N-Trifluoromethyl Amines and Azoles: An Underexplored Functional Group in the Medicinal Chemist's Toolbox

Article abstract of DOI:10.1021/acs.jmedchem.0c01457

Introducing trifluoromethyl groups is a common strategy to improve the properties of biologically active compounds. However, N-Trifluoromethyl moieties on amines and azoles are very rarely used. To evaluate their suitability in drug design, we synthesized a series of N-Trifluoromethyl amines and azoles, determined their stability in aqueous media, and investigated their properties. We show that N-Trifluoromethyl amines are prone to hydrolysis, whereas N-Trifluoromethyl azoles have excellent aqueous stability. Compared to their N-methyl analogues, N-Trifluoromethyl azoles have a higher lipophilicity and can show increased metabolic stability and Caco-2 permeability. Furthermore, N-Trifluoromethyl azoles can serve as bioisosteres of N-iso-propyl and N-Tert-butyl azoles. Consequently, we suggest that N-Trifluoromethyl azoles are valuable substructures to be considered in medicinal chemistry.

Full text of DOI:10.1021/acs.jmedchem.0c01457

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Article
N‑Trifluoromethyl Amines and Azoles: An Underexplored Functional
Group in the Medicinal Chemist’s Toolbox
Stefan Schiesser,* Hanna Chepliaka, Johanna Kollback, Thibaut Quennesson, Werngard Czechtizky,
and Rhona J. Cox
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ABSTRACT: Introducing trifluoromethyl groups is a common strategy to improve the
properties of biologically active compounds. However, N-trifluoromethyl moieties on amines
and azoles are very rarely used. To evaluate their suitability in drug design, we synthesized a
series of N-trifluoromethyl amines and azoles, determined their stability in aqueous media,
and investigated their properties. We show that N-trifluoromethyl amines are prone to
hydrolysis, whereas N-trifluoromethyl azoles have excellent aqueous stability. Compared to
their N-methyl analogues, N-trifluoromethyl azoles have a higher lipophilicity and can show
increased metabolic stability and Caco-2 permeability. Furthermore, N-trifluoromethyl azoles
can serve as bioisosteres of N-iso-propyl and N-tert-butyl azoles. Consequently, we suggest
that N-trifluoromethyl azoles are valuable substructures to be considered in medicinal
chemistry.
Chart 1. Examples of Reported Druglike Molecules
Containing an N-Trifluoromethyl Azole or Amine
INTRODUCTION
■
The introduction of a trifluoromethyl group is a popular strategy
to modulate the properties of biologically active molecules. For
example, exchanging a methyl for a trifluoromethyl can increase
metabolic stability and permeability, decrease the basicity of
proximal amines, or change the conformation of the molecule.1
Introducing a trifluoromethyl substituent can also lead to
increased potency via the formation of multipolar interactions
with carbonyl groups in a protein.2,3 Moreover, a trifluoromethyl
can interact with the hydroxyl group of tyrosine or with
carboxylic acid residues.4,5 The importance of trifluoromethyl
substituents in medicinal chemistry is further illustrated by the
seventy-two launched drugs that contain at least one
trifluoromethyl group.6 Sixty-nine of these drugs contain the
trifluoromethyl moiety attached to a carbon, and three contain
an O-trifluoromethyl. In contrast, no drug has been launched so
far with the trifluoromethyl group attached to nitrogen.6 This is
particularly surprising given, for example, the importance of
N‑methyl azoles and amines in medicinal chemistry. Further-
more, in the AstraZeneca internal compound collection, more
than 22 000 compounds contain an O-trifluoromethyl moiety,
whereas only one N-trifluoromethyl azole and nine N‑trifluor-
omethyl amines were listed prior to this study.7 However, a very
limited number of reports suggest that N-trifluoromethyl azoles,
at least, deserve a spot in the medicinal chemist’s toolbox. For
example, replacing the methyl substituent of the checkpoint
kinase 1 (CHK1) inhibitor SCH900776 (Chart 1) with
trifluoromethyl to obtain MU380 resulted in reduced
N‑dealkylation while keeping a comparable CHK1 potency.8
The notion that N-trifluoromethyl azoles can be important in
medicinal chemistry is further supported by the single
compound patent protecting sodium/glucose cotransporter 1
(SGLT1) inhibitor 1, suggesting that this compound has been of
significant interest.9 Despite these reports, N-trifluoromethyl
azoles are still considered an unusual pharmacophore as noted
by Samadder et al.8 Furthermore, compared to N-trifluoro-
methyl azoles, N‑trifluoromethyl amines have been even less
frequently used in medicinal chemistry1 with the cannabinoid
receptor modulator 2 being a rare example.10
The reluctance of medicinal chemists to use N-trifluoro-
methyl amines or azoles could be due to their historically
challenging synthesis. Until very recently, synthetic methods for
N-trifluoromethylation of amines generally required the use of
Received: August 21, 2020
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highly toxic HF-based reagents11 or thermally unstable
compounds like O-trifluoromethyl benzofuranium reagents12
(for an overview on methods to synthesize N-trifluoromethyl
azoles, see the end of the Results and Discussion section).
Whereas no significant improvement in the synthesis of
N‑trifluoromethyl azoles has been reported until a very recently
described synthesis specific to N-trifluoromethyl indoles13 (vide
infra), four operationally simple methods for the synthesis of
tertiary N-trifluoromethyl amines have been published in the last
four years (additionally, a synthesis of N‑trifluoromethyl
azepines was recently described14). All four methods generate
a thiocarbamoyl fluoride intermediate, which is then trans-
formed into the desired N-trifluoromethyl amine using silver(I)
fluoride. The Schoenebeck group reported the formation of the
thiocarbamoyl fluoride intermediate using (Me4N)SCF3,15
whereas Yu et al. generated this intermediate from difluor-
ocarbene.16 Liang et al.17 reported a procedure using Langlois’
reagent (CF3SO2Na), and Onida et al.18 used carbon disulfide
and (diethylamino)sulfur trifluoride (DAST). These strategies
have been used to synthesize interesting analogues of bio-
logically active compounds (examples shown in Chart 2).15,17
hydrolytically unstable,19−21 and there is a single report
mentioning the hydrolysis of N-ethyl-N-trifluoromethylani-
line.19 Pan suggested that reducing the electron density of the
nitrogen could result in more stable trifluoromethyl groups.22
However, there is no systematic study investigating the effect of
the electron density of the nitrogen on the stability of the
trifluoromethyl group. Moreover, whether the potentially
increased stability of electron-deficient N-trifluoromethyl
groups renders the compounds sufficiently stable for applica-
tions in medicinal chemistry is still in question.22,23
Furthermore, it is unknown whether piperazines, benzylamines,
and azoles, which are highly important substructures in
medicinal chemistry,24 are sufficiently stable in aqueous
media25 when bearing an N-trifluoromethyl group.
Additional to concerns over aqueous stability, the highly
electron-withdrawing trifluoromethyl group could potentially
result in other liabilities, such as inducing reactivity toward
physiological nucleophiles such as glutathione, resulting in
putative toxic conjugates.26 Moreover, key in vitro properties of
N-trifluoromethyl amines and azoles have not been investigated.
Consequently, a systematic investigation of their stability and
properties is highly desirable to assess their suitability for
medicinal chemistry, as already noted by Meanwell.1
Chart 2. Exemplary Structures Synthesized by the
Schoenebeck Group15 and Yi et al.17
We therefore synthesized an array of diverse compounds
containing a trifluoromethyl amine or azole and systematically
investigated their stability in aqueous media. Since very few
examples of secondary N-trifluoromethyl amines have been
published, with no general synthetic method available,12,27 we
excluded secondary N-trifluoromethyl amines from this study.
For stable compounds, we explored their in vitro properties, with
a view to making sound recommendations for using
N‑trifluoromethyl amines and azoles in drug design.
RESULTS AND DISCUSSION
■
Solution Stability of Tertiary N-Trifluoromethyl
Amines and Azoles. To evaluate whether N-trifluoromethyl
groups on amines and azoles are sufficiently stable to be useful
for medicinal chemistry, we investigated the stability of
piperazine 3, anilines 4−13, benzylamine 14, pyrazole 15, and
benzimidazole 16 (Figure 1). This set was selected to include
substructures that are often used in drug design.24 Anilines 4−
13 contain electron-donating and electron-withdrawing sub-
stituents to elucidate the influence of the electron density of the
nitrogen on the stability of the N-trifluoromethyl moiety.
However, while these new procedures improve the synthetic
access to at least tertiary N-trifluoromethyl amines, the
suitability of these groups in drug design is still in doubt due
to their questionable stability in aqueous media. For example
N,N-bisalkyl-N-trifluoromethylamines are described to be
Figure 1. Percentage of N-trifluoromethyl amine and azole left (relative to 0 h) after standing in a 2 mM water/dimethyl sulfoxide (DMSO) 4:1
solution at room temperature under air for 0.5, 6, and 24 h (based on their respective integrated absorption between 220 and 350 nm determined by
liquid chromatography).
B
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In many company compound collections, samples are stored
as 10 mM solutions in DMSO. Consequently, we started by
studying the stability of the compounds in DMSO. All
compounds investigated are stable as 10 mM solutions in neat
DMSO at room temperature under air without protection from
sunlight with no trace of decomposition observed by liquid
chromatography−mass spectrometry (LCMS) after 1 month. In
contrast, in a 2 mM DMSO/H2O 1:4 mixture,28 piperazine 3
and benzylamine 14 as well as electron-rich and electron-neutral
anilines 4−9 hydrolyze to a substantial extent within 0.5 h
(Figure 1, for an exemplary liquid chromatogram of aniline 4, see
Figure 2). Anilines 10 and 13, which bear strong electron-
Chart 3. (a) Structures of N-Trifluoromethyl Compounds
Synthesized to Determine Their Aqueous Stability and
Additional Key In Vitro Properties. (b) Hydrolysis of Aniline
20a Results in Formation of Carbamoyl Fluoride 26, Which
Can React with Glutathione to Yield Adduct 27
Figure 2. Exemplary liquid chromatogram of 4-methoxy-N-methyl-N-
trifluoromethyl aniline after standing in a 2 mM water/DMSO 4:1
solution at room temperature under air for 0, 0.5, and 6 h.
withdrawing substituents, are more stable toward hydrolysis
with 24 and 90% of parent detected after 24 h, respectively. Gas
chromatography-mass spectrometry (GCMS) analysis of all
hydrolysis reactions shows the formation of the corresponding
carbamoyl fluoride, which was further supported by fully
characterizing the hydrolysis product 17 of aniline 4 by
NMR.19,20,29 For N-trifluoromethyl azoles 15 and 16, no trace
of the hydrolyzed compound was detected by LCMS or by NMR
even after 1 month.
Table 1. Half-Life of N-Trifluoromethyl Compounds 18a−
25a at 25 °C in 0.1 M HCl Solution (pH 1.0), 20 mM Sodium
Phosphate Buffer (pH 7.4), and 20 mM Sodium Carbonate
Buffer (pH 10.0)
We next investigated whether the above-observed stability
trends can be used to predict the stability of N-trifluoromethyl
analogues of known bioactive compounds. To do so, we
determined the half-lives of compounds containing an
N‑trifluoromethyl piperazine (sildenafil analogue 18a) or an
electron-deficient aniline (sulfamethoxazole derivative 19a and
tetracaine derivative 20a) at pH 1.0, 7.4, and 10.0. We also
investigated two N-trifluoromethyl imidazoles (21a and 22a)
and three N-trifluoromethyl pyrazoles (23a, 24a, and 25a) with
the corresponding N-methyl analogues being inhibitors of the
hedgehog pathway,30,31 methionine aminopeptidase,32 inter-
half-life at 25 °C
#
pH 1.0 [d]
pH 7.4 [d]
pH 10.0 [d]
18a
19a
20a
21a
22a
23a
24a
25a
<1.3
0.2
<0.8
0.4
<0.5
0.3
<0.6
>72
>72
71
<0.6
>72
>72
>72
12
<0.6
>72
>72
>72
>72
>72
>72
17
leukin-1 receptor associated kinase 4 (IRAK4),33 b-rapidly
41
34
accelerated fibrosarcoma (BRAF)V600E
,
and pyridoxal-5′-
phosphate-dependent transaminase,35 respectively (Chart 3a).
Piperazine 18a and the two anilines 19a and 20a showed fast
hydrolysis at all three pH values investigated, with half-lives of
less than 1.5 days at 25 °C (Table 1). For 18a and 19a, the
corresponding secondary amine was detected as the major
product at all three pH values. For 20a, the corresponding
carbamoyl fluoride 26 was the main product at pH 1.0 with a
small amount of product where both the carbamoyl fluoride had
been further hydrolyzed to the secondary amine and the ester
bond cleaved. The latter compound was also the main product at
pH 7.4 and pH 10.0.
Formation of an electrophilic carbamoyl fluoride36,37 as a
potentially reactive hydrolysis product raises concerns in terms
of drug safety. To see if a carbamoyl fluoride is liable to react
with physiological nucleophiles we studied the reactivity of the
carbamoyl fluoride product 26 with glutathione. Carbamoyl
fluoride 26 disappeared in the buffer in the presence of
glutathione with a half-life of 66 min (compared to a half-life of
237 min in buffer without glutathione), and the corresponding
glutathione adduct 27 (Chart 3b) was clearly detected by
LCMS. The half-life of 66 min is in the range of half-lives
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Table 2. Overview of the Change in Key In Vitro Properties When Exchanging N-Methyl, N-iso-Propyl, N-tert-Butyl, and
f
N‑Trifluoromethyl Groups
a
Due to the limitations in the determination of log D7.4 using the shake-flask method, exact values for measured log D7.4 > 4 are given as >4.0.
b
c
Experimentally determined polar surface area. Apical to basolateral passive permeability across the Caco-2 cell monolayer in the presence of
inhibitors against the three major efflux transporters: P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug-associated
d
protein 2 (MRP2). Metabolic stability of the compound measured as the disappearance of the parent compound over time when incubated with
e
human liver microsomes. Due to the low stability of compounds 18a, 19a, and 20a in an aqueous environment, which would interfere with a
proper determination of their metabolic stability or Caco-2 permeability, no metabolic stability or Caco-2 permeability was determined for these
f
compounds or for their N-methyl analogues. Each experimental value is the mean of at least three independent replicates. The standard deviation is
given in brackets.
observed for typical covalent warheads like N-acrylamides,
vinylsulfonamides, and reactive electrophilic heterocycles.38−40
In contrast to the N-trifluoromethyl anilines and piperazine,
for all N-trifluoromethyl azoles investigated, no corresponding
carbamoyl fluoride or free azole was detected in aqueous media
at all three pH values studied. Furthermore, the determined half-
lives are in a range suitable for chemical development (Table
1).41 We investigated the reactivity of azoles 21a−25a toward
glutathione to exclude the possibility that these compounds have
intrinsic electrophilicity. For all N-trifluoromethyl imidazoles
and pyrazoles investigated, no trace of glutathione adduct was
detectable after incubation for 20 h at 37 °C in the presence of
glutathione.
chemistry due to their rapid hydrolysis and the formation of
electrophilic carbamoyl fluorides. In contrast, the investigated
N-trifluoromethyl azoles have excellent stability in aqueous
media and show no reactivity toward glutathione.
Comparison of Properties of N-Trifluoromethyl and
N‑Methyl Amines and Azoles. We next compared key in vitro
properties in medicinal chemistry (log D, experimentally
determined polar surface area (ePSA),42,43 permeability in
human epithelial colorectal adenocarcinoma cells (Caco-2), and
metabolic stability) for the N-trifluoromethyl compounds 18a−
25a and their N-methyl counterparts 18b−25b (Table 2). For
comparing the log D, we used the shake-flask method (termed
44
log D7.4) and chromatographically determined log D7.4
Consequently, our data suggests that tertiary N-trifluoro-
(chromlog D7.4), since both methods are often used in medicinal
methyl amines are likely to be of limited use in medicinal
chemistry. In the compounds investigated, the exchange of a
D
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methyl for trifluoromethyl leads to the expected higher
lipophilicity as proven by an increased log D7.4 and chrom-
log D7.4 and a decreased ePSA. Log D7.4 increases by on average
1.1 log units and chromlog D7.4 by 1.6 log units. However, the
extent of this change can vary significantly and is dependent on
both the individual compound and type of log D7.4 analysis used.
Changes in permeability and metabolic stability are less
consistent. The Caco-2 permeability, for example, can be
significantly increased as for 25a (p = 0.007). Stability to human
liver microsomes (HLMs) can be significantly increased for the
trifluoromethyl analogue as seen for 22a (p = 0.004) or
decreased as for 24a and 21a. The decreased metabolic stability
of the latter two compounds could be due to an increased
lipophilicity, rendering the potential metabolic soft spots
(benzylic methyl group in 21a and two methoxy ethers in
24a) more susceptible toward metabolism.
and 20b were synthesized by reductive amination of the
corresponding secondary amines.
Whereas N-trifluoromethyl pyrazole 15 is commercially
available, the N-trifluoromethyl benzimidazoles 16 and 22a
were synthesized in a two-step procedure. First, the
benzimidazoles 28 and 29 were transformed into their
N‑bromodifluoromethyl analogues, which were then converted
into the desired benzimidazoles 16 and 22a. Imidazole 21a and
pyrazoles 23a, 24a, and 25a were synthesized using
commercially available azole building blocks bearing the
N‑trifluoromethyl group via either Buchwald−Hartwig amina-
tion (23a) or an amide bond formation (24a and 25a) following
the procedures described for the synthesis of the N-methyl
analogues 21b, 23b, 24b, and 25b.30−35 The iso-propyl and tert-
butyl analogues of compounds 23a, 24a, and 25a were
synthesized following a similar procedure using the respective
azole building blocks. N-Methyl benzimidazole 20b was
Additionally, we investigated the change in pKa when
replacing an N-methyl with an N-trifluoromethyl group on
imidazoles and showed that having an N-trifluoromethyl group
decreases the basicity by at least 3 orders of magnitude (Table
3).
́
synthesized following the procedure described by Simeon et
al.47
CURRENT CHALLENGES AND NEED FOR
IMPROVEMENTS IN THE SYNTHESIS OF
N-TRIFLUOROMETHYL AZOLES
■
Table 3. Measured pKa Values of the Corresponding Acids for
a
Compounds 21a/21b and 22a/22b
Whereas mild conditions using nontoxic reagents have recently
been reported for the synthesis of tertiary N-trifluoromethyl
amines (vide supra),15−18 the synthesis of N-trifluoromethyl
azoles still mainly requires the use of highly toxic and nongreen
reagents.48 For example, the two-step procedure to synthesize
benzimidazole derivatives 22a and 16 relies on the use of
CF2Br2,49−53 a known ozone-depleting reagent.54 An alternative
approach is the alkylation of azoles using trifluoromethyl
iodide.55−58 However, for both approaches, strong bases like
NaH or KOtBu are generally required.
a
pKa,2 is likely to correspond to the pyridine motif of compounds 21a
This could explain why these methodologies have so far only
been applied to azoles bearing a very limited number of
functional groups and not to more druglike structures. An
alternative approach was described by Yagupolskii et al. where
they transformed 2-methyl-benzimidazole and benzotriazole
first into the corresponding dithiocarbamates using carbon
disulfide, followed by their conversion into the corresponding
N-trichloromethyl azoles using chlorine gas and carbon
tetrachloride, and finally into the desired N-trifluoromethyl
azoles using anhydrous HF.53,59 Kanie et al. showed that the
dithiocarbamate of indole could be directly converted into the
N-trifluoromethyl indole via the use of HF·TEA. However, this
strategy was not applied to azoles beyond indole. It still needs
the use of special equipment on larger scales and poses a safety
risk due to the need for a HF-based reagent.11 The Toste and
Togni groups reported on the synthesis of N-trifluoromethyl
(benz)triazoles, (benz)imidazoles, (benz)pyrazoles, and tetra-
zoles,60−62 but the required hypervalent iodine species are
reported to exhibit violent thermal decomposition60,63,64
severely limiting their use. Another approach was described by
the Beier group who reacted a trifluoromethyl azide either with
an alkyne or an enamine to the corresponding 1,2,3-triazole,65,66
which could then be further transformed into various
N‑trifluoromethyl pyrroles and imidazoles.25 However, the
need to use trifluoromethylazide could limit the use of this
strategy at least on scale.
and 21b.
Comparison of Properties of N-Trifluoromethyl, N‑iso-
Propyl, and N-tert-Butyl Azoles. The replacement of
N‑methyl with N-iso-propyl or N-tert-butyl is reported to on
average lead to similar increases in log D7.4 to those we see when
replacing an N-methyl with an N-trifluoromethyl moiety.45
Consequently, we investigated whether a trifluoromethyl group
on an azole could be a suitable bioisostere for an iso-propyl or a
tert-butyl group (Table 2). As expected from the large spread of
log D7.4 values when exchanging a methyl for a trifluoromethyl
group, trifluoromethyl-containing compounds can have lipo-
philicities similar to those of iso-propyl analogues (25a) or even
significantly higher than that of a tert-butyl moiety (23a and
24a). In our set of investigated compounds, the metabolic
stability of the trifluoromethyl compounds is either similar to
(23a and 25a) or lower (24a) than that of the iso-propyl and tert-
butyl compounds. Interestingly, the Caco-2 permeability is
significantly higher for the trifluoromethyl derivative 25a
compared to its iso-propyl analogue 25c (p = 0.01) despite
very similar log D7.4 and resembles more the Caco-2
permeability of the more lipophilic tert-butyl variant.
Synthesis. The synthesis of N-trifluoromethyl amines 5, 8,
14, 18a, and 20a is described by Scattolin et al. (Scheme 1),15
and their procedure was used with slight modifications46 to
access amines 3, 4, 6, 7, and 9−13. Compound 19a was
synthesized following the procedure of Liang et al.17 N-Methyl
analogue 18b is commercially available, and compounds 19b
Very recently, the Schoenebeck group reported on the
synthesis of N-trifluoromethyl hydrazine derivatives. Even
though these might be promising intermediates to access
E
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a
Scheme 1. Synthesis of N-Trifluoromethyl Compounds and N-Methyl, N-iso-Propyl, and N-tert-Butyl Analogues
a
Reagents and conditions: (a) 3, 4, 6, 9−13, 19a: corresponding amine, (NMe4)(SCF3), dichloromethane (DCM), rt, 15 min then AgF, rt, 4−9 h,
yields: 3: 37%, 4: 24%, 6: 76%, 7: 6%, 9: 58%, 10: 67%, 11: 42%, 12: 55%, 13: 37%. (b) 19a: NaCF3SO2, PPh3, MeCN, rt, 3 h, then, AgF, rt, 3.5 h,
8%. (c) 19b: HO(CH2O)nH, NaBH3CN, MeOH, rt to 40 °C, 23 h, 6%. 20b: HO(CH2O)nH, NaBH3CN, MeOH, rt to 40 °C, 23 h, 4%. (d) 16:
CF2Br2, NaH, NBu4Br, N,N-dimethylformamide (DMF), 0 °C to rt, 2 h. 22a: CF2Br2, NaH, NBu4Br, DMF, 0 °C to rt, overnight. (e) 16: NMe4F,
sulfolane, 60 °C, 12 h, 2% (over two steps). 22a: NMe4F, sulfolane, 60 °C, 12 h, 9% (over two steps). (f) 21a: 4-bromo-1-(trifluoromethyl)-1H-
imidazole, Pd(PPh3)4, Cs2CO3, 1,4-dioxane/H2O 3:1, 150 °C, 5 h, 40%. (g) 23a: 1-(trifluoromethyl)-1H-pyrazol-4-amine, XPhos Pd G3, K3PO4,
tBuOH, 80 °C, 15 h, 29%. 23c: 1-(iso-propyl)-1H-pyrazol-4-amine, XPhos Pd G3, K3PO4, tBuOH, 80 °C, 6 h, 10%. 23d: 1-(tert-butyl)-1H-pyrazol-
t
4-amine, XPhos Pd G3, K3PO4, BuOH, 80 °C, 6 h, 12%. (h) 24a: 1-(trifluoromethyl)-1H-pyrazol-4-amine, EDC·HCl, HOBt·xH2O, DCM, rt,
14 h, 80%. 24c: 1-(iso-propyl)-1H-pyrazol-4-amine, EDC·HCl, HOBt·xH2O, DCM, rt, 5 h, 26%. 24d: 1-(tert-butyl)-1H-pyrazol-4-amine, EDC·
HCl, HOBt·xH2O, DCM, rt, 23 h, 13%. (i) 25a: 1-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid, EDC·HCl, 4-dimethylaminopyridine (DMAP),
DMF, rt, overnight, 61%. 25c: 1-(iso-propyl)-1H-pyrazole-4-carboxylic acid, EDC·HCl, DMAP, DMF, rt, 6 h, 37%. 25d: 1-(tert-butyl)-1H-pyrazole-
4-carboxylic acid, EDC·HCl, DMAP, DMF, rt, 6 h, 29%.
various N-trifluoromethyl azoles, this strategy has so far only
been applied to the synthesis of N-trifluoromethyl indoles.13
Consequently, to encourage the use of N-trifluoromethyl
azoles in medicinal chemistry, novel synthetic procedures are
needed, which avoid the use of explosive, highly toxic, or
nongreen reagents. Furthermore, the introduction of the
trifluoromethyl moiety on azoles in a late-stage fashion using
mild conditions with high functional group tolerance is highly
desirable.
vitro properties of newly accessible compounds to judge their
suitability for biological applications.
In contrast, the N-trifluoromethyl moiety attached to azoles is
significantly more stable with no signs of hydrolysis for any
investigated azole. Additionally, none of the five investigated
N‑trifluoromethyl azoles showed any reactivity with glutathione,
despite the strongly electron-withdrawing trifluoromethyl
group. Exchanging an N-methyl for an N-trifluoromethyl moiety
can be a valuable strategy to increase the lipophilicity and Caco-
2 permeability and to modulate the metabolic stability of a
compound, depending on the desired profile and on the starting
point. Furthermore, we showed that an N-trifluoromethyl group
could serve as a bioisostere to N‑iso-propyl or N-tert-butyl azoles
with potentially improved Caco-2 permeability.
CONCLUSIONS
■
N-Trifluoromethyl amines and azoles are not frequently used in
medicinal chemistry,1,8 which could be due to their challenging
syntheses and unknown aqueous stability. With the recently
developed mild conditions for the synthesis of N‑trifluoro-
methyl amines using nontoxic reagents,15−18 we set out to
investigate the stability of N-trifluoromethyl amines in aqueous
media. We found that the stability of N‑trifluoromethyl amines
correlates with the electron density of the nitrogen, with
electron-deficient anilines being the most stable amines
investigated. However, even the electron-deficient anilines
investigated underwent significant hydrolysis within hours.
Furthermore, the formed hydrolysis product, the corresponding
N-carbamoyl fluoride, can react with physiological nucleophiles.
Consequently, N-trifluoromethyl amines might only be suitable
for very specific drug discovery endeavors. We therefore strongly
recommend stability studies to be conducted on N-trifluoro-
methyl amines made to ensure that any observed pharmaco-
logical effect is due to the N‑trifluoromethyl amine and not due
to hydrolysis products like the corresponding carbamoyl
fluoride or secondary amine. Furthermore, our study showcases
the need to rigorously investigate the aqueous stability and in
In recent years, underused moieties like sulfoximines,67
sulfonimidamides,68 and phosphine oxides69 have been
suggested as interesting substructures in medicinal chemistry.
As an addition to these moieties, we believe that N‑trifluoro-
methyl azoles can be a highly valuable substructure and deserve a
spot in the toolbox of medicinal chemists. We hope that our
study provides inspiration as to when an N‑trifluoromethyl
moiety on an azole can be considered and will facilitate the use of
this underused functional group.
EXPERIMENTAL SECTION
■
General Procedures. All reactions were performed in dried
reaction vessels under a N2 atmosphere. Reactions were monitored by
either LCMS (ESI+ and ESI−), GCMS (EI+) or analytical thin-layer
chromatography (TLC). TLC was performed on silica-plated glass
plates, using Merck silica gel grade 60 F254. Spots on TLC plates were
visualized by UV (λ = 254 nm) or by cerium ammonium molybdate
staining solution (2.5 g of ammonium molybdate tetrahydrate, 1 g of
cerium ammonium sulfate dihydrate in 10 mL of sulfuric acid and 90
mL of water) followed by heating at 640 °C for 10 s. For LCMS analysis,
F
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a GenTech Scientific Waters ACQ equipped with an Acquity ultra
performance liquid chromatography (UPLC) system, an HSS C18
column (1.8 μm, 2.1 mm × 50 mm), and an SQ2 detector was used.
Acetonitrile and water (modified either with 47 mM ammonia and 6.5
mM ammonium carbonate, pH 10, or with 1 mM ammonium formate
and 10 mM formic acid, pH 3) were used as mobile phases. For GCMS
analysis, an Agilent 7890A GCMS system equipped with an Agilent HP-
5MS column (0.25 μm, 30 mm × 0.25 mm), a 7683B Series injector, a
7683 autosampler, and a 5975C Insert MSD detector was used. For
automated flash column chromatography, a Biotage SP-4 system with
Biotage prepacked KP-SIL SNAP cartridges was used. For preparative
high-performance liquid chromatography (HPLC), a Waters Fraction
Lynx system with a Waters Acquity SQD and a Waters binary gradient
module 2525, with a flow of 60 mL/min at ambient temperature, was
used. The HPLC was equipped with either a Kromasil C8 column (10
μm, 250 mm × 20 mm, HPLC-system A), a Waters Sunfire C18 column
(5 μm, 19 mm × 150 mm, HPLC-system B), a Waters Xbridge C18
column (5 μm, 30 mm × 150 mm, HPLC-system C), a Waters Sunfire
C18 column (5 μm, 10 mm × 100 mm, HPLC-system D), a Waters
Sunfire C18 column (5 μm, 30 mm × 150 mm, HPLC-system E), or a
Waters BEH C18 column (1.7 μm, 2.1 mm × 50 mm, HPLC-system F).
For preparative SFC, a Waters Prep 100 SFC MS with a Waters mass
detector 3100, a TharSFC high-pressure pump, and a Waters
quaternary gradient pump 2545, with a flow of 100 g/min at 40 °C
and 120 bar, was used. The SFC system was equipped with either a
Waters BEH column (5 μm, 30 mm × 250 mm, SFC-system A), a
Phenomenex Luna Hilic column (3.5 μm, 3 mm × 100 mm, SFC-
system B), or a Waters Sunfire C18 column (5 μm, 19 mm × 150 mm,
SFC-system C). NMR spectra were recorded at an uncalibrated
temperature of 25 °C on a Bruker Avance III 500 spectrometer with a 5
mm QNP cryoprobe at a frequency of 500 MHz (1H), 125 MHz (13C),
or 471 MHz (19F), a Bruker Avance III with a 5 mm QNP cryoprobe at
a frequency of 600 MHz (1H) or 151 MHz (13C), or a Bruker Neo with
a 5 mm TCI probe with a cryofit at a frequency of 600 MHz (1H) or 151
MHz (13C). 13C NMRs and 19F NMRs were run in the proton
decoupled mode. Chemical shifts are reported in parts per million (δ)
and referenced from the residual protonium for 1H NMR [(CDCl3): δ
7.26 (CHCl3); DMSO-d6: δ 2.50 (DMSO-d5); CD2Cl2: δ 5.43
(CHDCl2)]. 13C NMR spectra are referenced from the carbon
reference of the solvent [(CDCl3: δ 77.2; DMSO-d6: δ 39.5; CD2Cl2:
δ 54.0)]. COSY, HSQC, and HMBC spectra were recorded to support
the structural assignments of the NMR signals. A Waters LCT Premiere
mass spectrometer coupled to a Waters Acquity UPLC was used to
record high-resolution mass spectra (HRMS). The Waters Acquity
UPLC was equipped with either a BEH C18 column (1.7 μm, 2.1 mm ×
50 mm, at 45 °C using a gradient from 5 to 90% acetonitrile in water
modified with 40 mM ammonia and 5 mM H2CO3, pH 10 within 2.5
min or 3 min) or a CSH C18 column (1.7 μm, 2.1 mm × 50 mm at 45
°C using a gradient from 5 to 90% acetonitrile in water modified with 10
mM formic acid and 1 mM ammonium formate, pH 3, within 2.5 min or
3 min). For purity analysis, a GenTech Scientific Waters ACQ equipped
with an Acquity UPLC system, an HSS C18 column (1.8 μm, 2.1 mm ×
50 mm), and an SQ2 detector with a wavelength range of 220−350 nm
was used. Acetonitrile and water (modified either with 47 mM
ammonia and 6.5 mM ammonium carbonate, pH 10, or with 1 mM
ammonium formate and 10 mM formic acid, pH 3) were used as mobile
phases. The purity of all final compounds is 95% or higher unless
otherwise stated.
dissolved in DCM (HPLC grade, 2.6 mL). The resulting solution was
added dropwise to tetramethylammonium trifluoromethanethiolate
(126 mg, 719 μmol, 2.0 equiv), and the resulting mixture was stirred at
room temperature. After 15 min, silver(I) fluoride (228 mg, 1.80 mmol,
5.0 equiv.) was added and stirring at room temperature was continued
until LCMS analysis showed complete conversion into the desired N-
trifluoromethyl amine (typically after 4−6 h). The reaction mixture was
purified using automated silica column chromatography with isocratic
100% pentane as the mobile phase.
1-Phenyl-4-(trifluoromethyl)piperazine (3). It was synthesized
according to the general procedure. Colorless solid (31 mg, 37%). 1H
NMR (500 MHz, CDCl3): δ = 7.33−7.26 (m, 2H), 6.97−6.88 (m, 3H),
3.27−3.20 (m, 4H), 3.12−3.06 (m, 4H). 13C NMR (126 MHz,
CDCl3): δ = 151.0 (s), 129.4 (s), 124.6 (q, 1JC−F = 256.2 Hz), 120.7 (s),
3
116.7 (s), 48.6 (s), 44.5 (q, JC−F = 2.8 Hz). 19F NMR (470 MHz,
CDCl3): δ = −68.3 (s). MS (EI): m/z calcd for C11H13F3N2 [M]+:
1
230.1. Found: 230.2. H, 13C, and 19F NMR are consistent with the
literature.17
4-Methoxy-N-methyl-N-(trifluoromethyl)aniline (4). It was synthe-
sized according to the general procedure. Colorless liquid (18 mg,
24%). 1H NMR (500 MHz, CDCl3): δ = 7.21 (d, 3JH−H = 8.7 Hz, 2H),
4
6.89−6.83 (m, 2H), 3.80 (s, 3H), 2.97 (q, JH−F = 1.2 Hz, 3H). 13C
NMR (126 MHz, CDCl3): δ = 158.4 (s), 135.8 (s), 127.5 (q, 3JC−F = 1.6
Hz), 124.2 (q, 1JC−F = 256.1 Hz), 114.4 (s), 55.6 (s), 36.9 (q, 3JC−F = 1.9
Hz). 19F NMR (471 MHz, CDCl3): δ = −61.5 (s). MS (EI): m/z calcd
for C9H10F3NO [M]+: 205.1. Found: 205.2. 1H, 13C, and 19F NMR are
consistent with the literature.16
N,4-Dimethyl-N-(trifluoromethyl)aniline (6).70 It was synthesized
according to the general procedure. Light yellow liquid (52 mg, 76%).
1H NMR (500 MHz, CDCl3): δ = 7.23−7.07 (m, 4H), 3.00 (q, 4JH−F
=
1.2 Hz, 3H), 2.34 (s, 3H). 13C NMR (126 MHz, CDCl3): δ = 140.4 (s),
136.4 (s), 129.9 (s), 125.4 (q, 3JC−F = 1.4 Hz), 123.7 (q, 1JC−F = 241.3
Hz), 36.6 (q, 3JC−F = 2.3 Hz), 21.1 (s). 19F NMR (471 MHz, CDCl3): δ
= −60.9 (s). 1H, 13C, and 19F NMR are consistent with the literature.16
N-Ethyl-N-(trifluoromethyl)aniline (7).70 It was synthesized accord-
1
ing to the general procedure. Light yellow liquid (18 mg, 26%). H
NMR (600 MHz, CDCl3): δ = 7.37−7.33 (m, 2H), 7.27−7.23 (m, 3H),
3.42−3.39 (m, 2H), 1.08 (t, 3JH−H = 7.2 Hz, 3H). 13C NMR (151 MHz,
CDCl3): δ = 140.9 (s), 129.3 (s), 126.9 (s), 126.8 (s), 123.7 (q, 1JC−F
=
254.7 Hz), 43.9 (s), 13.9 (s). 19F NMR (471 MHz, CDCl3): δ = −57.6
(s). 1H, 13C, and 19F NMR are consistent with the literature.11
1-(Trifluoromethyl)-1,2,3,4-tetrahydroquinoline (9). It was synthe-
sized according to the general procedure. Yellow liquid (42 mg, 21%).
1H NMR (500 MHz, CDCl3): δ = 7.22−7.04 (m, 3H), 7.03−6.89 (m,
1H), 3.55−3.41 (m, 2H), 2.80 (t, 3JH−H = 6.5 Hz, 2H), 2.07−1.91 (m,
2H). 13C NMR (126 MHz, CDCl3): δ = 137.7 (s), 129.5 (s), 128.3 (s),
126.8 (s), 123.2 (q, 1JC−F = 254.1 Hz), 122.9 (s), 119.9 (q, 3JC−F = 3.7
Hz), 43.9 (q, 3JC−F = 2.1 Hz), 27.3 (s), 22.1 (s). 19F NMR (470 MHz,
CDCl3): δ = −56.7 (s). MS (EI): m/z calcd for C10H10F3N [M]+:
1
201.1. Found: 201.2. H, 13C, and 19F NMR are consistent with the
literature.16
4-(N-Methyl-N-(trifluoromethyl)amino)benzonitrile (10). It was
synthesized according to the general procedure. Colorless liquid (48
1
mg, 67%). H NMR (500 MHz, CDCl3): δ = 7.64−7.63 (m, 2H),
7.26−7.25 (m, 2H), 3.24−3.03 (m, 3H). 13C NMR (126 MHz,
CDCl3): δ = 146.5 (s), 133.3 (s), 122.6 (q, 1JC−F = 257.8 Hz), 122.1 (q,
3JC−F = 2.3 Hz), 118.7 (s), 107.9 (s), 35.3 (q, 3JC−F = 2.3 Hz). 19F NMR
(470 MHz, CDCl3): δ = −58.8 (s). MS (EI): m/z calcd for C10H10F3N
[M]+: 200.1. Found: 200.1. 1H, 13C, and 19F NMR are consistent with
the literature.16
Materials. All solvents and chemicals were used as purchased
without further purification. Solvents for reactions were anhydrous
(≤50 ppm H2O) unless otherwise stated. Solvents for extraction and
chromatographic purification were of HPLC grade. Where necessary
(so noted), solvents where deoxygenated using three cycles of freeze,
pump for 1 min, and thaw. The synthesis of N-trifluoromethyl amines 5,
8, 14, 18a, and 20a is described by Scattolin et al.15 Compounds
21b,30,31 22b,47 23b,33 24b,34 25b,35 30,30,31 and 3133 were synthesized
according to literature procedures. Compound 18b is commercially
available.
4-Fluoro-N-methyl-N-(trifluoromethyl)aniline (11).70 It was syn-
thesized according to the general procedure. Yellow liquid (29 mg,
1
42%). H NMR (500 MHz, CDCl3): δ = 7.25−7.23 (m, 2H, CH),
7.06−7.02 (m, 2H, CH), 2.99 (q, 4JH−F = 1.1 Hz, 3H, CH3). 13C NMR
(126 MHz, CDCl3): δ = 161.2 (d, 1JC−F = 246.0 Hz, CHCF), 138.9 (d,
4JC−F = 3.2 Hz, NCCH), 127.6 (d, 3JC−F = 8.6 Hz, FCCHCH), 123.6 (q,
2
1JC−F = 257.4 Hz, CF3), 116.1 (d, JC−F = 22.6 Hz, FCCH), 36.8 (q,
3JC−F = 2.0 Hz, CH3). 19F NMR (471 MHz, CDCl3): δ = −61.3 (s, 3F,
CF3), −115.4 (s, 1F, CHCF).
General Procedure for the Synthesis of N-Trifluoromethyl
Amines. The respective secondary amine (360 μmol, 1.0 equiv) was
G
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3-Fluoro-N-methyl-N-(trifluoromethyl)aniline (12).70 It was syn-
thesized according to the general procedure. Colorless liquid (38 mg,
56%). 1H NMR (500 MHz, CDCl3): δ = 7.33−7.28 (m, 1H,
FCCHCH), 7.04−7.01 (m, 1H, NCCHCH), 6.97−6.90 (m, 2H,
m/z calcd for C12H12F3N3O3S + H+ [M + H]+: 336.0625. Found:
336.0637.
4-(Dimethylamino)-N-(5-methylisoxazol-3-yl)benzenesulfon-
amide (19b). A mixture of 4-(methylamino)-N-(5-methylisoxazol-3-
yl)benzenesulfonamide (100 mg, 374 μmol, 1.0 equiv) and
paraformaldehyde (22 mg, 0.75 mmol, 2.0 equiv) in methanol (7.5
mL) was stirred for 5 min. Sodium cyanoborohydride (94 mg, 1.5
mmol, 4.0 equiv) was then added, and the resulting mixture was stirred
at room temperature for 20 h. The mixture was then heated to 40 °C for
3 h. The reaction mixture was allowed to cool to room temperature and
was quenched with an aqueous saturated NaHCO3 solution (30 mL).
The product was extracted with DCM (1 × 50 mL); the organic phase
was washed with an aqueous saturated NaHCO3 solution (1 × 30 mL),
dried over MgSO4, and filtered, and the solvent was removed in vacuo.
The resulting residue was purified using preparative HPLC-system A
with a gradient from 20 to 55% acetonitrile in water/acetonitrile 95:5
(aqueous phase modified with 0.2% formic acid) within 25 min to give
4-(dimethylamino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide
(19b) as a yellow solid (4 mg, 2%). 1H NMR (500 MHz, DMSO-d6): δ
= 11.01 (s, 1H, NH), 7.61−7.58 (m, 2H, SCCH), 6.76−6.73 (m, 2H,
NCCHCH), 6.10 (s, 1H, OCCH), 2.98 (s, 6H, NCH3), 2.28 (s, 3H,
4
FCCH, FCCH), 3.05 (q, JH−F = 1.3 Hz, 3H, CH3). 13C NMR (126
MHz, CDCl3): δ = 163.0 (d, 1JC−F = 241.3 Hz, FCCH), 144.4 (d, 3JC−F
= 9.5 Hz, NCCH), 130.3 (d, 3JC−F = 9.2 Hz, FCCHCH), 123.2 (q, 1JC−F
4
4
= 259.6 Hz, CF3), 119.8 (dq, JC−F = 1.8 Hz, JC−F = 1.7 Hz,
NCCHCH), 112.8 (d, 2JC−F = 21.4 Hz, FCCHCH), 111.7 (dq, 2JC−F
=
F
23.5 Hz, 4JC−F = 1.7 Hz, NCCHCF), 36.2 (q, 3JC−F = 2.1 Hz, CH3). 19
NMR (471 MHz, CDCl3): δ = −60.3 (s, 3F, CF3), −111.7 (s, 1F,
CHCF).
N-Methyl-N,4-bis(trifluoromethyl)aniline (13).70 It was synthe-
sized according to the general procedure. Colorless liquid (32 mg,
36%). 1H NMR (500 MHz, CDCl3): δ = 7.62−7.60 (m, 2H, CF3CCH),
4
7.32−7.30 (m, 2H, NCCH), 3.11 (q, JH−F = 1.4 Hz, 3H, CH3). 13C
NMR (126 MHz, CDCl3): δ = 145.8 (s, NCCH), 127.4 (q, 2JC−F = 33.0
Hz, CF3C), 126.4 (q, 3JC−F = 3.6 Hz, CF3CCH), 124.1 (q, 1JC−F = 271.3
Hz, CF3), 123.2 (q, 4JC−F = 1.9 Hz, NCCH), 123.0 (q, 1JC−F = 257.4 Hz,
CF3), 35.8 (q, 3JC−F = 2.2 Hz, CH3). 19F NMR (471 MHz, CDCl3): δ =
−59.6 (s, 3F, CF3), −62.4 (s, 3F, CF3).
CCH3). 13C NMR (126 MHz, DMSO-d6): δ = 169.9 (CCH3), 158.0
(NHCN), 152.8 (NCCHCH), 128.4 (SCCH), 124.3 (CS), 110.9
(NCCHCH), 95.3 (OCCH), 40.4 (NCH3), 12.1 (CCH3). HRMS
(ESI): m/z calcd for C12H13F3N3O3S + H+ [M + H]+: 282.0907.
Found: 282.0918.
1-(Trifluoromethyl)-1H-benzo[d]imidazole (16). Dibromodi-
fluoromethane (1.6 mL, 18 mmol, 1.5 equiv) was added under
vigorous stirring to a suspension of 1H-benzo[d]imidazole (1.41 g, 11.9
mmol, 1.0 equiv), sodium hydride (60% dispersion in mineral oil, 480
mg, 12.0 mmol, 1.0 equiv), and tetrabutylammonium bromide (23 mg,
71 μmol, 0.4 mol %) in DMF (2.4 mL) at 0 °C. The reaction mixture
was gradually warmed to 25 °C within 2 h and stirred at 25 °C for 2 h.
Water (10 mL) was then added dropwise; the product was extracted
with diethylether (5 × 10 mL), and the combined organic phases were
washed with water (5 × 10 mL), dried over MgSO4, and filtered. The
solvent was removed in vacuo, and the resultant crude 4-(1-
(bromodifluoromethyl)-1H-benzo[d]imidazole) was dissolved in
sulfolane (HPLC grade, 2.8 mL). To this solution was added
tetramethylammonium fluoride (201 mg, 2.16 mmol, 0.2 equiv), and
the resulting mixture was stirred at 90 °C. After 12 h, the reaction
mixture was allowed to cool to room temperature and directly subjected
to automated flash column chromatography using a gradient from 0 to
10% ethyl acetate in heptane to give 1-(trifluoromethyl)-1H-benzo-
[d]imidazole (16) as a yellow powder (38 mg, 2% over two steps). 1H
NMR (500 MHz, DMSO-d6): δ = 8.87 (s, 1H), 7.91 (d, 3JH−H = 8.0 Hz,
1H), 7.46 (t, 3JH−H = 7.6 Hz, 1H), 7.40 (t, 3JH−H = 7.7 Hz, 1H), 7.29 (d,
3JH−H = 8.1 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δ = 143.7 (s),
2-(Dimethylamino)ethyl 4-(butyl(methyl)amino)benzoate For-
mate (20b). A solution of 2-(dimethylamino)ethyl 4-(butylamino)-
benzoate (100 mg, 378 μmol, 1.0 equiv) and paraformaldehyde (23 mg,
0.76 mmol, 2.0 equiv) in methanol (7.6 mL) was stirred at room
temperature for 5 min. Sodium cyanoborohydride (95 mg, 1.5 mmol,
4.0 equiv) was then added, and the reaction mixture was stirred at room
temperature for 20 h. The mixture was then heated to 40 °C for an
additional 3 h. The reaction mixture was then allowed to cool to room
temperature and was quenched by the addition of an aqueous saturated
NaHCO3 solution (30 mL). The reaction mixture was extracted with
DCM (1 × 50 mL); the organic phase was washed with an aqueous
saturated NaHCO3 solution (1 × 30 mL), dried over MgSO4, and
filtered, and the solvent removed in vacuo. The resultant residue was
purified using preparative HPLC-system B with a gradient from 5 to
95% acetonitrile in water (aqueous phase modified with 0.2% formic
acid) within 10 min to give 2-(dimethylamino)ethyl 4-(butyl(methyl)-
amino)benzoate formate (20b) as a pale yellow solid (7 mg, 5%). 1H
NMR (500 MHz, DMSO-d6): δ = 8.36 (s, 1H), 7.76−7.72 (m, 2H),
6.71−6.68 (m, 2H), 4.25 (t, 3JH−H = 5.8 Hz, 2H), 3.47−3.42 (m, 2H),
3.42−3.36 (m, 2H), 2.96 (s, 3H), 2.21 (s, 6H), 1.53−1.46 (m, 2H),
1.34−1.25 (m, 2H), 0.90 (t, 3JH−H = 7.4 Hz, 3H). 13C NMR (151 MHz,
DMSO-d6): δ = 165.8, 165.0, 152.3, 130.9, 115.4, 110.6, 61.7, 57.5,
51.1, 45.4, 38.0, 28.4, 19.6, 13.9. HRMS (ESI): m/z calcd for
C16H27N2O2 + H+ [M + H]+: 279.2073. Found: 279.2073.
141.1 (s), 130.2 (s), 125.4 (s), 124.5 (s), 120.8 (s), 115.0 (q, 1JC−F
=
252.3 Hz), 111.2 (s). 19F NMR (471 MHz, DMSO-d6): δ = −59.1 (s).
1H, 13C, and 19F NMR are consistent with the literature.71
4-(Methyl(trifluoromethyl)amino)-N-(5-methylisoxazol-3-yl)-
benzenesulfonamide (19a). A solution of 4-(methylamino)-N-(5-
methylisoxazol-3-yl)benzenesulfonamide (134 mg, 501 μmol, 1.0
equiv), triphenylphosphine (393 mg, 1.50 mmol, 3.0 equiv), and
sodium trifluoromethanesulfinate (117 mg, 750 μmol, 1.5 equiv) in
deoxygenated acetonitrile (2.5 mL) was stirred at room temperature.
After 165 min, silver(I) fluoride (285 mg, 2.25 mmol, 4.5 equiv) was
added and the resulting mixture was stirred at room temperature. After
3 h, the solvent was removed in vacuo and the resulting crude product
was purified using preparative HPLC-system A with a gradient from 20
to 55% acetonitrile in water/acetonitrile 95:5 (aqueous phase modified
with 0.2% formic acid) within 25 min to give 4-(methyl-
(trifluoromethyl)amino)-N-(5-methylisoxazol-3-yl)benzenesulfon-
amide (19a) as a yellow solid (14 mg, 8%) with a purity of 84%.72 1H
NMR (500 MHz, DMSO-d6): δ = 11.39 (s br, 1H, NH), 7.87−7.83 (m,
2H, SCCH), 7.47−7.43 (m, 2H, NCCHCH), 6.14 (q br, 4JH−H = 0.9
N-(2-Methyl-5-(1-(trifluoromethyl)-1H-imidazol-4-yl)phenyl)-4-
(pyridin-2-ylmethoxy)benzamide (21a). A mixture of (4-methyl-3-(4-
(pyridin-2-ylmethoxy)benzamido)phenyl)boronic acid30 (100 mg, 276
μmol, 1.0 equiv), Cs2CO3 (270 mg, 829 μmol, 3.0 equiv), Pd(PPh3)4
(48 mg, 42 μmol, 15 mol %), and 4-bromo-1-(trifluoromethyl)-1H-
imidazole (77 mg, 0.36 mmol, 1.3 equiv) in deoxygenated 1,4-dioxane
(1.4 mL) and deoxygenated water (460 μL) was stirred at 150 °C. After
5 h, the reaction mixture was allowed to cool to room temperature and
extracted with ethyl acetate (3 × 5 mL); the combined organic phases
were washed with an aqueous saturated NaCl solution (2 × 5 mL),
dried over MgSO4, filtered, and the solvent was removed in vacuo. The
resulting residue was purified using automated flash column
chromatography with a gradient from 5 to 50% ethyl acetate in
heptane to give N-(2-methyl-5-(1-(trifluoromethyl)-1H-imidazol-4-
yl)phenyl)-4-(pyridin-2-ylmethoxy)benzamide (21a) as a colorless
powder (50 mg, 40%). 1H NMR (500 MHz, DMSO-d6): δ = 9.83 (s,
1H, NH), 8.60−8.59 (m, 1H, NCHCH), 8.44 (s, 1H, NCHN), 7.98−
7.95 (m, 2H, CHCHCO), 7.85 (td, 3JH−H = 7.7 Hz, 4JH−H = 1.7 Hz, 1H,
NCHCHCH), 7.54 (d, 3JH−H = 7.8 Hz, 1H, NCHCHCHCH), 7.47 (d,
Hz, 1H, OCCH), 3.12 (q, 4JH−F = 1.6 Hz, 3H, NCH3), 2.30 (d, 4JH−H
=
0.8 Hz, 3H, CCH3). 13C NMR (126 MHz, DMSO-d6): δ = 170.3 (s,
CCH3), 157.6 (s, NHCN), 145.7 (s, NCCHCH), 135.5 (s, CS), 128.1
(s, SCCH), 122.4 (q, 1JC−F = 256.3 Hz, CF3), 122.3 (q, 4JC−F = 2.1 Hz,
NCCHCH), 95.4 (s, OCCH), 35.0 (q, 3JC−F = 2.0 Hz, NCH3), 12.1 (s,
CCH3). 19F NMR (471 MHz, DMSO-d6): δ = −57.4 (s). HRMS (ESI):
3
4JH−H = 1.0 Hz, 1H, CH3CCCH), 7.38 (d, JH−H = 8.2 Hz, 1H,
H
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CH3CCH), 7.36 (dd, 3JH−H = 7.4 Hz, 3JH−H = 5.0 Hz, 1H, NCHCH),
7.25 (dd, 3JH−H = 7.7 Hz, 4JH−H = 1.0 Hz, 1H, CH3CCHCH), 7.22 (s,
1H, CF3NCHC), 7.17−7.14 (m, 2H, OCCH), 5.28 (s, 2H, CH2), 2.28
(s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6): δ = 164.8 (s, C(O)),
160.8 (s, COCH2), 156.3 (s, OCH2C), 149.2 (s, NCHCH), 137.1 (s,
NCHCHCH), 136.9 (s, CH3C), 136.7 (q br, 3JC−F = 2.1 Hz, NCHN),
134.9 (s, CH3CCNH), 130.7 (s, CH3CCHCH), 130.7 (q, 3JC−F = 2.1
Hz, CF3NCHC), 130.4 (s, CF3NCHC), 129.7 (s, CHCHCO), 126.9
(s, C(O)C or CH3CCCH), 126.8 (s, C(O)C or CH3CCH), 126.4 (s,
CH3CCHCH), 125.1 (s, CH3CCHCHC), 123.1 (s, NCHCH), 121.8
NHCH), 7.49−7.48 (m, 1H, NCHCHCH), 6.82 (dd, 3JH−H = 4.4 Hz,
4JH−H = 1.7 Hz, 1H, NCHCHCH), 6.41 (dd, 3JH−H = 4.4 Hz, 3JH−H
=
2.5 Hz, 1H, NCHCHCH), 4.08−3.98 (m, 1H, NHCH), 2.36−2.27 (m,
1H, CH3NCH), 2.27 (s, 6H, NCH3), 2.05−2.02 (m, 2H, NHCHCH2),
1.94−1.89 (m, 2H, CH3NCHCH2), 1.45−1.37 (m, 2H, NHCHCH2),
1.35−1.27 (m, 2H, CH3NCHCH2). 13C NMR (126 MHz, DMSO-d6):
δ = 165.2 (s, HCOO of formate), 153.2 (s, NCNCNH), 152.9 (s,
NCNCNH), 137.0 (s, CF3NCH), 127.0 (s, CF3NCHC), 118.7 (q,
1JC−F = 260.6 Hz, CF3), 118.0 (s, NCHCHCH), 115.2 (s, CF3NNCH),
112.0 (s, NCHCHCHC), 108.5 (s, NCHCHCH), 100.8 (s,
NCHCHCH), 62.2 (s, CH3NCH), 48.5 (s, NHCH), 40.9 (s,
NCH3), 31.0 (s, CH3NCHCH2), 26.6 (s, NHCHCH2). 19F NMR
(471 MHz, DMSO-d6): δ = −59.3 (s, CF3). HRMS (ESI): m/z calcd
1
(s, NCHCHCHCH), 118.1 (q, JC−F = 264.6 Hz, CF3), 114.5 (s,
OCCH), 70.4 (s, CH2), 17.8 (s, CH3). 19F NMR (471 MHz, DMSO-
d6): δ = −51.8 (s, CF3). HRMS (ESI): m/z calcd for C24H19F3N4O2 +
H+ [M + H]+: 453.1533. Found: 453.1541.
for C18H23F3N8 + H+ [M + H]+: 409.2071. Found: 409.2075.
N4-((1r,4r)-4-(Dimethylamino)cyclohexyl)-N2-(1-(iso-propyl)-1H-
pyrazol-4-yl)pyrrolo[2,1-f ][1,2,4]triazine-2,4-diamine (23c). A mix-
ture of 1-(iso-propyl)-1H-pyrazol-4-amine (21 mg, 0.17 mmol, 2.0
equiv), tripotassium phosphate (90 mg, 0.43 mmol, 5.1 equiv), (1r,4r)-
N1-(2-chloropyrrolo[2,1-f ][1,2,4]triazin-4-yl)-N4,N4-dimethylcyclo-
hexane-1,4-diamine33 (25 mg, 85 μmol, 1.0 equiv), and XPhos Pd G3
4-(1-(Trifluoromethyl)-1H-benzo[d]imidazol-2-yl)thiazole (22a).
Dibromodifluoromethane (136 μL, 1.49 mmol, 1.5 equiv) was added
under vigorous stirring to a suspension of thiabendazole (200 mg,
994 μmol, 1.0 equiv), sodium hydride (60% dispersion in mineral oil, 44
mg, 1.1 mmol, 1.1 equiv), and tetrabutylammonium bromide (2 mg, 6
μmol, 0.4 mol %) at 0 °C in DMF (3.8 mL). The resulting reaction
mixture was gradually warmed to room temperature within 2 h and
stirred at room temperature overnight. An aqueous saturated NH4Cl
solution (5 mL) was added dropwise. The product was extracted with
diethylether (5 × 4 mL). The combined organic phases were washed
with water (5 × 4 mL), dried over MgSO4 and filtered; the solvent was
removed in vacuo; and the resultant crude product was subjected to
automated flash column chromatography with a gradient from 5 to 10%
ethyl acetate in heptane. The resultant product was dissolved in
sulfolane (HPLC grade, 2.4 mL), tetramethylammonium fluoride
(50 mg, 0.54 mmol, 0.5 equiv) was added, and the reaction mixture was
stirred at 60 °C. After 12 h, the reaction mixture was allowed to cool to
room temperature; the product was extracted with diethylether (3 × 5
mL); the combined organic phases were washed with water (1 × 5 mL),
dried over MgSO4, and filtered; and the solvent was removed in vacuo.
The crude material was purified using preparative HPLC-system C with
a gradient from 5 to 95% acetonitrile in water (aqueous phase modified
with 0.2% ammonia) within 10 min to give 4-(1-(trifluoromethyl)-1H-
benzo[d]imidazol-2-yl)thiazole (22a) as a colorless solid (25 mg, 9%
t
(14 mg, 17 μmol, 20 mol %) in deoxygenated BuOH (5 mL) was
stirred at 90 °C. After 8 h, additional tripotassium phosphate (90 mg,
0.43 mmol, 5.1 equiv) and XPhos Pd G3 (14 mg, 17 μmol, 20 mol %)
were added at room temperature, and stirring at 90 °C was continued
for an additional 14 h. The reaction mixture was then allowed to cool to
room temperature, and the solvent was removed in vacuo. The resulting
residue was purified using preparative HPLC-system C with a gradient
from 5 to 95% acetonitrile in water (aqueous phase modified with 0.2%
ammonia) within 10 min to give N4-((1r,4r)-4-(dimethylamino)-
cyclohexyl)-N2-(1-(iso-propyl)-1H-pyrazol-4-yl)pyrrolo[2,1-f ][1,2,4]-
triazine-2,4-diamine (23c) as a brown solid (3 mg, 9%). 1H NMR (500
MHz, DMSO-d6): δ = 8.39 (s, 1H, NHCCH), 7.85 (s, 1H, iPrNNCH),
7.67 (d, 3JH−H = 8.0 Hz, 1H, NHCH), 7.42 (s, 1H, iPrNCH), 7.35 (dd,
3JH−H = 2.3 Hz, 4JH−H = 1.8 Hz, 1H, NCHCHCH), 6.76 (dd, 3JH−H
=
4.4 Hz, 4JH−H = 1.7 Hz, 1H, NCHCHCH), 6.35 (dd, 3JH−H = 4.3 Hz,
3
3JH−H = 2.4 Hz, 1H, NCHCHCH), 4.42 (sept, JH−H = 6.8 Hz,
CHCH3), 4.07−3.99 (m, 1H, NHCH), 2.23−2.16 (m, 1H, CH3NCH),
2.20 (s, 6H, NCH3), 2.03−2.00 (m, 2H, NHCHCH2), 1.90−1.87 (m,
2H, CH3NCHCH2), 1.41−1.23 (m, 4H, NHCHCH2 and
over two steps). 1H NMR (500 MHz, DMSO-d6): δ = 9.34 (d, 4JH−H
=
2.0 Hz, 1H, NCHS), 8.55 (d, 4JH−H = 2.0 Hz, 1H, SCHC), 7.89−7.85
(m, 1H, CF3NCCHCHCHCH), 7.78−7.75 (m, 1H, CF3NCCH),
7.53−7.47 (m, 2H, CF3NCCHCHCH). 13C NMR (126 MHz, DMSO-
d6): δ = 155.4 (s, NCHS), 145.0 (s, NCCHS or NCN), 144.4 (s,
NCCHS or NCN), 141.6 (s, CF3NCCN), 131.9 (s, CF3NCCN), 125.9
(s, CF3NCCHCHCH or CF3NCCHCHCH), 125.0 (s,
CF3NCCHCHCH or CF3NCCHCHCH), 124.6 (s, SCHC), 120.5
(s, CF3NCCHCHCHCH), 118.9 (q, 1JC−F = 264.4 Hz, CF3), 112.6 (q,
4JC−F = 4.4 Hz, CF3NCCH). 19F NMR (470 MHz, DMSO-d6): δ =
−51.0 (s, CF3). HRMS (ESI): m/z calcd for C11H6F3N3S + H+ [M +
3
CH3NCHCH2), 1.40 (d, JH−H = 6.7 Hz, 6H, CHCH3). 13C NMR
(126 MHz, DMSO-d6): δ = 153.6 (NCNCNH), 153.0 (NCNCNH),
129.1 (iPrNCH), 123.7 (iPrNCHC), 117.4 (NCHCHCH), 115.3
(iPrNNCH), 112.1 (NCHCHCHC), 108.0 (NCHCHCH), 100.3
(NCHCHCH), 62.3 (CH3NCH), 57.5 (CHCH3), 48.4 (NHCH),
41.3 (NCH3), 31.3 (CH3NCHCH2), 29.5 (NHCHCH2), 27.0
(CHCH3). HRMS (ESI): m/z calcd for C20H30N8 + H+ [M + H]+:
383.2667. Found: 383.2642.
N2-(1-(tert-Butyl)-1H-pyrazol-4-yl)-N4-((1r,4r)-4-(dimethyl-
amino)cyclohexyl)pyrrolo[2,1-f ][1,2,4]triazine-2,4-diamine (23d). A
mixture of 1-(tert-butyl)-1H-pyrazol-4-amine (47 mg, 0.34 mmol, 2.0
equiv), tripotassium phosphate (181 mg, 853 μmol, 5.0 equiv), (1r,4r)-
N1-(2-chloropyrrolo[2,1-f ][1,2,4]triazin-4-yl)-N4,N4-dimethylcyclo-
hexane-1,4-diamine33 (50 mg, 0.17 mmol, 1.0 equiv), and XPhos Pd G3
H]+: 270.0308. Found: 270.0307.
N4-((1r,4r)-4-(Dimethylamino)cyclohexyl)-N2-(1-(trifluorometh-
yl)-1H-pyrazol-4-yl)pyrrolo[2,1-f ][1,2,4]triazine-2,4-diamine For-
mate (23a). A mixture of 1-(trifluoromethyl)-1H-pyrazol-4-amine
(51 mg, 0.34 mmol, 2.0 equiv), tripotassium phosphate (181 mg,
853 μmol, 5.0 equiv), (1r,4r)-N1-(2-chloropyrrolo[2,1-f][1,2,4]triazin-
4-yl)-N4,N4-dimethylcyclohexane-1,4-diamine33 (50 mg, 0.17 mmol,
1.0 equiv), and XPhos Pd G3 (29 mg, 34 μmol, 20 mol %) in
t
(29 mg, 34 μmol, 20 mol %) in deoxygenated BuOH (5 mL) was
stirred at 80 °C. After 6 h, the reaction mixture was allowed to cool to
room temperature and the solvent was removed in vacuo. The resulting
residue was purified using preparative SFC-system A with a gradient
from 35 to 40% MeOH/H2O 97:3 (modified with 50 mM ammonia)
within 11 min, followed by a second purification using HPLC-system E
with a gradient from 5 to 95% acetonitrile (modified with 0.1 M formic
acid) within 10 min, followed by a third purification using preparative
SFC-system B with a gradient from 27 to 32% MeOH (modified with
20 mM ammonia) to give N2-(1-(tert-butyl)-1H-pyrazol-4-yl)-N4-
((1r,4r)-4-(dimethylamino)cyclohexyl)pyrrolo[2,1-f ][1,2,4]triazine-
2,4-diamine (23d) as a yellow oil (8 mg, 12%). 1H NMR (500 MHz,
DMSO-d6): δ = 8.36 (s, 1H, NHCCH), 7.91 (d, 4JH−H = 0.8 Hz, 1H,
tBuNNCH), 7.66 (d, 3JH−H = 8.1 Hz, 1H, NHCH), 7.45 (d, 4JH−H = 0.7
Hz, 1H, tBuNCH), 7.36−7.35 (m, 1H, NCHCHCH), 6.76 (dd, 3JH−H
= 4.4 Hz, 4JH−H = 1.7 Hz, 1H, NCHCHCH), 6.35 (dd, 3JH−H = 4.4 Hz,
t
deoxygenated BuOH (5 mL) was stirred at 80 °C. After 15 h, the
reaction mixture was allowed to cool to room temperature and the
solvent was removed in vacuo. The resulting residue was purified using
automated flash column chromatography with a gradient from 5 to 30%
ethyl acetate in heptane (both mobile phases modified with 5%
ammonia in MeOH) followed by purification using preparative HPLC-
system D with a gradient from 5 to 95% MeCN in water (aqueous phase
modified with 0.1 M formic acid) within 8 min to give N4-((1r,4r)-4-
(dimethylamino)cyclohexyl)-N2-(1-(trifluoromethyl)-1H-pyrazol-4-
yl)pyrrolo[2,1-f ][1,2,4]triazine-2,4-diamine formate (23a) as a color-
less solid (20 mg, 29%). 1H NMR (500 MHz, DMSO-d6): δ = 8.97 (s,
1H, NHCCH), 8.36 (s, 1H, CF3NNCH), 8.32 (s, 1H, HCOO of
3
formate), 7.92 (s, 1H, CF3NCH), 7.88 (d, JH−H = 7.9 Hz, 1H,
I
https://dx.doi.org/10.1021/acs.jmedchem.0c01457
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
3JH−H = 2.4 Hz, 1H, NCHCHCH), 4.08−3.99 (m, 1H, NHCH), 2.21−
2.16 (m, 1H, CH3NCH), 2.19 (s, 6H, NCH3), 2.02−1.99 (m, 2H,
NHCHCH2), 1.89−1.86 (m, 2H, CH3NCHCH2), 1.51 (s, 9H, CCH3),
1.44−1.36 (m, 2H, NHCHCH2), 1.31−1.23 (m, 2H, CH3NCHCH2).
13C NMR (126 MHz, DMSO-d6): δ = 153.6 (NCNCNH), 153.0
temperature for 23 h. The solvent was then removed in vacuo, and the
resulting residue was purified using preparative HPLC-system B with a
gradient from 5 to 95% acetonitrile in water (aqueous phase modified
with 0.1 M formic acid) within 10 min to give N-(1-(tert-butyl)-1H-
pyrazol-4-yl)-2-(3,4-dimethoxyphenyl)acetamide (24d) as a brown
solid (9 mg, 14%). 1H NMR (500 MHz, DMSO-d6): δ = 10.06 (s, 1H,
NH), 7.89 (d, 4JH−H = 0.7 Hz, 1H, tBuNNCH), 7.42 (d, 4JH−H = 0.7 Hz,
(NCNCNH), 129.1 (tBuNCH), 123.7 (tBuNCHC), 117.4
(NCHCHCH), 115.3 (tBuNNCH), 112.1 (NCHCHCHC), 108.0
(NCHCHCH), 100.3 (NCHCHCH), 62.3 (CH3NCH), 57.5
(CCH3), 48.4 (NHCH), 41.3 (NCH3), 31.3 (CH3NCHCH2), 29.5
(CCH3), 27.0 (NHCHCH2). HRMS (ESI): m/z calcd for C21H32N8 +
H+ [M + H]+: 397.2823. Found: 397.2825.
t
4
1H, BuNCH), 6.90 (d, JH−H = 2.0 Hz, 1H, CH3OCCHC), 6.88 (d,
3JH−H = 8.2 Hz, 1H, CH3OCCHCH), 6.80 (dd, 3JH−H = 8.2 Hz, 4JH−H
=
2.0 Hz, 1H, CH3OCCHCH), 3.73 (s, 3H, CH3O), 3.71 (s, 3H, CH3O),
3.46 (s, 2H, CH2), 1.46 (s, 9H). 13C NMR (126 MHz, DMSO-d6): δ =
167.7 (CH2C(O)), 148.5 (CH3OC), 147.6 (CH3OC), 129.1
(tBuNCH), 128.5 (CCH2C(O)), 121.3 (CHCNH), 121.0
(CH3OCCHCH), 116.8 (tBuNNCH), 113.0 (CH3OCCHC), 111.8
(CH3OCCHCH), 57.9 (CCH3), 55.6 (OCH3), 55.4 (OCH3), 42.1
(CH2), 29.4 (CCH3). HRMS (ESI): m/z calcd for C17H23N3O3 + H+
[M + H]+: 318.1812. Found: 318.1808.
2-(3,4-Dimethoxyphenyl)-N-(1-(trifluoromethyl)-1H-pyrazol-4-
yl)acetamide (24a). A solution of 2-(3,4-dimethoxyphenyl)acetic acid
(39 mg, 0.20 mmol, 1.0 equiv), HOBt·xH2O (30 mg), and EDC·HCl
(38 mg, 0.20 mmol, 1.0 equiv) in DCM (1.4 mL) was stirred at room
temperature. After 30 min, 1-(trifluoromethyl)-1H-pyrazol-4-amine
(30 mg, 0.20 mmol, 1.0 equiv) was added, and the resulting reaction
mixture was stirred at room temperature for additional 14 h. Water (5
mL) was then added, and the product was extracted with ethyl acetate
(3 × 10 mL). The combined organic phases were washed with water (1
× 10 mL), dried over MgSO4 and filtered, and the solvent was removed
in vacuo. The crude product was purified using automated flash column
chromatography with a gradient from 5 to 50% ethyl acetate in heptane
to give 2-(3,4-dimethoxyphenyl)-N-(1-(trifluoromethyl)-1H-pyrazol-
1-(4-(4-(1-(Trifluoromethyl)-1H-pyrazole-4-carbonyl)piperazin-
1-yl)phenyl)ethan-1-one (25a). EDC·HCl (75 mg, 0.39 mmol, 1.0
equiv) and DMAP (48 mg, 0.39 mmol, 1.0 equiv) were added to a
solution of 1-(4-(piperazin-1-yl)phenyl)ethan-1-one (95 mg,
0.47 mmol, 1.2 equiv) and 1-(trifluoromethyl)-1H-pyrazole-4-carbox-
ylic acid (70 mg, 0.39 mmol, 1.0 equiv) in DMF (2.8 mL). The resulting
reaction mixture was stirred overnight at room temperature. The
solvent was then removed in vacuo, and the product was purified using
automated flash column chromatography with a gradient from 5 to 50%
ethyl acetate in heptane to give 1-(4-(4-(1-(trifluoromethyl)-1H-
pyrazole-4-carbonyl)piperazin-1-yl)phenyl)ethan-1-one (25a) as a
colorless powder (87 mg, 62%, as mixture of two rotamers as evident
1
4-yl)acetamide (24a) as a colorless powder (52 mg, 80%). H NMR
(500 MHz, DMSO-d6): δ = 10.49 (s, 1H, NH), 8.41−8.40 (m, 1H,
CF3NCH), 7.94−7.93 (m, 1H, CF3NNCH), 6.91 (d, 4JH−H = 1.9 Hz,
1H, CH3OCCHC), 6.90 (d, 3JH−H = 8.2 Hz, 1H, CH3OCCHCH), 6.81
(dd, 3JH−H = 8.2 Hz, 4JH−H = 2.0 Hz, 1H, CH3OCCHCH), 3.73 (s, 3H,
CH3O), 3.72 (s, 3H, CH3O), 3.54 (s, 2H, CH2). 13C NMR (126 MHz,
DMSO-d6): δ = 168.8 (s, CH2C(O)), 148.5 (s, CH3OC), 147.7 (s,
CH3OC), 136.7 (s, CF3NNCH), 127.8 (s, CCH2C(O)), 124.3 (q,
4JC−F = 1.6 Hz, CHCNH), 121.1 (s, CH3OCCHCH), 118.1 (s,
CF3NCH), 118.0 (q, 1JC−F = 261.2 Hz, CF3), 113.0 (s, CH3OCCHC),
111.8 (s, CH3OCCHCH), 55.4 (s, OCH3), 55.5 (s, OCH3), 41.9 (s,
CH2). 19F NMR (471 MHz, DMSO-d6): δ = −59.4 (s, CF3). HRMS
1
by two carbon signals for C(O)NCH2 in 13C NMR). H NMR (500
MHz, DMSO-d6): δ = 8.89 (s, 1H, CF3NCH), 8.21 (s, 1H,
CF3NNCH), 7.85−7.82 (m, 2H, C(O)CCH), 6.99−6.96 (m, 2H,
NCCH), 3.75−3.73 (m, 4H, C(O)NCH2), 3.46−3.44 (m, 4H,
CHCNCH2), 2.46 (s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6):
δ = 195.7 (s, CH3C(O)), 161.1 (s, NC(O)), 153.4 (s, NCCH), 144.1
(s, CF3NNCH), 130.8 (s, CF3NCH), 130.2 (s, NCCHCH), 126.9 (s,
C(O)CCHCH), 119.5 (s, C(O)CCHN), 117.7 (q, 1JC−F = 263.0 Hz,
CF3), 113.2 (s, NCCH), 46.6 (s, C(O)NCH2 of rotamer 1), 46.3 (s,
C(O)NCH2 of rotamer 2), 41.2 (s, CHCNCH2), 26.2 (s, CH3). 19F
NMR (471 MHz, DMSO-d6): δ = −59.2 (s, CF3). HRMS (ESI): m/z
(ESI): m/z calcd for C14H14F3N3O3 + H+ [M + H]+: 330.1060. Found:
330.1064.
2-(3,4-Dimethoxyphenyl)-N-(1-iso-propyl-1H-pyrazol-4-yl)-
acetamide (24c). A purple solution of 2-(3,4-dimethoxyphenyl)acetic
acid (47 mg, 0.24 mmol, 1.0 equiv), HOBt·xH2O (37 mg), 1-iso-propyl-
1H-pyrazol-4-amine (30 mg, 0.24 mmol, 1.0 equiv), and EDC·HCl (46
mg, 0.24 mmol, 1.0 equiv) in DCM (1.7 mL) was stirred at room
temperature overnight. The reaction mixture was diluted with water (5
mL) and extracted with ethyl acetate (3 × 10 mL). The combined
organic phases were washed with water (1 × 10 mL), dried over MgSO4
and filtered, and the solvent was removed in vacuo. The crude product
was purified using preparative SFC-system C with a gradient from 10 to
15% MeOH (modified with 20 mM ammonia) within 11 min to give 2-
(3,4-dimethoxyphenyl)-N-(1-iso-propyl-1H-pyrazol-4-yl)acetamide
(24c) as a colorless oil (20 mg, 27%). 1H NMR (500 MHz, DMSO-d6):
δ = 10.06 (s, 1H, NH), 7.86 (d br, 4JH−H = 0.5 Hz, 1H, iPrNNCH), 7.39
calcd for C17H17F3N4O2 + H+ [M + H]+: 367.1377. Found: 367.1367.
1-(4-(4-(1-iso-Propyl-1H-pyrazole-4-carbonyl)piperazin-1-yl)-
phenyl)ethan-1-one (25c). EDC·HCl (62 mg, 0.32 mmol, 1.0 equiv)
and DMAP (40 mg, 0.33 mmol, 1.0 equiv) were added to a solution of
1-(4-(piperazin-1-yl)phenyl)ethan-1-one (79 mg, 0.39 mmol,
1.2 equiv) and 1-(iso-propyl)-1H-pyrazole-4-carboxylic acid (50 mg,
0.32 mmol, 1.0 equiv) in DMF (1.8 mL), and the yellow solution was
stirred at room temperature. After 6 h the solvent was removed in vacuo
and the resulting residue purified using SFC-system A with a gradient
from 12 to 17% MeOH/H2O 97:3 (modified with 50 mM ammonia)
within 11 min, followed by a second purification using preparative
HPLC-system F with a gradient from 2 to 94% acetonitrile in water
(aqueous phase modified with 0.2% ammonia) within 10 min, to give 1-
(4-(4-(1-iso-propyl-1H-pyrazole-4-carbonyl)piperazin-1-yl)phenyl)-
4
i
4
(d, JH−H = 0.7 Hz, 1H, PrNCH), 6.91 (d, JH−H = 2.0 Hz, 1H,
3
1
CH3OCCHC), 6.88 (d, JH−H = 8.2 Hz, 1H, CH3OCCHCH), 6.80
ethan-1-one (25c) as a colorless solid (41 mg, 38%). H NMR (500
(dd, 3JH−H = 8.2 Hz, 4JH−H = 2.0 Hz, 1H, CH3OCCHCH), 4.42 (sept,
3JH−H = 6.7 Hz, 1H, CHCH3), 3.73 (s, 3H, CH3O), 3.71 (s, 3H,
MHz, DMSO-d6): δ = 8.15 (d, 4JH−H = 0.8 Hz, 1H, iPrNCH), 7.85−
4
i
7.82 (m, 2H, C(O)CCH), 7.72 (d, JH−H = 0.8 Hz, 1H, PrNNCH),
6.99−6.96 (m, 2H, NCCH), 4.53 (sept, 3JH−H = 6.7 Hz, 1H, CH3CH),
3.77−3.75 (m, 4H, C(O)NCH2), 3.44−3.42 (m, 4H, CHCNCH2),
CH3O), 3.46 (s, 2H, CH2), 1.35 (d, 3JH−H = 6.7 Hz, 6H, CHCH3). 13
C
NMR (126 MHz, DMSO-d6): δ = 167.7 (CH2C(O)), 148.5 (CH3OC),
147.6 (CH3OC), 129.1 (iPrNCH), 128.5 (CCH2C(O)), 121.3
(CHCNH), 121.0 (CH3OCCHCH), 118.0 (iPrNNCH), 113.0
(CH3OCCHC), 111.8 (CH3OCCHCH), 55.6 (OCH3), 55.5
(OCH3), 52.9 (CCH3), 42.1 (CH2), 22.6 (CCH3). HRMS (ESI):
m/z calcd for C16H21N3O3 + H+ [M + H]+: 304.1656. Found:
304.1671.
N-(1-(tert-Butyl)-1H-pyrazol-4-yl)-2-(3,4-dimethoxyphenyl)-
acetamide (24d). A solution of 2-(3,4-dimethoxyphenyl)acetic acid
(42 mg, 0.21 mmol, 1.0 equiv), HOBt·xH2O (33 mg), 1-(tert-butyl)-
1H-pyrazol-4-amine (30 mg, 0.22 mmol, 1.0 equiv), and EDC·HCl (41
mg, 0.21 mmol, 1.0 equiv) in DCM (1.5 mL) was stirred at room
3
2.46 (s, 3H, C(O)CH3), 1.43 (d, JH−H = 6.7 Hz, 6H, CH3CH). 13C
NMR (126 MHz, DMSO-d6): δ = 195.6 (CH3C(O)), 162.9 (NC(O)),
153.5 (NCCH), 138.8 (iPrNNCH), 130.2 (NCCHCH), 129.7
(iPrNCH), 126.8 (C(O)CCHCH), 115.8 (C(O)CCHN), 113.1
(NCCH), 53.3 (CHCH3), 46.5 (2C, C(O)NCH2 and CHCNCH2),
26.2 (C(O)CH3), 22.6 (CHCH3). HRMS (ESI): m/z calcd for
C19H24N4O2 + H+ [M + H]+: 341.1973. Found: 341.1969.
1-(4-(4-(1-tert-Butyl-1H-pyrazole-4-carbonyl)piperazin-1-yl)-
phenyl)ethan-1-one (25d). EDC·HCl (80 mg, 0.42 mmol, 1.0 equiv)
and DMAP (51 mg, 0.42 mmol, 1.0 equiv) were added to a solution of
1-(4-(piperazin-1-yl)phenyl)ethan-1-one (102 mg, 499 μmol, 1.2
J
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equiv) and 1-(tert-butyl)-1H-pyrazole-4-carboxylic acid (70 mg,
0.42 mmol, 1.0 equiv) in DMF (3.0 mL), and the yellow solution
was stirred at room temperature. After 6 h, the solvent was removed in
vacuo and the resulting residue was purified using preparative SFC-
system A with a gradient from 12 to 17% MeOH/H2O 97:3 (modified
with 50 mM ammonia) within 11 min to give 1-(4-(4-(1-tert-butyl-1H-
pyrazole-4-carbonyl)piperazin-1-yl)phenyl)ethan-1-one (25d) as a
taken and analyzed with LCMS. The area under the absorbance curve
between 220 and 350 nm of the remaining N‑trifluoromethyl amine at
the respective time point was calculated and divided by the area under
the absorbance curve between 220 and 350 nm of the N-trifluoromethyl
amine at t = 0 h.
Determination of stability at pH 1.0, 7.4, and 10.0. A 25 μM
solution of the respective compound in 0.1 M HCl solution (pH 1.0) or
20 mM sodium phosphate buffer (pH 7.4) or 20 mM sodium carbonate
buffer (pH 10.0) was incubated at 70 °C at 300 rpm using an Eppendorf
Thermomixer Comfort plate shaker. After 0, 2, 4, 8, and 24 h, an aliquot
(150 μL) was analyzed using a Waters Acquity UPLC H-Class/QDA
equipped with a Waters Xselect HSS T3 C18 column (2.5 μm, 2.1 mm
× 50 mm) with a gradient of 5−98% acetonitrile in water (modified
with 0.1% formic acid) within 4 min at 40 °C to determine the peak area
of the parent compound. The slope k is determined by linear regression
of the natural logarithm of the peak area of the parent compound vs
incubation time using Microsoft Excel. The half-life t1/2 is determined
using the following equation
1
colorless solid (43 mg, 29%). H NMR (600 MHz, DMSO-d6): δ =
4
8.13 (d, JH−H = 0.5 Hz, 1H, tBuNCH), 7.84−7.82 (m, 2H,
t
C(O)CCH), 7.74 (d br, 4JH−H = 0.5 Hz, 1H, BuNNCH), 6.97−6.95
(m, 2H, NCCH), 3.77−3.75 (m, 4H, C(O)NCH2), 3.43−3.41 (m, 4H,
CHCNCH2), 2.45 (s, 3H, C(O)CH3), 1.54 (s, 9H, NCCH3). 13C
NMR (151 MHz, DMSO-d6): δ = 195.7 (CH3C(O)), 163.1 (NC(O)),
153.5 (NCCH), 138.6 (tBuNNCH), 130.2 (NCCHCH), 128.7
(tBuNCH), 126.8 (C(O)CCHCH), 115.8 (C(O)CCHN), 113.1
(NCCH), 58.8 (NCCH3), 46.6 (2C, CHCNCH2 and C(O)NCH2),
29.4 (NCCH3), 26.1 (C(O)CH3). HRMS (ESI): m/z calcd for
C20H26N4O2 + H+ [M + H]+: 355.2129. Found: 355.2124.
Hydrolysis of 4-Methoxy-N-methyl-N-(trifluoromethyl)aniline (4)
to Form (4-Methoxyphenyl)(methyl)carbamic Fluoride (17). A
solution of 4-methoxy-N-methyl-N-(trifluoromethyl)aniline (4, 2 mg,
10 μmol) in DMSO-d6/D2O 1:4 (150 μL) was left standing under air at
room temperature. After 1 h, the reaction mixture was analyzed by
NMR and GCMS. Due to the partial double bond character of the N−
t1/2 = −0.693/k
The extrapolated half-life at 25 °C is calculated by taking the measured
half-life at 70 °C and assuming a factor of 2 change in reaction rate for
each 10 °C reduction in temperature.
Measuring Compound Reactivity against GSH. The reactivity
of the compounds against GSH was determined by the DMPK
department at Pharmaron. To do so, a 1 μM solution of the respective
compound was incubated in the presence of 4.6 mM glutathione in 0.02
M phosphate-buffered saline (pH 7.4) and 1 mM EDTA-Na2 at 37 °C.
Verapamil was used as an internal standard. The loss of the parent was
monitored by LCMS using a Waters UPLC ACQ-TQC equipped with
an Acquity UPLC system and a Waters Xselect HSS T3 C18 column
(2.5 μm, 2.1 mm × 50 mm). The slope k was determined by linear
regression of the natural logarithm of the area ratio (remaining parent
peak area normalized to verapamil peak area) of the parent drug vs
incubation time. The half-life was calculated using the following
equation:
1
C(O) bond, two rotamers in a ratio of 1:1 are observed in NMR. H
NMR (500 MHz, DMSO-d6/D2O 1:4): δ = 7.20 (d, 3JH−H = 8.9 Hz,
2H, CH), 7.18−7.14 (m, 2H, CH), 6.91−6.86 (m, 4H, CH), 3.68 (s,
3H, OCH3), 3.67 (s, 3H, OCH3), 3.15 (d, 4JH−F = 0.8 Hz, 3H, NCH3),
3.12 (d, 4JH−F = 0.7 Hz, 3H, NCH3). 13C NMR (126 MHz, DMSO-d6/
D2O 1:4): δ = 159.9 (s, CH3OC), 159.8 (s, CH3OC), 149.4 (d, 1JC−F
=
1
284.3 Hz, C(O)F), 149.2 (d, JC−F = 202.4 Hz, C(O)F), 136.0 (s,
NCCH), 135.2 (s, NCCH), 128.8 (s, OCCHCH), 128.5 (s,
OCCHCH), 116.5 (s, OCCH), 116.3 (s, OCCH), 57.2 (s, OCH3),
57.2 (s, OCH3), 40.3 (s, NCH3), 39.9 (s, NCH3).19F NMR (471 MHz,
DMSO-d6/D2O 1:4): δ = −17.9 (s, C(O)F), −20.5 (s, C(O)F). GCMS
(EI): m/z calcd for C9H10FNO2+ [M]+: 183.1. Found: 183.1.
Hydrolysis of 2-(Dimethylamino)ethyl 4-(butyl(trifluoromethyl)-
amino)benzoate (20a) to Form 2-(Dimethylamino)ethyl 4-(butyl-
(fluorocarbonyl)amino)benzoate (26). A solution of 2-
(dimethylamino)ethyl 4-(butyl(trifluoromethyl)amino)benzoate
(20a, 2 mg, 6 μmol) in DMSO-d6/D2O 1:4 (3.2 mL) was left standing
under air at room temperature. After 24 h, the solvent was removed in
vacuo and the reaction mixture was analyzed by NMR and LCMS. Due
to the partial double bond character of the N−C(O) bond, two
rotamers in a ratio of 0.4:1 are observed in the 19F NMR.73 1H NMR
t1/2 = −0.693/k
To rule out non-GSH-mediated loss of the parent, a control reaction of
the respective compound in buffer alone was investigated in parallel.
Additionally, to determining the loss of the parent, the potential
formation of the corresponding glutathione adduct was analyzed by
LCMS.
Measurements of Shake-Flask log D7.4 and Metabolic
Stability in Human Liver Microsomes. Log D7.4 measurements
and measurements of the metabolic stability in human liver microsomes
were performed according to Wernevik et al.74
3
(500 MHz, CD2Cl2): δ = 8.09 (d, JH−H = 8.4 Hz, 2H, C(O)CCH),
3
3
7.32 (d, JH−H = 8.0 Hz, 2H, NCCH), 4.45 (t, JH−H = 5.7 Hz, 2H,
CH2CH2O), 3.72 (t, 3JH−H = 7.5 Hz, 2H, C(O)NCH2), 2.77 (t, 3JH−H
=
Chromlog D7.4 Measurements. A 0.5 mM DMSO solution (1 μL)
of the compound was analyzed by LCMS using a Waters Acquity with a
BEH C18 column (1.7 μm, 50 mm × 2.1 mm) using a gradient from 0
to 100% acetonitrile/H2O 95:5 in acetonitrile/H2O 5:95 (adjusted
with ammonia to pH 7.4) within 1.76 min with a flow rate of 1.0 mL/
min at 40 °C. The retention factor k′ was calculated from the measured
retention time rt of the compound using the following formula:
5.6 Hz, 2H, CH2CH2O), 2.37 (s, 6H, NCH3), 1.62−1.51 (m, 2H,
CH2CH2CH3), 1.35 (sept, 3JH−H = 7.5 Hz, 2H, CH2CH2CH3), 0.91 (t,
3JH−H = 7.4 Hz, 3H, CH2CH3). 13C NMR (126 MHz, CD2Cl2): δ =
165.9 (s, C(O)O), 146.1 (d, 1JC−F = 298.7 Hz, C(O)F), 144.1 (s, C(O)
C), 131.3 (s, C(O)CCH), 130.2 (s, NCCH), 127.2 (s, NCCH), 63.4
(s, CH2CH2O), 58.1 (s, CH2CH2O), 51.9 (s, C(O)NCH2), 45.9 (s,
NCH3), 30.0 (s, CH2CH2CH3), 20.2 (s, CH2CH2CH3), 13.9 (s,
CH2CH2CH3). 19F NMR (471 MHz, CD2Cl2): δ = −14.1 (s, 1F, C(O)
F), −17.9 (s, 0.4F, C(O)F). HRMS (ESI): m/z calcd for C16H23FN2O3
+ H+ [M + H]+: 311.1766. Found: 311.1775.
Stability Study in DMSO. Solutions (10 mM) of the respective
compounds in DMSO were left standing at room temperature in glass
vials without any additional precautions against moisture or oxygen.
After 1 month, aliquots (10 μL) were taken out and analyzed with
LCMS.
Stability Study in H2O/DMSO 4:1. The respective compounds
were dissolved in DMSO and then mixed with water to prepare a 2 mM
stock solution in 4:1 H2O/DMSO. Due to the weak UV absorption of
N-trifluoromethyl pyrazole, a 10 mM solution of this compound was
used. The stock solutions were left standing at room temperature in
glass vials without any additional precautions against moisture or
oxygen. After 0, 0.5, 6, and 24 h and 1 month, aliquots (10 μL) were
k′ = (rt − t0)/t0
with t0 being the retention time of DMSO. Metoprolol, warfarin,
propranolol, chlorpromazine, and felodipine were used as standards. A
calibration curve by plotting the measured k′ of the standards vs their
literature known log D7.4 values was used to transform the determined
k′ of the sample into the chromlog D7.4.
ePSA measurements. A 1.2 mM DMSO solution of the respective
compound was analyzed by SFC using a Phenomenex Chirex 3014
column (4.6 mm × 50 mm) at 40 °C with 20 mM ammonium formate
in MeOH as the co-solvent. A gradient from 5 to 60% of the co-solvent
within 2 min with a flow rate of 4 mL/min was used. Antipyrene,
chlorpromazine, desipramine, pindolol, diclofenac, 3-nitrobenzoic acid,
bumetanide, and furosemide were used as calibration standards. A
calibration curve by plotting the measured retention times of the
K
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calibration standards vs their known ePSA values was used to transform
the measured retention time of the analyte into its ePSA value.
Caco-2 Measurements. Caco-2 measurements were performed by
the DMPK department at Pharmaron as described by Fredlund et al.75
pKa measurements. The pKa was determined spectrophotometri-
cally by Pion using a SiriusT3 following a procedure of Hossain et al.76
shareholders of AstraZeneca. H.C. and T.Q. declare no conflict
of interest.
ACKNOWLEDGMENTS
■
We would like to thank the analytical and separation science
teams at AstraZeneca Gothenburg for purification of final
compounds and analytical support. We also thank colleagues at
AstraZeneca and Pharmaron for measuring the in vitro
properties. Furthermore, we thank Matthew Perry and Martin
Hemmerling (both AstraZeneca Gothenburg) for critically
proofreading the manuscript and James Bird (AstraZeneca
Gothenburg) for fruitful discussions on the measured in vitro
properties. H.C. and T.Q. thank the Erasmus+ program for
fellowships during their internships at AstraZeneca.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01457.
■
sı
NMR spectra for novel compounds and purity analyses
(PDF)
Molecular formula strings and associated data (CSV)
AUTHOR INFORMATION
Corresponding Author
■
ABBREVIATIONS
■
au, arbitrary units; BCRP, breast cancer resistance protein;
BRAF, b-rapidly accelerated fibrosarcoma; chromlog D7.4,
chromatographically determined logarithm of distribution
coefficient at pH 7.4; Caco-2, human epithelial colorectal
adenocarcinoma cells; DAST, (diethylamino)sulfur trifluoride;
DMAP, 4-(dimethylamino)pyridine; EDC, N-(3-dimethylami-
nopropyl)-N′-ethylcarbodiimide; ePSA, experimental polar
surface area; GCMS, gas chromatography-mass spectrometry;
GSH, glutathione; HLM, human liver microsome; HOBt, 1-
hydroxybenzotriazole; IRAK4, interleukin-1 receptor associated
kinase 4; LCMS, liquid chromatography-mass spectrometry;
log D7.4, logarithm of distribution coefficient in 1-octanol water
at pH 7.4; MRP2, multidrug-associated protein 2; TEA,
triethylamine; UPLC, ultra-performance liquid chromatography
Stefan Schiesser − Department of Medicinal Chemistry, Research
and Early Development, Respiratory & Immunology,
̈
BioPharmaceuticals R&D, AstraZeneca, 43183 Molndal,
Sweden; orcid.org/0000-0002-8668-2844; Phone: +46 (0)
31 776 1430; Email: stefan.schiesser@astrazeneca.com
Authors
Hanna Chepliaka − Department of Medicinal Chemistry,
Research and Early Development, Respiratory & Immunology,
̈
BioPharmaceuticals R&D, AstraZeneca, 43183 Molndal,
Sweden; Department of Chemistry, Ludwig-Maximilians
̈
̈
Universitat Munchen, 81377 Munich, Germany
Johanna Kollback − Department of Medicinal Chemistry,
Research and Early Development, Respiratory & Immunology,
̈
BioPharmaceuticals R&D, AstraZeneca, 43183 Molndal,
Sweden; Department of Chemistry and Molecular Biology,
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https://pubs.acs.org/10.1021/acs.jmedchem.0c01457
Author Contributions
S.S. and R.J.C. conceived the project and wrote the majority of
the manuscript. S.S. led the project and synthesized some of the
investigated compounds. S.S., R.J.C., and W.C. designed the
compounds investigated and analyzed data. H.C., J.K., and T.Q.
synthesized some of the investigated compounds, supported the
analysis of the data, and wrote parts of the manuscript. All
authors have given approval to the final version of the
manuscript.
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Notes
The authors declare the following competing financial
interest(s): S.S., J.K., R.J.C, and W.C. are employees and
(7) Search performed on July 15th, 2018.
L
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