Synthesis, X-ray Structure Determination, and Comprehensive Photochemical Characterization of (Trifluoromethyl)diazirine-Containing TRPML1 Ligands

Article abstract of DOI:10.1021/acs.joc.0c02993

Potential (trifluoromethyl)diazirine-based TRPML1 ion channel ligands were designed and synthesized, and their structures were determined by single-crystal X-ray diffraction analysis. Photoactivation studies via 19F NMR spectroscopy and HPLC-MS analysis revealed distinct kinetical characteristics in selected solvents and favorable photochemical properties in an aqueous buffer. These photoactivatable TRPML activators represent useful and valuable tools for TRPML photoaffinity labeling combined with mass spectrometry.

Full text of DOI:10.1021/acs.joc.0c02993

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Article
Synthesis, X‑ray Structure Determination, and Comprehensive
Photochemical Characterization of (Trifluoromethyl)diazirine-
Containing TRPML1 Ligands
Kevin Schwickert, Michał Andrzejewski, Simon Grabowsky, and Tanja Schirmeister*
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ABSTRACT: Potential (trifluoromethyl)diazirine-based
TRPML1 ion channel ligands were designed and synthesized,
and their structures were determined by single-crystal X-ray
diffraction analysis. Photoactivation studies via 19F NMR spec-
troscopy and HPLC-MS analysis revealed distinct kinetical
characteristics in selected solvents and favorable photochemical
properties in an aqueous buffer. These photoactivatable TRPML
activators represent useful and valuable tools for TRPML
photoaffinity labeling combined with mass spectrometry.
INTRODUCTION
■
Mucolipidosis type IV (MLIV) is an autosomal recessive
lysosomal storage disease, which is clinically characterized by
severe neurodegenerative defects, profound cognitive impair-
ment, and eye abnormalities (e.g., corneal opacity, strabismus,
and progressive retinopathy).1−4 These hallmark symptoms are
often accompanied by other manifestations such as constitutive
achlorhydria and iron deficiency anemia.5,6 MLIV patients also
feature accumulated lysosomal inclusions containing different
lipids and proteins that appear in almost all tissues, as
demonstrated by electron microscopy.7−9 MLIV is caused by
mutations in the MCOLN1 gene, encoding mucolipin-1, a
member of the transient receptor potential mucolipin (TRPML)
cation channel subfamily.1,10,11 TRPML1 is a nonselective
cation channel consisting of a six-transmembrane helix core and
a large (∼100 kDa) extracytosolic/luminal domain (ELD) that
is exposed to the lysosomal lumen or the extracytosolic side of
the cell.12−15 The main function of the TRPML1 channel is the
Ca2+ ion transfer from the lysosome lumen to the cytoplasm, and
its activity is influenced by different Ca2+ concentrations and pH
values, therefore, playing a crucial role in vesicular transport,
exocytosis, and autophagy.13,14,16 Currently, there are no specific
treatment options for MLIV patients, particularly for those
carrying mutations in the MCOLN1 gene that result in complete
TRPML1 absence in cells.3,17 Chen et al. identified a new small-
molecule agonist of TRPML1 (MK6−83 (1), Figure 1),
demonstrating its restoring effect on specific TRPML1 channel
mutant isoforms and its ability to improve defects in
endolysosomal trafficking as well as heavy metal ion homeo-
stasis.17
Figure 1. Structures of known TRPML channel agonists (see references
for details).17−19
These findings suggest that the TRPML1 activator MK6−83
may serve as a basis for a small-molecule treatment approach
addressing a specific subgroup of MLIV patients.17 However, the
exact location of the binding site of MK6−83 could not yet be
determined, and currently, only cryo-EM structures of TRPML1
and TRPML3, respectively, bound with the nonselective and
(structurally) different agonist (ML-SA1 (2), Figure 1)17,19 are
available.20,21 It is not unambiguously clarified if MK6−83
addresses the same binding site within the channel and which
key interactions are underlying to explain its different TPRML
selectivity and activation profile compared to ML-SA1.22 For
rational structure-based design and development of new drug
Received: December 22, 2020
Published: April 9, 2021
https://doi.org/10.1021/acs.joc.0c02993
© 2021 American Chemical Society
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Figure 2. Design of new photoaffinity probes based on the known TRPML1 activator MK6−83.
a
Scheme 1. Initial Synthesis Route for the Introduction of the Piperidine Ring
a
Reagents and conditions: (a) ICl, CaCO3, MeOH/H2O, rt, overnight, quant; (b) Ac2O, 1,4-dioxane/H2O, 0 °C → rt, overnight, 99%.
molecules, an in-depth understanding of ligand-target recog-
nition and the interaction between the receptor protein and its
small-molecule ligand is essential.23,24 In the past decades,
photoaffinity labeling (PAL) has emerged as a very powerful tool
in medicinal chemistry and chemical biology for studying ligand-
target interactions and to provide additional information on the
binding pocket and the amino acid residues involved in drug
binding.25−30 This valuable technique exploits the intrinsic
property of ligands featuring a photoreactive group (PRG) to
produce a highly reactive species (e.g., a carbene), which, after
the formation of a particular noncovalent ligand−receptor
complex, covalently and irreversibly binds to proximate residues
within the binding site during photolysis by irradiation with a
specific wavelength of light.25,26,29,31 In this study, we focused on
the synthesis as well as structural determination of novel
(trifluoromethyl)diazirine-containing TRPML1 activators and
extensively investigated their photochemical insertion reactivity
in different solvents. These stable and useful photoactivatable
analogues of MK6−83 may allow the identification of the
binding site within the TRPML1 channel and thus make a
relevant contribution to the elucidation of the molecular
mechanisms involved in the specific ligand-protein interaction
between the TRPML1 activator and its target ion channel.
ties for the PAPs: (i) the incorporated photoreactive group
(PRG) should be as small as possible, minimizing the risk of
significantly altering the biological activity of the parent
molecule; (ii) the modified activator should exhibit relative
chemical stability in the dark at room temperature; (iii) cross-
linking should be specific and possible at higher wavelengths
(∼360 nm), reducing the risk of damage to targeted proteins.
Based on this discussion, the frequently used azido group was
not considered since the shorter wavelengths required for
excitation (λ < 300 nm) can cause significant damage to
biological molecules, and the nitrene intermediates and
rearranged ketenimines formed during photolysis can result in
decreased photoaffinity yields and nonspecific labeling.31−33
Consequently, we envisioned that the introduction of a
(trifluoromethyl)diazirine group would meet our criteria as it
represents a relatively small PRG among all commonly used
photophores. This keeps the structural changes of the parent
molecule to a minimum, and its physicochemical properties are
not substantially altered (contrary to, e.g., benzophenones).31,32
Furthermore, diazirines generate highly reactive carbenes upon
photoactivation with longwave (∼360 nm) UV light, and their
selective cross-linking property reduces the risk of nonspecific
binding due to the short lifetime of the reactive intermedi-
ate.28,31,32,34 Their relative chemical stability (e.g., under acidic
and basic conditions, toward nucleophiles and electrophiles)
enables easy handling and performance of demanding synthetic
transformations.26,32,35
RESULTS AND DISCUSSION
■
Design of the Photoaffinity Probes (PAPs). In designing
new photoaffinity probes (PAPs) for cross-linking experiments
with TRPML1 channels, we used the known TRPML1 activator
MK6−83 as the parent molecule.17 We started our design with
general considerations and requirements regarding the proper-
We decided to use the pre-existing phenyl ring of the parent
ligand MK6−83 as the most suitable substitution site for PRGs
(Figure 2) since structural modifications such as chlorine at the
5′-position of the aromatic core only slightly reduced efficacy on
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TRPML1 activation whereas larger substituents at the
thiophene ring and other residues than piperidine affected the
activity significantly.17 In addition, the central orientation of the
reactive group within the whole molecule should facilitate cross-
linking with a residue of the potential binding site. We further
assumed that generating two constitution isomers of the
diazirine-containing PAPs, in which the PRG is attached at
different positions along the aromatic core, would provide an
opportunity to perform binding site mapping and thus to
achieve an increased topological resolution of the cavity.31,35
NaHMDS43 as a strong base for the introduction of the Boc
group was applied to obtain 13b. Our initial synthetical
approach by this route involved the use of an acetyl protecting
group, which was introduced in one step in a reduction and
acetylation reaction starting from the nitro derivative 11a using
zinc dust in a mixture of glacial acid and acetic anhydride.
Unfortunately, the harsh acid or basic conditions required for its
deprotection led to the decomposition of the diazirinyl ring
(data not shown). Therefore, we selected the Boc group as a
comparatively more labile protective group, which proved to be
well suited for the established synthesis route since no ring
opening was observed during removal. For the preparation of
the key intermediate (trifluoromethyl)ketones 14a and 14b, the
bromo derivatives 13a and 13b were subjected to a
trifluoroacetylation reaction, which was achieved via NH-
deprotonation with potassium hydride44 at 0 °C, followed by
a subsequent halogen-metal exchange with BuLi at −78 °C
and treatment of the in situ formed organolithium compound
with 2,2,2-trifluoro-N-methoxy-N-methylacetamide (9, pre-
pared from trifluoracetic anhydride and N,O-dimethylhydroxyl-
amine hydrochloride46,47 (Scheme 3A)). The deprotonation
step in the trifluoroacetylation sequence was crucial since it
turned out that the halogen-metal exchange competed with NH-
abstraction, leading to a significant reduction in yield due to the
formation of the corresponding dehalogenated side-product.
The trifluoroacetylated compounds 14a and 14b were then
converted to oximes 15a and 15b with hydroxylamine
hydrochloride in pyridine/ethanol under reflux, which were
isolated as a mixture of E/Z-diastereomers (88% and 96% yields,
respectively) and subsequently tosylated with tosyl chloride
under basic conditions at 0 °C. The obtained O-tosyl oximes
were immediately used for the next step without further
purification because of their instability on silica and warming
during evaporation. The construction of the (trifluoromethyl)-
diazirine moiety on the aromatic core was realized by reaction of
the tosylated oximes with liquid ammonia at −78 °C to give
diaziridines 16a and 16b in 99% and 86% yields, respectively,
which were then oxidized with iodine/NEt3 in CH2Cl2 to
provide the photoreactive diazirines 17a and 17b. Deprotection
of the amino group using HCl (4.0 M in 1,4-dioxane) went
smoothly without affecting the diazirine ring, and the hydro-
chlorides 18a and 18b were isolated in quantitative yields.
Formation of the sulfonamide with 5-methylthiophene-2-
sulfonyl chloride in pyridine at 0 °C completed the synthesis,
and the final photoaffinity probes were obtained in good yields
(63% and 72%, respectively) and high purity (≥99%) as
confirmed by HPLC-MS analysis (Figures S1 and S2). The
synthesized compounds retain their stability even after several
months of storage in the dark at −21 °C under an argon
atmosphere. No decomposition of 3a and 3b could be observed,
providing both, the 4′- and 5′-(trifluoromethyl)diazirine-
substituted analogue, as stable and easy-to-handle reagents for
photoaffinity labeling purposes.
SYNTHESIS
■
Our initial approach to the target diazirines involved the
introduction of the piperidine residue in iodoaniline 6 via a
copper- or palladium-catalyzed N-arylation reaction.36−38 The
latter compound was synthesized in a sequence including
selective ortho-monoiodination with iodine monochloride39 and
acetylation using acetic anhydride and catalytic amounts of
sulfuric acid (Scheme 1).40
45
t
Unfortunately, multiple attempts to realize the amination
reaction of 6 were not satisfactory, as low conversion rates
associated with the formation of complex product mixtures were
observed in each case. Further optimization of this reaction was
not undertaken because 4 represented an expensive reactant,
and a different synthesis route was developed simultaneously
that was much more attractive from an economic point of view
and in terms of scalability. Retrosynthetic analysis in this
alternative approach identified 4- and 5-bromo-2-fluoro-1-
nitrobenzenes as inexpensive and commercially readily available
starting materials, thus bypassing transition metal-catalyzed N-
arylation in favor of nucleophilic aromatic substitution to
introduce the required piperidine ring (Scheme 2).
Scheme 2. Retrosynthetic Analysis for the Synthesis of
a
Diazirine 3a
a
FGI: Functional group interconversion.
Therefore, the final multistep syntheses of the new photo-
affinity probes MK6−83PRG1 (3a) and MK6−83PRG2 (3b)
started from commercially available 4- or 5-bromo-2-fluoro-1-
nitrobenzenes, in which a piperidine ring was introduced by the
application of a nucleophilic aromatic substitution using
Cs2CO3 as a base to give 11a and 11b in high yields (Scheme
3B).41
The nitro derivatives 11a and 11b were then reduced to
provide the corresponding anilines 12a and 12b using
SnCl2·2 H2O.41 Boc-protection of 12a was accomplished with
Boc2O in the presence of the Lewis acid Zn(ClO4)2·6 H2O as
the catalyst.42 Since this procedure was not suitable for the
protection of 12b due to longer reaction times and side reactions
such as biscarbamoylation, an alternative method using
With the HPLC-MS analysis, we also investigated the
fragmentation pattern of the synthesized diazirines to evaluate
their stability under electrospray ionization (ESI) MS/MS
conditions and to identify potential fragments in mass
spectrometry experiments that might occur after the photo-
affinity labeling procedure with proteins. For both compounds, a
high abundance of the parent ion [M + H]+ (m/z = 445.1) was
observed with only a minor amount of a fragment ion (m/z =
417) resulting from the loss of molecular nitrogen (Δm/z = 28)
(Figures S5 and S6, Schemes S1 and S2). Under collision-
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a
Scheme 3. Final Synthesis of the (Trifluoromethyl)diazirine-substituted TRPML1 Activators
a
Reagents and conditions: [A] (a) N,O-dimethylhydroxylamine hydrochloride, py, CH2Cl2, 0 °C, 2 h, 64%; [B] (a) piperidine, Cs2CO3, DMF, rt,
overnight, 99%; (b) SnCl2·2 H2O, EtOAc, rt, 1 h−overnight, 93−96%; (c) 12a Boc2O, Zn(ClO4)2·6 H2O (5 mol %), CH2Cl2, 45 °C, 10 h, 83%;
t
12b Boc2O, NaHMDS (1.9 M in THF), THF, rt, overnight, 60%; (d) (1) KH (30% suspension in mineral oil), 0 °C, 1 h; (2) BuLi (1.6 M in
pentane), 5, −78 °C, 6 h, 66−77%; (e) NH2OH·HCl, py/EtOH, 80 °C, 4−5 h, 88−96%; (f) (1) TsCl, Et3N, acetone; 0 °C, 3 h; (2) NH3 (l),
MTBE, −78 °C → rt, overnight, 86−99%; (g) I2, Et3N, CH2Cl2, 0 °C → rt, 2 h, 82−95%; (h) HCl (4.0 M in 1,4-dioxane), rt, 1−2 h, quant; (i) 5-
methylthiophene-2-sulfonyl chloride, py, 0 °C → rt, overnight, 63−72%.
induced dissociation (CID) conditions (MS/MS), the pre-
cursor ion was completely converted to the above-mentioned
fragment ion, which in the case of constitution isomer 3a was
accompanied by trace amounts of another product ion (4%, m/z
= 255.1) as a consequence of additional loss of the 5-
methylthiophene-2-sulfonyl moiety or cleavage of the entire
sulfonamide residue without extrusion of nitrogen (3%, m/z =
268.1). Fragmentation of diazirine 3b, on the other hand,
resulted in a concurrent appearance of a different product ion
with approximately 60% abundance (m/z = 240.0), in which the
whole sulfonamide fragment was presumably dissociated in
addition to the loss of nitrogen. The high abundance of the
precursor ions in the LC-MS mode, as well as the occurrence of
defined product ions under application of moderate collision
energy, indicates that the (trifluoromethyl)diazirines 3a and 3b,
respectively, exhibit outstanding structural integrity and stability
under ESI-MS conditions, which facilitates the identification of
labeled protein residues or amino acids after photoaffinity
labeling.
Figure 3. Crystal structures (ORTEP) of the studied compounds 3a
and 3b. Thermal ellipsoids are displayed with 50% probability.
Table 1. Selected Crystallographic Data for 3a and 3b
parameter
a (Å)
b (Å)
c (Å)
3a (MK6−83PRG1
)
3b (MK6−83PRG2
)
14.8659(2)
7.68040(10)
17.72280(10)
101.6910(10)
1981.54(4)
4
15.34810(10)
8.43370(10)
16.1087(2)
111.7130(10)
1937.19(4)
4
X-RAY STRUCTURE DETERMINATION
■
β (°)
Single-crystal X-ray diffraction data of 3a and 3b were collected
at 100 K on a Rigaku SuperNova diffractometer with MoKα
radiation using an Eos CCD detector. The structures were
solved and refined inside Olex248 with the SHELXT49 and the
SHELXL50 software, respectively (Figure 3 and Figure S4). Both
compounds crystallize in the monoclinic P21/c space group.
Selected crystallographic data for 3a and 3b are listed in Table 1.
Full experimental and crystallographic data are in Table S1.
In the context of quantum-crystallographic electron-density
research, the inter- and intramolecular bonding of biologically
active vinyl sulfone groups has been studied in detail.51,52
volume (Å3)
Z
ρcalc (g cm−3
)
1.490
1.524
Rint
R1
0.0362
0.0457
0.0543
0.0527
wR2
0.1372
0.1544
Moreover, the motif of an aziridine three-membered ring in
protease inhibitor model compounds has been scrutinized by
experimental quantum crystallography.53,54 Therefore, we had a
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Figure 4. Hirshfeld surfaces of (A) 3a and (B) 3b color coded with the property dnorm. The color range is −0.1773 (red) to 0 (white) to 1.4995 (blue).
Red dots represent short atom−atom contacts, and the symmetry-generated interaction partners outside the Hirshfeld surfaces are given.
Displacement ellipsoids and spheres are given at a 50% probability level. The pictures were generated with the software CrystalExplorer (see reference
for details).59
Scheme 4. Photoactivation Reaction of Diazirines 3a and 3b in Different Solvents
closer look at the three-membered diazirine ring motif here in
comparison to the aziridine motif, although disorder and
reduced resolution of the data prevented an electron-density
analysis (Figure S4). The N−N bond is quite short with
1.228(3)/1.218(2) Å in 3a/b, and the two C−N bonds are long,
being just below 1.50 Å. In the aziridine three-membered ring,
the C−N bonds are significantly shorter at 1.45 Å.53,54 The C−
N−N angles are between 65−66° and the N−C−N angle below
49° in both compounds 3a/b leading to an acute triangle,
whereas, in aziridine, all three angles are around 60°. These
geometric details point toward an asymmetric distribution of
electron density in the three-membered ring, an accumulation of
electron density in the N−N bond, and hence a different
reactivity than aziridine-containing biologically active com-
pounds.
to get an overall picture of the intermolecular interactions that
dominate the crystal packing in 3a and 3b, respectively (Figure
4; red dots highlight close intermolecular interactions). In 3a,
there is only a C−H···O hydrogen bond involving the sulfonyl
group, but the N−H group does not act as a hydrogen bonding
donor (Figure 4 (A)). Instead, there is a remarkably short
inverse diaziridine-CF3 intermolecular contact, basically a N···F
interaction between two electron-rich atoms, which deserves
further study in the context of recent discussions around halogen
and pnictogen bonding.58
In contrast, the crystal packing in 3b is dominated by the more
usual inverse N−H···Osulfonyl bonding motif one might expect
with a N−H hydrogen bonding donor group present in the
structure. Obviously, this N−H group is more accessible for
intermolecular interactions because of the meta- instead of para-
substitution of the phenyl ring in 3b. The diaziridine-CF3
contact is present in 3b, too, but less pronounced, and there is
no C−H···O hydrogen bond either. In summary, the difference
in substitution leads to significantly different crystal packings in
Since it is known that geometrical and electron-density
features of biologically active compounds in small-molecule
crystal structure packings can resemble those in biological
ensembles,55,56 we employed Hirshfeld Surface Analysis57 here
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Figure 5. 19F NMR spectra of a 5 mM solution of 3a in methanol-d4 (A) and cyclohexane-d12 (B) upon irradiation with UV light (λ = 365 nm) recorded
at different irradiation intervals. Spectra are shifted for better visualization, and signals of the diazirine, linear diazo isomer, and the corresponding
insertion product are highlighted in red, blue, and gray, respectively (see Scheme 4). For 19F NMR spectra of the photoinduced decay of the
constitution isomer 3b, see Figure S7 .
Figure 6. Photoinduced decay of diazirine 3a (c = 2 mM; λ = 365 nm) in methanol (A) and cyclohexane (B) investigated via HPLC (C18). Aliquots
were collected and analyzed at different time intervals of UV activation. The resulting peak areas (UV detection at λ = 254 nm) of the diazirine (red,
circles), linear diazo compound (blue, triangles), and the insertion product (gray, squares) in the chromatograms were integrated and plotted against
the irradiation time after normalization. Diazirine turnover and product formation can be fitted by first-order exponential functions, while the evolution
and subsequent decay of the linear diazo isomer can be expressed by a biexponential trend line.
Figure 7. Potentially different influence of the substitution pattern on the carbene stability in 20a and 20b explaining the heterogeneous reactivity and
product formation times of both constitution isomers.
both compounds, which might have consequences on their
activities.
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Figure 8. Investigation of the photoinduced decay of diazirines 3a (A) and 3b (B) (c = 50 μM; λ = 365 nm) in aqueous buffer (50 mM TRIS, 150 mM
NaCl, 2 mM CaCl2, 5 mM KCl, 5 mM MgCl2, 4 mM EDTA, and 250 mM sucrose) via reversed-phase HPLC. Aliquots were collected and analyzed at
different time intervals of UV activation. The resulting peak areas (UV detection at λ = 254 nm) of the diazirine (red, circles), linear diazo compound
(blue, triangles), and the insertion product (gray, squares) in the chromatograms were integrated and plotted against the irradiation time after
normalization. Diazirine turnover as well as the product formation can be fitted by first-order exponential functions, while the evolution and
subsequent decay of the linear diazo isomer can be expressed by a biexponential trend line.
results proved that diazirines 3a and 3b even readily undergo
rapid photolysis in a nonpolar environment suggesting that the
insertion reaction can also take place with hydrophobic amino
acid residues (e.g., leucine or valine) within the binding site. For
a more quantitative description and physicochemical character-
ization, the photochemical reactions of (trifluoromethyl)-
diazirines 3a and 3b in methanol and cyclohexane (c = 2 mM)
were additionally followed by HPLC-MS analysis. Since
photoaffinity labeling experiments are commonly accomplished
in aqueous media, we also investigated the photochemical
properties of these compounds in an aqueous buffer solution (c
= 50 μM), e.g., to define the reaction conditions required for
high-yield cross-linking of biological samples. Aliquots from the
irradiation experiment were taken after fixed periods of time and
subjected to LC-MS analysis, and the resulting peak areas (UV
detection at λ = 254 nm) of the diazirines 3a and 3b, linear diazo
compounds 19a/b, and the insertion products 23ab−25ab were
integrated. As expected, the obtained results agreed well with the
observations of the 19F NMR spectroscopy data (vide supra)
since similar irradiation times were required for the complete
photolysis of the starting material and the normalized integrated
peak areas confirmed the recorded signal intensities in the 19F
NMR experimental setup (Figure 6 and Figure S8).
KINETICAL CHARACTERIZATION OF THE
PHOTOCHEMICAL REACTION OF 3A AND 3B
■
Next, we investigated the kinetical photoactivation behavior of
the new TRPML photoaffinity probes in different media.34,60−62
Solutions of 3a and 3b in the appropriate (deuterated) solvent
(methanol(-d4), cyclohexane(-d12), and aqueous buffer as
carbene scavengers) were exposed to UV light (λ = 365 nm)
̈
delivered by a high-power UV LED (Opsytec Dr. Grobel
GmbH, Germany) for different irradiation intervals, 1H and 19
F
NMR, respectively, as well as HPLC-MS spectra were recorded
to monitor the course of the photoinduced diazirine decay (see
Scheme 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure S7, and
Figure S8). First, the photolysis reactions were performed in
deuterated methanol and cyclohexane (c = 5 mM), respectively,
and the signal changes of the fluorine signals (CF3 group) were
followed by 19F NMR spectroscopy at various time intervals.
Upon irradiation with UV light, diazirines 3a and 3b (δ = −68
ppm) completely underwent photolysis in methanol within 48
min (3a) and 78 min (3b) to form either the insertion products
21a/b (δ = −79 ppm) or the linear diazo isomers 19a/b (δ =
−60 ppm) (Figure 5 and Figure S7) as a result of photo-
isomerization which is in competition with the appearance of the
singlet carbene.
The observed photolytic characteristics and chemical shifts
are in accordance with studies and spectral data reported for
other aromatic (trifluoromethyl)diazirines.34,60,62 The linear
diazo compound was converted to the methyl ether on further
UV light exposure, as indicated by the subsequent decrease of
the corresponding 19F NMR signal under continued irradiation.
Remarkably, the photoactivation kinetics of 3a and 3b in
cyclohexane-d12 strikingly differed from the experiments in
methanolic solution since complete decay of the diazirines was
already observed after 30 s, while the lifetime of the linear diazo
compound relative to the diazirines proved to be significantly
longer (full conversion after 960 and 1320 s for 3a and 3b,
respectively). This deviation can be explained by different
abilities of the solvents to exert a stabilizing effect on the
intermediate formed singlet carbene. In several studies, it could
be shown that polar solvents like methanol preferably stabilize
the singlet carbene by the interaction of the empty p orbital with
nonbonding electrons of the solvent.63,64 Nevertheless, our
Parallel controls proved that the diazirines remained
unaffected in the absence of a light source (data not shown).
In all solvents, approximately first-order kinetics were obtained
for the photolytic diazirine decomposition, which can be
described by a typical exponential decay (eq 1).
N(t) = N0 × e−kt
(1)
The photoactivation half-life t1/2 and the time constant τ of the
diazirine derivatives 3a and 3b were calculated from the
resulting rate constant k according to eq 2 and are listed in Table
2.
ln(2)
k
t1/2
=
= τ × ln(2)
(2)
In cyclohexane and aqueous buffer, diazirine decomposition
over the course of light exposure occurred immediately within a
few seconds (t1/2 7−9 s), whereas prolonged irradiation times
were required in methanol (t1/2 12 and 34 min for 3a and 3b,
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sized. These MK6−83 analogues exhibited favorable phys-
icochemical properties since they efficiently underwent clean
photolysis to the desired insertion products, as demonstrated in
detailed photoactivation studies in various solvents. Further-
more, fast diazirine decay associated with short irradiation times
required in aqueous buffer additionally proves their suitability
for photoaffinity labeling procedures. They were also shown to
be stable during long-term storage at low temperatures in the
absence of light, which highlights their value as useful and
important photoreactive analogues of the TRPML1 activator
MK6−83. In their crystal structures, the differences in the
substitution pattern led to significant differences in the crystal
packing pattern involving an unusual diazirine-CF3 close
contact. Future investigations exploiting the advantageous
properties of the photoreactive analogues presented here will
provide detailed insights into TRPML1 modulation by the
synthetic agonist MK6−83. Biological functional analysis and
photoaffinity labeling experiments in combination with a
proteomics mass spectrometry approach can thus make a
significant contribution to elucidating the binding site within the
TRPML1 channel. This knowledge could be very important for
the structure-based design of new TRPML activators and helpful
to further explore the mechanism of TRPML activation by
synthetic molecules.
Table 2. Calculated Photoactivation Half-Lives and Time
Constants of the Synthesized Diazirines in Different
a
Solvents
b
methanol
cyclohexane
aqueous buffer
compound
t1/2 (min)
τ (min)
t1/2 (s)
τ (s)
t1/2 (s)
τ (s)
3a
3b
12
34
18
49
8
7
11
9
10
9
14
12
a
Distance from UV light source (λ = 365 nm): 2 cm (see
b
Experimental Section for details). c = 50 μM in 50 mM TRIS, 150
mM NaCl, 2 mM CaCl2, 5 mM KCl, 5 mM MgCl2, 4 mM EDTA, and
250 mM sucrose.
respectively), which is consistent with the results of the NMR
experiments. Interestingly, the curves and half-lives of diazirines
3a and 3b in methanolic solution illustrated substantial kinetical
differences indicating distinct photolytic properties between
both constitution isomers, which were not observed in
cyclohexane or aqueous solution. Presumably, the correspond-
ing photochemical properties of the diazirines 3a and 3b can be
attributed to a different stabilization of the intermediate
carbenes: the empty p orbital in the case of 20b can additionally
be stabilized by the electron-donating piperidine moiety in the
para position (Figure 7). In contrast, the electron deficiency in
20a cannot be compensated to the same extent, resulting in
higher reactivity and thus faster insertion. The carbene-
stabilizing effect is probably especially enhanced by the
methanolic solvent but is not as pronounced in the other
solvents since the reactive intermediates are directly scavenged
by solvent molecules. Similar observations have already been
reported in which substituent and solvent effects on carbene
reactions explained their different reactivity.64−67 The higher
reactivity of the carbenes toward water compared to methanol
can presumably be explained by the higher acidity and the
extended hydrogen bonding network of water.68
EXPERIMENTAL SECTION
■
Chemistry. All solvents and reagents were obtained from
commercial suppliers (Sigma-Aldrich, Acros Organics, Alfa Aesar,
TCI, or Apollo Scientific) and used without any prior purification, if not
stated otherwise. Anhydrous methyl tert-butyl ether, tetrahydrofuran,
and 1,4-dioxane were freshly distilled from sodium and benzophenone.
Dichloromethane was dried by distillation from calcium hydride.
Reactions under anhydrous conditions were performed under an argon
atmosphere using flame-dried glassware. Unless otherwise mentioned,
heating was carried out with an oil bath if needed. Reactions at −78 °C
were conducted in a dry ice/acetone cooling bath. Purifications by flash
chromatography were performed on silica gel (0.015−0.040 mm,
Machery-Nagel). Reaction progress was monitored by thin-layer
chromatography (TLC) using Machery-Nagel Alugram Xtra Sil G/
UV254 silica 60 plates. Compound visualization on these plates was
attained by radiation at 254 nm or by staining with Ehrlich’s reagent
(prepared from 4-(N,N-dimethylamino)benzaldehyde (1.6 g) and
concentrated HCl (80 mL) in methanol (120 mL)).
In an aqueous buffer, both modified TRPML activators
cleanly underwent photolysis at 365 nm within 90 s, and product
formation was completed after 360 s including a fast decay of the
linear diazo compound (Figure 8).
The short irradiation times required, combined with a
temporarily short-lived formation of the undesired linear diazo
compound, demonstrated efficient photoactivation of
(trifluoromethyl)diazirines 3a and 3b, respectively, highlighting
their suitability for photoaffinity labeling in an aqueous
environment since long irradiation periods exhibit an increased
risk of unspecific labeling (pseudoaffinity labeling) or even
damage of biological systems.31,32 These results also reveal the
ability of water to quench the highly reactive carbenes
immediately, which further contributes to minimizing unspecific
labeling: only molecules in a close environment to the binding
site of the protein will react covalently with amino acid residues,
whereas free and unbound molecules are directly scavenged by
the aqueous medium.32 In all photolysis experiments, no
characteristic products arising from hydrogen atom abstraction
(such as reduced derivatives of 3a and 3b) were observed,
suggesting that the involvement of triplet carbene intermediates
is rather unlikely. Indeed, it was previously noted that most of
the products developed during photolysis of aromatic diazirines
result from a singlet carbene state.69,70
1H and 13C NMR spectra were recorded on a Bruker Fourier 300, a
Bruker Avance-III HD (1H NMR, 300 MHz; 13C{1H} NMR, 75.5
MHz; 19F NMR, 282 MHz), or a Bruker Avance-II (1H NMR 400
MHz; 13C{1H} NMR 100.6 MHz; 19F NMR 376 MHz). Two-
dimensional NMR experiments (COSY, HSQC, and HMBC) were
used for assignments. All chemical shifts are referenced to the signal of
the residual solvent (CDCl3, 7.26 and 77.16 ppm; DMSO-d6, 2.50 and
39.52 ppm; methanol-d4, 3.31 and 49.00 ppm; cyclohexane-d12, 1.38
and 26.43 ppm for 1H NMR and 13C NMR, respectively) and reported
in parts per million (ppm) relative to tetramethylsilane (TMS). For 19F
NMR, chemical shifts are given in ppm relative to C(35Cl)2(37Cl)F. The
spectrometer was calibrated with α,α,α-trifluorotoluene in CDCl3
(−63.9 ppm). Multiplicities of the corresponding NMR signals are
given using the following abbreviations: br = broad, s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, and combinations
thereof. Infrared (IR) spectra were recorded on an FT-IR spectrometer
(Avatar, Thermo-Nicolet) with an ATR correction and are reported in
terms of frequency of absorption ν [cm−1].
̅
Analytical HPLC analysis was performed on an HP Agilent 1100
series HPLC system using an Agilent Poroshell 120 EC-C18 (150 × 2.10
mm, 4 μm) column at 40 °C oven temperature and a detection
wavelength of 254 nm. The mobile phase consisted of mixtures of
acetonitrile, Milli-Q-grade water, and 10% of a 0.1% solution of formic
acid in Milli-Q-grade water and was adapted to each separation problem
CONCLUSION
Two modified TRPML1 activators featuring a photoreactive
(trifluoromethyl)diazirine moiety were designed and synthe-
■
6176
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The Journal of Organic Chemistry
pubs.acs.org/joc
Article
(injection volume = 5−20 μL; flow rate = 0.5−0.7 mL min−1).
Electrospray ionization (ESI) mass spectra were recorded on an Agilent
1100 series LC/MSD ion trap spectrometer in the positive ion mode
(drying gas temperature = 350 °C, nebulizer pressure = 70 psi, capillary
voltage = 3500 V, drying gas (N2) = 12 L min−1). The purity of the final
compounds was confirmed by HPLC analysis and was higher than 95%
in all cases. Semipreparative HPLC was performed on a Varian PrepStar
system using an Agilent Zorbax PrepHT XDB C18 (150 mm × 21.2 mm,
5 μm) column at a detection wavelength of 254 nm. The mobile phase
consisted of mixtures of acetonitrile and Milli-Q-grade water and was
adapted to each separation problem (injection volume = 1−5 mL, flow
rate = 10−20 mL min−1). High-resolution masses (ESI-MS) were
recorded using an Agilent 6545 Q-TOF-MS instrument with a suitable
external calibrant. Melting points were determined in open capillaries
stirring overnight at room temperature, the reaction was quenched by
the addition of a KF (aq) solution (1 M, 100 mL) and stirred further 30
min. The mixture was extracted with EtOAc (2 × 250 mL). The
combined organic layers were washed with a KF (aq) solution (1 M, 1 ×
100 mL) and a solution of NaCl (aq) (saturated, 2 × 100 mL) and dried
over Na2SO4, and the solvent was removed under reduced pressure.
The title compound was obtained as a yellow-brown solid (7.65 g, 29.98
mmol, 93% yield), which was used without any further purification. Rf =
0.50 (SiO2, cHex/EtOAc = 5:1). Mp: 40−41 °C. 1H NMR, COSY (300
MHz, DMSO-d6): δ 6.98−6.89 (m, 2H, H-3Ph and H-5Ph), 6.70−6.60
(m, 1H, H-6Ph), 4.91 (s (br), 2H, NH2), 2.76−2.67 (m, 4H, 2 × H-2Pip
and 2 × H-6Pip), 1.70−1.57 (m, 4H, 2 × H-3Pip and 2 × H-5Pip), 1.56−
1.44 (m, 2H, 2 × H-4Pip) ppm. 13C{1H} NMR, HSQC, HMBC (75.5
MHz, DMSO-d6): δ 141.6 (C-2Ph), 139.9 (C-1Ph), 126.1 (C-5Ph), 121.9
(C-3Ph), 115.7 (C-6Ph), 107.0 (C-4Ph), 51.6 (C-2Pip and C-6Pip), 26.0 (C-
on a Kruss-Optronic KSP 1 N apparatus (heating rate = 1 °C/min).
̈
3Pip and C-5Pip), 23.7 (C-4Pip) ppm. IR (ATR): ν 3423, 3330, 2936,
X-ray diffraction was measured at 100 K on a Rigaku SuperNova
diffractometer with a CCD Eos detector using monochromated Mo Kα
(λ = 0.71073 Å) radiation. Single crystals suitable for crystallography
were obtained by recrystallization from a CH2Cl2 solution of diazirines
3a and 3b, respectively, with vapor diffusion of petroleum ether at room
temperature.
̅
1601, 1487, 1438, 1272, 1226, 1029, 929, 805 cm−1. HRMS (ESI-
+
QTOF) m/z: calcd for C11H16BrN2 [M + H]+, 255.0491/257.0471;
found, 255.0489/257.0468.
tert-Butyl [4-bromo-2-(piperidin-1-yl)phenyl]carbamate (13a).
This compound was synthesized according to a method by Bartoli et
al.42 To a solution of 4-bromo-2-(piperidin-1-yl)aniline (12a, 6.79 g,
26.61 mmol, 1.00 equiv) in CH2Cl2 (50 mL) were added Boc2O (8.71
g, 39.91 mmol, 1.50 equiv) and Zn(ClO4)2·6 H2O (496 mg, 1.33 mmol,
0.05 equiv). The mixture was stirred at 45 °C for 5 h in an oil bath, and
then an additional portion of Boc2O (8.71 g, 39.91 mmol, 1.50 equiv)
was added and stirred for further 5 h at 45 °C. After stirring overnight at
room temperature, water was added (100 mL), and the aqueous phase
was extracted with CH2Cl2 (3 × 100 mL). The combined organic
extracts were dried over Na2SO4 and concentrated under reduced
pressure. After purification by flash chromatography on silica gel (cHex/
EtOAc = 80:1), the title compound was obtained as a light-yellow solid
(7.85 g, 22.10 mmol, 83% yield). Rf = 0.53 (SiO2, cHex/EtOAc = 25:1).
Mp: 89−91 °C. 1H NMR, COSY (300 MHz, DMSO-d6): δ 7.74 (d, 3J =
8.6 Hz, 1H, H-6Ph), 7.72 (s, 1H, NH), 7.28−7.17 (m, 2H, H-3Ph and H-
5Ph), 2.72 (pseudo-t, J ≈ 5.2 Hz, 4H, 2 × H-2Pip and 2 × H-6Pip), 1.74−
1.58 (m, 4H, 2 × H-3Pip and 2 × H-5Pip), 1.58−1.49 (m, 2H, 2 × H-4Pip),
1.46 (s, 9H, C(CH3)3) ppm. 13C{1H} NMR, HSQC, HMBC (75.5
MHz, DMSO-d6): δ 152.3 (CO), 144.7 (C-2Ph), 132.4 (C-1Ph), 126.8
(C-5Ph), 123.5 (C-3Ph), 120.7 (C-6Ph), 114.7 (C-4Ph), 79.8 (C(CH3)3),
52.6 (C-2Pip and C-6Pip), 27.9 (C(CH3)3), 26.0 (C-3Pip and C-5Pip), 23.4
Note that all photolabile compounds were stored in the dark under
an argon atmosphere at −21 °C to prevent decomposition.
2,2,2-Trifluoro-N-methoxy-N-methylacetamide (9). This com-
pound was prepared according to reported procedures.46,47 To a
mixture of N,O-dimethylhydroxylamine hydrochloride (7.90 g, 80.99
mmol, 1.05 equiv) and trifluoroacetic anhydride (10.9 mL, 77.14 mmol,
1.00 equiv) in CH2Cl2 (200 mL) was added pyridine (18.5 mL, 229.2
mmol, 2.97 equiv) dropwise at 0 °C. The resulting mixture was stirred
for 2 h at 0 °C and then quenched with water (50 mL). The organic
layer was washed with water (1 × 50 mL), 1 M HCl (aq) (2 × 100 mL),
and a solution of NaCl (aq) (saturated, 1 × 100 mL) and dried over
Na2SO4, and the solvent was removed under reduced pressure to afford
the title compound (7.74 g, 49.27 mmol, 64% yield) as a colorless oil,
1
which was used for the next reaction without further purification. H
NMR (300 MHz, CDCl3): δ 3.70 (s, 3H, OCH3), 3.22 (s, 3H, NCH3)
ppm. 13C{1H} NMR (75.5 MHz, CDCl3): δ 156.8 (q, 2JCF = 37.3 Hz,
1
CF3C(O)), 116.2 (q, JCF = 286.4 Hz, CF3), 62.1 (OCH3), 32.7
(NCH3) ppm. IR (ATR): ν 1701, 1257, 1211, 1152, 1082, 983, 896,
̅
745, 662 cm−1.
The analytical data are consistent with those reported in the
literature.47
(C-4Pip) ppm. IR (ATR): ν 3360, 2934, 1714, 1502, 1446, 1396, 1245,
̅
1146, 1022, 925, 833 cm−1. HRMS (ESI-QTOF) m/z: calcd for
1-(5-Bromo-2-nitrophenyl)piperidine (11a). This compound was
synthesized according to a modified procedure by Yin et al.41 To a
mixture of 4-bromo-2-fluoro-1-nitrobenzene (8.00 g, 36.36 mmol, 1.00
equiv) and Cs2CO3 (15.04 g, 46.17 mmol, 1.27 equiv) in DMF (100
mL) was added piperidine (3.8 mL, 38.18 mmol, 1.05 equiv) at room
temperature. After stirring overnight, the solvent was evaporated under
reduced pressure, and the residue was dissolved in EtOAc (100 mL)
and water (100 mL). The aqueous phase was extracted with EtOAc (3
× 100 mL), and the combined organic phases were dried over Na2SO4
and concentrated under reduced pressure to obtain the title compound
(10.29 g, 36.09 mmol, 99% yield) as an orange solid. Rf = 0.65 (SiO2,
+
C16H24BrN2O2 [M + H]+, 355.1016/357.0995; found, 355.1010/
357.0991.
tert-Butyl [2-(Piperidin-1-yl)-4-(2,2,2-trifluoroacetyl)phenyl]-
carbamate (14a). Under an argon atmosphere, a solution of 13a
(4.69 g, 13.20 mmol, 1.00 equiv) in anhydrous THF (25 mL) was
added dropwise to a suspension of KH (30% suspension in mineral oil,
2.64 g, 19.80 mmol, 1.50 equiv) in anhydrous THF (75 mL) at 0 °C.
After 1 h at 0 °C, the reaction mixture was cooled down to −78 °C, and
a solution of tBuLi (1.6 M in pentane, 17.3 mL, 27.72 mmol, 2.10 equiv)
was carefully added dropwise via cannula. After 45 min, 2,2,2-trifluoro-
N-methoxy-N-methylacetamide (9, 4.15 g, 26.40 mmol, 2.00 equiv)
was added and stirring was continued for 6 h at −78 °C. The reaction
was quenched by the addition of a solution of NH4Cl (aq) (saturated,
20 mL). Water (50 mL) and EtOAc (100 mL) were added, the phases
were separated, and the aqueous layer was extracted with EtOAc (3 ×
100 mL). The combined organic phases were dried over Na2SO4, and
the solvent was removed under reduced pressure. After purification by
flash chromatography on silica gel (cHex/EtOAc = 80:1), the title
compound was obtained as a yellow solid (3.78 g, 10.15 mmol, 77%
1
cHex/EtOAc = 10:1). Mp: 60−61 °C. H NMR, COSY (300 MHz,
CDCl3): δ 7.63 (d, 3J = 8.7 Hz, 1H, H-3Ph), 7.22 (d, 4J = 2.0 Hz, 1H, H-
6Ph), 7.03 (dd, 3J = 8.7, 4J = 2.0 Hz, 1H, H-4Ph), 3.06−2.97 (m, 4H, 2 ×
H-2Pip and 2 × H-6Pip), 1.76−1.65 (m, 4H, 2 × H-3Pip and 2 × H-5Pip),
1.65−1.53 (m, 2H, 2 × H-4Pip) ppm. 13C{1H} NMR, HSQC, HMBC
(75.5 MHz, CDCl3): δ 147.8 (C-1Ph), 140.7 (C-2Ph), 128.1 (C-5Ph),
127.7 (C-3Ph), 123.9 (C-6Ph), 123.3 (C-4Ph), 52.8 (C-2Pip and C-6Pip),
25.8 (C-3Pip and C-5Pip), 23.9 (C-4Pip) ppm. IR (ATR): ν 2934, 2852,
̅
1591, 1548, 1498, 1332, 1295, 1231, 1135, 1033 cm−1. MS (ESI): m/z
calcd for [C11H13BrN2O2+H]+ ([M + H]+), 285.0/287.0; found, m/z =
285.2 (90%, [M + H]+)/287.0 (100%, [M + H]+).
c
1
yield). Rf = 0.42 (SiO2, Hex/EtOAc = 10:1). Mp: 110−111 °C. H
3
NMR, COSY (300 MHz, DMSO-d6): δ 8.19 (d, J = 8.7 Hz, 1H, H-
6Ph), 8.05 (s, 1H, NH), 7.80−7.72 (m, 2H, H-3Ph and H-5Ph), 2.76
(pseudo-t, J ≈ 5.2 Hz, 4H, 2 × H-2Pip and 2 × H-6Pip), 1.82−1.64 (m,
4H, 2 × H-3Pip and 2 × H-5Pip), 1.64−1.53 (m, 2H, 2 × H-4Pip), 1.50 (s,
9H, C(CH3)3) ppm. 13C{1H} NMR, HSQC, HMBC (75.5 MHz,
The analytical data are consistent with those reported in the
literature.71
4-Bromo-2-(piperidin-1-yl)aniline (12a). This compound was
synthesized according to a modified procedure by Yin et al.41 To a
solution of 11a (9.24 g, 32.40 mmol, 1.00 equiv) in EtOAc (250 mL)
was added SnCl2·2 H2O (24.80 g, 97.20 mmol, 3.00 equiv). After
2
DMSO-d6): δ 177.4 (q, JCF = 34.0 Hz, CF3C(O)), 151.3 (CO),
141.8 (C-2Ph), 140.8 (C-1Ph), 127.8 (C-5Ph), 122.7 (C-4Ph), 121.8 (C-
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3Ph), 116.4 (q, 1JCF = 292.1 Hz, CF3), 116.3 (C-6Ph), 80.7 (C(CH3)3),
52.8 (C-2Pip and C-6Pip), 27.7 (C(CH3)3), 26.0 (C-3Pip and C-5Pip), 23.2
(C-4Pip) ppm. 19F NMR (376 MHz, DMSO-d6): δ −71.12 (s, CF3)
(C-4Pip) ppm. 19F NMR (376 MHz, DMSO-d6): δ −74.98 (s, CF3)
ppm. IR (ATR): ν 3367, 2937, 2359, 1722, 1522, 1464, 1420, 1371,
̅
1215, 1146, 1095, 1027, 948, 831 cm−1. HRMS (ESI-QTOF) m/z:
calcd for C18H26F3N4O2+ [M + H]+, 387.2002; found, 387.2002.
tert-Butyl {2-(Piperidin-1-yl)-4-[3-(trifluoromethyl)-3H-diazirin-3-
yl]phenyl}carbamate (17a). Note that this reaction was performed in
the dark to avoid partial photolysis of the product (aluminum foil was
used to cover flasks and columns). To a solution of the diaziridine 16a
(1.00 g, 2.59 mmol, 1.00 equiv) in anhydrous CH2Cl2 (25 mL) was
added triethylamine (897 μL, 6.47 mmol, 2.50 equiv) and iodine (980
mg, 3.89 mmol, 1.50 equiv) at 0 °C. The reaction mixture was stirred 1
h at 0 °C and 1 h at room temperature. After complete conversion of the
starting material, the reaction was quenched by the addition of a
Na2S2O3 (aq) solution (1 m, 50 mL), and the aqueous layer was
extracted with CH2Cl2 (3 × 50 mL). The combined organic layers were
dried over Na2SO4, and the solvent was removed under reduced
pressure. After flash chromatography purification on silica gel (cHex/
EtOAc = 50:1), the title compound was obtained as a colorless solid
(948 mg, 2.47 mmol, 95% yield). Rf = 0.20 (SiO2, cHex/EtOAc = 50:1).
Mp: 46−48 °C. 1H NMR, COSY (300 MHz, DMSO-d6): δ 7.94 (d, 3J =
8.5 Hz, 1H, H-6Ph), 7.85 (s, 1H, NH), 7.02 (dd, 3J = 8.5, 4J = 1.8 Hz, 1H,
H-5Ph), 6.92 (d, 4J = 1.8 Hz, 1H, H-3Ph), 2.76−2.67 (m, 4H, 2 × H-2Pip
and 2 × H-6Pip), 1.72−1.59 (m, 4H, 2 × H-3Pip and 2 × H-5Pip), 1.57−
1.49 (m, 2H, 2 × H-4Pip), 1.47 (s, 9H, C(CH3)3) ppm. 13C{1H} NMR,
HSQC, HMBC (75.5 MHz, DMSO-d6): δ 152.2 (CO), 143.3 (C-
ppm. IR (ATR): ν 2936, 2359, 1738, 1697, 1593, 1519, 1442, 1223,
̅
1190, 1127, 1051, 908 cm−1. HRMS (ESI-QTOF) m/z: calcd for
C18H24F3N2O3+ [M + H]+, 373.1734; found, 373.1733.
tert-Butyl (E/Z)-{2-(Piperidin-1-yl)-4-[2,2,2-trifluoro-1-
(hydroxyimino)ethyl]phenyl}carbamate (15a). To a solution of 14a
(2.00 g, 5.37 mmol, 1.00 equiv) in pyridine/EtOH (37.5 mL, 2:1 v/v)
was added hydroxylamine hydrochloride (448 mg, 6.44 mmol, 1.20
equiv), and the mixture was stirred for 5 h at 80 °C in an oil bath. After
complete conversion of the starting material, the reaction mixture was
cooled, and the solvent was removed under reduced pressure. The
residue was dissolved in EtOAc (100 mL), washed with water (2 × 50
mL) and a solution of NaCl (aq) (saturated, 2 × 50 mL), and dried over
Na2SO4, and the solvent was removed under reduced pressure. After
purification by flash chromatography on silica gel (cHex/EtOAc = 8:1),
the title compound was obtained as a mixture of (E/Z)-diastereomers
(colorless solid, 1.84 g, 4.75 mmol, 88% yield). Rf = 0.33 and 0.36 (SiO2,
1
cHex/EtOAc = 5:1). Mp: 143−144 °C. H NMR, COSY (300 MHz,
DMSO-d6): δ 12.90 and 12.66 (2 × s, 1H, NOH, both isomers), 7.95
and 7.93 (2 × d, J = 8.5 Hz, 1H, H-6Ph, both isomers), 7.88 and 7.85 (2
× s, 1H, NH, both isomers), 7.32−7.14 (m, 2H, H-3Ph and H-5Ph), 2.73
(pseudo-q, J ≈ 4.9 Hz, 4H, 2 × H-2Pip and 2 × H-6Pip), 1.73−1.60 (m,
4H, 2 × H-3Pip and 2 × H-5Pip), 1.60−1.50 (m, 2H, 2 × H-4Pip), 1.48 (s,
9H, C(CH3)3) ppm. 13C{1H} NMR, HSQC, HMBC (75.5 MHz,
DMSO-d6): δ 152.2 (CO), 144.3 (q, 2JCF = 28.1 Hz, CF3CNOH, one
isomer), 144.0 (q, 2JCF = 30.8 Hz, CF3CNOH, other isomer), 142.4 and
142.2 (C-2Ph, both isomers), 134.80 and 134.76 (C-1Ph, both isomers),
124.9 (C-5Ph, one isomer), 124.7 (C-4Ph, one isomer), 124.5 (C-5Ph,
other isomer), 121.3 (q, 1JCF = 274.0 Hz, CF3, one isomer), 120.9 (C-
3Ph, one isomer), 120.7 (C-4Ph, other isomer), 120.4 (C-3Ph, other
isomer), 118.6 (q, 1JCF = 283.0 Hz, CF3, other isomer), 117.9 (C-6Ph),
80.04 and 80.00 (C(CH3)3, both isomers), 52.9 (C-2Pip and C-6Pip),
27.9 (C(CH3)3), 26.2 (C-3Pip and C-5Pip), 23.4 (C-4Pip) ppm. 19F NMR
(376 MHz, DMSO-d6): δ −62.80 and −65.58 (s, CF3, both isomers)
1
2Ph), 135.0 (C-1Ph), 122.8 (C-5Ph), 122.0 (q, JCF = 274.8 Hz, CF3),
121.6 (C-4Ph), 119.1 (C-6Ph), 118.4 (C-3Ph), 80.2 (C(CH3)3), 52.6 (C-
2Pip and C-6Pip), 28.0 (q, JCF = 39.8 Hz, Cqdiazirine), 27.9 (C(CH3)3),
2
26.1 (C-3Pip and C-5Pip), 23.4 (C-4Pip) ppm. 19F NMR (282 MHz,
DMSO-d6): − 65.88 (CF3) ppm. IR (ATR): ν 3357, 2932, 1723, 1584,
̅
1523, 1421, 1364, 1245, 1143, 1046, 979, 830 cm−1. HRMS (ESI-
+
QTOF) m/z: calcd for C18H24F3N4O2 [M + H]+, 385.1846; found,
385.1848.
2-(Piperidin-1-yl)-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]aniline
hydrochloride (18a). Note that this reaction was performed in the dark
to avoid partial photolysis of the product (aluminum foil was used to
cover flasks and columns). Under an argon atmosphere, diazirine 17a
(1.20 g, 3.12 mmol, 1.00 equiv) was submitted in a round-bottom flask
and a hydrogen chloride solution (4.0 M in 1,4-dioxane, 15 mL) was
added at room temperature. After stirring for 2 h, the solvent was
removed under reduced pressure and the residue dried in a fine vacuum
to obtain the title compound (998 mg, 3.11 mmol, quant) as a colorless
solid, which was used without further purification in the next step. Rf =
ppm. IR (ATR): ν 3346, 2940, 2360, 1720, 1701, 1523, 1369, 1238,
̅
1192, 1153, 1023, 964 cm−1. HRMS (ESI-QTOF) m/z: calcd for
C18H25F3N3O3+ [M + H]+, 388.1843; found, 388.1839.
tert-Butyl {2-(Piperidin-1-yl)-4-[3-(trifluoromethyl)diaziridin-3-
yl]phenyl}carbamate (16a). Under an argon atmosphere, to a solution
of the oxime 15a (1.22 g, 3.14 mmol, 1.00 equiv) in acetone (50 mL)
and triethylamine (1.3 mL, 9.42 mmol, 3.00 equiv) was added a
solution of p-toluenesulfonyl chloride (658 mg, 3.45 mmol, 1.10 equiv)
in acetone (5 mL) dropwise at 0 °C. After stirring for 3 h at 0 °C, the
solvent was removed under reduced pressure (no warming!) to give the
crude O-tosyl oxime as a yellow solid, which was immediately used for
the next step without further purification. Note that the product is not
stable upon warming and on silica, so it is crucial to avoid further
purification and warming during evaporation to obtain higher yields.
To a cooled Schlenk flask containing liquid ammonia (approximately
50 mL) under an argon atmosphere at −78 °C was added a suspension
of the crude O-tosyl oxime in anhydrous MTBE (25 mL) dropwise. The
mixture was stirred for 5 h at −78 °C and warmed to room temperature
overnight (side arm of the Schlenk flask opened to allow ammonia to
evaporate slowly). The resulting suspension was diluted with MTBE
(50 mL) and water (50 mL) and extracted with MTBE (3 × 50 mL).
The combined organic layers were dried over Na2SO4 and concentrated
under reduced pressure to obtain the diaziridine 16a as a colorless solid
(1.21 g, 3.13 mmol, 99% yield). Rf = 0.38 (SiO2, cHex/EtOAc = 5:1).
Mp: 122−123 °C. 1H NMR, COSY (300 MHz, DMSO-d6): δ 7.87 (d,
1H, J = 8.3 Hz, H-6Ph), 7.81 (s, 1H, NH), 7.30−7.21 (m, 2H, H-3Ph and
H-5Ph), 4.02 (d, J = 8.3 Hz, 1H, NHdiaziridine), 3.89 (d, J = 8.3 Hz, 1H,
NHdiaziridine), 2.79−2.68 (m, 4H, 2 × H-2Pip and 2 × H-6Pip), 1.74−1.60
(m, 4H, 2 × H-3Pip and 2 × H-5Pip), 1.59−1.50 (m, 2H, 2 × H-4Pip), 1.48
(s, 9H, C(CH3)3) ppm. 13C{1H} NMR, HSQC, HMBC (75.5 MHz,
DMSO-d6): δ 152.3 (CO), 142.4 (C-2Ph), 134.1 (C-1Ph), 126.4 (C-
c
1
0.41 (SiO2, Hex/EtOAc = 10:1). Mp: decomposition ≥ 200 °C. H
NMR, COSY (300 MHz, methanol-d4): δ 7.34−7.26 (m, 2H, H-3Ph
and H-5Ph), 7.14 (d, 3J = 8.3 Hz, 1H, H-6Ph), 3.54 (pseudo-t, J ≈ 5.6 Hz,
4H, 2 × H-2Pip and 2 × H-6Pip), 2.20−2.05 (m, 4H, 2 × H-3Pip and 2 ×
H-5Pip), 1.86−1.70 (m, 2H, 2 × H-4Pip) ppm. 13C{1H} NMR, HSQC,
HMBC (75.5 MHz, methanol-d4): δ 141.5 (C-1Ph), 132.0 (C-2Ph),
129.8 (C-5Ph), 123.5 (q, 1JCF = 273.7 Hz, CF3), 121.8 (C-3Ph), 121.3 (C-
6Ph), 121.2 (C-4Ph), 56.3 (C-2Pip and C-6Pip), 29.2 (q, 2JCF = 40.6 Hz,
CqDiazirin), 24.8 (C-3Pip and C-5Pip), 22.6 (C-4Pip) ppm. 19F NMR (376
MHz, methanol-d4): δ −68.72 (s, CF3) ppm. IR (ATR): ν 3418, 3154,
̅
2400, 1655, 1524, 1317, 1244, 1148, 1137, 997, 820 cm−1. HRMS (ESI-
+
QTOF) m/z: calcd for C18H24F3N4O2 [M + H]+, 285.1322; found,
285.1315.
5-Methyl-N-{2′-(piperidin-1-yl)-4′-[3-(trifluoromethyl)-3H-diazir-
in-3-yl]phenyl}thiophene-2-sulfonamide (MK6−83PRG1, 3a). Note
that this reaction was performed in the dark to avoid partial photolysis
of the product (aluminum foil was used to cover flasks and columns).
Under an argon atmosphere, 5-methylthiophene-2-sulfonyl chloride
(574 mg, 2.92 mmol, 1.00 equiv) was added dropwise to a solution of
18a (935 mg, 2.92 mmol, 1.00 equiv) in anhydrous pyridine (10 mL) at
0 °C. The mixture was stirred 1 h at 0 °C and at room temperature
overnight. The solvent was removed under reduced pressure and the
residue dissolved in EtOAc (100 mL). The organic layer was washed
with a NH4Cl (aq) solution (saturated, 1 × 50 mL) and a solution of
NaCl (aq) (saturated, 2 × 50 mL), dried over Na2SO4, and
concentrated under reduced pressure. After purification by column
chromatography on silica gel (cHex/EtOAc = 30:1), the title compound
1
4Ph), 124.6 (C-5Ph), 124.2 (q, JCF = 278.9 Hz, CF3), 120.5 (C-3Ph),
118.2 (C-6Ph), 79.9 (C(CH3)3), 57.1 (q, JCF = 34.8 Hz, Cqdiaziridine),
2
52.9 (C-2Pip and C-6Pip), 27.9 (C(CH3)3), 26.2 (C-3Pip and C-5Pip), 23.5
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was isolated as a light-yellow solid (824 mg, 1.85 mmol, 63% yield). In
general, no further purification was required in this step. However, if
necessary, preparative HPLC purification (C18, eluent A = Milli-Q-
grade water, eluent B = acetonitrile, isocratic (20:80) over 90 min with a
flow rate of 12.5 mL min−1 and detection at 254 nm) can be performed.
Rf = 0.15 (SiO2, cHex/EtOAc = 20:1). Mp: 92−94 °C. 1H NMR, COSY
(400 MHz, DMSO-d6): δ 9.07 (s, 1H, NH), 7.47 (d, 3J = 8.6 Hz, 1H, H-
6Ph),72 7.47 (d, 3J = 3.7 Hz, 1H, H-3Thio),72 7.03 (ddd, 3J = 8.6 Hz, 4J =
2.2 Hz, J = 1.0 Hz, 1H, H-5Ph), 6.89 (d, 4J = 2.2 Hz, 1H, H-3Ph), 6.86
(dd, 3J = 3.7 Hz, 4J = 1.1 Hz, 1H, H-4Thio), 2.59−2.52 (m, 4H, 2 × H-2Pip
and 2 × H-6Pip), 2.45 (d, 4J = 1.1 Hz, 3H, CH3), 1.67−1.57 (m, 4H, 2 ×
H-3Pip and 2 × H-5Pip), 1.52−1.42 (m, 2H, 2 × H-4Pip) ppm. 13C{1H}
NMR, HSQC, HMBC (100.6 MHz, DMSO-d6): δ 148.1 (C-2Thio),
145.6 (C-2Ph), 136.9 (C-5Thio), 133.8 (C-1Ph), 133.1 (C-3Thio), 126.4
(C-4Thio), 123.8 (C-4Ph), 122.8 (C-5Ph), 121.9 (q, 1JCF = 274.8 Hz, CF3),
HMBC (75.5 MHz, DMSO-d6): δ 144.3 (C-1Ph), 138.6 (C-2Ph), 121.0
(C-3Ph), 118.5 (C-4Ph), 116.0 (2C, C-5Ph and C-6Ph), 51.7 (C-2Pip and
C-6Pip), 26.1 (C-3Pip and C-5Pip), 23.9 (C-4Pip) ppm. IR (ATR): ν 3443,
̅
3343, 2932, 2794, 1593, 1495, 1379, 1229, 1200, 843, 795 cm−1. HRMS
+
(ESI-QTOF) m/z: calcd for C11H16BrN2 [M + H]+, 255.0491/
257.0471; found, 255.0493/257.0473.
tert-Butyl [5-Bromo-2-(piperidin-1-yl)phenyl]carbamate (13b).
This compound was synthesized according to a method by Kelly et
al.43 Under an argon atmosphere, a solution of NaHMDS (1.9 M in
THF, 25.0 mL, 47.02 mmol, 2.00 equiv) was added dropwise to a
solution of the aniline 12b (6.00 g, 23.51 mmol, 1.00 equiv) in
anhydrous THF (25 mL) at room temperature. After stirring for 15
min, a solution of Boc2O (5.13 g, 23.51 mmol, 1.00 equiv) in anhydrous
THF (5 mL) was added, and the reaction mixture was stirred overnight.
NH4Cl (aq) (saturated, 20 mL) was added to quench the reaction, the
solvent was removed under reduced pressure, and the aqueous layer was
extracted with EtOAc (3 × 100 mL). The combined organic layers were
dried over Na2SO4, concentrated under reduced pressure, and purified
by flash chromatography on silica gel (cHex/EtOAc = 80:1) to obtain
the title compound as a red oil, which crystallizes upon freezing (5.04 g,
120.7 (C-6Ph), 119.2 (C-3Ph), 52.8 (C-2Pip and C-6Pip), 27.9 (q, 2JCF
=
39.9 Hz, Cqdiazirine), 25.6 (C-3Pip and C-5Pip), 23.4 (C-4Pip), 15.1 (CH3)
ppm. 19F NMR (376 MHz, DMSO-d6): δ −65.79 (CF3) ppm. IR
(ATR): ν 2938, 2855, 1505, 1440, 1342, 1245, 1153, 1123, 1022, 905,
̅
823 cm−1. HRMS (ESI-QTOF) m/z: calcd for C18H20F3N4O2S2+ [M +
H]+, 445.0974; found, 445.0972. HPLC analysis: retention time = 2.51
min; peak area = 99.80%; eluent A = Milli-Q-grade water; eluent B =
CH3CN; eluent C = 0.1% solution of formic acid in Milli-Q-grade
water; isocratic (10:80:10) over 8 min with a flow rate of 0.7 mL min−1
and detection at 254 nm; injection volume = 5 μL; column temperature
= 40 °C.
c
14.19 mmol, 60% yield). Rf = 0.53 (SiO2, Hex/EtOAc = 20:1). Mp:
83−85 °C. 1H NMR, COSY (300 MHz, DMSO-d6): δ 8.03 (d, 4J = 2.3
Hz, 1H, H-6Ph), 7.80 (s, 1H, NH), 7.16 (dd, 3J = 8.5, 4J = 2.3 Hz, 1H, H-
4Ph), 7.10 (d, 3J = 8.5 Hz, 1H, H-3Ph), 2.69 (pseudo-t, J ≈ 5.2 Hz, 4H, 2 ×
H-2Pip and 2 × H-6Pip), 1.71−1.58 (m, 4H, 2 × H-3Pip and 2 × H-5Pip),
1.58−1.48 (m, 2H, 2 × H-4Pip), 1.47 (s, 9H, (C(CH3)3) ppm. 13C{1H}
NMR, HSQC, HMBC (75.5 MHz, DMSO-d6): δ 152.2 (CO), 141.8
(C-2Ph), 134.6 (C-1Ph), 125.3 (C-4Ph), 122.7 (C-3Ph), 120.6 (C-6Ph),
116.5 (C-5Ph), 80.2 (C(CH3)3), 52.8 (C-2Pip and C-6Pip), 27.9
(C(CH3)3), 26.1 (C-3Pip and C-5Pip), 23.4 (C-4Pip) ppm. IR (ATR): ν
1-(4-Bromo-2-nitrophenyl)piperidine (11b). This compound was
synthesized according to a modified procedure by Yin et al.41 To a
mixture of 4-bromo-1-fluoro-2-nitrobenzene (20.00 g, 90.91 mmol,
1.00 equiv) and Cs2CO3 (44.43 g, 136.37 mmol, 1.50 equiv) in DMF
(200 mL) was added piperidine (9.4 mL, 95.45 mmol, 1.05 equiv) at
room temperature. After stirring for 1 h, the solvent was evaporated
under reduced pressure, and the residue was dissolved in EtOAc (200
mL) and a NaCl (aq) solution (saturated, 200 mL). The aqueous phase
was extracted with EtOAc (3 × 200 mL), and the combined organic
layers were washed with a solution of NaCl (aq) (saturated, 1 × 200
mL), dried over Na2SO4, and concentrated under reduced pressure to
obtain the title compound (25.80 g, 90.87 mmol, 99% yield) as a bright
red oil, which crystallizes upon freezing to give red crystals. Rf = 0.49
̅
3443, 3343, 2932, 2814, 2358, 1718, 1593, 1495, 1437, 1228, 1026, 843,
796 cm−1. HRMS (ESI-QTOF) m/z: calcd for C16H24BrN2O2+ [M +
H]+, 355.1016/357.0995; found, 355.1016/357.0997.
tert-Butyl [2-(Piperidin-1-yl)-5-(2,2,2-trifluoroacetyl)phenyl]-
carbamate (14b). Under an argon atmosphere, a solution of 13b
(4.74 g, 13.34 mmol, 1.00 equiv) in anhydrous THF (25 mL) was
added dropwise to a suspension of KH (30% suspension in mineral oil,
2.68 g, 20.01 mmol, 1.50 equiv) in anhydrous THF (100 mL) at 0 °C.
After 1 h at 0 °C, the reaction mixture was cooled down to −78 °C and a
solution of tBuLi (1.9 M in pentane, 14.7 mL, 28.01 mmol, 2.10 equiv)
was carefully added dropwise via cannula. After 60 min, 2,2,2-trifluoro-
N-methoxy-N-methylacetamide (9, 4.19 g, 26.68 mmol, 2.00 equiv)
was added and stirring was continued for 6 h at −78 °C. The reaction
was quenched by the addition of a solution of NH4Cl (aq) (saturated,
25 mL). Water (50 mL) and EtOAc (100 mL) were added, the phases
were separated, and the aqueous layer was extracted with EtOAc (3 ×
100 mL). The combined organic phases were dried over Na2SO4, and
the solvent was removed under reduced pressure. After purification by
flash chromatography on silica gel (cHex/EtOAc = 30:1), the title
compound was obtained as a light-yellow solid (3.26 g, 8.76 mmol, 66%
yield). Note that the product may be in equilibrium with its hydrate
form (≈ 3.0−1.5:1, determined by 1H NMR spectroscopy). Rf = 0.18
(SiO2, cHex/EtOAc = 20:1). Mp: 106−107 °C. 1H NMR, COSY (300
MHz, DMSO-d6): δ 8.21 (d, 4J = 2.3 Hz, 1H, H-6Ph), 8.04 (s, 1H, NH),
7.73 (ddq, 3J = 8.6 Hz, 4J = 2.3 Hz, J = 1.4 Hz, 1H, H-4Ph), 7.22 (d, 3J =
8.6 Hz, 1H, H-3Ph), 3.03−2.93 (m, 4H, 2 × H-2Pip and 2 × H-6Pip),
1.80−1.62 (m, 4H, 2 × H-3Pip and 2 × H-5Pip), 1.62−1.52 (m, 2H, 2 ×
H-4Pip), 1.47 (s, 9H, C(CH3)3) ppm. 13C{1H} NMR, HSQC, HMBC
(75.5 MHz, DMSO-d6): δ 178.0 (q, 2JCF = 33.5 Hz, CF3C(O)), 152.8
(CO), 152.0 (C-2Ph), 131.4 (C-1Ph), 127.0 (C-4Ph), 123.9 (C-6Ph),
c
1
(SiO2, Hex/EtOAc = 20:1). Mp: 51−52 °C. H NMR, COSY (300
MHz, DMSO-d6): δ 7.98 (d, 4J = 2.5 Hz, 1H, H-3Ph), 7.69 (dd, 3J = 8.9
Hz, 4J = 2.5 Hz, 1H, H-5Ph), 7.23 (d, 3J = 8.9 Hz, 1H, H-6Ph), 3.01−2.89
(m, 4H, 2 × H-2Pip and 2 × H-6Pip), 1.65−1.45 (m, 6H, 2 × H-3Pip, 2 ×
H-5Pip and 2 × H-4Pip) ppm. 13C{1H} NMR, HSQC, HMBC (75.5
MHz, DMSO-d6): δ 145.2 (C-1Ph), 142.5 (C-2Ph), 136.1 (C-5Ph), 127.6
(C-3Ph), 123.3 (C-6Ph), 111.4 (C-4Ph), 52.1 (C-2Pip and C-6Pip), 25.4 (C-
3Pip and C-5Pip), 23.3 (C-4Pip) ppm. IR (ATR): ν 2933, 2814, 1598,
̅
1506, 1448, 1324, 1281, 1224, 1127, 1022, 828 cm−1. MS (ESI): m/z
calcd for [C11H13BrN2O2+H]+ ([M + H]+), 285.0/287.0; found, m/z =
285.1 (87%, [M + H]+)/287.0 (100%, [M + H]+).
The analytical data are consistent with those reported to the
literature.73
5-Bromo-2-(piperidin-1-yl)aniline (12b). This compound was
synthesized according to a modified procedure by Yin et al.41 To a
solution of 11b (10.00 g, 35.21 mmol, 1.00 equiv) in EtOAc (250 mL)
was added SnCl2·2 H2O (27.00 g, 105.63 mmol, 3.00 equiv) at room
temperature. After stirring for 1 h, the reaction was quenched by the
addition of a KF (aq) solution (2 M, 200 mL) and stirred further 30
min. The phases were separated, and the aqueous layer was extracted
with EtOAc (3 × 100 mL). The combined organic extracts were washed
with a KF (aq) solution (1 M, 1 × 100 mL) and a solution of NaCl (aq)
(saturated, 2 × 100 mL), dried over Na2SO4, and concentrated under
reduced pressure. The title compound was obtained as a yellow solid
(8.64 g, 33.86 mmol, 96% yield), which was used without any further
purification. Rf = 0.43 (SiO2, cHex/EtOAc = 20:1). Mp: 71−73 °C. 1H
NMR, COSY (300 MHz, DMSO-d6): δ 6.82 (d, 4J = 2.3 Hz, 1H, H-
6Ph), 6.76 (d, 3J = 8.3 Hz, 1H, H-3Ph), 6.63 (dd, 3J = 8.3, 4J = 2.4 Hz, 1H,
H-4Ph), 4.96 (s (br), 2H, NH2), 2.69 (pseudo-t, J ≈ 5.1 Hz, 4H, 2 × H-
1
122.4 (C-5Ph), 119.8 (C-3Ph), 116.7 (q, JCF = 292.0 Hz, CF3), 79.8
(C(CH3)3), 51.3 (C-2Pip and C-6Pip), 28.0 (C(CH3)3), 25.5 (C-3Pip and
C-5Pip), 23.6 (C-4Pip) ppm. 19F NMR (282 MHz, DMSO-d6): δ −71.13
(CF3) ppm. IR (ATR): ν 2945, 1731, 1699, 1599, 1527, 1442, 1239,
̅
1138, 1127, 1103, 917, 767, 734, 657 cm−1. HRMS (ESI-QTOF) m/z:
calcd for C18H24F3N2O3+ [M + H]+, 373.1734; found, 373.1732.
tert-Butyl (E/Z)-{2-(Piperidin-1-yl)-5-[2,2,2-trifluoro-1-
(hydroxyimino)ethyl]phenyl}carbamate (15b). To a solution of 14b
(3.06 g, 8.22 mmol, 1.00 equiv) in pyridine/EtOH (60 mL, 2:1 v/v)
was added hydroxylamine hydrochloride (685 mg, 9.86 mmol, 1.20
2
Pip and 2 × H-6Pip), 1.63 (pseudo-p, J ≈ 5.5 Hz, 4H, 2 × H-3Pip and 2 ×
H-5Pip), 1.55−1.43 (m, 2H, 2 × H-4Pip). 13C{1H} NMR, HSQC,
6179
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equiv), and the mixture was stirred for 4 h at 80 °C in an oil bath. After
complete conversion of the starting material, the reaction mixture was
cooled, and the solvent removed under reduced pressure. The residue
was dissolved in EtOAc (100 mL), washed with a solution of NH4Cl
(aq) (saturated, 1 × 50 mL), water (1 × 50 mL), and a solution of NaCl
(aq) (saturated, 1 × 50 mL), and dried over Na2SO4, and the solvent
was removed under reduced pressure. The title compound was
obtained as a mixture of (E/Z)-diastereomers (colorless solid, 3.07 g,
7.92 mmol, 96% yield). Rf = 0.32 (SiO2, cHex/EtOAc = 5:1). Mp: 69−
72 °C. 1H NMR, COSY (300 MHz, DMSO-d6): δ 12.95 and 12.67 (2 ×
s, 1H, NOH, both isomers), 7.98 and 7.95 (2 × d, 4J = 2.0 Hz, 1H, H-
6Ph, both isomers), 7.77 and 7.75 (2 × s, 1H, NH, both isomers), 7.21
and 7.18 (2 × d, 3J = 8.2 Hz, 1H, H-3Ph, both isomers), 7.15−7.08 (m,
1H, H-4Ph), 2.76 (pseudo-q, J ≈ 4.8 Hz, 4H, 2 × H-2Pip and 2 × H-6Pip),
1.73−1.59 (m, 4H, 2 × H-3Pip and 2 × H-5Pip), 1.59−1.48 (m, 2H, 2 ×
H-4Pip), 1.46 (2 × s, 9H, C(CH3)3, both isomers) ppm. 13C{1H} NMR,
HSQC, HMBC (75.5 MHz, DMSO-d6): δ 152.4 (CO), 144.49 and
144.36 (C-2Ph, both isomers), 144.41 (q, 2JCF = 28.1 Hz, CF3CNOH,
one isomer) and 144.40 (q, 2JCF = 31.0 Hz, CF3CNOH, other isomer),
132.8 and 132.7 (C-1Ph, both isomers), 126.0 (C-5Ph, one isomer),
123.5 and 122.9 (C-4Ph, both isomers), 122.0 (C-5Ph, other Isomer),
121.2 (q, 1JCF = 274.2 Hz, CF3, one isomer), 120.34 and 120.31 (C-3Ph,
both isomers), 119.2 and 118.58 (C-6Ph, both isomers), 118.56 (q, 1JCF
= 283.0 Hz, CF3, both isomers), 79.9 and 79.8 (C(CH3)3, both
isomers), 52.6 and 52.5 (C-2Pip and C-6Pip, both isomers), 27.9
(C(CH3)3), 26.0 (C-3Pip and C-5Pip), 23.5 (C-4Pip) ppm. 19F NMR (376
MHz, DMSO-d6): δ −62.78 and −65.86 (s, CF3, both isomers) ppm. IR
added triethylamine (2.2 mL, 15.79 mmol, 2.50 equiv) and iodine (2.39
g, 9.47 mmol, 1.50 equiv) at 0 °C. The reaction mixture was stirred 1 h
at 0 °C and 1 h at room temperature. After complete conversion of the
starting material, the reaction was quenched by the addition of a
Na2S2O3 (aq) solution (1 m, 50 mL) and the aqueous layer was
extracted with CH2Cl2 (3 × 100 mL). The combined organic layers
were dried over Na2SO4 and the solvent was removed under reduced
pressure. After flash chromatography purification on silica gel (cHex/
EtOAc = 100:1), the title compound was obtained as a bright yellow
solid (2.00 g, 5.20 mmol, 82% yield). Rf = 0.33 (SiO2, cHex/EtOAc =
1
50:1). Mp: 56−58 °C. H NMR, COSY (300 MHz, DMSO-d6): δ
7.82−7.74 (m, 2H, H-6Ph and NH), 7.22 (d, 3J = 8.4 Hz, 1H, H-3Ph),
6.86 (dd, 3J = 8.3, 4J = 2.4 Hz, 1H, H-4Ph), 2.72 (pseudo-t, J ≈ 5.2 Hz,
4H, 2 × H-2Pip and 2 × H-6Pip), 1.71−1.58 (m, 4H, 2 × H-3Pip and 2 ×
H-5Pip), 1.58−1.49 (m, 2H, 2 × H-4Pip), 1.47 (s, 9H, C(CH3)3) ppm.
13C{1H} NMR, HSQC, HMBC (75.5 MHz, DMSO-d6): δ 152.3 (C
O), 144.5 (C-2Ph), 133.6 (C-1Ph), 122.6 (C-5Ph), 122.0 (q, 1JCF = 274.8
Hz, CF3), 121.3 (C-3Ph), 121.1 (C-4Ph), 116.2 (C-6Ph), 80.2 (C(CH3)3),
2
52.5 (C-2Pip and C-6Pip), 28.1 (q, JCF = 39.9 Hz, Cqdiazirine), 27.9
(C(CH3)3), 26.0 (C-3Pip and C-5Pip), 23.5 (C-4Pip) ppm. 19F NMR (282
MHz, DMSO-d6): −65.85 (CF3) ppm. IR (ATR): ν 3356, 2940, 1729,
̅
1525, 1440, 1366, 1252, 1148, 1106, 1049, 989, 894, 806 cm−1. HRMS
+
(ESI-QTOF) m/z: calcd for C18H24F3N4O2 [M + H]+, 385.1846;
found, 385.1847.
2-(Piperidin-1-yl)-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]aniline
hydrochloride (18b). Note that this reaction was performed in the dark
to avoid partial photolysis of the product (aluminum foil was used to
cover flasks and columns). Under an argon atmosphere, the diazirine
17b (1.20 g, 3.12 mmol, 1.00 equiv) was submitted in a round-bottom
flask and a hydrogen chloride solution (4.0 M in 1,4-dioxane, 15 mL)
was added at room temperature. After stirring for 1 h, the solvent was
removed under reduced pressure and the residue dried in a fine vacuum
to obtain the title compound (1.00 g, 3.12 mmol, quant) as a yellow oil,
which was used without further purification in the next step. Rf = 0.59
(ATR): ν 3331, 2938, 1681, 1522, 1444, 1369, 1235, 1150, 1128, 1105,
̅
1023, 978, 821, 719 cm−1. HRMS (ESI-QTOF) m/z: calcd for
C18H25F3N3O3+ [M + H]+, 388.1843; found, 388.1844.
tert-Butyl {2-(Piperidin-1-yl)-5-[3-(trifluoromethyl)diaziridin-3-
yl]phenyl}carbamate (16b). Under an argon atmosphere, to a solution
of the oxime 15b (3.05 g, 7.87 mmol, 1.00 equiv) in acetone (80 mL)
and triethylamine (3.3 mL, 23.61 mmol, 3.00 equiv) was added a
solution of p-toluenesulfonyl chloride (1.65 g, 8.66 mmol, 1.10 equiv)
in acetone (10 mL) dropwise at 0 °C. After stirring for 3 h at 0 °C, the
solvent was removed under reduced pressure (no warming!) to give the
crude O-tosyl oxime as a yellow solid, which was immediately used for
the next step without further purification. Note that the product is not
stable upon warming and on silica, so it is crucial to avoid further
purification and warming during evaporation to obtain higher yields.
To a cooled Schlenk flask containing liquid ammonia (approximately
100 mL) under an argon atmosphere at −78 °C was added a suspension
of the crude O-tosyl oxime in anhydrous MTBE (75 mL) dropwise. The
mixture was stirred for 6 h at −78 °C and warmed to room temperature
overnight (side arm of the Schlenk flask opened to allow ammonia to
evaporate slowly). The resulting suspension was diluted with MTBE
(100 mL) and water (100 mL) and extracted with MTBE (3 × 100
mL). The combined organic layers were dried over Na2SO4 and
concentrated under reduced pressure to obtain the diaziridine 16b as a
colorless amorphous solid (2.61 g, 6.75 mmol, 86% yield). Rf = 0.33
(SiO2, cHex/EtOAc = 5:1). 1H NMR, COSY (300 MHz, DMSO-d6): δ
8.10 (d, 4J = 1.8 Hz, 1H, H-6Ph), 7.77 (s, 1H, NH), 7.25−7.12 (m, 2H,
H-3Ph and H-4Ph), 4.02 (d, 3J = 8.2 Hz, 1H, NHdiaziridine), 3.88 (d, 3J = 8.2
Hz, 1H, NHdiaziridine), 2.73 (pseudo-t, J ≈ 5.2 Hz, 4H, 2 × H-2Pip and 2 ×
H-6Pip), 1.73−1.60 (m, 4H, 2 × H-3Pip and 2 × H-5Pip), 1.60−1.49 (m,
2H, 2 × H-4Pip), 1.48 (s, 9H, C(CH3)3) ppm. 13C{1H} NMR, HSQC,
HMBC (75.5 MHz, DMSO-d6): δ 152.3 (CO), 143.7 (C-2Ph), 132.8
(C-1Ph), 127.9 (C-5Ph), 124.2 (q, 1JCF = 278.6 Hz, CF3), 123.1 (C-4Ph),
120.3 (C-3Ph), 118.6 (C-6Ph), 79.8 (C(CH3)3), 57.3 (q, 2JCF = 34.8 Hz,
Cqdiaziridine), 52.7 (C-2Pip and C-6Pip), 27.9 (C(CH3)3), 26.1 (C-3Pip and
C-5Pip), 23.5 (C-4Pip) ppm. 19F NMR (282 MHz, DMSO-d6): δ −74.98
1
(SiO2, cHex/EtOAc = 10:1). H NMR, COSY (300 MHz, methanol-
d4): δ 7.58 (d, 3J = 8.7 Hz, 1H, H-3Ph), 6.94 (d, 4J = 2.2 Hz, 1H, H-6Ph),
6.80 (dd, 3J = 8.7 Hz, 4J = 2.2 Hz, 1H, H-4Ph), 3.52−3.43 (m, 4H, 2 × H-
2Pip and 2 × H-6Pip), 2.15−2.01 (m, 4H, 2 × H-3Pip and 2 × H-5Pip),
1.83−1.69 (m, 2H, 2 × H-4Pip) ppm. 13C{1H} NMR, HSQC, HMBC
(75.5 MHz, methanol-d4): δ 139.9 (C-1Ph), 134.6 (C-2Ph), 131.1 (C-
1
5Ph), 123.4 (q, JCF = 274.0 Hz, CF3), 123.4 (C-3Ph), 119.7 (C-4Ph),
2
118.8 (C-6Ph), 56.2 (C-2Pip and C-6Pip), 29.1 (q, JCF = 40.6 Hz,
CqDiazirin), 25.1 (C-3Pip and C-5Pip), 23.0 (C-4Pip) ppm. 19F NMR (282
MHz, methanol-d4): δ −68.06 (s, CF3) ppm. IR (ATR): ν 3323, 3191,
̅
1955, 1609, 1519, 1441, 1286, 1220, 1151, 1120, 1012, 872, 854, 723
+
cm−1. HRMS (ESI-QTOF) m/z: calcd for C13H16F3N4 [M + H]+,
285.1322; found, 285.1323.
5-Methyl-N-{2′-(piperidin-1-yl)-5′-[3-(trifluoromethyl)-3H-diazir-
in-3-yl]phenyl}thiophene-2-sulfonamide (MK6−83PRG2, 3b).
Note that this reaction was performed in the dark to avoid partial
photolysis of the product (aluminum foil was used to cover flasks and
columns). Under an argon atmosphere, 5-methylthiophene-2-sulfonyl
chloride (610 mg, 3.10 mmol, 1.00 equiv) was added dropwise to a
solution of 18b (994 mg, 3.10 mmol, 1.00 equiv) in anhydrous pyridine
(25 mL) at 0 °C. The mixture was stirred for 1 h at 0 °C and at room
temperature overnight. The solvent was removed under reduced
pressure and the residue dissolved in EtOAc (100 mL). The organic
layer was washed with a NH4Cl (aq) solution (saturated, 2 × 100 mL)
and a solution of NaCl (aq) (saturated, 1 × 100 mL), dried over
Na2SO4 and concentrated under reduced pressure. After purification by
column chromatography on silica gel (cHex/EtOAc = 30:1), the title
compound was isolated as a light-yellow solid (991 mg, 2.23 mmol, 72%
yield). In general, no further purification was required in this step.
However, if necessary, preparative HPLC purification (C18, eluent A,
Milli-Q-grade water; eluent B, acetonitrile, isocratic (30:70) over 90
min with a flow rate of 12.5 mL min−1 and detection at 254 nm) can be
performed. Rf = 0.21 (SiO2, cHex/EtOAc = 10:1). Mp: 107−109 °C. 1H
NMR, COSY (400 MHz, DMSO-d6): δ 9.15 (s, 1H, NH), 7.39 (d, 3J =
3.8 Hz, 1H, H-3Thio), 7.19 (d, 3J = 8.4 Hz, 1H, H-3Ph),74 7.19 (dd, 4J =
2.3, J = 0.8 Hz, 1H, H-6Ph),74 6.89 (dd, 3J = 8.4 Hz, 4J = 2.3 Hz, 1H, H-
ppm. IR (ATR): ν 3258, 2936, 1725, 1581, 1524, 1443, 1368, 1231,
̅
1211, 1140, 1104, 1049, 1024, 819, 709 cm−1. HRMS (ESI-QTOF) m/
z: calcd for C18H26F3N4O2+ [M + H]+, 387.2002; found, 387.2005.
tert-Butyl ({2-(Piperidin-1-yl)-5-[3-(trifluoromethyl)-3H-diazirin-
3-yl]phenyl}carbamate (17b). Note that this reaction was performed
in the dark to avoid partial photolysis of the product (aluminum foil was
used to cover flasks and columns). To a solution of the diaziridine 16b
(2.44 g, 6.31 mmol, 1.00 equiv) in anhydrous CH2Cl2 (70 mL) was
6180
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Article
4Ph), 6.88 (dd, 3J = 3.8 Hz, 4J = 1.4 Hz, 1H, H-4Thio), 2.66−2.59 (m, 4H,
2 × H-2Pip and 2 × H-6Pip), 2.47 (d, 4J = 1.1 Hz, 3H, CH3), 1.66−1.56
bridge Crystallographic Data Centre, 12 Union Road, Cam-
bridge CB2 1EZ, UK; fax: +44 1223 336033.
(m, 4H, 2 × H-3Pip and 2 × H-5Pip), 1.52−1.42 (m, 2H, 2 × H-4Pip
)
ppm. 13C{1H} NMR, HSQC, HMBC (100.6 MHz, DMSO-d6): δ 148.0
(C-2Thio), 147.1 (C-2Ph), 137.0 (C-5Thio), 132.9 (C-3Thio), 131.8 (C-
1Ph), 126.3 (C-4Thio), 123.5 (C-4Ph), 122.4 (C-5Ph), 122.1 (C-3Ph),
121.8 (q, 1JCF = 274.9 Hz, CF3), 119.3 (C-6Ph), 52.5 (C-2Pip and C-6Pip),
AUTHOR INFORMATION
Corresponding Author
■
Tanja Schirmeister − Institute of Pharmaceutical and
Biomedical Sciences, Johannes Gutenberg University Mainz,
55128 Mainz, Germany; Email: schirmei@uni-mainz.de
27.8 (q, JCF = 39.9 Hz, Cqdiazirine), 25.5 (C-3Pip and C-5Pip), 23.5 (C-
2
4
Pip), 15.1 (CH3) ppm. 19F NMR (376 MHz, DMSO-d6): δ −65.81
(CF3) ppm. IR (ATR): ν 3245, 2947, 2811, 1515, 1436, 1337, 1228,
̅
1144, 1021, 921, 906, 822, 806, 721 cm−1. HRMS (ESI-QTOF) m/z:
Authors
calcd for C18H20F3N4O2S2 [M + H]+, 445.0974; found, 445.0974.
+
Kevin Schwickert − Institute of Pharmaceutical and Biomedical
Sciences, Johannes Gutenberg University Mainz, 55128 Mainz,
Germany; orcid.org/0000-0002-2575-0380
Michał Andrzejewski − Department of Chemistry, Biochemistry
and Pharmaceutical Sciences, University of Bern, 3012 Bern,
Switzerland
HPLC analysis: retention time = 4.87 min; peak area = 99.60%; eluent
A = Milli-Q-grade water; eluent B = CH3CN; eluent C = 0.1% solution
of formic acid in Milli-Q-grade water; isocratic (20:70:10) over 8 min
with a flow rate of 0.7 mL min−1 and detection at 254 nm; injection
volume = 5 μL; column temperature = 40 °C.
Simon Grabowsky − Department of Chemistry, Biochemistry
and Pharmaceutical Sciences, University of Bern, 3012 Bern,
Switzerland; orcid.org/0000-0002-3377-9474
PHOTOCHEMICAL KINETIC STUDIES AND
INSTRUMENTATION
■
The photoactivation studies were performed according to
procedures as reported by Brunner et al.34 and Seifert et al.60
using a high-power UV LED (λmax = 365 nm; UV LED smart;
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c02993
̈
Opsytec Dr. Grobel GmbH) with the “standard” optic for a
Notes
small spot diameter and a focused beam profile. Please refer to
the homepage of the UV light source supplier (https://www.
opsytec.com/products/uv-led-light-sources) for further details.
Irradiation experiments were conducted within a ventilated
fume hood in quartz cuvettes (d = 10 mm) including a tiny
magnetic stirring bar placed 2 cm from the light source for very
effective photoactivation. Under these conditions, no further
cooling was necessary to maintain ambient temperature since
the development of heat by the LED light source is negligible. A
typical reaction setup is shown in Figure S9 (Supporting
Information). For the analysis of the photolytic decay via 19F
NMR spectroscopy, (trifluoromethyl)diazirines 3a and 3b were
dissolved in methanol-d4 or cyclohexane-d12 (c = 5 mM), and
after different irradiation intervals, samples were transferred and
measured in 5 mm NMR amber glass tubes (type: 507-HP-AT-7
by Norell) before irradiation was continued. In HPLC-MS
photolysis experiments, aliquots of 50 μL were collected from
solutions of the diazirines 3a and 3b in selected solvents
(HPLC-MS grade; c = 2 mm (methanol and cyclohexane) or c =
50 μM (aqueous buffer)), diluted (1:10 v/v) with acetonitrile/
water (1:1 v/v), filtered through PTFE syringe filters (d = 13
mm, 0.2 μm pore size) and subsequently injected (injection
volume = 5−10 μL) in the HPLC-MS system described above.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Johannes C. Liermann and Nadine Schenk
(Department of Chemistry, Johannes Gutenberg University
Mainz) for NMR spectroscopy, Dr. Christopher Kampf
(Department of Chemistry, Johannes Gutenberg University
Mainz) for high-resolution mass spectrometry, and Mergim
Meta and Tobias Ott for assisting with syntheses.
REFERENCES
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ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.0c02993.
HPLC traces of key target compounds 3a and 3b, details
about the crystal structures, the fragmentation pattern of
diazirines 3a and 3b, 19F NMR and LC-MS kinetic data of
photolysis of compound 3b, and 1H and 13C NMR spectra
for all synthesized compounds (PDF)
Accession Codes
CCDC 2046164−2046165 contain the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/data_request/cif, or by email-
ing data_request@ccdc.cam.ac.uk, or by contacting The Cam-
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