydrolase inhibitors.
To facilitate the rapid and inexpensive discovery of bioactive
compounds, several laboratories have focused on the develop-
ment of large libraries of oligomeric compounds displayed on
hydrophilic TentaGel beads.1–6 These can be screened for
ligands to a protein of interest at a fraction of the cost of a
functional, automated high-throughput screen. However, the
chemical diversity of these libraries is limited by the avail-
ability of reactions efficient enough to produce the extremely
high yields needed for the production of high quality libraries
by split and pool solid-phase synthesis. In particular, hetero-
cyclic compounds, which are so commonly found in bioactive
small molecules, are underrepresented in such libraries.7,8
Here we report facile methods for the solid-phase synthesis of
six-membered 1,3,4-oxadiazin-5-one and five-membered 1,3,4-
oxadiazol-2-one rings (Fig. 1A) using exceedingly efficient reac-
tions suitable for the construction of combinatorial libraries
by split and pool synthesis. These units are found in several
bioactive molecules9–17 and the latter is a source of serine
hydrolase (SH) inhibitors.18–22 For example, compound 7600
Fig. 1 Structures and function of the target molecules of this study. (A)
General structures of the two types of heterocycles created here. (B)
Structures of two 1,3,4-oxadiazol-2-ones reported to be inhibitors of
serine hydrolases.
and CAY10499 (Fig. 1B) are potent inhibitors of hormone-
sensitive lipases (HSLs) that function via a covalent, but reversible,
mechanism.19,20,23,24
This work proceeded from our recent observation that
2-substituted-1,3,4-oxadiazin-5(6H)-ones (R = H in Fig. 1A) were
often formed as a side product when resin-bound N-bromo-
acylated aryl or hetero-aromatic acyl hydrazides were treated
with a primary amine.25,26
While this was originally encountered as an unwanted side
reaction during the synthesis of N-azapeptoid chains, a useful
synthesis of 2-substituted-1,3,4-oxadiazin-5(6H)-one-capped
oligomers was effected by simply replacing the primary amine
with diisopropylethylamine (DIEA), which provided exclusively
the cyclic product (Fig. 1A; R = H).26 Since enantiomerically
pure 2-bromo carboxylic acids are readily available from amino
acids,27 this chemistry should provide access to a variety of
chiral, 2,6-disubstituted 1,3,4-oxadiazin-5-ones. To test this idea,
the peptoid sequence (Nbsa-Nmea-Nmea) [Nbsa = 4-(2-amino-
aDepartment of Chemistry, School of Natural Sciences, Shiv Nadar University, Dadri,
Uttar Pradesh-201314, India
bDepartments of Chemistry and Cancer Biology, Scripps Research Institute,
Scripps Florida, 130 Scripps Way, #3A2, Jupiter, Fl 33458, USA.
E-mail: kodadek@scripps.edu
†Electronic supplementary information (ESI) available: Detailed experimental
procedures, HPLC chromatograms, MALDI TOF MS spectra and NMR spectra of
compounds. See DOI: 10.1039/c4ob01883d
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ethyl)benzenesulfonamide; Nmea = 2-methoxyethylamine] was carbodiimide (DIC) (10 equiv.) in DMF at 37 °C for 1 h. The
synthesized on the solid support using the standard peptoid beads were washed and then treated with a 1 M DIEA solution
sub-monomer synthesis28 (Fig. 2A). After acylation with in N-methylpyrrolidinone (NMP) for ∼15 hours at 60 °C, which
bromoacetic acid, the terminal bromide was displaced with led to efficient formation of the 6-membered oxadiazinone
different aryl and hetero-aromatic acyl hydrazides using our ring-containing compounds 6–13 (Fig. 2A), as evidenced by
previously reported method.25 The resin-bound acyl hydrazides their HPLC traces and mass spectra (Fig. 2B and ESI, Fig. S7†).
were then treated with different chiral 2-substituted-2-bromo-
Several efficient synthetic routes for the solution phase
acetic acids (1–5) (10 equiv.) in the presence of diisopropyl- synthesis of 1,3,4-oxadiazol-2-one moieties are known in the
Fig. 2 Solid-phase synthesis of chiral, 6-substituted 2-aryl-1,3,4-oxadiazin-5-ones. (A) Synthetic scheme. (B) HPLC trace of crude compound 6
after release from the solid support.
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literature.29–31 To incorporate 1,3,4-oxadiazol-2-one moieties screened for reversible covalent inhibitors that derive high
on the solid phase, we followed a similar sequence of reactions selectivity for a particular SH through a combination of the
as discussed above for compounds 6–13. After incorporating a 1,3,4-oxadiazol-2-one ring coupling with the active site and
peptoid linker (Nbsa-Nmea-Nmea) and different acyl hydra- the oligomer binding to a surface nearby that might be
zides, the acyl hydrazide-bound resin was treated with 4-nitro- unique to the particular SH of interest. However, the com-
phenyl chloroformate (5 equiv.) in the presence of DIEA (7 pounds that have been well-characterized in this regard, such
equiv.) in dichloromethane (DCM) at room temperature for as compound 7600, contain the heterocycle fused directly to
3 hours (Fig. 3A). The beads were washed with DMF and DCM a phenyl ring, whereas our chemistry most naturally places
and then treated with 1 M DIEA for ∼15 hours at 60 °C, which an acetamide group on the C-terminal side of the ring. There-
resulted in the highly efficient formation of the 5-membered fore, we set out to evaluate whether this acetamide-containing
oxadiazolone ring-containing compounds (14–18) (purity unit can serve as a general unit for the inhibition of SHs. For
>95%; see Fig. 3B and the ESI, Fig. S8†).
this purpose, we set out to synthesize compounds 19–36
As mentioned above, the 1,3,4-oxadiazol-2-one ring can (Fig. 4) using the method discussed above. All were obtained
function as the core unit of a reversible covalent inhibitor of in excellent yield and purity with the exception of 36,
SHs. A pan-selective, moderate activity covalent modifier of which places a methoxy group at the 5-position as in com-
SHs is of interest to us for use as a “warhead” with which to pound 7600 and CAY 10499. In this case, unfortunately, the
cap libraries of oligomers. These libraries could then be only product obtained was 37, indicating that an oxygen
Fig. 3 Solid-phase synthesis of 1,3,4-oxadiazol-2-ones. (A) Synthetic scheme. (B) HPLC trace of crude compound 14 after release from the solid
support.
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Fig. 4 Structures of various 1,3,4-oxadiazol-2-ones prepared. Com-
pound 36 was not obtained from the Fig. 3 protocol. Instead, only com-
pound 37 was obtained.
Fig. 5 Inhibition of SHs by 1,3,4-oxadiazol-2-ones. (A) ABPP Gel-based
inhibition assay of compounds 19–35 using a HeLa cell lysate. The lysate
(1 mg mL−1 total protein) was incubated with 100 μM of each compound
(19–35) for 30 minutes. Rhodamine fluorophosphonate (FP-Rh) (100
nM) was then added and the reaction mixture was incubated for
substituent at the 5-position strongly inhibits the ring-closing
reaction.
To evaluate the activity of these molecules, they were ana- 10 minutes before being quenched with
a denaturing buffer. The
samples were subjected to SDS-PAGE and the rhodamine-labeled
protein bands were visualized using a Typhoon gel scanner. (B) A similar
experiment as in part A was conducted except that five purified, recom-
binant SH enzymes (2 µM each) were employed in place of the cell
lysate. In this case the compounds were employed at a concentration of
lysed using an activity-based protein-profiling (ABPP) gel-based
assay32 in which the compound being tested was asked to
inhibit coupling of SH enzymes in a HeLa cell lysate to a fluore-
scently-labelled fluorophosphonate.33 Thus, a diminution of
labelling represents engagement of the SH active site with the 200 µM.
compound. Since these species may be reversible covalent
inhibitors, they were pre-incubated with the enzymes for
lead compounds could yet arise from this collection or similar
30 minutes prior to addition of the labelled fluorophos-
phonate, after which the reactions were allowed to proceed for
10 minutes before being quenched. Control experiments
demonstrated that this was well within the linear range of the
labelling reaction (not shown). As shown in Fig. 5A, none of
the compounds except 35 had a noticeable effect on the SHs
that can be visualized by ABPP in this assay at a concentration
of 100 µM. 35 reduced the activity of most of the visible SHs by
about 50% at this concentration. To complement these data,
we also analyzed the effect of compounds 19–35 (200 µM) on
five purified SHs using the same assay. Again, compound 35
inhibited all the five of the SHs studied (Fig. 5B). Many of the
compounds had no effect on any of the enzymes, but several
inhibited FAM108b selectively, particularly compound 28.
In summary, we have developed efficient protocols for the
solid-phase synthesis of oligomers terminated with diverse
2-R1-1,3,4-oxadiazin-5(6R)-one and 2-R1-1,3,4-oxadiazol-2-one
scaffolds. Several of the latter class of molecules were tested
for SH inhibitor activity using an ABPP assay, revealing com-
pound 35 as a weak, pan-selective SH inhibitor (Fig. 5) All of
the other compounds studied failed to show significant activity
at 200 µM with the SHs analysed, except for some that inhib-
ited FAM108b (Fig. 5B). Of course, there are a great many SHs
that have yet to be tested against this suite of compounds, so
species. Future work will focus on the creation of complex
combinatorial libraries capped with these species, which may
provide a source of highly selective SH inhibitors.
Acknowledgements
This work was supported by a grant from the NIH (GM090294).
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