Discovery of a subnanomolar and selective spirocyclic agonist of the glucocorticoid receptor

Article abstract of DOI:10.1016/j.ejmech.2018.10.032

Pure diastereomeric spirocyclic analogs of fluorocortivazol were conveniently prepared by a short and efficient synthetic sequence recently developed in our laboratory. The structures and conformations of several key products were confirmed by single crystal X-ray diffraction analysis. Conformational assignments were also supported by DFT calculations. Biological evaluation led to the identification of a highly potent hGR agonist with excellent anti-inflammatory effects in the subnanomolar range. All tested compounds from this series were also selective versus the progesterone receptor.

Full text of DOI:10.1016/j.ejmech.2018.10.032

European Journal of Medicinal Chemistry 161 (2019) 354e363
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of a subnanomolar and selective spirocyclic agonist of the
glucocorticoid receptor
,
b
c
a
d
d
Eduard Badarau a , Frederic Robert , Stephane Massip , Florian Jakob , Simon Lucas ,
ꢀ ꢀ
ꢀ
d
d
d
a, *
ꢀ
Daniela Friebe , Stephanie Hennen , Sven Frormann , Leon Ghosez
a Univ. Bordeaux, European Institute of Chemistry and Biology (IECB), 2 Rue Robert Escarpit, 33607, Pessac, France
b
ꢀ
^
Univ. Bordeaux, Institute of Chemistry & Biology of Membranes & Nano-objects (CBMN), Allee Geoffroy Saint Hilaire, Bat B14, 33600, Pessac, France
c
^
ꢀ
Univ. Bordeaux, Institute of Molecular Sciences (ISM), Batiment A12, 351 cours de la Liberation, 33405, TALENCE Cedex, France
d Grunenthal GmbH, Zieglerstr. 6, 52078, Aachen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Pure diastereomeric spirocyclic analogs of fluorocortivazol were conveniently prepared by a short and
efficient synthetic sequence recently developed in our laboratory. The structures and conformations of
several key products were confirmed by single crystal X-ray diffraction analysis. Conformational as-
signments were also supported by DFT calculations. Biological evaluation led to the identification of a
highly potent hGR agonist with excellent anti-inflammatory effects in the subnanomolar range. All tested
compounds from this series were also selective versus the progesterone receptor.
Received 30 May 2018
Received in revised form
11 October 2018
Accepted 12 October 2018
Available online 15 October 2018
© 2018 Elsevier Masson SAS. All rights reserved.
Keywords:
Glucocorticoids
Fluorocortivazol
Spirocyclic analogues
hGR agonist
1. Introduction
overpasses the GR monomer transcriptional transrepression, a new
therapeutic window could be exploited for unmet medical needs
Glucocorticoids (GCs) are potent therapeutic agents currently
used for the treatment of allergic and inflammatory diseases. The
currently accepted paradigm is that the therapeutic benefit of glu-
cocorticoids is highly correlated with their transrepression versus
transactivation effects. Transrepression occurs when the complex of
GCs and their receptor (GR) inhibits post-transcriptional activity of
other transcriptional factors, such as activator protein 1 (AP-1) and
such as SIRS and sepsis [1]. Identifying new “super agonists” able to
satisfy these demands could therefore be a new interesting
approach to potential anti-inflammatory drugs.
We recently designed a new class of potential ligands of
glucocorticoid receptor [2] which are spirocyclic analogs of the
compounds MK disclosed by Ali et al. [3] (Scheme 1). The spi-
rocyclic substructure was expected to increase the conformational
pool while keeping the key hydroxyl pharmacophore in the same
environment, as indicated by the good superposition of the refer-
ence MK molecule on the spirocyclic target (Fig. 1).
Earlier examples of spirocyclic corticoids did not imply a modi-
fication of the core scaffold but consisted in the replacement of a
carbonyl group by a spirolactone aiming at producing potent
aldosterone antagonists devoid of undesired endocrinological side
effects. These derivatives were highly potent binders for the MR, AR
and PR [4] but they bound weakly to the GR [5].
We have recently reported a detailed study of a practical syn-
thetic route to this new class of spirocyclic ligands. The observation
of an IC50 of 27 nM for a representative example in this series [2]
prompted us to prepare more examples and study their biological
activities in more detail.
the nuclear factor-kappa B (NFkB) which are known to induce in-
flammatory effects. On the contrary, transactivation will trigger un-
desired side effects. Moreover, GCs also interact with many other
steroid receptors (MR - mineralocorticoid, PR - progesterone, ER -
estrogen, AR - androgen receptors) thus leading to other side effects.
The development of new anti-inflammatory drugs with enhanced
therapeutical benefit will thus require the synthesis of selective GR
ligands with a better dissociating profile.
A more recent concept postulated by Libert and coll. suggests
that if the GR homodimer-mediated transactivation greatly
* Corresponding author.
E-mail address: l.ghosez@iecb.u-bordeaux.fr (L. Ghosez).
https://doi.org/10.1016/j.ejmech.2018.10.032
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

E. Badarau et al. / European Journal of Medicinal Chemistry 161 (2019) 354e363
355
Scheme 1. Design of new potential spiro-corticoids.
Scheme 2. Synthesis of the spirodiketones 4. NaH, HCOOEt in Et2O, 48 h, rt; (b) methyl
vinyl ketone, DBU, TEA/THF, 12 h, rt, 91%; (c) morpholine: AcOH (1:1)/DMSO, 24 h, rt,
63%; (d) ethylene glycol, PTSA (0.05 eq.)/PhH/12 h, reflux, 25%.
The construction of the pyrazole ring was performed in two
steps: formylation of the protected ketone 4 followed by treate-
ment with p-fluorophenyl hydrazine to yield the tricyclic pyrazole 6
in 72% yield (Scheme 3). Hydrolysis of 6 delivered ketone 7c in a
good 83% yield.
A
comparative DFT conformational analysis (M06-2X/6-
311 þ G(2d,p) level in chloroform) was performed on the homol-
ogous series of ketones 7 (n ¼ 1, 2 and 3). Contrarily to the 6-
membered derivative which has two low-energy conformations
in a 1:1 ratio [2], for the more rigid five-membered ketone 7a
(Fig. 2) the major (73%) conformation resembles that of the six-
membered ketone in the crystal. Surprisingly, the seven-
membered ketone 7c (Fig. 2) exhibited a preference (68%) for a
twist-boat conformation rather than the expected twist-chair [2].
The full details of the conformational analyses are described in the
supplementary material.
Ketone 7c and the lower homologs, ketones 7a and 7b synthe-
sized as described earlier [2], were converted into the
target alcohols by addition of phenylmagnesium bromide and
benzyl magnesium bromide. The selection of a benzyl and a phenyl
group resulted from our previous results, which showed that hy-
drophobic moieties led to favourable interactions with the GR. For
example, we have previously identified that a benzyl moiety trig-
gered an IC50 of 27 nM in the case of the 6-membered analogue.
The addition reactions yielded mixtures of diastereoisomeric
alcohols, which could be separated. (Scheme 4, Table 1). At this
stage the formation of two diastereoisomers was a bonus since they
were easily separated.
Fig. 1. Example of superposition between the reference ligand MK (green) and a
designed spirocyclic derivative (violet, n ¼ 2, R ¼ Bn). (For interpretation of the refer-
ences to colour in this figure legend, the reader is referred to the Web version of this
article.)
2. Results and discussion
2.1. Synthesis of the target alcohols
We had already optimized the synthetic route for the 5- and 6-
membered analogues [2]. We were also interested in testing the
higher homologues which should show a higher conformational
flexibility, possibly favouring a better interaction with the target
protein. The synthetic route was similar to that reported for the
lower homologues (Scheme 2). The commercially available cyclo-
heptanone was formylated by treatment with sodium hydride and
ethyl formate in dry diethyl ether. Base-catalyzed addition of 1 to
methyl vinyl ketone followed by treatment of the Michael adduct 2
with a 1:1 mixture of morpholine and acetic acid delivered the
desired spirocyclic intermediate 3. The selective protection of the
saturated ketone was first conducted under the experimental
conditions optimized for the 6-membered analogue: treatment of 3
with ethylene glycol in the presence of 1 eq. PTSA and of molecular
sieves at room temperature. This gave a poor yield (z10%) of the
desired monoketal 4 while the lower homologue had led to a 72%
yield in the same experimental conditions. A slightly better yield
(25%) of 4 was obtained after treatment of 3 in a refluxing mixture
of ethylene glycol/benzene (Dean-Stark) containing 0.05 eq. of
PTSA.
Scheme 3. Construction of the pyrazole ring. (a): NaH, HCOOEt, MeOH/toluene, 48 h,
rt; (b) 4-F-Ph-NHNH2, AcONa/AcOH, 24 h, rt, (over two steps, 6: 64%); (c) HCl/THF, 1.5 h,
65 ꢀC, (7c: 83%).

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E. Badarau et al. / European Journal of Medicinal Chemistry 161 (2019) 354e363
Table 1
Reaction of organometallics with ketones 7.
n
R
Diastereoisomers (yield %)
1
2
2
3
3
Ph
Ph
Bn2
Ph
Bn
8a (12%)
8b (56%)
8c (81%)
8d (10%)
8e (20%)
not isolated
9b (27%)
9c (13%)
9d (38%)
9e (18%)
Fig. 2. Conformational analysis of the 5- and 7-membered ketones 10a and 10c.
In our previous report [2] we had assigned the preferred con-
formations of diastereoisomeric alcohols 8 and 9 by DFT calcula-
tions. The conformational assignment was also supported by an X-
ray diffraction analysis conducted on only one diastereoisomer
(R ¼ 1-propynyl). Now we have been able to obtain suitable crystals
for both diastereoisomers 8b and 9b (R ¼ Ph). Our initial hypothesis
concerning the conformation of all diastereoisomers was thus
confirmed: an axial hydroxyl group and the bulkier alkyl or aryl
substituent in equatorial position (Fig. 3). Crystals of 7-membered
analogues (8e and 9d) suitable for X-ray diffraction analyses
could also be obtained. They also confirmed that both isomers
adopt the conformation with an axial hydroxyl group as in the case
of the related 5- and 6-membered alcohols (Fig. 4).
Fig. 3. Crystal structures of tertiary alcohols 8b and 9b.
The spatial orientation of the hydroxyl group was expected to be
an important factor for the interaction with the glucocorticoid re-
ceptor. It seemed thus important to confirm that the ligands
conserve their conformations in physiological conditions. We
therefore studied the conformational profile of the phenyl-
substituted tertiary alcohols by DFT in water (M06-2X/6-
311 þ G(2d,p) level). As expected, in both alcohols 8b and 9b, the
favoured conformations were those with an equatorial phenyl
group (higher A value: 3 vs 0.87 kcal/mol), This conformation was
further stabilized as a result of a p-stacking interaction between the
phenyl and pyrazole ring. For 8b in aqueous solution, 97% of the
conformational population corresponded to conformers with an
equatorial phenyl group but differing by the conformation around
the N-Ar bond. One of these conformers was found in the crystal
structure of 8b. The situation is similar for the other diastereo-
isomer 9b, the conformers with an equatorial phenyl group rep-
resenting 90% of the conformational population (Fig. 5). The
calculations also revealed some stabilization of these conformers
Fig. 4. Crystal structures of tertiary alcohols 8e and 9d.
by the p-stacking between the phenyl group and the pyrazole ring.
The same factors were also responsible for the stabilization of the
major (98%) conformation of the more rigid cyclopentanol 8a.
Surprisingly, for the more flexible cycloheptanols 8d and 9d, the
more stable twist-chair conformations with an equatorial phenyl
group were also predominant (Fig. 6).
2.2. Synthesis of the dehydrated analogues
We also synthesized a series of dehydrated analogues to
Fig. 5. Representative conformers for the alcohols 8b (97% population) and 9b (90%
population).
investigate whether the hydroxyl group was essential for the
interaction with the GR. The mixture of diastereomeric alcohols 8b
and 9b obtained from the reaction of ketone 7b with phenyl-
magnesium bromide was treated with triflic acid for 10 min in
refluxing toluene to yield the expected elimination product 10a
(Scheme 5). In order to offer a possible additional H-bonding in the
binding site, we decided to introduce an OH group on the aromatic
substituent. The reaction of the Grignard reagent derived from 4-
Scheme 4. Reaction of organometallics with ketones 7a-c. (a) RMgBr/THF, 12 h, 0 ꢀC to
r.t. (see Table 1 for details).

E. Badarau et al. / European Journal of Medicinal Chemistry 161 (2019) 354e363
357
Scheme 6. Synthesis of
a-substituted analogues 13 and 14. (a) (i) LHMDS (1 eq.),
Fig. 6. Representative conformers for the alcohols 8a (98% population), 9c (97.5%
population) and 9c (97% population).
DMPU (1.2 eq.), BnBr (1.2 eq.)/THF, 12 h, ꢁ78 ꢀC to r.t. (R ¼ Bn, 11a (12%, isolated as 2/1
mix of diastereoisomers), 12a (14%); (ii) LDA (1.2eq.), propargyl bromide (1.5 eq.)/THF,
12 h, ꢁ78 ꢀC to r.t. (R ¼ propargyle, 11b (18%), 12b (10%); (b) DIBAL-H (3 eq.)/THF, 3 h
then recrystallization in ACN (R ¼ Bn, 13a (55%); R ¼ propargyl, 13b (18%)); (c) LAH (1.2
eq.)/THF, 2 h, 0 ꢀC (R ¼ Bn, 14a, 56%).
bromo-anisole yielded the expected alcohol but the subsequent
treatment with BBr3 aiming at cleaving the methyl group only
yielded the dehydration product 10c. We thus decided to use a
trialkylsilyl protecting group which could be easily cleaved under
non acidic conditions. It was anticipated that a trimethylsilyl group
would not stand the conditions of the first step. We thus selected
the more stable triisopropylsilyl (TIPS) ethers. The halogen ex-
change step with sec-butyl lithium yielded the reactive lithiated
nucleophiles. The corresponding diastereomeric mixture of ad-
ducts 8 and 9 was reacted with TBAF and subsequently with BBr3 to
yield the elimination product 10b (Scheme 5).
discussed by several other groups [6e8]. It is generally accepted
that the enolate reactivity relies on its stage of aggregation, less
substituted enolate (thus more aggregated) should be less reactive.
The p- p interaction between the enolate and the alkylating agent
could also be an important factor in the polyalkylation process [9].
The structures of diastereoisomer 13a and 13b were confirmed
by X-ray diffraction analyses. In both cases the preferred confor-
mation was that with an axial hydroxyl group and an equatorial
vicinal substituent (Fig. 7).
2.3. Synthesis of
a-substituted analogues
2.4. Biological studies
We expected the
s
-position of the ketone 7b easy to modulate,
in spite of the sterical hindrance imposed by the spirocyclic moiety.
However, classic anionic conditions employing sodium hydride or
LDA (1.2e3 eq.) and benzyl bromide as alkylating agents (1.2e3
eq.), at low temperature, conducted to full recovery of the starting
material. De-aggregating the lithium enolates by coordinating the
metal with N,N0-dimethylpropyleneurea (DMPU), a less toxic
equivalent of the well-known coordinating reagent hexamethyl-
phosphoramide (HMPA) delivered low yields of the alkylated
products along with the recovery of the remaining starting material
(Scheme 6). Interestingly, in the case of propargyl bromide, the best
experimental conditions were LDA (1.2 eq) in THF at ꢁ78 ꢀC, while
adding the DMPU additive showed only traces of the desired
product. Surprisingly, dialklylated compounds 12 were also iso-
lated. They were characterized by NMR, MS and also analysed by X-
rays diffraction (Fig. 7). The mono-vs poly-alkylation issue was
A study of the binding affinity to the hGR was performed using
[3H]dexamethasone as radioligand (see supporting information for
details on binding measurements). Only pure diastereoisomeric al-
cohols were tested. In most cases their 3D structure had been
assigned by X-rays diffraction studies (vide supra). As shown in
Table 2, a clear trend was observed: compounds having the hy-
droxyl group parallel to the axis of the central heterocyclic scaffold
were all good GR binders with IC50 values in the nM range. Except for
compound 8d, all other diastereoisomers, which have the hydroxyl
moiety orthogonal to the axis of the main scaffold showed a low
affinity for the glucocorticoid receptor. The best GR ligand identi-
fied in this study was compound 9b which showed a subnanomolar
affinity (IC50 ¼ 0.41 nM). This derivative revealed 30-fold higher
affinity than prednisolone (IC50 ¼ 13 nM), a classical reference GR
Scheme 5. Synthesis of dehydrated analogues 10a-c. For X ¼ H: (a) PhMgBr/THF; (b)
Tf2O/PhCH3, 10 min, reflux (15% over two steps); for X ¼ OH: (a) 4-OTIPS-bromo-
benzene, n-BuLi; (b) i: TBAF/THF, 5 min, 0 ꢀC; ii: BBr3/DCM, 1 h, 0 ꢀC (36% over three
steps); for X ¼ OMe: (a) 4-OMe-phenylmagnesium chloride/THF, 1h, 0 ꢀC; (b) BBr3/
DCM, 1 h, 0 ꢀC (20% over two steps).
Fig. 7. Crystal structures of dibenzylated product 12a and of the isolated reduction
products 13a and 13b. H-atoms were omitted for clarity.

358
E. Badarau et al. / European Journal of Medicinal Chemistry 161 (2019) 354e363
Table 2
Binding affinities (see supporting information for details) of the new compounds to
hGR.
R1
R2
n
Cpd
IC50 (nM)
Cpd
IC50 (nM)
Ph
Ph
Bn2
Ph
Bn
H
H
H
H
H
1
2
2
3
3
2
2
2
8a
8b
8c
8d
>1000
1012
5729
9.5
e
e
9b
9c
9d
9e
e
0.41
27
6
89
e
Fig. 8. Crystal structure of compound 10c and superposition of crystal structures of 9b
(green), 9d (blue) and 10c (yellow). (For interpretation of the references to colour in
this figure legend, the reader is referred to the Web version of this article.)
H
Bn
Propargyl
Bn
8e
130
13a
13b
14a
13 nM
>1000
>1000
>1000
H
H
e
e
e
e
spirocycles were devoid of any affinity for this closely related steroid
receptor.
The compound with highest hGR affinity identified in this series,
compound 9b, was selected for functional analyses to confirm its
anti-inflammatory effects on NF-kB-mediated cytokine secretion.
prednisolone
Inhibition of IL6 release was investigated in vitro in IL1b-induced
inflamed human pulmonary epithelial A549 cells and repression
of TNFa secretion was analysed ex vivo in lipopolysaccharide (LPS)-
Ar ¼ 4-F-Ph.
IC50 values represent the average of 2e3 independent experiments run in triplicate
for which SD ꢂ 20%.
stimulated rat whole blood. (Table 5). In line with the results from
binding assays to the human GR, compound 9b is very potent in the
human A549 IL6 inhibition assay with roughly 10-fold higher po-
tency compared to prednisolone, whereas in the rat whole blood
assay the potency of compound 9b is 10-fold lower, but comparable
to prednisolone, which might be explained by a species difference
human versus rat or by a higher plasma protein binding of com-
pound 9b, thus a lowered free fraction. It was not within the scope
of the current study to further investigate this finding. It can be
concluded that compound 9b is a highly affine and highly potent
hGR agonist with excellent anti-inflammatory effects in the sub-
nanomolar range.
ligand included in the same experiment. Increasing the size of the
hydroxyl-carrying ring induced a 15-fold decrease of affinity.
However, the higher homologue 9d still remained a highly potent
hGR ligand (IC50 ¼ 6 nM).
Unexpectedly, even if 50-fold less active than the corresponding
tertiary alcohol 9b, the dehydrated compounds 10a,b still showed a
binding affinity in the low nM range (10e20 nM), similar to pred-
nisolone (IC50 ¼ 13 nM) (Table 3). The corresponding methyl ether
10c showed a much weaker activity resulting perhaps from the
replacement of H by a bulkier methyl group. An X-ray diffraction
was performed on 10c. Superposition between the crystal structure
of 10c and the crystal structures of the potent derivatives 9b and 9d
shows that all these derivatives share the same aromatic/hydro-
phobic pharmacophore (Fig. 8). In the case of compound 10c, the
absence of a hydroxyl group, presumably responsible for a H-
bonding within the binding site, decreases its affinity 50-fold for
hGR.
3. Conclusions
Spirocyclic glucocorticoids were designed, synthesized and
evaluated for their activity towards hGR. Pure diastereoisomers
were tested for their binding to the hGR. This led to the identifi-
cation of novel highly active glucocorticoid ligands. The best ligand
identified so far in this series, compound 9b, had an excellent IC50
of 0.4 nM, 30-fold more potent than prednisolone, the classical
reference GR ligand used in the same experiment (IC50 ¼ 13 nM).
Moreover, the same compound 9b revealed highly potent anti-
inflammatory effects in in vitro and ex vivo cytokine release ex-
periments. The corresponding diastereoisomer 8b was devoid of
activity for hGR (IC50 ¼ 1012 nM). Preliminary results also showed
that this new class of hGR binders were selective over the PR. The
dehydrated analogue (10a) of the most potent compound identified
so far (9b) showed a 50-fold lower affinity for the hGR (10b,
A subset of ligands was also evaluated for their binding affinity
to the progesterone receptor (PR). As shown in Table 4, these
Table 3
Binding affinities (see supporting information for details) to the GR of the dehydrated
compounds 10.
Table 4
Affinities for the human glucocorticoid and the progesterone receptor.
Cpd
IC50 (nM) GR
IC50 (nM) PR
9b
9c
9d
10a
0.41
27
6
20
13
780
X
Cpd
IC50 (nM)
>1000
>1000
>1000
>1000
H
10a
10b
10c
20
10
135
4-OH
4-OMe
prednisolone
IC50 values represent the average of 2e3 different independent experiments run in
triplicate for which SD ꢂ 20%.
IC50 values represent the average of 2e3 different independent experiments run in
triplicate for which SD ꢂ 20%.

E. Badarau et al. / European Journal of Medicinal Chemistry 161 (2019) 354e363
359
Table 5
solvent was evaporated, the residue diluted with DCM (200 ml) and
washed with ammonium chloride (sat. soln., 2 ꢃ 100 ml) and so-
dium chloride (sat. soln., 1 ꢃ 100 ml). The organic solvent was dried
over magnesium sulfate and evaporated under vacuum. The crude
product was purified by flash-chromatography (eluent: PE/EtOAc 7/
3) to give the desired addition derivative 2 as a colorless oil (24.7 g).
In vitro/ex vivo biological evaluation.
Cpd
IC50 (nM)
Rat whole blood
IC50 (nM)
Human A549
9b
4.0
4.6
0.5
5.8
prednisolone
C
12H18O3; Light yellow liquid; Yield: 91%; M ¼ 210.27 g/mol; IR
IC50 values represent the average of 2e3 independent experiments run in triplicate
for which SD ꢂ 20%.
(ZnSe)
n
¼ 2926, 1722, 1685, 1360, 1236, 1072 cmꢁ1
;
1H NMR
(300 MHz, CDCl3) d 1.47e1.54 (m, 7H),1.70e2.01 (m, 5H), 2.24e2.54
(m, 5H), 5.82 (ls, 1H); 13C NMR (75 MHz, CDCl3)
d 27.4 (CH2), 28.2
IC50 ¼ 20 nM). This seems to indicate the importance of the H-bond
generated by the hydroxyl moiety present on the ligand. However,
as the affinity is still in the nM range, this could be an opportunity
for the medicinal chemist to keep essential features for a nM po-
tency, while removing the chemical complexity represented by an
additional asymmetric center.
We believe that these preliminary synthetic and biological
studies open a new window for the discovery of new potent and
selective glucocorticoid ligands.
(CH2), 30.0 (CH2), 31.3 (CH2), 33.3 (CH2), 36.7 (CH2), 37.2 (CH2), 40.1
(CH3), 126.7 (CH), 172.0 (Cq), 195.2 (Cq), 200.1 (Cq); HRMS: calcd. for
C12H19O3 211.13287, found 211.13289 (0.090 ppm).
4.2.2. spiro[5.6]dodec-4-ene-3,12-dione (3)
Under inert atmosphere, morpholine (9.36 ml, 1 eq.) and acetic
acid (6.2 ml, 1 eq.) were added to a solution of the previously ob-
tained Michel adduct 2 (22.6 g, 1 eq.) in DMSO (150 ml). The
mixture was vigorously stirred overnight at room temperature,
then, after the complete consumption of the starting material,
slowly added to sodium carbonate (satd. soln., 200 ml). The or-
ganics were extracted with ethyl acetate (4 ꢃ 100 ml), then washed
with ammonium chloride (150 ml) before drying over magnesium
sulfate. The solvent was evaporated under reduced pressure and
the crude product was purified by flash chromatography (PE/EtOAc
7/3) to give the desired annulated compound 3 (13 g).
4. Experimental
4.1. General experimental
The infrared (IR) spectra were recorded on a Bruker IRFT IFS 55
spectrometer on zinc selenide plates, unless stated otherwise.
1H NMR and 13C NMR were recorded with Bruker Avance II 300
(at 300.18 MHz and 75.48 MHz, respectively) in CDCl3-d1 and
DMSO‑d6, using tetramethylsilane (TMS) as an internal standard.
Chemical shifts are reported in parts per million (ppm) and the
coupling constants are reported in units of Hertz [Hz]. Multiplicities
were abbreviated as follows: singlet (s), doublet (d), triplet (t),
quartet (q).
Low resolution mass spectra (MS)/High resolution mass spectra
(HRMS) were recorded with a ThermoElectron LCQ Advantage and
a ThermoElectron Exactive spectrometer, respectively.
Melting points were recorded using a Buchi B-540 apparatus
and are uncorrected.
C
12H16O2; light-yellow oil; yield: 63%; M ¼ 192.25 g/mol; IR
(ZnSe)
(300 MHz, CDCl3)
n
¼ 2937, 1696, 1682, 1455, 1346, 1152, 1138 cmꢁ1
;
1H NMR
d 1.40e1.91 (m, 9H), 2.16e2.50 (m, 4H),
2.59e2.68 (m, 1H), 5.91e5.99 (m, 1H), 6.67e6.74 (m, 1H); 13C NMR
(75 MHz, CDCl3) 24.3 (CH2), 26.5 (CH2), 30.1 (CH2), 31.6 (CH2), 33.9
d
(CH2), 36.4 (CH2), 40.1 (CH2), 53.3 (Cq), 129.3 (CH), 150.9 (CH), 198.4
(Cq), 212.3 (Cq); HRMS: calcd. for C12H17O2 193.12231, found
193.12250 (1.003 ppm).
4.2.3. 1,4-dioxadispiro[4.0.5-6.5-5]hexadec-7-en-9-one (4)
Under nitrogen atmosphere, to a solution of the spiro-derivative
3 (1 g, 1 eq.) in benzene were successively added ethylene glycol
(3 ml, 10 eq.) and PTSA (0.05 g, 0.05 eq.). The mixture was refluxed
for 12 h with water distillation using a Dean-Stark trap. The solvent
was removed under vacuum, the residue was dissolved in sodium
bicarbonate (aq. satd. soln., 30 ml), then the organics extracted with
DCM (3 ꢃ 30 ml). The combined organic layers were washed with
brine (100 ml), dried over magnesium sulfate and the solvent was
evaporated under vacuum. The desired ketal was isolated by flash-
chromatography (PE/EtOAc 15/5) in mixture with an unidentified
product (1/2 ratio as showed by H NMR).
Flash chromatography was performed on Merck 40e70 nM
(230e400 mesh) silica gel under air pressure. Thin-layer chroma-
tography (TLC) was carried out on Merck silica gel 60 F254 pre-
coated plates. Visualization was made with ultraviolet light
(
l
¼ 254/365 nm) or using an aqueous potassium permanganate
staining solution.
4.2. Experimental details and characterization data
4.2.1. 2-Oxo-1-(3-oxobutyl)cycloheptanecarbaldehyde (2)
C
14H20O3; light-yellow visc. oil; yield: 25%; M ¼ 236,14g/mol; IR
Sodium hydride (60% dispersion in mineral oil, 10.5 g) was
added under nitrogen to dry diethyl ether (400 ml). Subsequently,
ethyl formate (11.68 g, 13 ml, 1.2 eq.) and then cyclohexanone (15 g,
12.6 ml, 1 eq.) were added and the mixture was stirred for 48 h at
room temperature. The orange mixture was slowly poured on ice-
cold water (200 ml), the aqueous layer separated and washed
with diethyl ether (2 ꢃ 200 ml) before being acidified with
hydrogen chloride (aq. soln., 6N). The organic materials were
extracted with diethyl ether (3 ꢃ 150 ml), the combined organic
layers were dried over magnesium sulfate and the solvent was
(ZnSe)
CDCl3)
n
d
¼ 2933, 1682, 1455, 1225, 1050 cmꢁ1
;
1H NMR (300 MHz,
1.42e1.79 (m, 10H), 1.84e1.98 (m, 1H), 2.05e2.19 (m, 1H),
2.26e2.52 (m, 2H), 3.76e3.91 (m, 4H), 5.84 (d, J ¼ 10.4 Hz, 1H), 6.83
(dd, J ¼ 10.4 Hz, J ¼ 1.4 Hz, 1H); 13C NMR (75 MHz, CDCl3)
d 21.1
(CH2), 22.5 (CH2), 27.1 (CH2), 27.6 (CH2), 32.6 (CH2), 33.3 (CH2), 34.0
(CH2), 46.8 (Cq), 64.5 (CH2), 64.7 (CH2), 114.0 (Cq), 127.5 (CH), 155.5
(CH), 199.7 (Cq); HRMS: calcd. for C14H21O3 237.14852, found
237.14902 (2.104 ppm).
4.2.4. 9-Oxo-1,4-dioxadispiro[4.0.56.55]hexadec-10-ene-8-
carbaldehyde (5)
evaporated to conduct to the desired a-formylated cyclohexanone 1
as a light-yellow liquid subsequently used without further purifi-
cation. The crude compound 1 (18 g, 1 eq.) was dissolved in THF
(130 ml), then DBU (2 ml, 0.1 eq.), TEA (2.69 ml, 0.1 eq.) and methyl
acrylate (16.1 ml, 1.5 eq.) were consecutively added to the mixture
and stirred for 24 h at room temperature under inert atmosphere.
After the complete consumption of the starting material, the
Under inert atmosphere, the previously obtained ketal 4 (1.4 g, 1
eq.) was dissolved in dry toluene (50 ml) and the mixture cooled
to ꢁ40 ꢀC. Ethyl formate (1.9 ml, 4 eq.) and then sodium hydride
(60% dispersion in oil, 0.94 g, 4 eq.) were subsequently added before
slowly adding methanol (0.1 ml, 0.5 eq.) at low temperature over
15 min. The mixture was stirred for 30 min at low temperature,

360
E. Badarau et al. / European Journal of Medicinal Chemistry 161 (2019) 354e363
then left to slowly come back to room temperature and stirred for
two additional hours. After the complete consumption of the
starting material, the mixture was slowly poured onto ice-cold
water (100 ml), the organic layer was separated and washed two
additional times with water (2 ꢃ 75 ml). The combined aqueous
layers were subsequently acidified to with hydrogen chloride (aq.
soln., 6N) before extracting the organic materials with diethyl ether
(3 ꢃ 100 ml). The combined organic layers were dried over mag-
nesium sulfate and the solvent evaporated to give a light yellow oil
(turning brown on standing) and was subsequently used without
further purification.
(CH), 116.2 (CH), 125.0 (CH), 125.2 (CH), 132.8 (CH), 135.6 (Cq), 136.1
(Cq), 138.1 (CH), 159.9 (Cq), 163.1 (Cq), 213.2 (Cq); HRMS: calcd. for
C13H20FN2O 311.15542, found 311.15486 (ꢁ1.793 ppm).
4.2.6.1. 1-(4-fluorophenyl)-10-phenyl-spiro[4H-indazole-5,20-cyclo-
hexane]-10-ol. Under inert atmosphere and at 0 ꢀC, the previously
obtained ketone 7b (0.3 g, 1 eq.) was dissolved in dry THF (10 ml).
Phenylmagnesium bromide (1M soln. in THF, 1.02 ml, 3 eq.) was
slowly added at low temperature and the mixture was stirred 2
additional hours at 0 ꢀC until the complete conversion of the
starting material. The reaction was quenched by addition of
ammonium chloride (satd. soln. 30 ml) and the organic-soluble
materials extracted with ethyl acetate (3 ꢃ 50 ml). The organic
phase was dried over magnesium sulfate and concentrated under
vacuum. The solid residue was triturated with diethyl ether to give
a white powder which was further crystallized in acetonitrile to
conduct to the (5R, 8R)-diastereoisomer (0.180 g, 48%), as
confirmed by X-rays. The recovered filtrate was subsequently
evaporated to conduct to a solid residue, further purified by flash-
chromatography. The (5R, 8S)-diastereoisomer (as showed by
crystallization in ethanol) was eluted first (PE/EtOAc 9/1, 0.103 g,
27%), then the remaining (5R, 8R)-diastereoisomer (0.031 g, 8%).
C
15H20O4; Light yellow oil; Yield: 89%; M ¼ 264.32 g/mol; IR
(ZnSe)
(300 MHz, CDCl3)
n
¼ 2933, 1641, 1576, 1444, 1415, 1192, 1043 cmꢁ1
;
1H NMR
d 1.43e1.71 (m, 8H), 1.75e1.85 (m, 2H), 2.32 (dd,
J ¼ 14.7 Hz, J ¼ 1.4 Hz, 1H), 2.68 (dd, J ¼ 14.0 Hz, J ¼ 0.8 Hz, 1H),
3.87e3.99 (m, 4H), 5.98 (d, J ¼ 10.3 Hz, 1H), 6.79 (dd, J ¼ 10.3 Hz, J ¼
1.7 Hz, 1H), 7.42 (s, 1H), 13.67 (ls, 1H); 13C NMR (75 MHz, CDCl3)
d
21.2 (CH2), 22.3 (CH2), 27.5 (CH2), 29.0 (CH2), 32.6 (CH2), 32.7
(CH2), 48.3 (Cq), 64.5 (CH2), 64.9 (CH2), 106.7 (Cq), 113.6 (Cq), 126.3
(CH), 153.7 (CH), 166.7 (CH), 189.4 (Cq); HRMS: calcd. for C15H27O4
265.14344, found 265.14418 (2.807 ppm).
4.2.5. 1-(4-fluorophenyl)spiro[4H-indazole-5,20-cycloheptane]-10-
one-ethylene-acetal (6)
At room temperature, sodium acetate (0.190 g, 1.2 eq.) and 4-F-
phenylhydrazine (0.37 g, 1.2 eq.) were added to a solution of the
4.2.7. 1-(4-fluorophenyl)-10-phenyl-spiro[4H-indazole-5,20-
cyclohexane]-10-ol (8b)
(5R, 8R)-diastereoisomer:
C24H23FN2O; colourless crystals;
previously-obtained
a
-formylation product 5 (0. 5 g, 1 eq.) in acetic
Mp ¼ 234e236 ꢀC; Yield: 56%; M ¼ 374.45 g/mol; IR (ZnSe)
acid (10 ml). The mixture was stirred until the complete con-
sumption of the starting material (approx. 1.5 h), then the reaction
was quenched by addition of sodium carbonate (sat. soln. 100 ml).
The aqueous mixture was washed with ethyl acetate (3 ꢃ 100 ml),
the combined organic layers were dried over magnesium sulfate
and the solvent evaporated under reduced pressure. The crude
product was subsequently purified by flash-chromatography
(eluent: PE/EtOAc 8/2) to conduct to the desired indazole deriva-
tive 6.
n
¼ 2922, 1513, 837 cmꢁ1
; d 1.49e1.66
1H NMR (300 MHz, DMSO)
(m, 5H), 1.79e1.88 (m, 2H), 2.46 (d, J ¼ 16.4 Hz, 1H), 2.83 (d,
J ¼ 16.4 Hz, 1H), 4.85 (s, 1H), 6.28 (d, J ¼ 10.3 Hz, 1H), 6.46 (d,
J ¼ 10.4 Hz, 1H), 7.06 (t, J ¼ 7.2 Hz, 1H), 7.17 (t, J ¼ 7.5 Hz, 2H),
7.27e7.37 (m, 5H), 7.50 (d, J ¼ 7.4 Hz, 2H) (1H masked by the DMSO
signal); 13C NMR (75 MHz, DMSO)
d 21.0 (CH2), 21.4 (CH2), 27.0
(CH2), 34.0 (CH2), 34.2 (CH2), 44.4 (Cq), 75.2 (Cq), 114.5 (CH), 115.7
(Cq), 115.9 (CH), 116.2 (CH), 124.8 (CH), 124.9 (CH), 125.9 (CH), 126.6
(2CH), 127.0 (2CH), 135.3 (Cq), 136.3 (CH), 137.8 (CH), 147.0 (Cq),
158.9 (Cq), 162.2 (Cq); MS: m/z ¼ 375.2 [M þ H]þ; HRMS: calcd. for
C
21H23FN2O2; light brown viscous oil; yield: 72%; M ¼ 354.42 g/
mol; IR (ZnSe)
CDCl3)
n
¼ 2922, 1517, 1225, 1076 cmꢁ1; 1H NMR (300 MHz,
C24H24FN2O 375.1867, found 375.1863 (ꢁ1.2371 ppm).
d
1.45e1.72 (m, 8H), 1.82e1.87 (m, 2H), 2.74 (d, J ¼ 15.9 Hz,
1H), 2.92 (d, J ¼ 15.9 Hz, 1H), 3.95e4.02 (m, 4H), 6.01 (d, J ¼ 10.2 Hz,
4.2.8. 1-(4-fluorophenyl)-10-phenyl-spiro[4H-indazole-5,20-
cyclohexane]-10-ol (9b)
1H), 6.39 (d, J ¼ 10.2, 1H), 7.11e7.16 (m, 2H), 7.42e7.46 (m, 2H); 13
C
NMR (75 MHz, CDCl3)
d
21.1 (CH2), 21.6 (CH2), 25.7 (CH2), 27.4 (CH2),
(5R, 8S)-diastereoisomer: C24H23FN2O; colourless crystals;
32.0 (CH2), 32.8 (CH2), 49.0 (Cq), 64.6 (CH2), 64.9 (CH2), 113.6 (CH),
114.3 (Cq), 115.5 (Cq), 116.0 (CH), 116.3 (CH), 125.2 (CH), 125.3 (CH),
136.6 (CH), 136.7 (Cq), 138.7 (CH), 159.9 (Cq), 163.2 (Cq); HRMS:
calcd. for C21H24FN2O2 355.18163, found 355.18277 (3.202 ppm).
Mp ¼ 173e175 ꢀC; Yield: 27%; M ¼ 374.45 g/mol; IR (ZnSe)
n
d
¼ 3369, 2936, 1513, 1265, cmꢁ1
;
1H NMR (300 MHz, CDCl3)
1.56e1.77 (m, 5H), 1.86e1.97 (m, 2H), 2.56 (dd, J ¼ 4.1 Hz,
J ¼ 9.5 Hz,1H), 2.64 (d, J ¼ 15.9 Hz, 1H), 2.90 (d, J ¼ 15.9 Hz,1H), 6.25
(d, J ¼ 10.2 Hz, 1H), 6.41 (d, J ¼ 10.3 Hz, 1H), 7.07e7.18 (m, 3H),
7.23e7.33 (m, 5H), 7.46e7.49 (m, 2H); 13C NMR (75 MHz, CDCl3)
4.2.6. 1-(4-fluorophenyl)spiro[4H-indazole-5,20-cycloheptane]-10-
one (7c)
d
20.1 (CH2), 21.0 (CH2), 25.1 (CH2), 30.3 (CH2), 33.7 (CH2), 45.5 (Cq),
The ketal 6 was deprotected by adding hydrogen chloride (aq.
soln. 6N, 6 ml) to a solution of indazole 6 (0.4 g,1 eq.) in THF (12 ml).
The reaction was conducted at 65 ꢀC for 1.5 h, and then quenched
by pouring onto a solution of sodium bicarbonate (10% aq. soln.
100 ml). The aqueous solution was washed with ethyl acetate
(3 ꢃ 100 ml), then the combined organic layers were dried over
magnesium sulfate and the solvent evaporated under vacuum. The
desired ketone 7c was isolated as a light yellow oil after purification
on flash chromatography (eluent PE/EtOAc 8/2).
76.7 (Cq), 114.3 (CH), 115.0 (Cq), 115.9 (CH), 116.2 (CH), 125.1 (CH),
125.2 (CH), 127.1 (CH), 127.1 (2CH), 127.5 (2CH), 136.4 (Cq), 137.9
(CH), 146.3 (Cq), 159.9 (Cq), 163.2 (Cq); MS: m/z ¼ 375.2 [M þ H]þ;
HRMS: calcd. for
C24H24FN2O 375.18672, found 375.18586
(ꢁ2.287 ppm).
4.2.8.1. 1-(4-fluorophenyl)-10-phenyl-spiro[4H-indazole-5,20-cyclo-
heptane]-10-ol. Phenylmagnesium bromide (1M in THF, 0.97 ml,
0.97 mmol, 3 eq.) was slowly added at 0 ꢀC to a solution of ketone
7b (0.1 g, 0.32 mmol, 1 eq.) in dry THF (10 ml). After 2 h at low
temperature, the reaction was quenched by addition of water
(20 ml), and the organics extracted with ethyl acetate (3 ꢃ 20 ml).
The recovered organic layers were dried over magnesium sulfate,
concentrated under vacuum and further purified by flash chro-
matography (PE/EtOAc 9/1). The (5R, 8S)-diastereoisomer was
eluted firstly (38%), then the (5R, 8R)-diastereoisomer (10%),
C
19H19FN2O; Light yellow visc. oil; Yield: 83%; M ¼ 310.37 g/mol;
IR (ZnSe)
CDCl3)
n
¼ 2930, 1703, 1516, 1222, 841 cmꢁ1; 1H NMR (300 MHz,
d
1.40e1.71 (m, 6H), 1.80e1.84 (m, 2H), 2.52e2.68 (m, 3H),
3.23 (d, J ¼ 16.1 Hz, 1H), 5.87 (d, J ¼ 10.0 Hz, 1H), 6.45 (dd, J ¼
10.0 Hz, J ¼ 0.5 Hz, 1H), 7.07e7.13 (m, 2H), 7.37e7.43 (m, 2H); 13C
NMR (75 MHz, CDCl3)
d 24.3 (CH2), 26.6 (CH2), 27.7 (CH2), 30.2
(CH2), 35.4 (CH2), 39.9 (CH2), 54.3 (Cq), 114.8 (Cq), 115.9 (CH), 115.9

E. Badarau et al. / European Journal of Medicinal Chemistry 161 (2019) 354e363
361
followed by the recovered starting material (20%).
7.45e7.50 (m, 3H); 13C NMR (75 MHz, CDCl3)
d
21.0 (CH2), 22.5
(CH2), 25.5 (CH2), 26.5 (CH2), 31.1 (CH2), 35.9 (CH2), 44.7 (CH2), 48.8
(Cq), 77.3 (Cq), 113.7 (CH), 115.2 (Cq), 116.1 (CH), 116.4 (CH), 125.2
(CH), 125.3 (CH), 126.8 (CH), 128.5 (2CH), 131.5 (2CH), 136.9 (Cq),
137.5 (Cq), 138.4 (CH), 138.7 (CH), 160.0 (Cq), 163.3 (Cq); HRMS:
calcd. for C26H28FN2O 403.2180, found 403.21873 (1.765 ppm).
4.2.9. 1-(4-fluorophenyl)-10-phenyl-spiro[4H-indazole-5,20-
cycloheptane]-10-ol (8d)
(5R,
8R)-diastereoisomer:
C
25H25FN2O;
White
solid;
Mp ¼ 130e131 ꢀC; Yield: 10%; M ¼ 388.48 g/mol; IR (ZnSe)
n
d
¼ 3333, 2923, 1515, 1222, 839 cmꢁ1
;
1H NMR (300 MHz, CDCl3)
1.52e1.73 (m, 6H), 1.85e2.11 (m, 4H), 2.49e2.59 (m, 1H), 2.64 (d,
4.2.13. 1'-(4-fluorophenyl)-2-phenyl-10,40-dihydrospiro-[cyclohex-
2-ene-1,50-indazole] (10a)
J ¼ 16.1 Hz, 1H), 2.81 (d, J ¼ 16.1 Hz, 1H), 6.15 (dd, J ¼ 10.4 Hz, 1H),
6.34 (dd, J ¼ 10.3 Hz, J ¼ 0.5 Hz, 1H), 7.08e7.17 (m, 3H), 7.23e7.28
(m, 3H), 7.34e7.38 (m, 2H), 7.46e7.49 (m, 2H); 13C NMR (75 MHz,
Dry toluene (10 ml) was added to a crude mixture of diaster-
oisomers 8b-9b (obtained from 0.5 g of ketone 7b as described
above). Triflic anhydride (0.7 ml, 4.2 mmol, 2.5 eq.) was subse-
quently added to the solution and the mixture was heated at 100 ꢀC
under stirring for 10 min. After cooling to room temperature, the
mixture was diluted with ethyl acetate (25 ml) and poured onto
sodium bicarbonate (satd. soln., 30 ml). Theo organic materials
were extracted with ethyl acetate (3 ꢃ 50 ml), dried over magne-
sium sulfate and concentrated under vacuum. The crude product
was purified by flash chromatography (PE/EtOAc 95/5) to conduct
to the desired elimination product (15%) along with an unidentified
product.
CDCl3)
d 20.8 (CH2), 21.9 (CH2), 27.0 (CH2), 28.8 (CH2), 30.8 (CH2),
41.6 (CH2), 49.2 (Cq), 79.8 (Cq), 115.0 (CH), 116.0 (CH), 116.3 (CH),
116.5 (Cq), 125.1 (CH), 125.2 (CH), 126.6 (CH), 126.7 (2CH), 127.7
(2CH), 135.4 (CH), 136.1 (Cq), 138.6 (CH), 148.4 (Cq), 163.2 (Cq); MS
m/z ¼ 389.2 [MþH]þ; HRMS: calcd. for C25H28FN2O 389.20285,
found 389.20136 (ꢁ2.590 ppm).
4.2.10. 1-(4-fluorophenyl)-10-phenyl-spiro[4H-indazole-5,20-
cycloheptane]-10-ol (9d)
(5R,
8S)-diastereoisomer:
C
25H25FN2O;
White
solid;
Mp ¼ 145e147 ꢀC; Yield: 38%; M ¼ 388.48 g/mol; IR (ZnSe)
C
24H21FN2; light yellow oil; Yield: 15%; M ¼ 356.43 g/mol; IR
n
d
¼ 3331, 2922, 1521, 1229, 846 cmꢁ1
;
1H NMR (300 MHz, CDCl3)
(ZnSe)
n
¼ 2932, 1515, 1224 cmꢁ1
;
1H NMR (300 MHz, CDCl3)
1.50e2.04 (m, 9H), 2.12e2.22 (m, 1H), 2.55e2.65 (m, 1H), 2.82 (q,
d 1.60e1.70 (m, 3H), 1.80e1.88 (m, 1H), 2.12e2.22 (m, 2H), 2.49 (d,
J ¼ 15.9 Hz, 2H), 6.14 (dd, J ¼ 10.3 Hz, J ¼ 0.6 Hz, 1H), 6.31 (d, J ¼
J ¼ 16.3 Hz, 1H), 2.97 (d, J ¼ 16.3 Hz, 1H), 5.85 (t, J ¼ 3.9 Hz, 1H), 6.02
10.3 Hz, 1H), 7.06e7.21 (m, 6H), 7.25e7.30 (m, 2H), 7.44e7.47 (m,
(d, J ¼ 10.0 Hz, 1H), 6.32 (d, J ¼ 10.0 Hz, 1H), 7.03e7.24 (m, 8H),
2H); 13C NMR (75 MHz, CDCl3)
d
20.7 (CH2), 22.0 (CH2), 26.1 (CH2),
7.30e7.36 (m, 2H); 13C NMR (75 MHz, CDCl3)
d 18.3 (CH2), 26.2
27.1 (CH2), 33.4 (CH2), 38.9 (CH2), 48.3 (Cq), 79.4 (Cq), 113.2 (CH),
115.4 (Cq), 115.9 (CH), 116.2 (CH), 125.1 (CH), 125.2 (CH), 126.9 (CH),
127.0 (CH), 127.2 (CH), 136.5 (Cq), 137.8 (CH), 138.9 (CH), 148.0 (Cq),
159.9 (Cq), 163.1 (Cq); HRMS: calcd. for C25H28FN2O 389.20237,
found 389.20341 (2.677 ppm).
(CH2), 30.8 (CH2), 36.1 (CH2), 41.0 (Cq), 113.8 (CH), 115.4 (Cq), 116.0
(CH), 116.3 (CH), 125.3 (CH), 125.4 (CH), 126.4 (CH), 127.8 (2CH),
128.8 (CH),128.8 (2CH),136.1 (Cq),138.3 (CH),140.8 (CH),142.7 (Cq),
144.0 (Cq), 160.0 (Cq), 163.2 (Cq); HRMS: calcd. for C24H22FN2
357.17615, found 357.17532 (ꢁ2.333 ppm).
4.2.10.1. 10-benzyl-1-(4-fluorophenyl)spiro[4H-indazole-5,20-cyclo-
heptane]-10-ol. The title compound was obtained following the
same experimental method employed for the synthesis of com-
pound 8d-9d, after 2h of reaction at 0 ꢀC. Purification by flash-
chromatography (eluent: 9/1 PE/EtOAc) delivered the (5R, 8R)-
diastereoisomer followed by the (5R, 8S)-diastereoisomer. The
secondly eluted diastereoisomer was crystallized in ACN and sub-
mitted to X- Rays diffractions in order to confirm the proposed
stereochemistry.
4.2.14. 4-[1-(4-fluorophenyl)spiro[4H-indazole-5,60-cyclohexene]-
10-yl]phenol (10b)
Under inert atmosphere, (4-bromophenoxy)-triisopropylsilane
(0.56 g, 1.69 mmol, 2 eq.) was dissolved in dry THF (15 ml) and the
mixture cooled at ꢁ78 ꢀC before slowly adding a solution of n-butyl
lithium (1.5 M in THF, 1.25 ml, 1.85 mmol, 2.2 eq.). After 30 min of
stirring at ꢁ78 ꢀC, a solution of the ketone 7b (0.25 g, 0.84 mmol, 1
eq.) in dry THF (5 ml) was added and the mixture was allowed to
slowly come back to room temperature overnight. The reaction was
quenched by addition of water (100 ml) and the organic materials
were extracted with ethyl acetate (3 ꢃ 100 ml) before removing the
solvent under vacuum. The crude product was purified by flash-
chromatography (PE/EtOAc 9/1 to 8/2) to deliver the desired
addition product. Starting material was also recovered during the
purification (30%).
4.2.11. 10-benzyl-1-(4-fluorophenyl)spiro[4H-indazole-5,20-
cycloheptane]-10-ol (8e)
(5R,
8S)-diastereoisomer:
C
25H25FN2O;
White
solid;
Mp ¼ 144e146 ꢀC; Yield: 20%; M ¼ 402.50 g/mol; IR (ZnSe)
n
¼ 3574, 2926, 1515, 1222, 1079, 839 cmꢁ1
;
1H NMR (300 MHz,
CDCl3)
d
1.41e1.87 (m, 10H), 2.52 (d, J ¼ 13.5 Hz, 1H), 2.75 (d, J ¼
C
33H43FN2O2Si; Colorless vis. oil; Yield: 57%; M ¼ 546.79 g/mol;
16.6 Hz, 1H), 3.12 (d, J ¼ 13.5 Hz, 1H), 3.28 (d, J ¼ 16.6 Hz, 1H), 5.93
(d, J ¼ 10.3 Hz, 1H), 6.47 (d, J ¼ 10.3 Hz, 1H), 7.14e7.35 (m, 7H),
IR (ZnSe)
(300 MHz, CDCl3)
n
¼ 3380, 2948, 1601, 1517, 1269, 835 cmꢁ1
;
1H NMR
d
1.04 (d, J ¼ 6.8 Hz, 18H), 1.12e1.29 (m, 3H),
7.45e7.50 (m, 3H); 13C NMR (75 MHz, CDCl3)
d
20.0 (CH2), 21.1
1.6e1.83 (m, 8H), 2.41e2.52 (m,1H), 2.51 (d, J ¼ 16.3 Hz,1H), 3.10 (d,
J ¼ 16.3 Hz,1H), 6.24 (q, J ¼ 10.3 Hz, 2H), 6.69 (d, J ¼ 8.7 Hz, 2H), 7.09
(t, J ¼ 8.6 Hz, 2H), 7.22 (s, 1H), 7.27e7.32 (m, 4H); 13C NMR (75 MHz,
(CH2), 27.0 (CH2), 27.7 (CH2), 31.4 (CH2), 35.7 (CH2), 45.6 (CH2), 48.6
(Cq), 63.5 (CH2) 77.0 (Cq), 114.6 (CH), 116.1 (CH), 116.4 (CH), 125.3
(CH), 125.4 (CH), 126.7 (CH), 128.5 (2CH), 131.4 (2CH), 135.6 (CH),
137.4 (Cq), 139.0 (CH); MS m/z ¼ 403.2 [MþH]þ; HRMS: calcd. for
CDCl3)
d 12.7 (3CH), 18.0 (6CH3), 21.5 (CH2), 21.8 (CH2), 27.6 (CH2),
34.9 (CH2), 35.8 (CH2), 44.9 (Cq), 76.6 (Cq), 115.5 (CH), 115.8 (CH),
116.1 (CH), 116.5 (Cq), 118.5 (2CH), 125.1 (CH), 125.2 (CH), 127.8
(2CH), 135.2 (CH), 135.9 (Cq), 138.0 (CH), 138.3 (Cq), 154.8 (Cq), 159.9
(Cq), 163.1 (Cq); HRMS: calcd. for C33H44FN2O2Si 547.31506, found
547.31572 (1.206 ppm).
C
26H28FN2O 403.21855, found 403.21711 (ꢁ2.252 ppm).
4.2.12. 10-benzyl-1-(4-fluorophenyl)spiro[4H-indazole-5,20-
cycloheptane]-10-ol (9e)
(5R,
8R)-diastereoisomer:
C
25H25FN2O;
White
solid;
The previously obtained alcohol (0.09 g, 0.0.164 mmol, 1 eq.)
was dissolved in THF (3 ml) and the solution was cooled to 0 ꢀC in
an ice-bath before slowly adding a solution of TBAF (1M in DCM,
0.5 ml, 0.5 mmol, 3 eq.). After 5 min the TLC indicated the complete
consumption of the starting material. The reaction was quenched
Mp ¼ 127e129 ꢀC; Yield: 18%; M ¼ 402.50 g/mol; IR (ZnSe)
n
¼ 3390, 2926, 1514, 1225, 1079, 838 cmꢁ1
;
1H NMR (300 MHz,
CDCl3)
d 1.48e1.71 (m, 9H), 2.02e2.11 (m, 1H), 2.73e3.02 (m, 4H),
6.36 (d, J ¼ 10.3 Hz, 1H), 6.47 (d, J ¼ 10.3 Hz, 1H), 7.13e7.35 (m, 7H),

362
E. Badarau et al. / European Journal of Medicinal Chemistry 161 (2019) 354e363
by addition of sodium bicarbonate (20% aq. soln. 20 ml) and the
organics extracted by EtOAc (3 ꢃ 20 ml). The combined organic
layers were dried over magnesium sulfate and further concentrated
under vacuum to deliver a crude product. This was subsequently
purified by flash-chromatography (DCM/MeOH 9/1), to conduct to
the desired deprotected phenol. The isolated fraction was further
triturated in diethyl ether to deliver a white powder, subsequently
filtered on a glass-sintered funnel and dried under vacuum.
two steps); M ¼ 386.46 g/mol; IR (ZnSe)
n
¼ 2933, 1609, 1517, 1284,
1.69e1.77 (m, 3H),
1247, 838 cmꢁ1 1H NMR (300 MHz, CDCl3)
;
d
1.85e1.93 (m, 1H), 2.21e2.26 (m, 2H), 2.58 (d, J ¼ 16.3 Hz, 1H), 3.07
(d, J ¼ 16.3 Hz, 1H), 3.76 (s, 3H), 5.90 (t, J ¼ 3.9 Hz, 1H), 6.06 (d, J ¼
10.0 Hz, 1H), 6.41 (d, J ¼ 10.0 Hz, 1H), 6.77 (d, J ¼ 8.8 Hz, 2H),
7.12e7.18 (m, 1H), 7.22 (d, J ¼ 8.8 Hz, 2H), 7.33 (s, 1H), 7.41e7.46 (m,
2H); 13C NMR (75 MHz, CDCl3)
d 18.3 (CH2), 26.2 (CH2), 30.7 (CH2),
35.9 (CH2), 41.0 (Cq), 55.3 (CH3), 113.2 (2CH), 113.8 (CH), 115.5 (Cq),
116.0 (CH), 116.3 (CH), 125.3 (CH), 125.4 (CH), 127.9 (CH), 129.8
(2CH), 135.2 (Cq), 136.0 (Cq), 138.4 (CH), 140.9 (CH), 143.4 (Cq), 158.2
(Cq), 160.0 (Cq), 163.2 (Cq); HRMS: calcd. for C25H24FN2O 387.18672,
found 387.18647 (ꢁ0.641 ppm).
C
24H23FN2O2; White solid; Mp ¼ 182e184 ꢀC; Yield: 76%;
M ¼ 390.45 g/mol; IR (ZnSe)
n
¼ 3401, 2973, 1663, 1612, 1517, 1229,
1021 cmꢁ1
; d 1.54e1.91 (m, 8H),
1H NMR (300 MHz, CDCl3)
2.38e2.44 (m, 2H), 3.29 (t, J ¼ 16.5 Hz, 1H), 6.19 (s, 2H), 6.53 (d, J ¼
8.7 Hz, 2H), 7.03 (t, J ¼ 8.6 Hz, 2H), 7.15e7.31 (m, 6H); 13C NMR
(75 MHz, CDCl3)
d
21.3 (CH2), 21.6 (CH2), 27.7 (CH2), 35.1 (CH2), 35.6
4.2.16. 60-benzyl-1-(4-fluorophenyl)spiro[4H-indazole-5,20-cyclo-
hexane]-10-one (11a)
(CH2), 44.6 (Cq), 76.3 (Cq), 113.5 (2CH), 115.9 (CH), 116.2 (CH), 116.3
(CH),117.1 (Cq),125.6 (CH),125.7 (CH),128.0 (2CH),134.7 (CH),136.6
(Cq), 136.8 (Cq), 137.2 (CH), 155.3 (Cq), 160.1 (Cq), 163.4 (Cq); HRMS:
calcd. for C24H24FN2O2 391.18163, found 391.18276 (2.882 ppm).
The previously obtained phenol (0.1 g, 0.25 mmol, 1 eq.) was
dissolved in dry DCM (2 ml) then the mixture was cooled to ꢁ78 ꢀC
before slowly adding boron tribromide (1M in DCM, 0.3 ml,
0.3 mmol, 1.2 eq.). The reaction was continued at low temperature
for 1 h, and then quenched with sodium bicarbonate (20% aq. soln.,
10 ml). The organic materials were extracted with DCM (3 ꢃ 10 ml),
the combined organic layers were dried over magnesium sulfate
and the crude product purified by flash-chromatography (PE/EtOAc
15/5) to deliver the dehydrated product 10b.
Under nitrogen atmosphere and at low temperature (ꢁ78 ꢀC),
LHMDS (1M soln, in THF, 0.7 ml, 0.67 mmol, 1 eq.) and then DMPU
(0.1 ml, 0.84 mmol, 1.2 eq.) were subsequently added to a solution
of the starting ketone 7b (0.2 g, 0.67 mmol, 1 eq.) in dry THF (5 ml).
The mixture was stirred at low temperature for 1 h, prior to addi-
tion of benzyl bromide (0.08 ml, 0.67 mmol, 1 eq.). The reaction was
allowed to slowly come back to room temperature over 12 h under
stirring, then quenched by addition of water (10 ml). The organic
materials were extracted with ethyl acetate (3 ꢃ 10 ml) and sub-
sequently concentrated under vacuum. The crude product was
purified by flash-chromatography (PE/AcOEt 9/1). The dibenzylated
product was eluted first followed by the mono-benzylated deriva-
tive as a mixture of 2/1 diastereoisomers.
C
24H21FN2O; White solid; Mp ¼ 77e79 ꢀC; Yield: 83%;
M ¼ 372.43 g/mol; IR (ZnSe)
n
¼ 3320, 2933, 1609, 1517, 1265,
(as indicated by H NMR, mixture of diastereoisomers A/B 2/1):
1229 cmꢁ1
;
1H NMR (300 MHz, CDCl3)
d
1.68e1.91 (m, 4H),
C
25H23FN2O; colorless visc. oil; yield 14%, M ¼ 386.46 g/mol; IR
2.20e2.24 (m, 2H), 2.57 (d, J ¼ 16.3 Hz, 1H), 3.05 (d, J ¼ 16.3 Hz, 1H),
5.85 (t, J ¼ 3.9 Hz, 1H), 6.04 (d, J ¼ 10.0 Hz, 1H), 6.35 (dd, J ¼ 10.0 Hz,
J ¼ 0.5 Hz, 1H), 6.62e6.65 (m, 2H), 6.83 (s, 1H), 7.07e7.14 (m, 4H),
(ZnSe)
CDCl3)
n
¼ 2937, 1711, 1517, 1225, 1083 cmꢁ1
;
1H NMR (300 MHz,
d
1.49e2.12 (m, 8H), 2.39e2.49 (m, 2.5H), 2.79e3.05 (m, 2H),
3.16e3.28 (m, 2H), 3.45 (d, J ¼ 16.2 Hz, 1HA), 6.14 (d, J ¼ 10.0 Hz,
1HA), 6.20 (d, J ¼ 10.1 Hz, 1HB), 6.48 (d, J ¼ 10.1 Hz, 1HB), 6.50 (d,
J ¼ 10.0 Hz, 1HA), 7.12e7.31 (m, 10H), 7.39e7.48 (m, 4H); 13C NMR
7.33 (s, 1H), 7.36e7.41 (m, 2H); 13C NMR (75 MHz, CDCl3)
d 18.3
(CH2), 26.1 (CH2), 30.7 (CH2), 36.0 (CH2), 41.0 (Cq), 113.6 (CH), 114.7
(2CH), 115.5 (Cq), 116.1 (CH), 116.4 (CH), 125.5 (CH), 125.6 (CH), 127.7
(CH), 129.9 (2CH), 134.7 (Cq), 136.5 (Cq), 138.3 (CH), 141.3 (CH), 143.3
(Cq), 154.9 (Cq), 160.1 (Cq), 163.4 (Cq); HRMS: calcd. for C24H22FN2O
373.171068, found 373.16948 (ꢁ4.26 ppm).
(75 MHz, CDCl3)
d for major diastereoisomer A: 21.5 (CH2), 28.0
(CH2), 34.8 (CH2), 35.7 (CH2), 41.1 (CH2), 48.6 (CH), 52.1 (Cq), 116.1
(CH), 116.4 (CH), 116.9 (Cq), 117.2 (CH), 125.1 (CH), 125.3 (CH), 126.1
(CH), 128.4 (2CH), 129.2 (2CH), 130.5 (CH), 135.6 (Cq), 138.1 (CH),
140.5 (Cq), 160.1 (Cq), 163.3 (Cq), 210.5 (Cq); MS m/z ¼ 387.3
[MþH]þ; HRMS: calcd. for C25H24FN2O 387.18672, found 387.18806
(3.466 ppm).
4.2.15. 1-(4-fluorophenyl)-1'-(4-methoxyphenyl)spiro[4H-
indazole-5,60-cyclohexene] (10c)
Under inert atmosphere, iPrMgCl (2M in THF, 0.84 ml,
3.55 mmol, 2.1 eq.) was added at ꢁ20 ꢀC to a solution of 4-iodo-
anisole (0.4 g, 1.69 mmol, 1 eq.) in dry THF (10 ml). After 1 h of
stirring at ꢁ20 ꢀC, a solution of ketone precursor 7b (0.5 g,
1.69 mmol) in dry THF (3 ml) was slowly added. The temperature
was allowed to come back to room temperature and the mixture
stirred overnight. The reaction was quenched by addition of sodium
bicarbonate (20% aq. soln., 50 ml), then extracted with ethyl acetate
(3 ꢃ 50 ml). The combined organic layers were dried over magne-
sium sulfate, the solvent evaporated under reduced pressure. The
crude product was purified by flash chromatography (Cy/EtOAc 8/
2) to deliver the addition product (25%) along with recovery of the
starting material (50%). The obtained alcohol (0.1 g, 0.25 mmol, 1
eq.) was subsequently dissolved in dry DCM (2 ml), then the
mixture was cooled to ꢁ78 ꢀC before slowly adding boron tri-
bromide (1M in DCM, 0.3 ml, 0.3 mmol, 1.2 eq.). The reaction was
continued at low temperature for 1 h, and then quenched with
sodium bicarbonate (20% aq. soln., 10 ml). The organic materials
were extracted with DCM (3 ꢃ 10 ml), the combined organic layers
dried over magnesium sulfate and the crude product purified by
flash-chromatography (PE/EtOAc 15/5) to deliver the dehydrated
product 10c (85%).
4.2.17. 60,60-dibenzyl-1-(4-fluorophenyl)spiro[4H-indazole-5,20-
cyclohexane]-10-one (12a)
The title compound was obtained during the synthesis of the
compound 11a as indicated above.
C
32H29FN2O; white solid; m.p. 188e190C Yield: 14%;
M ¼ 476.58 g/mol; IR (ZnSe) ¼ 2933, 1689, 1517, 1222, 1079,
838 cmꢁ1; 1H NMR (300 MHz, CDCl3)
1.48e1.55 (m,1H),1.67e1.89
n
d
(m, 5H), 2.52 (d, J ¼ 13.2 Hz, 1H), 2.64 (d, J ¼ 6.3 Hz, 1H), 2.69 (d, J ¼
8.8 Hz, 1H), 2.96 (d, J ¼ 15.7 Hz,1H), 3.23 (d, J ¼ 13.2 Hz,1H), 3.35 (d,
J ¼ 13.2 Hz, 1H), 5.54 (d, J ¼ 10.0 Hz, 1H), 6.36 (d, J ¼ 10.0 Hz, 1H),
7.08e7.16 (m, 6H), 7.20e7.33 (m, 6H), 7.39e7.44 (m, 3H); 13C NMR
(75 MHz, CDCl3) d 16.4 (CH2), 29.1 (CH2), 29.4 (CH2), 32.2 (CH2), 43.9
(CH2), 44.4 (CH2), 51.0 (Cq), 54.5 (Cq), 113.8 (Cq), 115.2 (CH), 116.1
(CH), 116.4 (CH), 125.1 (CH), 125.2 (CH), 126.6 (CH), 126.8 (CH), 128.2
(2CH), 128.2 (2CH), 131.3 (2CH), 131.3 (2CH), 134.2 (CH), 135.9 (Cq),
137.1 (Cq), 137.8 (Cq), 138.3 (CH), 160.0 (Cq), 163.3 (Cq), 217.4 (Cq);
HRMS: calcd. for
C32H30FN2O 477.23367, found 477.23231
(ꢁ2.846 ppm).
4.2.18. 60-benzyl-1-(4-fluorophenyl)spiro[4H-indazole-5,20-
cyclohexane]-10-ol (13a)
C
25H23FN2O; White solid; Mp ¼ 126e128 ꢀC; Yield: 20% (over
Under inert atmosphere and at ꢁ78 ꢀC, DIBAL-H (1M soln. in

E. Badarau et al. / European Journal of Medicinal Chemistry 161 (2019) 354e363
363
cyclohexane, 0.23 ml, 0.23 mmol, 3 eq.) was slowly added to a so-
lution of the starting ketone 11a (0.03 g, 0.08 mmol, 1 eq.) in dry
THF (3 ml). The mixture was stirred at low temperature for 2 h,
then, after the complete consumption of the starting material, the
reaction was quenched with water (10 ml). The organics were
extracted with ethyl acetate (3 ꢃ 10 ml) and the combined organic
layers were concentrated under vacuum. The crude product was
purified by flash-chromatography (PE/EtOAc 7/3) to give the
desired alcohol.
2.15e2.25 (m, 2H), 2.45 (d, J ¼ 16.2 Hz, 1H), 2.45e2.62 (m, 2H),
2.91e3.02 (m, 1H), 3.42 (d, J ¼ 16.2 Hz, 1H), 6.12 (d, J ¼ 10.0 Hz, 1H),
6.52 (d, J ¼ 10.0 Hz, 1H), 7.11e7.17 (m, 2H), 7.40e7.46 (m, 3H); 13C
NMR (75 MHz, CDCl3)
d 19.2 (CH2), 21.3 (CH2), 27.9 (CH2), 34.5
(CH2), 40.9 (CH2), 45.9 (CH), 51.9 (Cq), 69.7 (CH), 82.7 (Cq), 116.1
(CH), 116.4 (CH), 116.8 (Cq), 117.5 (CH), 125.1 (CH), 125.3 (CH), 130.0
(CH), 135.5 (Cq), 138.0 (CH), 160.0 (Cq), 163.3 (Cq); HRMS: calcd. for
C21H19FN2NaO 357.13736, found 357.13660 (ꢁ2.14 ppm).
C
25H25FN2O; White solid; Mp ¼ 203e205 ꢀC; Yield: 77%;
4.2.21. 1-(4-fluorophenyl)-60-prop-2-ynyl-spiro[4H-indazole-5,20-
cyclohexane]-10-ol (13b)
The title compound was obtained following the same experi-
mental protocol used for the synthesis of compound 13a starting
from 0.05 g of the corresponding ketone.
M ¼ 388.48 g/mol; IR (ZnSe)
n
¼ 3342, 2922, 1517, 1218, 992 cmꢁ1
;
1H NMR (300 MHz, CDCl3)
d 1.36e1.70 (m, 7H), 1.83e1.94 (m, 1H),
2.43 (d, J ¼ 16.0 Hz, 1H), 2.55e2.70 (m, 2H), 2.92 (d, J ¼ 16.0 Hz, 1H),
3.41 (ls, 1H), 6.06 (d, J ¼ 10.1 Hz, 1H), 6.38 (d, J ¼ 10.1 Hz, 1H),
7.12e7.24 (m, 7H), 7.32e7.39 (m, 3H); 13C NMR (75 MHz, CDCl3)
C
21H21FN2O; White solid; Mp ¼ 173e175 ꢀC; Yield: 18%;
M ¼ 336.40g/mol; IR (ZnSe)
n
¼ 3302, 2930, 1517, 1225 cmꢁ1
;
1H
d
21.5 (CH2), 25.4 (CH2), 29.7 (CH2), 30.5 (CH2), 39.8 (CH2), 40.5
(CH), 41.2 (Cq), 71.9 (CH), 115.8 (CH), 115.8 (Cq), 116.1 (CH), 116.4
(CH), 125.1 (CH), 125.2 (CH), 126.0 (CH), 128.4 (2CH), 129.1 (2CH),
135.7 (CH), 137.2 (Cq), 138.3 (CH), 140.8 (Cq), 163.3 (Cq); HRMS:
calcd. for C25H26FN2O 389.20237, found 389.20147 (ꢁ2.31 ppm).
NMR (300 MHz, CDCl3)
d
1.39e1.68 (m, 5H), 1.93 (t, J ¼ 2.7 Hz, 1H),
2.11 (d, J ¼ 5.8 Hz, 1H), 2.19 (t, J ¼ 2.6 Hz, 1H), 2.22 (t, J ¼ 3.0 Hz, 1H),
2.42 (d, J ¼ 16.0 Hz, 1H), 2.96 (d, J ¼ 16.0 Hz, 1H), 3.64 (d, J ¼ 5.2 Hz,
1H), 6.09 (d, J ¼ 10.1 Hz, 1H), 6.45 (d, J ¼ 10.1 Hz, 1H), 7.13e7.19 (m,
2H), 7.38 (s, 1H), 7.42e7.47 (m, 2H); 13C NMR (75 MHz, CDCl3)
d 21.3
4.2.19. 60,60-dibenzyl-1-(4-fluorophenyl)spiro[4H-indazole-5,20-
cyclohexane]-10-ol (14a)
(CH2), 22.8 (CH2), 25.3 (CH2), 29.6 (CH2), 30.2 (CH2), 38.2 (CH), 41.2
(Cq), 69.6 (CH), 71.5 (CH), 83.4 (Cq), 115.9 (Cq), 115.9 (CH), 116.1 (CH),
116.4 (CH), 125.0 (CH), 125.1 (CH), 135.5 (CH), 137.2 (Cq), 138.3 (CH),
160.0 (Cq), 163.3 (Cq); HRMS: calcd. for C21H22FN2O 337.171068,
found 337.17086 (ꢁ0.62 ppm).
Under inert atmosphere and at 0 ꢀC, LiAlH4 (0.01 g, 0.23 mmol,
1.2 eq.) was added in one portion to a mixture of the starting ketone
12a (0.09 g, 0.19 mmol, 1 eq.) in dry THF (3 ml). The mixture was
stirred for 2 h at 0 ꢀC, and then the reaction was quenched by
addition of water (1 ml). After dilution with ethyl acetate (10 ml),
the precipitated salts were filtrated on a celite pad. Water was
subsequently added (10 ml) and the organics were extracted with
ethyl acetate (3 ꢃ 10 ml). The combined organic layers were dried
over magnesium sulfate and concentrated under vacuum. The
crude product was purified by flash-chromatography (PE/EtOAc 8/
2) to conduct to the desired alcohol.
Acknowledgments
This project was generously supported by Grünenthal GmbH
and IECB. We also thank the staff members of the technical plat-
form of IECB and Grünenthal GmbH for their availability and their
skills.
C
32H31FN2O; White solid; Mp ¼ 124e126 ꢀC; Yield: 56%;
¼ 3390, 2933, 1517, 1225, 1072 cmꢁ1
;
Appendix A. Supplementary data
M ¼ 478.60 g/mol; IR (ZnSe)
n
1H NMR (300 MHz, CDCl3)
d
0.88 (td, J ¼ 13.3 Hz, J ¼ 4.0 Hz, 1H), 1.14
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2018.10.032.
(td, J ¼ 13.7 Hz, J ¼ 3.1 Hz, 1H), 1.36e1.43 (m, 1H), 1.48 (d, J ¼ 3.5 Hz,
1H), 1.64 (dd, J ¼ 14.0 Hz, J ¼ 2.3 Hz, 1H), 1.81 (dd, J ¼ 13.7 Hz, J ¼
2.4 Hz, 1H), 1.88e2.05 (m, 1H), 2.05 (d, J ¼ 13.2 Hz, 1H), 2.98e3.31
(m, 4H), 3.38 (d, J ¼ 3.2 Hz, 1H), 5.34 (d, J ¼ 10.0 Hz, 1H), 6.42 (d, J ¼
10.0 Hz, 1H), 7.10e7.32 (m, 12H), 7.40, 7.46 (m, 3H); 13C NMR
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(75 MHz, CDCl3) d 16.4 (CH2), 23.0 (CH2), 29.9 (CH2), 33.0 (CH2), 37.4
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