Tetrahydroindazole inhibitors of CDK2/cyclin complexes

Article abstract of DOI:10.1016/j.ejmech.2021.113232

Over 50 tetrahydroindazoles were synthesized after 7-bromo-3,6,6-trimethyl-1-(pyridin-2-yl)-5,6,7,7a-tetrahydro-1H-indazol-4(3aH)-one (3) was identified as a hit compound in a high throughput screen for inhibition of CDK2 in complex with cyclin A. The activity of the most promising analogues was evaluated by inhibition of CDK2 enzyme complexes with various cyclins. Analogues 53 and 59 showed 3-fold better binding affinity for CDK2 and 2- to 10-fold improved inhibitory activity against CDK2/cyclin A1, E, and O compared to screening hit 3. The data from the enzyme and binding assays indicate that the binding of the analogues to a CDK2/cyclin complex is favored over binding to free CDK2. Computational analysis was used to predict a potential binding site at the CDK2/cyclin E1 interface.

Full text of DOI:10.1016/j.ejmech.2021.113232

European Journal of Medicinal Chemistry 214 (2021) 113232
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Tetrahydroindazole inhibitors of CDK2/cyclin complexes
a
a
a
c
b
€
Jae Chul Lee , Kwon Ho Hong , Andreas Becker , Joseph S. Tash , Ernst Schonbrunn ,
Gunda I. Georg a
,
*
a Department of Medicinal Chemistry, Institute for Therapeutics Discovery and Development, College of Pharmacy, University of Minnesota, 717 Delaware
Street, SE, Minneapolis, MN, 55455, USA
b Drug Discovery Department, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL, 33612, USA
c Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS, 66160, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Over 50 tetrahydroindazoles were synthesized after 7-bromo-3,6,6-trimethyl-1-(pyridin-2-yl)-5,6,7,7a-
tetrahydro-1H-indazol-4(3aH)-one (3) was identified as a hit compound in a high throughput screen for
inhibition of CDK2 in complex with cyclin A. The activity of the most promising analogues was evaluated
by inhibition of CDK2 enzyme complexes with various cyclins. Analogues 53 and 59 showed 3-fold better
binding affinity for CDK2 and 2- to 10-fold improved inhibitory activity against CDK2/cyclin A1, E, and O
compared to screening hit 3. The data from the enzyme and binding assays indicate that the binding of
the analogues to a CDK2/cyclin complex is favored over binding to free CDK2. Computational analysis
was used to predict a potential binding site at the CDK2/cyclin E1 interface.
Received 19 October 2020
Received in revised form
22 January 2021
Accepted 24 January 2021
Available online 31 January 2021
Keywords:
Tetrahydroindazoles
CDK2
© 2021 Elsevier Masson SAS. All rights reserved.
Cyclins
Kinase inhibitors
1. Introduction
[11], whereas Speedy 1 is about 20-fold overexpressed in the testes.
Cyclin A1 knockout mice show meiotic arrest associated with
Cyclin-dependent kinases (CDKs) are
a
family of serine-
reduction of CDK2 activation [12]. In addition, CDK2ꢀ/ꢀ knockout
mice are viable but sterile [10]. Therefore, inhibitors specific for the
CDK2/cyclin A1 or CDK2/Speedy 1 complexes may have potential as
non-hormonal male contraceptive agents [9,13].
threonine protein kinases that regulate progression of cells
through the cell cycle [1] and have additional roles such as regu-
lation of transcription, DNA damage repair, and epigenetic func-
tions [2]. There are 21 kinases, named CDK, all of which possess a
conserved ATP-binding site, a PSTAIRE-like cyclin-binding domain
and an activating T-loop [3]. Activity of CDKs is regulated through
multiple mechanisms including association with their partner
protein cyclins, and positive and negative phosphorylation events.
CDKs have become attractive therapeutic targets for cancer
chemotherapy [4]. Several CDK-cyclin complexes are involved in
cell cycle control. Among them, the CDK2/cyclin A complex is
required for both DNA synthesis, mitosis, meiosis and controls the
G1-to S-phase checkpoint [5]. In the area of developing new male
non-hormonal contraceptive agents [6e8], targeting cell cycle
regulatory processes is a promising approach, which may be
achievable via inhibition of the CDK2/cyclin A1 complex or CDK2/
Speedy 1 [9]. Spermatogenesis is highly dependent on CDK2 for
meiosis [10], and cyclin A1 is a testis-specific isoform of cyclin A
Inhibitors of kinases can be classified into four major types [14].
Type I and II inhibitors are ATP-competitive and recognize the
active (DFG-in) and inactive conformation (DGF-out), respectively.
Type III inhibitors are allosteric inhibitors that bind outside of the
ATP-binding site, and the fourth type are covalent inhibitors that
typically target a residue in the ATP site. Most reported CDK in-
hibitors possess a broad specificity profile, but new generation CDK
inhibitors have been developed with high potency and selectivity.
The FDA has approved palbociclib, abemaciclib, and ribociclib, three
selective CDK4/CDK6 inhibitors for the treatment of hormone re-
ceptor positive, human epidermal growth factor receptor 2 nega-
tive, advanced or metastatic breast cancer [4]. The common core
structural feature of all three compounds is a N-(5-(piperazin-1-
ylmethyl)pyridin-2-yl)pyrimidin-2-amine. In addition, the FDA
has given alvocidid (Fig. 1) Orphan Drug designation for the
* Corresponding author.
E-mail address: georg@umn.edu (G.I. Georg).
https://doi.org/10.1016/j.ejmech.2021.113232
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
treatment of acute myeloid leukemia. Alvocidib, TP-1287 and vor-
uciclib are derivatives of the plant-derived flavone rohitukine [15].
Fig. 1 shows the structures of CDK2 inhibitors that have been
investigated in clinical trials in the past (highest level shown) or are
currently in clinical trials. However, these inhibitors are inhibiting
multiple kinases in addition to CDK2. A selective CDK2 inhibitor is
not yet available that could be a candidate for investigation as a
male contraceptive agent. For a summary of the CDK targets for the
compounds shown in Fig. 1 see Ref. [9]. As shown in Fig. 1, there a
several structural classes of CDK2 inhibitors that target the ATP
binding site (type 1 inhibitors). 7-Hydroxystaurosporine is an
analog of the natural product staurosporine. (R)-roscovitine and
fadraciclib are 9H-purine-2,6-diamines. The hinge-binding ami-
nopyrimidine core structure is common to PF-06873600, milciclib,
R-547, ZK-304709 and roniciclib. AT7519, FN-1501 and PHA-793887
are based on a 4-amino-1H-pyrazole-3-carboxamide core struc-
ture. AT7519, FN01501 and PHA-793887 are 4-amino-1H-pyrazole-
Fig. 2. Structures of hit compound 3, HSP90 inhibitor SNX-5422, and an opioid re-
ceptor agonist.
2. Screening
We screened 100,000 compounds for inhibitors of the human
CDK2/cyclin A (also named A2) complex (cyclin A is an isoform of
cyclin A1 that can be functionally expressed in E. coli) [17]. Staur-
osporine was used as the positive control in all assays. Among the
hit compounds identified, compound 3 (Fig. 2) inhibited the
3-carboxamides. Dinaciclib,
a 5-(piperidin-1-yl)pyrazolo[1,5-a]
pyrimidin-7-amine, is an inhibitor of CDK1/2/5/9, that is currently
in phase III clinical trials for several cancers [16]. Compounds BMS-
387032 and PHA-690509 are N-acylated thiazol-2-amines. Cour-
oupitine B is a (E)-[2,30-biindolinylidene]-20,3-dione. A type II in-
hibitor of CDK2, K03861, is an aminopyrimidinephenyl urea (Fig. 1),
but this inhibitor is also promiscuous and not selective for CDK2.
Some CDK2 inhibitors with increased selectivity have been iden-
tified but none of those have progressed to a clinical trial [9]. In
general, they are structurally similar to those shown in Fig. 1.
enzymatic activity of CDK2/cyclin A with a Ki value of 2.3 mM. The
3,6,6-trimethyl-1-phenyl-6,7-dihydro-1H-indazol-4(5H)-one scaf-
fold of hit 3, appeared promising for a hit to lead campaign since a
number of bioactivities have been reported for this privileged
scaffold [18e21]. This includes HSP90 inhibitor SNX-5422 [21],
which is in phase III clinical trials for cancer chemotherapy [22],
and opioid receptor agonists (Fig. 2) [23]. Furthermore, indazoles
Fig. 1. Structures of CDK2 inhibitors in clinical trials and structure of type II inhibitor K03861.
2

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
have been identified previously as kinase inhibitors [24e28]. Here,
we report structure-activity relationship studies based on hit
compound 3 in our efforts to obtain potent and selective CDK2/
cyclin inhibitors.
cyclohexane-1,3-dione, OeC-isomerization, tetrahydroindazole
formation and bromination of 26 to yield analogue 27 (Scheme 6).
Modifications at the N1 position with heteroaromatic sub-
stituents are shown in Scheme 7. 2-Hydrazinylpyrimidine 28 was
prepared by the reaction of 2-chloropyrimidine with hydrazine
[35]. Ring formation and bromination of 29 provided imidazole
analogue 30. Next, a pyrazine group was introduced at N1. 2-
Hydrazinylpyrazine 31 was prepared from 2-chloropyrazine [36].
Tetrahydroindazole formation and subsequent bromination of 32
afforded compound 33. A 4-pyridyl group was also introduced at
the N1 position. 4-Hydrazinylpyridine hydrochloride 34 was pre-
pared from 4-chloropyridine hydrochloride by reaction with hy-
drazine. Ring formation and bromination reaction of 35 furnished
compound 36.
3. Chemistry
As shown in Scheme 1, compound 3 was obtained by reaction of
commercially available 2-acetyl-5,5-dimethy1-cyclohexane-1,3-
dione (1) with 2-hydrazinopyridine and subsequent bromination
of intermediate 2 [18]. Modification was initiated at C7 since the C7
allylic bromine offered a facile functional group to generate new
analogues to probe steric and electronic influences on activity. A
fluoro group was introduced into the C7 position using silver
fluoride to furnish compound 4. The 7-methoxy derivative 5 was
obtained using silver nitrate and MeOH [18]. The 7-hydroxy com-
pound 6 was prepared from 3 using silver nitrate and water. Sub-
The 3-methylpyridin-2-yl analogue 40 was synthesized in the
same way (Scheme 8). Bromination of 38 provided both the 7-
bromo analogue 39 and the 5-bromo analogue 40. The 5-
methylpyridine-2-yl analogue 43 was synthesized in a similar
fashion through hydrazination of 2-bromo-5-methyl pyridine, tet-
sequent chlorination of compound
6 provided the 7-chloro
analogue 7 [29]. The 7-methyl analogue 8 was prepared by the
reaction of compound 2 with methyl iodide in the presence of n-
butyl lithium [30]. A dimethylamino group was introduced by re-
action of 3 with dimethylamine under microwave conditions to
afford 7-dimethylamino analogue 9 [18]. The phenylthio group was
introduced at C7 to provide compound 10, which was oxidized and
furnished sulfone 11. 7-Azido analogue 12 was obtained by sub-
stitution reaction of 3 with sodium azide [31]. Hydrogenation of the
azido group afforded 7-amino derivative 13.
Modifications at C6 were performed using 5-substituted cyclo-
hexane-1,3-diones 14a-14d to provide compounds 18a-18d
(Scheme 2). First, cyclohexenyl acetates 15a-15d were prepared
from 14a-14d using acetyl chloride and pyridine. 2-
Acetylcyclohexane-1,3-diones 16a -16d were obtained through
OeC isomerization of enol esters 15a-15d in the presence of AlCl3
[32]. Tetrahydroindazoles 17a-17d were then prepared by reaction
of 16a -16d with 2-hydrazinopyridine in EtOH. Finally, bromination
of 17a-17d using NBS provided analogues 18a-18d.
We next prepared the ring-enlarged cyclic amides 19 and 20 by
Schmidt reaction of compound 2 using sodium azide and poly-
phosphoric acid [33]. Bromination of 20 furnished compound 21 in
35% yield (Scheme 3).
Reduction of compound 3 afforded alcohol 22 and oxime 23 was
obtained by the reaction of compound 3 with hydroxylamine
(Scheme 4) [18].
To explore modifications at the C3 position, we prepared
demethyl analogue 25. In a one-flask reaction under microwave
conditions, 5,5-dimethylcyclohexane-1,3-dione, 1,1-dimethoxy-
N,N-dimethylmethanamine, and 2-hydrazinopyridine provided
compound 24. Subsequent bromination afforded compound 25
(Scheme 5) [34].
rahydroindazole
42
formation,
and
bromination.
3-
Trifluoromethylpyridin-2-yl analogues 46 and 47 were synthe-
sized similarly, starting from 2-bromo-3-(trifluoromethyl)pyridine.
5-Chloropyridin-2-yl analogue 49 was synthesized using 5-chloro-
2-hydrazinopyridine as the starting material. 6-Chloropyridazin-3-
yl analogue 51 was synthesized from commercially available 3-
chloro-6-hydrazinopyridazine.
A thiazole group was introduced at the N1 position using 2-
hydrazinothiazole furnishing analog 53 (Scheme 9). Similarly, a
benzothiazole group was also introduced at the N1 position (55). As
a nonaromatic substituent, a cyanoethyl group was introduced at
N1 furnishing analogue 57. Pyrimidine-4-yl analogue 59 and 5-
trifluoromethylpyridine-2-yl compound 61 were synthesized as
other analogues modified at N1. The 6-methoxypyridin-2-yl
analogue 64 was prepared starting from 2-bromo-6-
methoxypyridine, which was converted to 2-hydrazineyl-6-
methoxypyridine 62 (NH2NH2$H2O, 120 ꢁC, 3.5 h, 41%) and then
converted in two steps to analog 64. 5-Nitropyridin-2-yl compound
66 was synthesized using 2-hydrazinyl-5-nitropyridine as the
starting material. Subsequent reduction of the nitro group in 66
yielded pyridinoaniline 67.
A 4-nitrophenylsulfonyl group was introduced at N1 (Scheme
10). Intermediate 68 was synthesized by the reaction of 2-acetyl-
5,5-dimethyl-cyclohexane-1,3-dione with hydrazine. Then 4-
nitrobenzenesulfonyl chloride was reacted with 68 to furnish
compound 69. Finally, bromination provided analogue 70.
For further modification at N1, the amino group of compound 67
was coupled with monomethyl malonate and sulfamoyl chloride to
afford compounds 71 and 72, respectively (Scheme 11). Meth-
anesulfonyl chloride and acetyl chloride were also coupled to the
amino group of compound 67 to provide analogues 73 and 74. The
naphthalene-2-yl compound 76 was prepared using naphthalene-
Another modification at C3 was made by introducing an isobutyl
group. The procedure includes O-acylation of 5,5-dimethyl
Scheme 1. Synthesis of compounds 2e13. Reagents and conditions: 2: (a) 2-hydrazino-pyridine, EtOH, 80 ꢁC, 4 h, 84%. 3: (b) N-bromosuccinimide, CHCl3, 60 ꢁC, 6 h, 61%. 4: c) AgF,
CaF2, acetonitrile, 80 ꢁC, 22 h, 12%. 5: (c) AgNO3. MeOH/acetone, rt, 5 days, 21%. 6: (c) AgNO3, MeOH/H2O/acetone, rt, 20 h, 70%; 7: (d) from 6 with SOCl2, pyridine, rt, 2 h, 70%. 8: (e)
from 2 with MeI, n-BuLi, ꢀ78 ꢁC to rt, 16.5 h, 25%; 9: (c) dimethylamine, THF, microwave at 40 ꢁC, 38 h, 21%. 10: (c) sodium thiophenate, DMF, rt, 7 h, 79%; (f) 11: from 10 with Oxone,
H2O/MeOH, rt, 3 h, 68%. 12: (c) NaN3, 80% EtOH, 90e100 ꢁC, 24 h, 43%; 13: (g) from 12 with H2, 10% Pd/C, EtOH, rt, 4 h, 73%.
3

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
Scheme 2. Synthesis of analogues 18a-18d. Reagents and conditions: (a) acetyl chloride, pyridine, CHCl3, rt; (b) AlCl3, CH2Cl2, rt; (c) 2-hydrazinopyridine, EtOH, 80 ꢁC; (d) NBS,
CHCl3, 60 ꢁC.
Scheme 3. Synthesis of cyclic amides 19e21. Reagents and conditions: (a) sodium azide, polyphosphoric acid, 100 ꢁC, 4.5 h, 17% for 19, 24% for 20; (b) NBS, CHCl3, 60 ꢁC, 6 h, 35% (20
to 21).
determined from dose-response curves using the phosphorylated
CDK2/cyclin A complex. Steady-state kinetic analysis of the parent
compound indicated ATP-competitive inhibition with a Ki value of
2.3 0.2 mM (Fig. S1).
Modifications at the C7 position: Analogues carrying chloro- or
azido-groups at C7 showed activities similar to 3 (compounds 2,
4e13 in Table 1). In the case of compound 2, which is devoid of a C7
substituent, the activity was reduced by more than 50-fold. Pre-
sumably, electron-withdrawing groups in this position provide
more favorable binding interactions with the target site.
Modifications at the C6 position. Change of the 6-dimethyl
moiety and introduction of single methyl, isopropyl or phenyl
groups (compounds 18a-18d in Table 1) did not improve inhibitory
activity. The trans isomers of methyl and isopropyl substituted
compounds 18b and 18c were more potent than their corre-
sponding cis isomers. The phenyl analogue 18d was inactive. Thus,
a small alkyl group (Me) and trans configuration seem important
for inhibition.
Scheme 4. Synthesis of analogues 22 and 23. Reagents and conditions: (a) CeCl3,
NaBH4, MeOH/THF, ꢀ40 to ꢀ10 ꢁC, 30 min, 20%; (b) H2NOH$HCl, NaOAc, 1,4-dioxane,
reflux, 6 h, 48%.
2-yl hydrazine hydrochloride (Scheme 12).
4. Inhibition of CDK2 kinase activity-structure activity
relationship
Modifications at C4 (carbonyl): For carbonyl modification,
hydrogen bond donor groups were introduced replacing the
The in vitro inhibitory activities of the analogues were
Scheme 5. Synthesis of analogue 25. Reagents and conditions: (a) AcOH/H2O, microwave at 200 ꢁC, 5 min, 54%; (b) NBS, CHCl3, 60 ꢁC, 4 h, 29%.
Scheme 6. Introducing an isobutyl group at C3. Reagents and conditions: (a) isovaleryl chloride, pyridine, CHCl3, rt, 2 h, 80%; (b) AlCl3, CH2Cl2, rt, 2 h, 51%; (c) 2-hydrazinopyridine,
EtOH, 80 ꢁC, 2 h, 95%; (d) NBS, CHCl3, 60 ꢁC, 40 min, 58%.
4

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
Scheme 7. Reagents and conditions: 30: (a) NH2NH2, pyridine, rt, 2.5 h, 41%; (b) EtOH, 80 ꢁC, 2 h, 95%; (c) NBS, CHCl3, 60 ꢁC, 3 h, 50%. 33: (a) 35% NH2NH2$H2O, 65 ꢁC, 12 h, 75%; (b)
EtOH, 80 ꢁC, 2 h, 62%; (c) NBS, CHCl3, 60 ꢁC, 2.5 h, 59%. 36: (a) i) NaOH/H2O, extraction with Et2O, ii) NH2NH2$H2O, EtOH, 100 ꢁC, 16 h, 37%; (b) pyridine, EtOH, 80 ꢁC, 2 h, 98%; (c) NBS,
CHCl3, 60 ꢁC, 24 h, 93%.
Scheme 8. Reagents and conditions: 39 and 40: (a) NH2NH2$H2O, EtOH, 105 ꢁC, 40 h, 36%; (b) EtOH, 80 ꢁC, 2.5 h, 30%; (c) NBS, CHCl3, 60 ꢁC, 6.5 h, 39% for 39, 17% for 40. 43: (a)
NH2NH2$H2O, EtOH, 105 ꢁC, 5 days, 79%; (b) EtOH, 80 ꢁC, 2.5 h, 83%; (c) NBS, CHCl3, 60 ꢁC, 2 h, 81%. 46 and 47: (a) NH2NH2$H2O, EtOH, 105 ꢁC, 29 h, 53%; (b) pyridine, EtOH, 80 ꢁC,
3 h, 22%; (c) NBS, CHCl3, 60 ꢁC, 2.5 h, 11% for 46, 10% for 47. 49: (b) EtOH, 80 ꢁC, 3 h, 83%; (c) NBS, CHCl3, 60 ꢁC, 2 h, 60%. 51: b) EtOH, 80 ꢁC, 3 h, 73%; (c) NBS, CHCl3, 60 ꢁC, 2 h, 23%.
Scheme 9. Synthesis of N1-modified analogues. Reaction conditions: (a) EtOH, 80 ꢁC, 2e3 h; (b) NBS, CHCl3, 60 ꢁC, 1.5e5 h; (c) SnCl2e2H2O, EtOH, 70 ꢁC, 15 min, 62%.
Scheme 10. Reagents and conditions: (a) NH2NH2$H2O, EtOH, 80 ꢁC, 4 h, 88%; (b) TEA, CH2Cl2, 0 ꢁC to rt, 5 h, 75%; (c) NBS, CHCl3, 60 ꢁC, 4 h, 56%.
carbonyl group with hydroxyl and oxime groups (Table 1). Com-
pounds 22 and 23 displayed only weak inhibitory activity, indi-
cating that the hydrogen bonding acceptor characteristics of the
carbonyl group are important for binding.
Modifications at C4/C5: Cyclic amides were prepared to inves-
tigate the effect of a hydrogen bond donor next to the carbonyl
group. Both compounds 19 and 20 were inactive, but bromo com-
pound 21 was moderately active (Table 2). This result shows that a
hydrogen bond donor at this position does not increase the activity.
This result might be related to the structural deformation due to the
size of the ring (7-membered lactam ring). 5-Br-substituted com-
pounds 40, 46, and 47 showed low activities (Table 1).
5

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
Table 2
CDK2/cyclin A inhibitory activities of C4/C5-modified analogues.
Compound
R1-R3
IC50 (mM)
Scheme 11. Reagents and conditions: 71: (a) monomethyl malonate, EDC$HCl, HOBT,
DIPEA, CH2Cl2, rt, 24 h, 69%; 72: (a) sulfamoyl chloride, dimethylacetamide, rt, 20 h,
89%. 73: (a) methanesulfonyl chloride, pyridine, CH2Cl2, rt, 23 h, 88%; 74: (a) acetyl
chloride, TEA, CH2Cl2, rt, 1.5 h, 92%.
19
20
21
R1 ¼ ꢀ(CO)-, R2 ¼ eNHe, R3 ¼ H
R1 ¼ eNHe, R2 ¼ ꢀ(CO)-, R3 ¼ H
R1 ¼ eNHe, R2 ¼ ꢀ(CO)-, R3 ¼ Br
>1000
>1000
12
3
with better inhibitory activities. The methyl group seemed to be
important at this position (Table 1).
4.1. Modifications at N1
Many different heterocyclic groups were introduced to modify
the N1 position. Among the synthesized analogues, several ana-
logues showed similar or slightly better activities than 3 (IC50
:
10.5e1.5 M) (Table 3).
m
Compounds with relatively high activity against CDK2/cyclin A
were tested against CDK2 complexes with different isoforms of
cyclins (Fig. 3). Despite the low activity of 18b-cis, it was included in
this assay to investigate the role of stereochemistry in the inhibi-
tion of the CDK2 complexes. Compounds exerted significant inhi-
bition against CDK2 complexes with specific isoforms of cyclins at
Scheme 12. Introducing a naphthalene-2-yl group at N1. Reagents and conditions: (a)
pyridine, EtOH, 80 ꢁC, 2 h, 79%; (b) NBS, CHCl3, 60 ꢁC, 3 h, 68%.
Modifications at C3: Deletion of the C3 methyl group (24) or
introduction of a 3-isobutyl group (27) did not provide analogues
1 mM while 18b-cis did not exhibit significant inhibitory activity
Table 1
CDK2/cyclin A inhibitory activities of analogues with modified R groups on C3, C4, C5, C6, C7 and N1.
Compound
R1
R2
R3
R4
R5
R6
IC50
(mM)
3
2
4
5
6
7
8
9
10
11
12
13
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
isobutyl
Me
Me
Me
Me
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
OH
¼ NOH
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
¼ O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
Br
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
H, H
Me, H
Me, H
isopropyl, H
isopropyl, H
Ph, H
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Br
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
CF3
CF3
2.6 0.1
110
21 0.5
46
5
OMe
OH
Cl
Me
NMe2
SPh
SO2Ph
N3
NH2
Br
Br
Br
Br
Br
Br
Br
Br
H
2
110 12
4.9 0.3
200 16
630 11
12
1
107
5
4.5 0.2
350 36
18a
18b-trans
18b-cis
18c-trans
18c-cis
18d
22
23
24
25
27
130
6
4.8 0.1
31.0 0.9
130
7
470 80
>1000
560 63
59
3
>1000
Br
Br
Br
H
Br
H
160 10
820 660
>1000
180 20
460 50
230 20
~50
39
40
46
47
Staurosporine
6

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
against all tested CDK complexes. Cyclins A1, E, and O are highly
expressed in testis, implying a potential role in spermatogenesis
[37]. The IC50 values against CDK2 with cyclins A, A1, E, and O are
shown in Table 4.
suggest that the binding pocket in CDK2 could be altered by cyclins
A1, E, and O to enhance the affinity of CDK2 for these compounds.
This pocket might be generated by complex formation of CDK2
with these cyclins. The pocket shapes may vary according to cyclin
isoform. Based on the data from the enzyme assays and the binding
assay (Tables 4 and 5), it was concluded that the binding of the
analogues to a complex of CDK2 with a cyclin is favored compared
to binding to free CDK2.
To obtain insights about their activity against cell growth,
antiproliferation assay of 3, 53, and 59 against MCF-7 cell line were
performed. None of these compounds showed antiproliferative
activities up to 200 mM. In addition, since 3 is an analogue of SNX-
5422, an HSP90 inhibitor, compound 3 was tested against HSP90
When compared with CDK2/cyclins A, A1, and O, the tested
compounds exerted enhanced activity against CDK2/cyclin
E
(Table 4) with IC50 values of 27, 2, and 9 nM against CDK2/cyclin E
for 3, 53, 59, respectively. Cyclin E (cyclins E1/E2) is responsible for
pairing of chromosomes, the stability of telomere, and the locali-
zation of CDK2 in male meiosis [38]. The specific expressions pat-
terns of these cyclins during spermatogenesis in the testis offer
opportunities to develop a safe male contraceptive agent selec-
tively aiming at a target in the testis.
However, the binding affinities (Kd) of compounds 3, 53 and 59
for CDK2 in the absence of cyclins are in the low micromolar range
but did not exhibit any activity (data not shown).
(5e15 mM, Table 5). This binding assay is based on the competition
4.2. MDCK cell permeability assay
of test compounds and an immobilized ligand for the ATP binding
site so that the results can represent a compound’s affinity for the
ATP binding pocket or an allosteric site that can induce confor-
mational changes to alter the ATP binding pocket. The results
To investigate whether the inactivity of these compounds in the
antiproliferation assay was due to poor membrane permeability,
lead compound 3 was tested to determine its cell permeability
Table 3
CDK2/cyclin A inhibitory activities of N1-modified analogues.
Compound
R
IC50
(
mM)
Compound
R
IC50 (mM)
3
2.6 0.1
59
1.5 0.03
30
33
9.3 0.2
61
64
6.7 0.1
97
8
2.1 0.3
36
39
10.5 0.2
66
67
3.1 0.2
4.6 0.2
>1000
43
46
7.5 0.8
460 50
70
71
>1000
2.7 0.06
49
51
53
3.0 0.2
72
73
74
2.4 0.05
19 0.3
26
1
2.2 0.3
3.6 0.03
55
57
>1000
>1000
76
37
4
Commercial
56 1.4
7

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
Fig. 3. Inhibition of CDK complexes by 3 and its analogues at 1 mM.
Table 4
IC50 values for 3 and analogues 53 and 59 against CDK2 complexes with cyclin A, cyclin A1, cyclin E, and cyclin O.
CDK2/cyclin
3 IC50
(
mM)
53 IC50
(
m
M)
59 IC50
(
m
M)
Staurosporine IC50 (mM)
CDK2/cyclin A
CDK2/cyclin A1
CDK2/cyclin E
CDK2/cyclin O
0.0860
0.134
0.0274
0.108
0.0619
0.0243
0.0023
0.0331
0.120
0.0464
0.00890
0.0416
0.000720
0.00114
0.00164
0.00148
Table 5
Binding affinity of 3 and analogues 53 and 59 for CDK2.
FTMap [40] and then selected the protein-protein interface be-
tween CDK2 and cyclin E for the docking simulations because the
experimental result had shown that the compounds exerted much
higher affinity for CDK2 in the presence of cyclins compared to
CDK2. In addition, the ATP binding site and other potential binding
sites did not show significant differences between CDK2 and CDK2/
cyclin E1. We then performed the docking simulations of com-
pounds 3, 53, and 59 with the selected binding sites (Fig. 5). The
predicted binding poses of these compounds at the CDK2/cyclin E1
interface are shown in Fig. 5. In CDK2, they occupy the binding site
formed by Arg126, Ala149, Arg150, Gly153, Val154, Tyr179, and
Tyr180 (Fig. 5 a, b, and c) and form a H-bond interaction with
Arg150 through the C4 carbonyl oxygen. The binding site in CDK2 is
formed by a conformational change of the activation loop of CDK2
from an active conformation to an inactive conformation (Fig. 5a).
This site is occupied by the activation loop of CDK2 and disappears
when CDK2 forms a complex with cyclins. The MM-GBSA relative
binding free energies of 3, 53, and 59 for CDK2 are ꢀ37.3 kcal/
mol, ꢀ41.7 kcal/mol, and ꢀ39.3 kcal/mol. In the CDK2/cyclin E1
complex, they occupy a binding site in the interface of CDK2 and
cyclin E1, formed by Val156, Glu172, Cys177, Lys178, Tyr179, Ser181,
Ala183, Trp227, Pro228, Met233, Asp270, Pro271, and Asn272 of
CDK2 and Leu251 and Leu252 of cyclin E1 (Fig. 5 d, e, and f). The
MM-GBSA relative binding free energies of 3, 53, and 59 for CDK2/
cyclin E1 are ꢀ44.0 kcal/mol, ꢀ45.0 kcal/mol, ꢀ45.5 kcal/mol,
3 Kd (
15
m
M)
53 Kd (
4.8
mM)
59 Kd (
4.5
mM)
CDK2
using the MDCK cell line (Table 6). A high-permeability compound
(metoprolol) and two moderate-permeability compounds (digoxin
and imatinib) were used as references. Compound 3 exerted high
permeability (Papp (A-B)), better than digoxin and imatinib. However,
3 also exhibited a higher rate in the assay for Papp (B-A) than digoxin
and imatinib, but not as high as metoprolol. Compound 3 showed a
lower efflux ratio compared to the reference compounds. The re-
covery rates of 3 for both assays (AP-BL and BL-AP) were approxi-
mately 50% that might result from compound 3 being trapped in
the lysosome [39]. The data suggests that compound 3 should be
cellularly active and does not explain why this compound does not
inhibit MCF-7 cell proliferation.
4.3. Computational simulations
In order to obtain insights into possible binding modes for
compounds 3, 53, and 59, we performed computational simula-
tions using the crystal structures of CDK2 and of the CDK2/cyclin E1
complex (Fig. 4). We first searched for potential binding sites using
Table 6
Permeability results of test compounds in MDCK cell line.
Compound
Papp (A-B)
Papp (B-A)
Efflux ratio
Recovery (%) AP-BL
Recovery (%) BL-AP
(10ꢀ6, cm/s)
(10ꢀ6, cm/s)
Metoprolol
Digoxin
Imatinib
3
26.9
1.08
7.69
20.1
21.2
5.41
6.50
9.52
0.79
5.01
0.84
0.47
90.7
77.1
56.6
52.4
90.4
83.7
69.9
49.8
8

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
Fig. 4. FT-mapping of the protein-protein interface region of CDK2 interacting with cyclins: (a) CDK2 with ATP (PDB ID: 1B38) with the activation loop in the inactive conformation
(orange, mapping solvents (magenta) in the blue circle) and (b) a complex of CDK2 with cyclin E1 (cyan) and dinaciclib (PDB ID: 5L2W) with the activation loop in the active
conformation (blue, mapping solvents (red) in the yellow circle).
respectively. In CDK2/cyclin E1, the C4 carbonyl oxygen did not
form any H-bonding interaction in the binding site. In the predicted
binding modes in CDK2/cyclin E1, the nitrogen in the hetero-
aromatic ring at the N1 position of compound 59 forms a H-
bonding interaction with Ser181 of CDK2 while compound 3 cannot
form such a H-bond (pyrimidine of 59 vs pyridine of 3). This could
explain why compound 59 exerts higher potency against CDK2/
cyclin E1 than compound 3. The predicted binding modes of
compounds 3, 53, and 59 in CDK2 and CDK2/cyclin E1 are consis-
tent with their affinities for CDK2 and CDK2/cyclin E1, suggesting
that these two pockets in the interface domain of CDK2/cyclins
might be the potential pockets for these compounds and that the
CDK2/cyclin E1 is predicted to be the preferred one based on the
calculations of the relative free binding energies. We also per-
formed the same docking simulations with the ATP bind sites but
did not find any differences between them that would explain the
observed disparities in binding affinities for CDK2 and the CDK2/
cyclin E1 complex.
analogues had significantly superior potency over the parent
compound against CDK2/cyclin A. Nine compounds were tested for
inhibition of 12 CDK/cyclin complexes. Compounds 3, 53, 59 dis-
played significant inhibitory activities against CDK2/cyclin E with
IC50 values of 27, 2, and 9 nM respectively. However, the binding
affinities (Kd) of compounds 3, 53 and 59 for CDK2 in the absence of
cyclins were in the low micromolar range. The three compounds
did not inhibit MCF-7 cancer cell proliferation. Lead compound 3
was tested to determine its cell permeability using the MDCK cell
line. The data suggested that compound 3 could be cellularly active,
but the low recovery rates of 3 for both assays (AP-BL and BL-AP) of
approximately 50% suggested that compound 3 might being trap-
ped in the lysosome. As compounds 3, 53 and 59 inhibit the
enzymatic activity of the activated CDK2/cyclin A complex, phos-
phorylated in vitro by CDK-activating kinase 1 [17]), but shows less
binding potential towards free CDK2, these compounds may
interact with the intact CDK2/cyclin complexes at a site different
from the ATP site. CDK2 undergoes large conformational changes
upon interaction with cyclins, and it is conceivable that these
compounds exert inhibitory activity through interactions with a
conformer of CDK2 induced by its interaction with cyclins. Co-
crystal structures of CDK2-inhibitor complexes have been deter-
mined for numerous ATP site directed inhibitors including com-
pounds that bind to a recently identified allosteric pocket [41,42].
While co-crystal structures of CDK2 were readily obtained for other
hit compounds [17], which all bind to the ATP site of CDK2, hit 3 and
its analogues failed to show binding potential in co-crystallization
and in-diffusion experiments. In order to gain insights into
possible binding modes for compounds 3, 53, and 59, we performed
computational simulations using the crystal structures of CDK2 and
the CDK2/cyclin E1 complex. The docking studies indicated that the
compounds could bind at the interface between CDK2 and cyclin E1
and that the predicted binding mode based on the CDK2/cyclin E1
complex is favored over the one generated from CDK2.
5. Conclusion
Compound 3, a 4,5,6,7-tetrahydroindazole, was identified as a
CDK2/cyclin
A inhibitor from a high throughput screening
campaign of a library of 100,000 diverse compounds. SAR studies
with over 50 analogues based on hit compound 3 were performed
by modifying eight different positions. The in vitro inhibitory ac-
tivities of the analogues were determined from dose-response
curves using the phosphorylated CDK2/cyclin A complex. Replac-
ing the C7 bromine with electron-withdrawing substituents Cl and
N3 provided compounds with activity similar to 3, whereas the
introduction of C7 electron donating groups reduced potency.
Removal of the C6 gem-dimethyl group and attachment of larger C6
groups (isopropyl and phenyl) reduced activity. The 6,7-trans
monomethyl analogue of 18 retained activity and was 10-fold
more active than the corresponding cis analogues. Reduction of
the C4 carbonyl to the alcohol reduced activity >200-fold and
oxime formation reduced activity 30-fold. Schmidt reaction-
generated seven-membered amide analogue 21 had 30-fold
reduced activity. Deletion of the C3 methyl group or introduction
of a 3-isobutyl group did not provide analogues with better
inhibitory activities. 5-Br-substituted compounds showed low ac-
tivities. Different heterocyclic groups were introduced to modify
the N1 position. Several analogues showed similar or in the case of
53 and 59 slightly better activities than 3. In summary, none of the
6. Experimental section
General procedures. Commercially available chemicals were
used as purchased without further purification. All solvents were
dried before use. All reactions with air- or moisture-sensitive re-
agents were carried out under nitrogen atmosphere. The 1H NMR
spectra were obtained at 400 MHz. For 1H NMR spectra, chemical
shifts were given in parts per million (ppm) and were referenced to
tetramethyl silane (TMS) peak as an internal standard or the
9

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
Fig. 5. Predicted binding modes of 3 (cyan), 53 (brown), and 59 (green) in CDK2 (a, b and c) and the complex of CDK2 (green) with cyclin E1 (gray) (d, e and f).
residual solvent peak. 13C NMR spectra were recorded at 100 MHz.
Chemical shifts were reported in ppm and were referenced to the
appropriate residual solvent peak. High-resolution mass spec-
trometry (HRMS) was performed by the University of Minnesota
mass spectroscopy facility. Column chromatography was per-
formed on silica gel (SiliaFlash P60 230e400 mesh).
Hill slope coefficient. Non-linear regression analysis was performed
using SigmaPlot (Systat Software). The IC50 data were generated by
conduction three independent assays.
6.1. Enzyme assays with CDK2 cyclin complexes was performed by
Reaction Biology
Biochemical methods. Reagents and compounds for
biochemical experiments were purchased from SigmaeAldrich
unless otherwise indicated. Expression, purification and activa-
tion of the CDK2/cyclin A complex were performed as described
[41]. Protein concentration was determined using the Coomassie
reagent from BioRad with bovine serum albumin as a standard.
Enzyme assay. The synthetic peptide PKTPKKAKKL [43] served
as a substrate for the activated CDK2/cyclin A complex, and the
formation of ADP from ATP was coupled to the oxidation of NADH
using pyruvate kinase (PK) and lactate dehydrogenase (LDH),
monitored at 340 nm [17]. HTS and dose-response assays were
carried out in 384-well plates at room temperature in 50 mM Tris/
HCl buffer (pH 7.5) containing 10 mM MgCl2, 0.24 mM NADH, 5 mM
DTT, 6 U mLꢀ1 LDH, 10 U mLꢀ1 PK, 1 mM phosphoenolpyruvate, 5%
(v/v) DMSO, and 0.01 mg/mL activated CDK-cyclin A complex
(specific activity 6 U/mg). Inhibitor was added to the mixture, and
the reaction was started by the addition of 0.15 mM ATP and
0.5 mM peptide substrate. IC50 values were obtained by fitting the
data to equation (1):
Reaction Biology protocol: The kinase assay method is briefly
described below. The substrates histone H1 (20
cyclins, RB protein (3 M) for CDK4/cyclin D1 and CDK6/cyclin D1,
myelin basic protein (20 M) for CDK7/cyclin H, peptide substrate,
(KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC, 20 M) for
CDK9/cyclin T1 and ATP (10 M) were added to the base reaction
mM) for most CDK/
m
m
m
m
buffer containing 20 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM
EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT,
and 1% DMSO. Cofactors were added to the substrate solution in the
base reaction buffer followed by CDK complexes. Compound solu-
tions in DMSO were added to the kinase reaction mixture, and then
the reaction mixture was incubated at rt for 20 min. After that, 33P-
ATP was added to the reaction mixture. Following incubating the
reaction mixture at rt for 2 h, reactions were spotted on P81 ion
exchange paper and the kinase activity was detected by filter-
binding method. The kinase profiling was carried out at 1 mM
single dose in duplicate. The IC50 data were determined at a 10-
dose single assay.
A
A0
1
6.2. Kinase binding assay for Kd determining was performed by
DiscoverX
¼
(equation 1)
½Iꢂ
n
1 þ ðIC
Þ
50
where A/A0 is the activity remaining relative to the control without
DiscoverX protocol: The kinase binding assay was performed
inhibitor (A0), [I] is the concentration of the inhibitor, and n is the
according to the reported method [44]. For the assay, an E. coli host
10

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
from the BL21 strain was used to prepare kinase-tagged T7 phage
strains. Following growing to log-phase, E. coli were infected with
T7 phage, and then incubated with shaking at 32 ꢁC until lysis. Cell
debris was removed from the lysates by centrifugation and filtra-
tion. Affinity resins for kinase binding assay were prepared by
treating streptavidin-coated magnetic beads with biotinylated
small molecule ligands for 30 min at room temperature. To remove
unbound ligands and to reduce non-specific binding, the liganded
beads were treated with excess biotin and washed with blocking
buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT).
Binding reactions were carried out by combining a kinase, liganded
affinity beads, and test compounds in 1x binding buffer (20% Sea-
Block, 0.17x PBS, 0.05% Tween 20, 6 mM TDD).
Kd values were measured with an 11-point, 3-fold compound
dilution series with three DMSO control points. Following acoustic
transfer of compounds in 100% DMSO, the compounds were diluted
directly into the assays maintaining the final concentration of
DMSO was 0.9%. Binding reactions were performed in poly-
propylene 384-well plate with each well with a final volume of
0.02 mL. Following 1 h incubation of the assay plate at rt with
shaking, the affinity beads were washed with wash buffer (1x PBS,
0.05% Tween 20). After that, the beads were resuspended in elution
buffer (1x PBS, 0.05% Tween 20, 0.5 mM non-biotinylated affinity
ligand) and incubated at rt with shaking for 30 min. The kinase
concentrations in the eluates were then measured by qPCR. The
binding constants (Kd values) were calculated with a standard
dose-response curve using the Hill equation shown below. The
possible effects of the aggregation of the molecules of hemoglobin
on its dissociation curves [45].
(10 mM HEPES, pH 7.4) to the wells in the receiver plate (basolateral
compartment). The assay was performed in duplicate. The rate of
drug transport in the basolateral to apical direction was measured
by adding 235
controls to the receiver plate wells (basolateral compartment) and
adding 75 L of HBSS (10 mM HEPES, pH 7.4) to the Transwell in-
serts (apical compartment). Time 0 samples were prepared by
transferring 50 L of 1 M working solution to wells of the 96-
deepwell plate, followed by the addition of 200 L cold methanol
mL of 1 mM working solutions of a test compound and
m
m
m
m
containing appropriate internal standards (100 nM alprazolam,
200 nM labetalol, 200 nM caffeine and 200 nM diclofenac).
Following incubating the plates at 37 ꢁC for 2 h, 50
mL samples from
donor sides (apical compartment for Ap/Bl flux, and basolateral
compartment for Bl/Ap) and receiver sides (basolateral
compartment for Ap/Bl flux, and apical compartment for Bl/Ap)
were transferred to wells of a new 96-well plate. Then, 200 mL cold
methanol containing appropriate internal standards (100 nM al-
prazolam, 200 nM labetalol, 200 nM caffeine and 200 nM diclofe-
nac) to the wells of the new 96-well plate. Samples were mixed by
vortexing for 5 min, and then centrifuged at 3220 g for 40 min.
100 mL of the supernatants were mixed with an appropriate volume
of ultra-pure water before LC-MS/MS analysis. For the measure-
ment of Lucifer Yellow leakage after 2 h transport, the stock solu-
tion of Lucifer yellow in water was prepared and then diluted with
HBSS (10 mM HEPES, pH 7.4) to prepare the final concentration of
100
Transwell insert (apical compartment), and 300
HEPES, pH 7.4) to the wells in the receiver plate (basolateral
compartment). The plates were Incubated at 37 ꢁC for 30 min. 80
mM. 100
mL of the Lucifer yellow solution was added to each
m
L of HBSS (10 mM
mL
of samples from the apical and basolateral wells (using the baso-
lateral access holes) were transferred to wells of new 96 wells
plates. The Lucifer Yellow fluorescence (to monitor monolayer
integrity) signal was measured in a fluorescence plate reader
(excitation at 485 nm excitation and emission at 530 nm). The
MDCK cell monolayers were checked for integrity and were found
to be intact. The assay was performed in duplicate.
signal ꢀ background
Response ¼ background þ
The Hill Slope was set to ꢀ1.
(equation 2)
Slope
KdHill
1 þ DoseHill
Slope
Curves were fitted using a non-linear least squares fit with the
Levenberg-Marquardt algorithm [46]. The assay was performed in
duplicate.
6.5. Experimental protocol for molecular modeling
6.3. Antiproliferation assay
CDK2 (PDB ID: 1B38) and CDK2/cyclin E1 (PDB ID: 5L2W) crystal
structures were imported from the PDB data bank (www.rcsb.org).
FTMap solvent mapping was performed at ftmap.bu.edu [40]. Im-
ported PDB structures were prepared for the computational sim-
ulations by assigning bond orders and H atoms, generating zero-
order bonds to metals and metal binding states, removing waters,
and reassigning H-bond networks followed by performing
restrained minimization of H atoms. Grids were generated in the
protein-protein interface domain using Glide: van der Waals radius
scaling factor 1.0, partial charge cutoff 0.25, and the grid box
dimension of 15 Å ꢃ 15 Å ꢃ 15 Å. Ligands were prepared for the
docking simulations using LigPrep [47]. Docking simulations were
performed in the SP mode followed by the XP mode using Glide
[48]. MM-GBSA relative binding free energy calculations of the
compounds in the target proteins were performed with Prime [49].
Upon harvesting MCF-7 cells, 5000 cells were plated out in each
well of 96-well plates. Following overnight incubation at 37 ꢁC in an
incubator with 5% CO2, cells were inoculated with various con-
centrations of compounds. Compounds 3, 53, and 59 were tested at
8 different concentrations ranges from 0.00256
Staurosporine (IC50 ¼ 282 nM) was used as a positive control at 8
different concentrations ranges from 0.000256 M to 20 M. After
the cells were incubated in the incubator (37 ꢁC, 5% CO2) for 72 h,
mM to 200 mM.
m
m
10 mL of alamarBlue was added in each well of the plates. Following
90 min incubation at 37 ꢁC, optical density of fluorescence (exci-
tation at 530 nm, emission at 590 nm) from each well was recorded
to determine the population of the proliferating cells.
6.4. MDCK assay (performed by Pharmaron)
Pharmaron protocol: The MDCK plate from the incubator was
washed twice with pre-warmed HBSS (10 mM HEPES, pH 7.4), and
then incubated at 37 ꢁC for 30 min. The stock solutions of a test
compound and controls were diluted in DMSO to prepare 0.2 mM
solutions, and further diluted with HBSS (10 mM HEPES, pH 7.4) to
6.6. Chemistry
3,6,6-Trimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-indazol-
4(5H)-one (2) [18]. Commercially available 2-acetyl-5,5-dimethy1-
cyclohexane-1,3-dione (1, 500 mg, 2.74 mmo1), and 2-hydrazino-
pyridine (300 mg, 2.75 mmo1) were dissolved in ethanol (15 m1).
The solution was stirred at 80 ꢁC for 4 h. After evaporation of the
solvent, the residue was purified by silica column chromatography
(EtOAc:hexanes 1:1) to afford compound 2 (586 mg, 84%) as a white
prepare 1
in the incubation system was 0.5%. The rate of drug transport in the
apical to basolateral direction was measured by adding 75 L of
M working solutions of test compound and controls to the
Transwell inserts (apical compartment) and adding 235 L of HBSS
mM working solutions. The final concentration of DMSO
m
1
m
m
solid. Mp ¼ 125e127 ꢁC; 1H NMR (400 MHz, CDCl3)
d 8.45 (ddd,
11

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
J ¼ 4.9,1.9, 0.8 Hz,1H), 7.94 (dt, J ¼ 8.3, 0.9 Hz,1H), 7.83 (ddd, J ¼ 8.3,
7.4,1.8 Hz,1H), 7.23 (ddd, J ¼ 7.3, 4.9,1.0 Hz,1H), 3.30 (s, 2H), 2.55 (s,
EtOAc:hexanes 1:2) to afford compound 6 as a white solid (57 mg,
70%). Mp ¼ 130e133 ꢁC; 1H NMR (400 MHz, CDCl3)
d 8.46 (d,
3H), 2.40 (s, 2H), 1.14 (s, 6H); 13C NMR (100 MHz, CDCl3)
d
194.1,
J ¼ 5.0 Hz, 1H), 8.04 (d, J ¼ 8.3 Hz, 1H), 7.94 (td, J ¼ 7.8, 1.6 Hz, 1H),
7.32 (t, J ¼ 6.2 Hz, 1H), 5.70 (s, 1H), 4.67 (s, 1H), 2.86 (d, J ¼ 16.0 Hz,
1H), 2.55 (s, 3H), 2.23 (d, J ¼ 16.0 Hz, 1H), 1.27 (s, 3H), 1.07 (s, 3H);
152.7, 150.6, 150.2, 147.7, 138.6, 121.7, 118.0, 115.6, 52.5, 39.0, 35.4,
28.6, 13.5; HRMS (ESI) calcd for [C15H17N3O þ H]þ 256.1450, found
256.1453.
13C NMR (100 MHz, CDCl3)
d 194.2, 151.8, 150.9, 150.3, 147.4, 139.8,
7-Bromo-3,6,6-trimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (3) [18]. To a solution of compound 2 (300 mg,
1.18 mmol) in CHCl3 (8 mL) was added N-bromosuccinimide
(209 mg, 1.18 mmol) and the mixture was stirred at 60 ꢁC for 6 h.
After cooling the reaction mixture, CH2Cl2 (10 mL) was added. The
solution was washed with water (2 ꢃ 10 mL), dried over anhydrous
Na2SO4, and evaporated under reduced pressure. The residue was
purified by silica column chromatography (EtOAc:hexanes 1:5) to
122.2, 117.2, 116.1, 68.7, 48.8, 38.5, 26.7, 25.1, 13.4; HRMS (ESI) calcd
for [C15H17N3O2 þ Na]þ 294.1213, found 294.1202.
7-Chloro-3,6,6-trimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (7). To a solution of compound 6 (30 mg,
0.11 mmol) and pyridine (36
added thionyl chloride (16
m
m
L, 0.44 mmol) in Et2O (0.5 mL) was
L, 0.22 mmol) at 0 ꢁC. The reaction
mixture was stirred at room temperature for 2 h. The mixture was
then poured to ice-cold H2O (5 mL) and extracted with EtOAc
(10 mL). The organic layer was washed with H2O (5 mL), brine
(2 ꢃ 5 mL), NH4Cl (2 ꢃ 5 mL) and dried over anhydrous Na2SO4.
After evaporation under reduced pressure, the residue was purified
by silica column chromatography (EtOAc:hexanes 1:2) to furnish
compound 7 as a white solid (22 mg, 70%). Mp ¼ 141e146 ꢁC; 1H
afford compound
3
as
a
white solid (238 mg, 61%).
Mp ¼ 149e154 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.50 (ddd, J ¼ 4.9,
1.8, 0.8 Hz, 1H), 8.00 (d, J ¼ 8.3 Hz, 1H), 7.85 (ddd, J ¼ 8.3, 7.4, 1.9 Hz,
1H), 7.27 (ddd, J ¼ 7.4, 4.8, 1.0 Hz, 1H), 6.39 (d, J ¼ 1.3 Hz, 1H), 2.87
(dd, J ¼ 16.9, 0.8 Hz, 1H), 2.55 (s, 3H), 2.27 (dd, J ¼ 16.8, 1.4 Hz, 1H),
1.40 (s, 3H), 1.26 (s, 3H); 13C NMR (100 MHz, CDCl3)
d
192.9, 152.6,
NMR (400 MHz, CDCl3)
d
8.50 (dd, J ¼ 4.9, 1.2 Hz, 1H), 8.00 (d,
150.1, 149.6, 147.9, 138.7, 122.0, 116.6, 115.4, 52.9, 48.7, 39.7, 29.6,
25.4, 13.5; HRMS (ESI) calcd for [C15H16BrN3O þ H]þ 334.0555,
found 334.0546.
J ¼ 8.3 Hz, 1H), 7.86 (ddd, J ¼ 8.3, 7.5, 1.9 Hz, 1H), 7.27 (ddd, J ¼ 7.3,
5.0, 0.9 Hz, 1H), 6.22 (d, J ¼ 1.0 Hz, 1H), 2.93 (d, J ¼ 16.8 Hz, 1H), 2.55
(s, 3H), 2.26 (dd, J ¼ 16.8, 1.2 Hz, 1H), 1.37 (s, 3H), 1.20 (s, 3H); 13C
7-Fluoro-3,6,6-trimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (4) [18]. To a solution of compound 3 (150 mg,
0.449 mmol) in acetonitrile (7 mL) were added silver fluoride
(114 mg, 0.898 mmol) and calcium fluoride (140 mg, 1.80 mmol).
The mixture was stirred at 80 ꢁC for 22 h. After cooling the reaction
mixture, the precipitate was filtered and the filtrate was evaporated
under reduced pressure. The crude product was purified by silica
column chromatography (EtOAc:hexanes 1:2) to afford compound
4 as a white solid (13 mg, 12%). Mp ¼ 91e99 ꢁC; 1H NMR (400 MHz,
NMR (100 MHz, CDCl3) d 193.1, 152.5, 150.1, 148.7, 147.9, 138.8, 122.1,
116.9, 115.3, 59.6, 47.8, 39.9, 27.7, 26.3, 13.5; HRMS (ESI) calcd for
[C15H16ClN3O þ Na]þ 312.0874, found 312.0843.
3,6,6,7-Tetramethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-inda-
zol-4(5H)-one (8). To a solution of compound 2 (70 mg, 0.27 mmol)
in THF (1 mL) was slowly added n-BuLi (175 mL, 0.28 mmol, 1.6 M
solution in hexanes) at ꢀ78 ꢁC. The reaction mixture was stirred
at ꢀ78 ꢁC for 30 min and further stirred at 0 ꢁC for 1 h. After the
mixture was cooled down to ꢀ78 ꢁC, methyl iodide (21
mL,
CDCl3)
d
8.52 (ddd, J ¼ 4.8, 1.8, 0.7 Hz, 1H), 8.00 (d, J ¼ 8.3 Hz, 1H),
0.33 mmol) was added. The mixture was stirred at ꢀ78 ꢁC for
30 min and further stirred at room temperature for 16 h. The
mixture was then poured into cold brine (0.5 mL) and extracted
with EtOAc (5 mL). The organic layer was washed with brine
(2 ꢃ 5 mL) and dried over anhydrous Na2SO4. After evaporation
under reduced pressure, the residue was purified by preparative
TLC (silica gel, EtOAc:hexanes 1:2) to afford compound 8 as a pale
yellow solid (19 mg, 25%). Mp ¼ 80e83 ꢁC; 1H NMR (400 MHz,
7.86 (ddd, J ¼ 8.2, 7.4, 1.8 Hz, 1H), 7.28 (ddd, J ¼ 7.4, 4.9, 1.0 Hz, 1H),
6.28 (d, J ¼ 48.2 Hz, 1H), 2.86 (d, J ¼ 16.6 Hz, 1H), 2.57 (s, 3H), 2.27
(dd, J ¼ 16.5, 1.0 Hz, 1H), 1.32 (d, J ¼ 2.0 Hz, 3H), 1.05 (s, 3H); 13C
NMR (100 MHz, CDCl3)
d 193.5, 152.2, 150.0, 148.2, 144.7, 138.8,
122.4, 118.4, 115.4, 88.2, 86.4, 47.7, 39.2, 39.0, 25.4, 25.0, 13.4; HRMS
(ESI) calcd for [C15H16FN3O þ Na]þ 296.1170, found 296.1173.
7-Methoxy-3,6,6-trimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (5) [18]. A solution of 0.1 N silver nitrate in
methanol (1.45 mL, 0.145 mmol) was added to a solution of com-
pound 3 (24.2 mg, 0.0724 mmol) in acetone (1.45 mL). After stirring
for 24 h, 1.75 ml of 0.1 N silver nitrate solution in methanol was
added. The reaction mixture was stirred for 4 days and then filtered.
The solvent was evaporated and the residue was purified by pre-
parative TLC (silica gel, EtOAc:hexanes 1:2) to afford compound 5 as
a white solid (4.3 mg, 21%). Mp ¼ 99e104 ꢁC; 1H NMR (400 MHz,
CDCl3)
d
8.50 (ddd, J ¼ 4.9, 1.8, 0.8 Hz, 1H), 7.94 (dt, J ¼ 8.3, 0.8 Hz,
1H), 7.83 (ddd, J ¼ 8.3, 7.4, 1.9 Hz, 1H), 7.22 (ddd, J ¼ 7.3, 4.9, 1.0 Hz,
1H), 3.36 (d, J ¼ 18.2 Hz, 1H), 3.24 (d, J ¼ 18.2 Hz, 1H), 2.55 (s, 3H),
2.35 (q, J ¼ 7.1 Hz, 1H), 1.17 (d, J ¼ 7.1 Hz, 3H), 1.15 (s, 3H), 1.00 (s,
3H); 13C NMR (100 MHz, CDCl3)
d 197.4, 152.7, 150.5, 149.5, 147.7,
138.6, 121.6, 117.2, 115.5, 52.8, 38.3, 37.8, 29.0, 23.5, 13.5, 10.2; HRMS
(ESI) calcd for [C16H19N3O þ Na]þ 292.1420, found 292.1413.
7-(Dimethylamino)-3,6,6-trimethyl-1-(pyridin-2-yl)-6,7-
CDCl3)
d
8.48 (ddd, J ¼ 4.9, 1.8, 0.7 Hz, 1H), 7.98 (d, J ¼ 8.3 Hz, 1H),
dihydro-1H-indazol-4(5H)-one (9) [18]. To a solution of com-
pound 3 (60 mg, 0.18 mmol) in THF (1.8 mL) was added 30%
dimethylamine in ethanol (3.6 mL). The mixture was stirred at
40 ꢁC with 300 W power in a microwave apparatus for 38 h and
then evaporated. The residue was diluted with CH2Cl2 (10 mL),
washed with water (5 mL) and brine (2 ꢃ 5 mL), dried over anhy-
drous Na2SO4, and evaporated. The residue was purified by pre-
parative TLC (silica gel, EtOAc:hexanes 1:1) to afford compound 9 as
a pale yellow solid (11 mg, 21%). Mp ¼ 59e67 ꢁC; 1H NMR
7.86 (ddd, J ¼ 8.2, 7.4, 1.8 Hz, 1H), 7.27 (ddd, J ¼ 7.4, 4.8, 1.0 Hz, 1H),
5.32 (s, 1H), 3.37 (s, 3H), 2.87 (d, J ¼ 16.8 Hz, 1H), 2.55 (s, 3H), 2.14
(dd, J ¼ 16.8, 0.7 Hz, 1H), 1.26 (s, 3H), 1.02 (s, 3H); 13C NMR
(100 MHz, CDCl3)
d 194.4, 153.0, 150.2, 149.8, 147.5, 138.8, 122.3,
117.6, 116.1, 77.8, 59.1, 47.9, 40.2, 26.2, 25.8, 13.5; HRMS (ESI) calcd
for [C16H19N3O2 þ Na]þ 308.1369, found 308.1364.
7-Hydroxy-3,6,6-trimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (6) [18]. A solution of 0.1 N silver nitrate in H2O
(6.0 mL, 0.60 mmol) was added to a solution of compound 3
(100 mg, 0.299 mmol) in acetone (6.0 mL). After stirring for 15 h,
3.0 mL of 0.1 N silver nitrate solution in H2O was added. The re-
action mixture was then stirred for 5 h. The reaction mixture was
filtered through Celite and evaporated to remove acetone. The re-
sidual water solution was extracted with EtOAc (3 ꢃ 10 mL). The
organic layer was dried over anhydrous Na2SO4 and evaporated.
The residue was purified by preparative TLC (silica gel,
(400 MHz, CDCl3)
d
8.46 (ddd, J ¼ 4.8, 1.8, 0.8 Hz, 1H), 7.94 (d,
J ¼ 8.3 Hz, 1H), 7.85 (ddd, J ¼ 8.2, 7.4, 1.8 Hz, 1H), 7.26 (m, 1H), 4.67
(s, 1H), 2.82 (d, J ¼ 17.3 Hz, 1H), 2.57 (s, 3H), 2.25 (s, 6H), 2.11 (d,
J ¼ 17.3 Hz, 1H), 1.23 (s, 3H), 1.02 (s, 3H); 13C NMR (100 MHz, CDCl3)
d
194.9, 152.8, 150.1, 150.0, 147.5, 138.6, 122.1, 118.9, 116.2, 64.6, 48.7,
44.3, 40.7, 27.8, 27.0, 13.6; HRMS (ESI) calcd for [C17H22N4O þ H]þ
299.1866, found 299.1868.
3,6,6-Trimethyl-7-(phenylthio)-1-(pyridin-2-yl)-6,7-dihydro-
12

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
1H-indazol-4(5H)-one (10) [18].
A
solution of compound
3
for the formation of 3-acyloxy-2-cyclohexenones. (Compounds
15a-15e are also commercially available.) Acetyl chloride (1.02 mL,
14.3 mmol) was added to a solution of cyclohexane-1,3-dione (14a,
1.00 g, 8.92 mmol) and pyridine (1.08 ml, 13.4 mmol) in CHCl3
(31 mL). The reaction mixture was stirred at room temperature for
16 h. The mixture was then washed with H2O (30 mL), 0.1 N HCl
(30 mL), saturated NaHCO3 (30 mL) and H2O (30 mL), dried over
anhydrous Na2SO4, and evaporated. The residue was purified by
silica column chromatography (EtOAc:hexanes 1:2) to afford
compound 15a as a yellowish oil (790 mg, 58%). 1H NMR (400 MHz,
(160 mg, 0.48 mmol), and sodium thiophenate (78 mg, 0.60 mmol)
in dry DMF (10 ml) was stirred at room temperature for 7 h. The
reaction mixture was then poured into brine (10 mL) and extracted
with CH2Cl2 (2 ꢃ 5 mL). The combined organic layer was washed
with brine (2 ꢃ 10 mL), dried over anhydrous Na2SO4 and evapo-
rated. The residue was purified by silica column chromatography
(EtOAc:hexanes 1:5) to furnish compound 10 as a solid (138 mg,
79%). Mp ¼ 60e88 ꢁC; 1H NMR (400 MHz, CDCl3)
d 8.25 (ddd,
J ¼ 4.8, 1.6, 0.8 Hz, 1H), 7.77 (d, J ¼ 8.2 Hz, 1H), 7.74 (dd, J ¼ 7.0,
1.8 Hz, 1H), 7.26e7.24 (m, 2H), 7.15e7.11 (m, 4H), 5.82 (d, J ¼ 1.1 Hz,
1H), 2.99 (dd, J ¼ 17.1, 0.6 Hz, 1H), 2.53 (s, 3H), 2.18 (dd, J ¼ 17.1,
1.2 Hz, 1H), 1.30 (s, 3H), 1.22 (s, 3H); 13C NMR (100 MHz, CDCl3)
CDCl3)
d
5.91 (t, J ¼ 1.2 Hz, 1H), 2.53 (td, J ¼ 3.1, 1.2 Hz, 2H), 2.41 (t,
J ¼ 6.7 Hz, 2H), 2.22 (s, 3H), 2.06 (quin, J ¼ 6.5 Hz, 2H); 13C NMR
(100 MHz, CDCl3)
d 199.6, 169.7, 167.4, 117.6, 36.7, 28.3, 21.3, 21.2.
d
193.4, 152.7, 150.3, 150.0, 147.4, 138.5, 134.3, 132.5, 128.6, 127.5,
5-Methyl-3-oxocyclohex-1-en-1-yl Acetate (15b). Prepared
from 5-methylcyclohexane-1,3-dione (14b, 1.50 g, 11.9 mmol) ac-
cording to the general procedure. Yield: 75% (1.50 g); colorless oil.
121.8, 117.5, 115.7, 52.1, 48.6, 40.3, 28.1, 27.7, 13.6; HRMS (ESI) calcd
for [C21H21N3OS þ Na]þ 386.1298, found 386.1301.
3,6,6-Trimethyl-7-(phenylsulfonyl)-1-(pyridin-2-yl)-6,7-
1H NMR (400 MHz, CDCl3)
d
5.90 (d, J ¼ 1.7 Hz, 1H), 2.49 (m, 2H),
dihydro-1H-indazol-4(5H)-one (11) [18]. To a solution of com-
pound 10 (98 mg, 0.27 mmol) in MeOH (2.5 mL) was slowly added a
solution of oxone (826 mg, 1.34 mmol) in H2O (2.5 mL) at 0 ꢁC. The
reaction mixture was stirred at room temperature for 3 h and then
diluted with H2O (10 mL). The mixture was extracted with CH2Cl2
(3 ꢃ 10 mL) and the combined organic layer was washed with brine
(2 ꢃ 10 mL), dried over anhydrous Na2SO4 and evaporated. The
residue was purified by preparative TLC (silica gel, EtOAc:hexanes
1:1) to afford compound 11 as a white solid (72 mg, 68%).
2.33 (m, 2H), 2.22 (s, 3H), 2.12 (m, 1H), 1.11 (d, J ¼ 6.3 Hz, 3H); 13C
NMR (100 MHz, CDCl3)
21.3, 20.8.
d 199.6, 169.1, 167.4, 117.2, 45.0, 36.4, 29.1,
5-Isopropyl-3-oxocyclohex-1-en-1-yl Acetate (15c). Prepared
from 5-isopropylcyclohexane-1,3-dione (14c, 1.00 g, 6.49 mmol)
according to the general procedure. Yield: 53% (674 mg); colorless
oil. 1H NMR (400 MHz, CDCl3)
d
5.90 (s, 1H), 2.50 (dd, J ¼ 16.0,
3.8 Hz, 1H), 2.43 (m, 2H), 2.23 (s, 3H), 2.14 (dd, J ¼ 16.0, 13.3 Hz, 1H),
1.97 (m, 1H), 1.63 (m, 1H), 0.95 (s, 3H), 0.93 (s, 3H); 13C NMR
Mp ¼ 160e164 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.33 (ddd, J ¼ 4.9,
(100 MHz, CDCl3)
21.3, 19.5.
d 200.0, 169.7, 167.4, 117.2, 40.8, 40.3, 32.3, 31.8,
1.8, 0.8 Hz, 1H), 7.59 (ddd, J ¼ 8.3, 7.4, 1.9 Hz, 1H), 7.38 (d, J ¼ 8.3 Hz,
1H), 7.33 (m, 3H), 7.18e7.10 (m, 3H), 6.29 (s, 1H), 3.65 (d, J ¼ 18.0 Hz,
1H), 2.58 (s, 3H), 2.27 (d, J ¼ 18.0 Hz, 1H), 1.66 (s, 3H), 1.19 (s, 3H);
5-Oxo-1,2,5,6-tetrahydro-[1,1′-biphenyl]-3-yl Acetate (15d).
Prepared from 5-phenylcyclohexane-1,3-dione (14d, 2.00 g,
10.6 mmol) according to the general procedure. Yield: 76% (1.85 g);
13C NMR (100 MHz, CDCl3)
d 193.2, 152.4, 150.5, 147.0, 142.4, 139.0,
138.5, 133.7, 128.6, 128.3, 122.1, 119.2, 115.6, 67.9, 48.7, 40.1, 30.8,
27.9, 13.6; HRMS (ESI) calcd for [C21H21N3O3S þ Na]þ 418.1196,
found 418.1214.
white solid. Mp ¼ 181e188 ꢁC; 1H NMR (400 MHz, CDCl3)
d 7.35 (m,
2H), 7.27 (m, 3H), 6.01 (d, J ¼ 2.2 Hz, 1H), 3.44 (m, 1H), 2.88 (ddd,
J ¼ 17.9, 11.1, 2.3 Hz, 1H), 2.67 (m, 3H), 2.23 (s, 3H); 13C NMR
7-Azido-3,6,6-trimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (12). A mixture of compound 3 (250 mg,
0.748 mmol) and sodium azide (194 mg, 2.98 mmol) in 80% EtOH
(5 mL) was stirred at 90e100 ꢁC for 24 h, and then evaporated. The
residue was purified by preparative TLC (silica gel, EtOAc:hexanes
1:2) to afford compound 12 as a yellowish solid, which was further
purified by recrystallization from 60% EtOH (4 mL) to give pure
compound 12 as a white solid (95 mg, 43%). Mp ¼ 125e131 ꢁC; 1H
(100 MHz, CDCl3) d 198.6, 168.7, 167.4, 142.1, 128.9, 127.3, 126.7,
117.4, 43.8, 39.6, 36.1, 21.3.
5,5-Dimethyl-3-oxocyclohex-1-en-1-yl 3-Methylbutanoate
(15e). Isovaleryl chloride (1.14 mL, 9.27 mmol) was added to a so-
lution of 5,5-dimethylcyclohexane-1,3-dione (1.00 g, 7.13 mmol)
and pyridine (692 ml, 8.56 mmol) in CHCl3 (25 mL). The reaction
mixture was stirred at room temperature for 2 h. The mixture was
then washed with H2O (30 mL), 0.1 N HCl (30 mL), and saturated
NaHCO3 (30 mL), dried over anhydrous Na2SO4, and evaporated.
The residue was purified by silica column chromatography (EtOA-
c:hexanes 1:1) to afford compound 15e as a colorless oil (1.28 g,
NMR (400 MHz, CDCl3)
d
8.49 (ddd, J ¼ 4.9, 1.8, 0.8 Hz, 1H), 8.04 (dt,
J ¼ 8.3, 0.8 Hz, 1H), 7.87 (ddd, J ¼ 8.3, 7.4, 1.9 Hz, 1H), 7.28 (ddd,
J ¼ 7.3, 4.8, 1.0 Hz, 1H), 5.53 (d, J ¼ 0.8 Hz, 1H), 2.77 (dd, J ¼ 16.6,
0.6 Hz, 1H), 2.55 (s, 3H), 2.18 (dd, J ¼ 16.6, 1.1 Hz, 1H), 1.32 (s, 3H),
80%). 1H NMR (400 MHz, CDCl3)
d 5.89 (s, 1H), 2.41 (s, 2H), 2.34 (d,
1.07 (s, 3H); 13C NMR (100 MHz, CDCl3)
d
193.2, 152.3, 150.1, 147.7,
J ¼ 7.1 Hz, 2H), 2.27 (s, 2H), 2.16 (m, 1H), 1.11(s, 6H), 1.01 (d,
147.0,139.0,122.1,117.7,114.8, 63.4, 47.9, 39.7, 26.7, 26.0,13.5; HRMS
(ESI) calcd for [C15H16N6O þ Na]þ 319.1278, found 319.1286.
7-Amino-3,6,6-trimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (13). To a solution of compound 12 (20 mg,
0.068 mmol) in EtOH (0.7 mL) was added 10% Pd/C (2 mg). The
reaction mixture was stirred under a H2 atmosphere at room
temperature for 4 h. The mixture was filtered through Celite and
concentrated under reduced pressure. The residue was purified by
preparative TLC (silica gel, MeOH: CH2Cl2 1:9) to afford compound
13 as a pale yellow solid (13 mg, 73%). 1H NMR (400 MHz, CDCl3)
J ¼ 6.6 Hz, 6H); 13C NMR (100 MHz, CDCl3)
d 199.4, 169.7, 168.2,
116.6, 50.8, 43.3, 42.3, 33.2, 28.2, 25.7, 22.3.
2-Acetylcyclohexane-1,3-dione (16a); general procedure for
the formation of 2-acylcyclohexane-1,3-diones. To a suspension
of anhydrous aluminum chloride (1.35 g, 10.1 mmol) in 1,2-
dichloroethane (15 mL) was added a solution of 3-oxocyclohex-1-
en-1-yl acetate (15a, 780 mg, 5.06 mmol) in 1,2-dichloroethane
(2 mL). The reaction mixture was stirred at room temperature for
2 h. The mixture was then poured into a mixture of ice (8 g) and
concentrated HCl (8 mL) and extracted with CHCl3 (4 ꢃ 17 mL). The
combined organic layer was washed with H2O (30 mL), dried over
anhydrous Na2SO4, and evaporated. The residue was purified by
silica column chromatography (EtOAc:hexanes 1:2) to afford
compound 16a as a colorless oil (687 mg, 54%) [50]. 1H NMR
d
8.48 (ddd, J ¼ 4.9, 1.8, 0.8 Hz, 1H), 7.99 (dt, J ¼ 8.3, 0.8 Hz, 1H), 7.88
(ddd, J ¼ 8.3, 7.4, 1.9 Hz, 1H), 7.27 (ddd, J ¼ 7.3, 5.0, 1.0 Hz, 1H), 4.27
(s, 1H), 2.83 (d, J ¼ 16.7 Hz, 1H), 2.55 (s, 3H), 2.19 (dd, J ¼ 16.7 Hz,
1H), 2.19 (br s, 2H), 1.26 (s, 3H), 1.02 (s, 3H); 13C NMR (100 MHz,
CDCl3)
d
193.9, 153.8, 152.3, 150.3, 147.8, 139.0, 121.9, 116.3, 115.7,
(400 MHz, CDCl3)
d
2.67 (t, J ¼ 6.4, 2H), 2.61 (s, 3H), 2.49 (t, J ¼ 6.6,
53.7, 47.7, 38.3, 27.2, 26.5, 13.5; HRMS (ESI) calcd for
2H), 1.98 (quin, J ¼ 6.5, 2H); 13C NMR (100 MHz, CDCl3)
d 203.1,
[C15H18N4O þ H]þ 271.1553, found 271.1538.
198.6, 195.4, 113.4, 38.6, 33.3, 28.8, 19.0.
3-Oxocyclohex-1-en-1-yl Acetate (15a). General procedure
2-Acetyl-5-methylcyclohexane-1,3-dione (16b). Prepared
13

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
from 5-methyl-3-oxocyclohex-1-en-1-yl acetate (15b, 1.40 g,
8.33 mmol) according to the general procedure. Yield: 33%
(456 mg); pale yellow solid. Mp ¼ 44e46 ꢁC; 1H NMR (400 MHz,
292.1420, found 292.1431.
3-Methyl-6-phenyl-1-(pyridin-2-yl)-6,7-dihydro-1H-indazol-
4(5H)-one (17d). Prepared from 2-acetyl-5-phenylcyclohexane-
1,3-dione (16d, 290 mg, 1.26 mmol) according to the general pro-
cedure. Yield: 75% (285 mg); white solid. Mp ¼ 163e179 ꢁC; 1H
CDCl3)
d
2.70 (ddd, J ¼ 17.8, 4.0, 2.1 Hz, 1H), 2.61 (s, 3H), 2.57 (dd,
J ¼ 12.5, 2.0 Hz,1H), 2.38 (dd, J ¼ 17.9, 11.0 Hz,1H), 2.21 (m, 2H), 1.09
(d, J ¼ 6.3 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d
202.9, 198.1, 195.3,
NMR (400 MHz, CDCl3)
d
8.38 (d, J ¼ 4.8 Hz, 1H), 7.94 (d, J ¼ 8.3 Hz,
112.9, 46.8, 41.1, 26.4, 20.8, 14.2; HRMS (ESI) calcd for
1H), 7.82 (td, J ¼ 4.0, 1.6 Hz, 1H), 7.40e7.28 (m, 5H), 7.20 (dd, J ¼ 7.3,
4.9 Hz, 1H), 3.93 (dd, J ¼ 17.3, 4.0 Hz, 1H), 3.52 (m, 1H), 3.41 (dd,
J ¼ 17.4, 11.4 Hz, 1H), 2.86 (dd, J ¼ 16.3, 12.5 Hz, 1H), 2.75 (dd,
[C9H12O3 þ H]þ 169.0865, found 169.0863.
2-Acetyl-5-isopropylcyclohexane-1,3-dione (16c). Prepared
from 5-isopropyl-3-oxocyclohex-1-en-1-yl acetate (15c, 600 mg,
3.06 mmol) according to the general procedure. Yield: 68%
J ¼ 16.3, 3.8 Hz, 1H), 2.58 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 193.3,
152.5, 150.8, 150.4, 147.8, 143.1, 138.6, 128.8, 127.1, 127.0, 121.8, 118.8,
115.5, 45.6, 42.2, 33.1, 13.6; HRMS (ESI) calcd for [C19H17N3O þ Na]þ
326.1264, found 326.1266.
(406 mg); yellowish oil. 1H NMR (400 MHz, CDCl3)
d 2.68 (ddd,
J ¼ 17.8, 4.0, 2.4 Hz, 1H), 2.60 (s, 3H), 2.58 (dt, J ¼ 16.5, 3.0 Hz, 1H),
2.44 (dd, J ¼ 17.8, 11.9 Hz, 1H), 2.20 (dd, J ¼ 16.3, 12.7 Hz, 1H), 1.87
(m, 1H), 1.60 (m, 1H), 0.95 (s, 3H), 0.94 (s, 3H); 13C NMR (100 MHz,
7-Bromo-3-methyl-1-(pyridin-2-yl)-6,7-dihydro-1H-indazol-
4(5H)-one (18a). General bromination procedure. A mixture of 3-
methyl-1-(pyridin-2-yl)-6,7-dihydro-1H-indazol-4(5H)-one (17a,
100 mg, 440 mmo1) and N-bromosuccinimide (102 mg, 0.572
mmo1) in CHCl3 (4 mL) was stirred at 60 ꢁC for 20 min. After cooling
the reaction mixture to room temperature, the solvent was
removed by evaporation. The residue was purified by silica column
chromatography (EtOAc:hexanes 1:2) to afford compound 18a as a
white solid (73 mg, 54%). Mp ¼ decompose at 292 ꢁC; 1H NMR
CDCl3)
d 202.9, 198.6, 195.7, 112.9, 42.7, 37.5, 37.1, 31.6, 28.7, 19.3;
HRMS (ESI) calcd for [C11H16O3 þ H]þ 197.1178, found 197.1185.
2-Acetyl-5-phenylcyclohexane-1,3-dione (16d). Prepared from
5-phenyl-3-oxocyclohex-1-en-1-yl acetate (16d, 500 mg,
2.17 mmol) according to the general procedure. Yield: 25%
(127 mg); white solid. Mp ¼ 100e104 ꢁC; 1H NMR (400 MHz, CDCl3)
d
7.38e7.35 (m, 2H), 7.34e7.21 (m, 3H), 3.37 (m, 1H), 2.91 (m, 2H),
2.80 (m, 1H), 2.69 (dd, J ¼ 16.5, 12.4 Hz, 1H), 2.65 (s, 3H); 13C NMR
(400 MHz, CDCl3)
d
8.50 (ddd, J ¼ 4.9, 1.9, 0.8 Hz, 1H), 7.99 (dt,
(100 MHz, CDCl3)
d
203.0, 197.8, 194.5, 141.7, 129.0, 127.3, 126.5,
J ¼ 8.3, 0.9 Hz, 1H), 7.86 (ddd, J ¼ 8.3, 7.4, 1.9 Hz, 1H), 7.27 (ddd,
J ¼ 7.4, 4.8, 1.0 Hz, 1H), 6.73 (t, J ¼ 3.2 Hz, 1H), 2.99 (m, 1H), 2.67 (m,
1H), 2.59 (m, 1H), 2.56 (m, 1H), 2.55 (s, 3H); 13C NMR (100 MHz,
113.0, 45.8, 40.5, 36.8, 28.8; HRMS (ESI) calcd for HRMS (ESI) calcd
for [C14H14O3 þ H]þ 231.1021, found 231.1028.
3-Methyl-1-(pyridin-2-yl)-6,7-dihydro-1H-indazol-4(5H)-
one (17a). General procedure for the formation of 4,5,6,7-
tetrahydroindazoles. A mixture of 2-acetylcyclohexane-1,3-dione
(16a, 350 mg, 2.27 mmo1) and 2-hydrazinopyridine (263 mg, 2.41
mmo1) in ethanol (13 mL) was stirred at 80 ꢁC for 2 h. The solvent
was then removed by evaporation. The residue was purified by
silica column chromatography (EtOAc:hexanes 3:1) to afford
CDCl3) d 193.2, 152.5, 150.3, 148.9, 147.9, 138.8, 122.1, 118.0, 115.3,
40.8, 35.3, 33.6, 13.6; HRMS (ESI) calcd for [C13H12BrN3O þ H]þ
306.0242, found 306.0244.
7-Bromo-3,6-dimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (18b). 6,7-Cis and trans isomers were prepared
from
3,6-dimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-indazol-
4(5H)-one (17b, 100 mg, 0.414 mmol) according to the general
procedure. Yield: cis-isomer; 37% (49 mg), trans-isomer; 27%
(36 mg); pale yellow solids. Cis-isomer: mp ¼ 100e105 ꢁC; 1H NMR
compound 17a as
a
pale yellow solid (405 mg, 79%).
Mp ¼ 189e195 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.44 (dd, J ¼ 4.9,
1.0 Hz, 1H), 7.93 (d, J ¼ 8.3 Hz, 1H), 7.83 (ddd, J ¼ 8.2, 7.4, 1.8 Hz, 1H),
7.22 (ddd, J ¼ 7.3, 4.9, 0.9 Hz,1H), 3.43 (t, J ¼ 6.2 Hz, 2H), 2.55 (s, 3H),
2.52 (t, J ¼ 6.5 Hz, 2H), 2.17 (quin, J ¼ 6.4 Hz, 2H); 13C NMR
(400 MHz, CDCl3)
d
8.50 (ddd, J ¼ 4.9, 1.9, 0.8 Hz, 1H), 7.99 (dt,
J ¼ 8.3, 0.9 Hz, 1H), 7.86 (ddd, J ¼ 8.3, 7.4, 1.9 Hz, 1H), 7.27 (ddd,
J ¼ 7.4, 4.8, 1.0 Hz, 1H), 6.55 (d, J ¼ 2.4 Hz, 1H), 2.63 (dd, J ¼ 16.5,
11.8 Hz,1H), 2.54 (s, 3H), 2.48 (m, 1H), 2.40 (ddd, J ¼ 16.5, 3.3,1.0 Hz,
(100 MHz, CDCl3)
d 194.7, 152.6, 151.6, 150.5, 147.7, 138.6, 121.7,
118.9, 115.6, 38.4, 25.2, 23.6, 13.6; HRMS (ESI) calcd for
1H), 1.30 (d, J ¼ 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 193.1,
[C13H13N3O þ Na]þ 250.0951, found 250.0960.
152.5, 150.2, 150.1, 147.9, 138.8, 122.1, 117.8, 115.4, 49.6, 43.2, 36.3,
19.7, 13.5; HRMS (ESI) calcd for [C14H14BrN3O þ H]þ 320.0398,
found 320.0390. Trans-isomer: mp ¼ 118e122 ꢁC; 1H NMR
3,6-Dimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-indazol-
4(5H)-one (17b). Prepared from 2-acetyl-5-methylcyclohexane-
1,3-dione (16b, 358 mg, 2.13 mmol) according to the general pro-
cedure. Yield: 65% (331 mg); pale yellow solid. Mp ¼ 111e113 ꢁC; 1H
(400 MHz, CDCl3)
d
8.50 (ddd, J ¼ 4.9, 1.9, 0.8 Hz, 1H), 7.99 (dt,
J ¼ 8.3, 0.9 Hz, 1H), 7.85 (ddd, J ¼ 8.3, 7.5, 1.9 Hz, 1H), 7.27 (ddd,
J ¼ 7.4, 4.9,1.0 Hz,1H), 6.50 (dd, J ¼ 2.3, 0.9 Hz,1H), 3.18 (dd, J ¼ 16.8,
4.6 Hz, 1H), 2.93 (m, 1H), 2.55 (s, 3H), 2.42 (ddd, J ¼ 16.9, 2.5, 1.1 Hz,
NMR (400 MHz, CDCl3)
d
8.46 (ddd, J ¼ 4.9, 1.8, 0.8 Hz, 1H), 7.93 (dt,
J ¼ 8.3, 0,9 Hz, 1H), 7.83 (ddd, J ¼ 8.3, 7.4, 1.9 Hz, 1H), 7.23 (ddd,
J ¼ 7.3, 4.8, 1.0 Hz, 1H), 3.67 (dd, J ¼ 17.9, 4.5 Hz, 1H), 2.93 (dd,
J ¼ 17.9, 10.2 Hz, 1H), 2.54 (s, 3H), 2.54 (dd, J ¼ 15.5, 3.1 Hz, 2H), 2.41
1H), 1.21 (d, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 192.8,
152.5, 150.2, 147.9, 147.4, 138.7, 122.1, 117.0, 115.3, 45.3, 42.1, 39.8,
18.9, 13.6; HRMS (ESI) calcd for [C14H14BrN3O þ H]þ 320.0398,
found 320.0393.
(m, 1H), 2.29 (dd, J ¼ 15.6, 11.7 Hz, 1H), 1.20 (d, J ¼ 6.5 Hz, 3H); 13
C
NMR (100 MHz, CDCl3) d 194.4,152.6,151.3,150.3,147.7,138.6,121.7,
118.7, 115.6, 46.8, 33.2, 31.4, 21.2, 13.6; HRMS (ESI) calcd for
7-Bromo-6-isopropyl-3-methyl-1-(pyridin-2-yl)-6,7-
dihydro-1H-indazol-4(5H)-one (18c). 6,7-Cis and trans isomers
were prepared from 6-isopropyl-3-methyl-1-(pyridin-2-yl)-6,7-
dihydro-1H-indazol-4(5H)-one (17c, 200 mg, 0.743 mmol) ac-
cording to the general procedure. Yield: cis-isomer; 26% (34 mg),
trans-isomer; 33% (42 mg); pale yellow solid. Cis-isomer:
[C14H15N3O þ Na]þ 264.1107, found 264.1115.
6-Isopropyl-3-methyl-1-(pyridin-2-yl)-6,7-dihydro-1H-inda-
zol-4(5H)-one
(17c).
Prepared
from
2-acetyl-5-
isopropylcyclohexane-1,3-dione (16c, 330 mg, 1.68 mmol) accord-
ing to the general procedure. Yield: 93% (421 mg); pale yellow solid.
Mp ¼ 93e96 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.46 (dd, J ¼ 4.8,
mp ¼ 127e131 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.51 (ddd, J ¼ 4.9,
0.7 Hz, 1H), 7.92 (d, J ¼ 8.3 Hz, 1H), 7.84 (t, J ¼ 7.8 Hz, 1H), 7.23 (dd,
J ¼ 7.3, 4.9 Hz, 1H), 3.62 (dd, J ¼ 17.9, 4.4 Hz, 2H), 2.98 (dd, J ¼ 17.9,
11.4 Hz, 1H), 2.57 (dd, J ¼ 13.2, 9.8 Hz, 1H), 2.54 (s, 3H), 2.33 (dd,
J ¼ 16.1, 12.9 Hz, 1H), 2.09 (m, 1H), 1.77 (m, 1H), 1.02 (d, J ¼ 2.4 Hz,
1.8, 0.7 Hz, 1H), 8.00 (dt, J ¼ 8.4, 0.8 Hz, 1H), 7.86 (ddd, J ¼ 8.3, 7.5,
1.9 Hz, 1H), 7.27 (ddd, J ¼ 7.4, 4.9, 1.0 Hz, 1H), 6.72 (d, J ¼ 1.9 Hz, 1H),
2.72 (ddd, J ¼ 17.2, 3.4, 1.0 Hz,1H), 2.56 (m, 1H), 2.54 (s, 3H),1.85 (m,
1H), 1.12 (d, J ¼ 6.2 Hz, 3H), 1.07 (d, J ¼ 6.4 Hz, 3H); 13C NMR
3H), 1.00 (d, J ¼ 2.4 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d
194.8,
(100 MHz, CDCl3) d 193.3, 152.6, 150.2, 150.1, 147.9, 138.8, 122.0,
152.6, 151.8, 150.2, 147.8, 138.6, 121.7, 118.9, 115.7, 42.7, 42.2, 31.9,
118.0, 115.4, 48.4, 46.6, 40.1, 30.5, 20.5, 20.0, 13.5; HRMS (ESI) calcd
28.8, 19.8, 19.6, 13.6; HRMS (ESI) calcd for [C16H19N3O þ Na]þ
for [C16H18BrN3O þ Na]þ 370.0525, found 370.0516. Trans-isomer:
14

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
mp ¼ 76e84 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.51 (ddd, J ¼ 4.9, 1.8,
7-Bromo-3,6,6-trimethyl-1-(pyridin-2-yl)-4,5,6,7-
0.7 Hz, 1H), 7.99 (d, J ¼ 8.3 Hz, 1H), 7.86 (ddd, J ¼ 8.3, 7.4, 1.9 Hz, 1H),
7.27 (ddd, J ¼ 7.4, 4.8, 1.0 Hz, 1H), 6.91 (dd, J ¼ 2.2, 1.2 Hz, 1H), 3.17
(dd, J ¼ 17.0, 4.6 Hz, 1H), 2.72 (ddd, J ¼ 17.0, 2.5, 1.3 Hz, 1H), 2.54 (s,
3H), 2.38 (m, 1H), 1.68 (m, 1H), 1.02 (d, J ¼ 6.0 Hz, 3H), 1.01 (d,
tetrahydro-1H-indazol-4-ol (22) [18]. To a solution of compound 3
(50 mg, 0.15 mmol) in MeOH/THF (3 mL/0.3 mL) was added
CeCl3$7H2O (84 mg, 0.22 mmol). After the mixture was cooled
to ꢀ40 ꢁC, NaBH4 (7 mg, 0.19 mmol) was added. The reaction
mixture was stirred at ꢀ10 ꢁC for 30 min and then quenched with
saturated NH4Cl (2 mL). After MeOH and THF were evaporated, the
aqueous layer was extracted with EtOAc (3 ꢃ 5 mL). The combined
organic layer was washed with water (2 ꢃ 10 mL) and brine
(2 ꢃ 10 mL), dried over anhydrous Na2SO4, and evaporated under
reduced pressure. The residue was purified by preparative TLC
(silica gel, EtOAc:hexanes 1:1) to afford compound 22 as a pale
yellow solid (10 mg, 20%). Mp ¼ 65e70 ꢁC; 1H NMR (400 MHz,
J ¼ 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 193.0, 152.6, 150.0,
148.0, 147.8, 138.8, 122.1, 117.9, 115.3, 52.1, 43.8, 39.1, 30.1, 21.6, 21.5,
13.5; HRMS (ESI) calcd for [C16H18BrN3O þ Na]þ 370.0525, found
370.0524.
7-Bromo-3-methyl-6-phenyl-1-(pyridin-2-yl)-6,7-dihydro-
1H-indazol-4(5H)-one (18d). Prepared from 3-methyl-6-phenyl-1-
(pyridin-2-yl)-6,7-dihydro-1H-indazol-4(5H)-one (17d, 200 mg,
0.743 mmol) according to the general procedure. Yield: 7% (8 mg);
yellow solid. 1H NMR (400 MHz, CDCl3)
d
8.47 (ddd, J ¼ 4.9, 1.9,
CDCl3)
d
8.43 (ddd, J ¼ 4.9, 1.8, 0.8 Hz, 1H), 7.87 (d, J ¼ 8.3 Hz, 1H),
0.8 Hz, 1H), 7.97 (dt, J ¼ 8.3, 0.9 Hz, 1H), 7.82 (ddd, J ¼ 8.3, 7.4, 1.9 Hz,
1H), 7.28e7.18 (m, 5H), 6.95 (dd, J ¼ 2.4, 1.0 Hz, 1H), 4.09 (m, 1H),
3.47 (dd, J ¼ 17.3, 5.4 Hz,1H), 2.95 (ddd, J ¼ 17.2, 2.4,1.1 Hz,1H), 2.56
7.77 (ddd, J ¼ 8.3, 7.4, 1.8 Hz, 1H), 7.16 (ddd, J ¼ 7.3, 4.9, 1.0 Hz, 1H),
6.18 (s, 1H) 4.92 (t, J ¼ 8.4 Hz, 1H), 2.42 (s, 3H), 1.98 (d, J ¼ 8.7 Hz,
2H), 1.30 (s, 3H), 1.16 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 153.1,
(s, 3H); 13C NMR (100 MHz, CDCl3)
d
192.4, 152.4, 150.2, 147.9, 147.3,
148.8, 147.6, 141.3, 138.4, 120.9, 119.1, 114.8, 63.4, 55.4, 42.4, 38.2,
30.6, 24.0, 13.0; HRMS (ESI) calcd for [C15H18BrN3O þ Na]þ
358.0525, found 358.0529.
140.0, 138.7, 128.9, 127.6, 127.4, 122.0, 118.1, 115.1, 49.6, 44.2, 40.4,
13.6; HRMS (ESI) calcd for [C19H16BrN3O þ H]þ 382.0555, found
382.0569.
3,7,7-Trimethyl-1-(pyridin-2-yl)-4,6,7,8-tetrahydropyrazolo
[4,3-b]azepin-5(1H)-one (19) and 3,7,7-Trimethyl-1-(pyridin-2-
7-Bromo-3,6,6-trimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one Oxime (23) [18]. A mixture of compound 3
(150 mg, 0.449 mmol), hydroxylamine hydrochloride (156 mg,
2.24 mmol) and sodium acetate (221 mg, 2.69 mmol) in 1,4-dioxane
(25 mL) was refluxed for 4 h. Additional hydroxylamine hydro-
chloride (75 mg, 1.08 mmol) and sodium acetate (111 mg,
1.35 mmol) were added and then the mixture was refluxed for 2 h.
After cooling, the mixture was poured into ice water (80 mL) and
extracted with CH2Cl2 (2 ꢃ 50 mL). The combined organic layer was
washed with brine (2 ꢃ 50 mL), dried over anhydrous Na2SO4, and
evaporated under reduced pressure. The residue was purified by
preparative TLC (silica gel, EtOAc:hexanes 1:4) to afford compound
23 as a white solid (76 mg, 48%). Mp ¼ 177e181 ꢁC; 1H NMR
yl)-5,6,7,8-tetrahydropyrazolo[4,3-c]azepin-4(1H)-one (20).
A
mixture of compound 2 (400 mg, 1.57 mmol) and polyphosphoric
acid (6.7 g) was vigorously stirred at 100 ꢁC for 1.5 h. Sodium azide
(102 mg, 1.57 mmol) was then added portionwise with stirring to
the homogenized solution at 100 ꢁC for 2 h. The solution was
vigorously stirred at 100 ꢁC for 1 h. After cooling, the solution was
poured into crushed ice (67 g) and the mixture was diluted with
NH4OH solution (H2O: conc. NH4OH ¼ 1:1, 12 mL). The solution
having some precipitation was left at room temperature for 19 h.
The slurry was filtered, washed with H2O and dried. The crude
compound was purified by silica column chromatography (EtOA-
c:hexanes 3:1) to afford compound 19 (73 mg, 17%) and 20 (103 mg,
24%) as white solids. Compound 19: mp ¼ 217e219 ꢁC; 1H NMR
(400 MHz, CDCl3)
d
8.69 (br s, 1H), 8.47 (dd, J ¼ 4.9, 1.1 Hz, 1H), 7.95
(d, J ¼ 8.4 Hz, 1H), 7.81 (ddd, J ¼ 8.4, 7.3, 1.8 Hz, 1H), 7.20 (ddd,
J ¼ 7.3, 4.9, 0.9 Hz, 1H), 6.36 (d, J ¼ 1.0 Hz, 1H), 3.11 (d, J ¼ 17.1 Hz,
(400 MHz, CDCl3)
d
8.40 (ddd, J ¼ 4.9, 1.8, 0.9 Hz, 1H), 7.84e7.76 (m,
1H), 2.56 (d, J ¼ 17.1 Hz,1H), 2.50 (s, 3H), 1.41 (s, 3H), 1.18 (s, 3H); 13
C
1H), 7.42 (br s, 1H), 7.17 (ddd, J ¼ 6.9, 4.9, 1.5 Hz, 1H), 3.29 (s, 2H),
NMR (100 MHz, CDCl3) d 152.9, 151.1, 147.7, 147.4, 143.6, 138.5, 121.2,
2.50 (s, 2H), 2.29 (s, 3H), 1.19 (s, 6H); 13C NMR (100 MHz, CDCl3)
114.7, 113.3, 54.1, 37.0, 32.7, 29.7, 25.6, 15.0; HRMS (ESI) calcd for
d
172.8, 153.6,147.3, 141.0, 138.3, 132.4, 121.0, 120.0, 115.9, 48.5, 40.5,
[C15H17BrN4O þ H]þ 349.0664, found 349.0658.
34.2, 29.3, 11.0; HRMS (ESI) calcd for [C15H18N4O þ H]þ 271.1559,
6,6-Dimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-indazol-
4(5H)-one (24). To a solution of 5,5-dimethylcyclohexane-1,3-
dione (700 mg, 4.99 mmol) in dimethylformamide dimethyl
found 271.1562. Compound 20: mp ¼ 149e154 ꢁC; 1H NMR
(400 MHz, CDCl3)
d
8.45 (dt, J ¼ 4.8, 1.3 Hz, 1H), 7.83 (m, 2H), 7.24
(dd, J ¼ 8.8, 4.5 Hz, 1H), 6.06 (br s, 1H), 3.27 (s, 2H), 3.00 (d,
acetal (DMFDMA, 796
mL, 5.99 mmol) were added 2-hydrazino
J ¼ 5.8 Hz, 2H), 2.54 (s, 3H), 1.10 (s, 6H); 13C NMR (100 MHz, CDCl3)
pyridine (561 mg, 5.14 mmol), H2O (12 mL) and AcOH (743
m
L,
d
168.8, 153.0, 151.9, 147.5, 144.4, 138.5, 122.0, 117.6, 115.2, 53.0, 38.8,
13.0 mmol). The mixture, contained in a sealed vial, was submitted
to microwave irradiation for 5 min at 200 ꢁC. After cooling the re-
action mixture, the precipitate was filtered and washed with H2O.
The crude compound was purified by silica column chromatog-
raphy (EtOAc:hexanes 1:2) to afford compound 24 (648 mg, 54%) as
37.2, 26.9, 13.7; HRMS (ESI) calcd for [C15H18N4O þ H]þ 271.1559,
found 271.1569.
8-Bromo-3,7,7-trimethyl-1-(pyridin-2-yl)-5,6,7,8-
tetrahydropyrazolo[4,3-c]azepin-4(1H)-one (21). To a solution of
compound 20 (70 mg, 0.26 mmol) in CHCl3 (1.8 mL) was added N-
bromosuccinimide (60 mg, 0.34 mmol) and the mixture was stirred
at 60 ꢁC for 6 h. After cooling the reaction mixture, CH2Cl2 (10 mL)
was added. The solution was washed with water (10 mL) and brine
(10 mL), dried over anhydrous Na2SO4, and evaporated under
reduced pressure. The residue was purified by preparative TLC
(silica gel, EtOAc:hexanes 4:1) to afford compound 21 as a pale
yellow solid (42 mg, 35%). Mp ¼ 193e195 ꢁC; 1H NMR (400 MHz,
a yellow solid. Mp ¼ 111e116 ꢁC; 1H NMR (400 MHz, CDCl3)
d 8.48
(dd, J ¼ 4.9, 1.7 Hz, 1H), 8.07 (s, 1H), 7.99 (d, J ¼ 8.2 Hz, 1H), 7.86 (td,
J ¼ 4.1, 1.8 Hz, 1H), 7.27 (ddd, J ¼ 7.3, 4.9, 0.8 Hz, 1H), 3.35 (s, 2H),
2.43 (s, 2H), 1.16 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 193.4, 152.7,
150.0, 147.7, 138.7, 138.6, 122.1, 120.5, 115.7, 52.1, 38.8, 35.5, 28.6;
HRMS (ESI) calcd for [C14H15N3O þ Na]þ 264.1107, found 264.1120.
7-Bromo-6,6-dimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (25). Prepared from compound 24 (100 mg,
0.414 mmol) according to the general procedure for bromination.
Yield: 29% (39 mg); white solid. Mp ¼ 158e161 ꢁC; 1H NMR
CDCl3)
d
8.49 (ddd, J ¼ 4.9, 1.7, 0.9 Hz, 1H), 7.90e7.82 (m, 2H), 7.30
(ddd, J ¼ 6.8, 5.0, 1.7 Hz, 1H), 6.76 (d, J ¼ 1.48 Hz, 1H), 6.01 (br d,
J ¼ 5.24 Hz,1H), 3.95 (dd, J ¼ 14.7, 2.8 Hz,1H), 2.86 (ddd, J ¼ 14.7, 7.3,
1.5 Hz,1H), 2.58 (s, 3H), 1.36 (s, 3H), 1.30 (s, 3H); 13C NMR (100 MHz,
(400 MHz, CDCl3)
d
8.53 (ddd, J ¼ 4.8, 1.7, 0.7 Hz, 1H), 8.04 (s, 1H),
8.03 (d, J ¼ 9.2 Hz, 1H), 7.89 (ddd, J ¼ 8.2, 7.5, 1.8 Hz, 1H), 7.31 (ddd,
J ¼ 7.4, 4.9, 0.9 Hz, 1H), 6.40 (s, 1H), 2.89 (dd, J ¼ 16.9, 0.7 Hz, 2H),
2.31 (dd, J ¼ 16.9, 1.3 Hz, 2H), 1.42 (s, 3H), 1.26 (s, 3H); 13C NMR
CDCl3)
d 166.0, 153.7, 153.3, 147.6, 142.4, 138.9, 122.4, 118.3, 113.8,
56.6, 48.6, 36.4, 29.4, 22.9, 15.1; HRMS (ESI) calcd for
[C15H17BrN4O þ H]þ 349.0664, found 349.0646.
(100 MHz, CDCl3) d 192.1, 152.6, 149.0, 147.9, 138.9, 138.4, 122.4,
15

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
119.4, 115.5, 52.3, 48.4, 39.7, 29.6, 25.4; HRMS (ESI) calcd for
[C14H14BrN3O þ Na]þ 342.0212, found 342.0185.
48.9 mmol) was added dropwise 2-chloropyrazine (779 mL,
8.73 mmol) at 65 ꢁC. The reaction mixture was stirred at 65 ꢁC for
12 h. After cooling, the reaction mixture was extracted with 10%
isopropyl alcohol/CH2Cl2 (5 ꢃ 10 mL). The combined organic layer
was dried over anhydrous Na2SO4 and evaporated to give com-
pound 31 as a brown solid (718 mg, 75%). 1H NMR (400 MHz,
5,5-Dimethyl-2-(3-methylbutanoyl)cyclohexane-1,3-dione
(16e). Prepared from compound 15e (1.10 g, 4.90 mmol) according
to the general procedure for the formation of 2-acylcyclohexane-
1,3-diones. Yield: 51% (558 mg); yellow oil. 1H NMR (400 MHz,
CDCl3)
d
2.92 (d, J ¼ 6.9 Hz, 2H), 2.54 (s, 2H), 2.36 (s, 2H), 2.14 (m,
DMSO‑d6)
d
8.08 (d, J ¼ 1.4 Hz, 1H), 7.91 (dd, J ¼ 2.7, 1.5 Hz, 1H), 7.89
1H), 1.08(s, 6H), 0.97 (d, J ¼ 6.7 Hz, 6H); 13C NMR (100 MHz, CDCl3)
(br s, 1H), 7.68 (d, J ¼ 2.8 Hz,1H), 4.20 (br s, 2H); 13C NMR (100 MHz,
d
205.0, 198.2, 195.1, 112.2, 52.7, 48.6, 47.1, 30.6, 28.2, 25.6, 22.7;
DMSO‑d6) d 157.4, 141.3, 131.8, 131.1.
HRMS (ESI) calcd for [C13H20O3 þ H]þ 225.1491, found 225.1487.
3-Isobutyl-6,6-dimethyl-1-(pyridin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (26). Prepared from compound 16e (450 mg,
2.01 mmol) according to the general procedure for the formation of
4,5,6,7-tetrahydroindazoles. Yield: 95% (567 mg); pale orange solid.
3,6,6-Trimethyl-1-(pyrazin-2-yl)-6,7-dihydro-1H-indazol-
4(5H)-one (32). Prepared from compound 31 (185 mg, 1.68 mmol)
and 2-acetyl-5,5-dimethy1-cyclohexane-1,3-dione (1, 300 mg, 1.65
mmo1) according to the general procedure for the formation of
4,5,6,7-tetrahydroindazoles. Yield: 62% (261 mg); yellow solid.
Mp ¼ 89e93 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.45 (ddd, J ¼ 4.9, 1.8,
Mp ¼ 179e186 ꢁC; H NMR (400 MHz, CDCl3)
d
9.32 (d, J ¼ 1.4 Hz,
1
0.8 Hz,1H), 7.96 (dt, J ¼ 8.3, 0.8 Hz,1H), 7.83 (ddd, J ¼ 8.3, 7.4, 1.9 Hz,
1H), 7.23 (ddd, J ¼ 7.3, 4.9,1.0 Hz,1H), 3.30 (s, 2H), 2.83 (d, J ¼ 7.2 Hz,
2H), 2.39 (s, 2H), 2.15 (m, 1H), 1.14 (s, 6H), 0.97 (d, J ¼ 6.7 Hz, 6H);
1H), 8.51 (d, J ¼ 2.6 Hz, 1H), 8.40 (dd, J ¼ 2.6, 1.4 Hz, 1H), 3.27 (s, 2H),
2.56 (s, 3H), 2.41 (s, 2H), 1.16 (s, 6H); 13C NMR (100 MHz, CDCl3)
d
193.8, 151.4, 151.2, 148.9, 141.8, 141.3, 138.2, 118.5, 52.4, 38.8, 35.4,
13C NMR (100 MHz, CDCl3)
d
193.8, 153.6, 152.8, 150.6, 147.7, 138.6,
28.6, 13.5; HRMS (ESI) calcd for [C14H16N4O þ H]þ 257.1402, found
121.6, 117.8, 115.9, 52.7, 39.0, 36.4, 35.3, 28.6, 28.0, 22.5; HRMS (ESI)
257.1411.
calcd for [C18H23N3O þ Na]þ 320.1733, found 320.1732.
7-Bromo-3,6,6-trimethyl-1-(pyrazin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (33). Prepared from compound 32 (100 mg,
0.390 mmol) according to the general procedure for bromination.
Yield: 59% (77 mg); white solid. Mp ¼ 140e150 ꢁC; 1H NMR
7-Bromo-3-isobutyl-6,6-dimethyl-1-(pyridin-2-yl)-6,7-
dihydro-1H-indazol-4(5H)-one (27). Prepared from compound 26
(150 mg, 0.504 mmol) according to the general procedure for
bromination. Yield: 58% (110 mg); yellowish oil. 1H NMR (400 MHz,
(400 MHz, CDCl3)
d
9.36 (d, J ¼ 1.3 Hz, 1H), 8.56 (d, J ¼ 2.5 Hz, 1H),
CDCl3)
d
8.51 (ddd, J ¼ 4.8, 1.9, 0.8 Hz, 1H), 8.01 (dt, J ¼ 8.3, 0.9 Hz,
8.46 (dd, J ¼ 2.5, 1.4 Hz, 1H), 6.18 (d, J ¼ 1.2 Hz, 1H), 2.88 (d,
J ¼ 16.8 Hz,1H), 2.56 (s, 3H), 2.29 (dd, J ¼ 16.9,1.3 Hz,1H),1.40 (s, 3H),
1H), 7.85 (ddd, J ¼ 8.3, 7.4, 1.9 Hz, 1H), 7.27 (ddd, J ¼ 7.4, 4.9, 1.0 Hz,
1H), 6.41 (d, J ¼ 1.4 Hz, 1H), 2.87 (dd, J ¼ 16.8, 0.9 Hz, 2H), 2.83 (dd,
J ¼ 7.2, 2.2 Hz, 2H), 2.26 (dd, J ¼ 16.9, 1.4 Hz, 2H), 2.15 (m, 1H), 1.39
(s, 3H), 1.26 (s, 3H), 0.98 (d, J ¼ 1.4 Hz, 3H), 0.97 (d, J ¼ 1.4 Hz, 3H);
1.26 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 192.5, 151.1, 150.3, 148.7,
142.2, 141.4, 137.9, 117.2, 52.1, 48.7, 39.7, 29.6, 25.4, 13.5; HRMS (ESI)
calcd for [C14H15BrN4O þ H]þ 335.0507, found 335.0498.
13C NMR (100 MHz, CDCl3)
d
192.6, 153.5, 152.7, 149.5, 147.8, 138.7,
4-Hydrazinylpyridine Hydrochloride (34). 4-Chloropyridine
hydrochloride (3g, 20 mmol) was dissolved in 2 N NaOH (12 mL).
The neutralized 4-chloropyridine was extracted with Et2O
(4 ꢃ 12 mL) and the combined organic layer was dried over
anhydrous Na2SO4 and evaporated. The residue was dissolved in
EtOH (7 mL) with 80% hydrazine hydrate (4.26 g, 68 mmol). The
mixture was stirred for 16 h at 100 ꢁC. After cooling, the EtOH was
evaporated. To the residue was added MeOH (10 mL) and the
resulting slurry was filtered to afford compound 34 as a light brown
solid (1.08 g, 37%). Mp ¼ 205e217 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
122.0, 116.5,115.6, 53.0, 48.9, 39.6, 36.3, 29.6, 27.9, 25.4, 22.5; HRMS
(ESI) calcd for [C18H22BrN3O þ H]þ 376.1024, found 376.1010.
2-Hydrazinylpyrimidine (28). To
a
solution of 2-
chloropyrimidine (1.0 g, 8.7 mmol) in pyridine (22 mL) was
added dropwise anhydrous hydrazine (3.56 mL, 113 mmol) at 0 ꢁC.
After adding additional pyridine (11 mL), the mixture was stirred at
room temperature for 2.5 h. Pyridine and hydrazine were removed
under reduced pressure and then H2O (6 mL) was added to the
residue. The resulting white precipitate was filtered and dried to
give compound 28 as a white solid (392 mg, 41%). Mp ¼ 93e107 ꢁC;
d
8.83 (br s, 1H), 8.03 (d, J ¼ 5.2 Hz, 2H), 6.79 (d, J ¼ 5.6 Hz, 2H), 4.59
1H NMR (400 MHz, DMSO‑d6)
d
8.28 (d, J ¼ 4.6 Hz, 2H), 8.10 (br s,
(br s, 2H); 13C NMR (100 MHz, DMSO‑d6)
d
158.1, 143.4, 105.5.
1H), 6.57 (t, J ¼ 4.7 Hz, 1H), 4.12 (br s, 2H); 13C NMR (100 MHz,
3,6,6-Trimethyl-1-(pyridin-4-yl)-6,7-dihydro-1H-indazol-
4(5H)-one (35). A mixture of 2-acetyl-5,5-dimethy1-cyclohexane-
1,3-dione (1, 350 mg, 1.92 mmo1), 4-hydrazino-pyridine hydro-
DMSO‑d6)
d 164.3, 157.8, 110.4.
3,6,6-Trimethyl-1-(pyrimidin-2-yl)-6,7-dihydro-1H-indazol-
4(5H)-one (29). Prepared from compound 28 (123 mg, 1.12 mmol)
and 2-acetyl-5,5-dimethy1-cyclohexane-1,3-dione (1, 200 mg, 1.10
mmo1) according to the general procedure for the formation of
4,5,6,7-tetrahydroindazoles. Yield: 95% (268 mg); pale yellow solid.
chloride (285 mg, 1.96 mmo1) and pyridine (233 mL, 2.88 mmol) in
ethanol (15 m1) was stirred at 80 ꢁC for 2 h. The solvent was then
removed by evaporation. The residue was purified by silica column
chromatography (MeOH:CH2Cl2 1:9) to afford compound 35 as a
pale yellow solid (478 mg, 98%). Mp ¼ 105e111 ꢁC; 1H NMR
Mp ¼ 130e139 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.81 (d, J ¼ 4.8 Hz,
2H), 7.27 (t, J ¼ 4.8 Hz,1H), 3.30 (s, 2H), 2.60 (s, 3H), 2.42 (s, 2H),1.16
(400 MHz, CDCl3)
d
8.74 (dd, J ¼ 4.8, 1.5 Hz, 2H), 7.51 (dd, J ¼ 4.7,
(s, 6H); 13C NMR (100 MHz, CDCl3)
d
194.0, 158.7, 156.9, 152.2, 151.5,
1.6 Hz, 2H), 2.92 (s, 2H), 2.55 (s, 3H), 2.43 (s, 2H), 1.14 (s, 6H); 13C
118.9, 118.8, 52.3, 39.4, 35.4, 28.6, 13.7; HRMS (ESI) calcd for
NMR (100 MHz, CDCl3) d 193.2, 151.2, 151.1, 149.3, 145.4, 118.2, 116.5,
[C14H16N4O þ H]þ 257.1402, found 257.1397.
52.2, 37.9, 35.9, 28.4, 13.4; HRMS (ESI) calcd for [C15H17N3O þ H]þ
7-Bromo-3,6,6-trimethyl-1-(pyrimidin-2-yl)-6,7-dihydro-
1H-indazol-4(5H)-one (30). Prepared from compound 29 (100 mg,
0.390 mmol) according to the general procedure for bromination.
Yield: 50% (65 mg); white solid. Mp ¼ 147e158 ꢁC; 1H NMR
256.1450, found 256.1454.
7-Bromo-3,6,6-trimethyl-1-(pyridin-4-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (36). Prepared from compound 35 (222 mg,
0.870 mmol) according to the general procedure for bromination.
Yield: 93% (270 mg); white solid. Mp ¼ 168e173 ꢁC; 1H NMR
(400 MHz, CDCl3)
d
8.86 (d, J ¼ 4.8 Hz, 2H), 7.32 (t, J ¼ 4.8 Hz, 1H),
6.26 (d, J ¼ 1.3 Hz, 1H), 2.89 (dd, J ¼ 16.9, 0.8 Hz, 1H), 2.61 (s, 3H),
(400 MHz, CDCl3)
d
8.80 (dd, J ¼ 4.7, 1.5 Hz, 1H), 7.77 (dd, J ¼ 4.6,
2.30 (dd, J ¼ 16.9, 1.4 Hz, 1H), 1.41 (s, 3H), 1.26 (s, 3H); 13C NMR
1.6 Hz,1H), 4.97 (d, J ¼ 1.1 Hz, 1H), 2.89 (dd, J ¼ 17.1, 0.8 Hz,1H), 2.55
(100 MHz, CDCl3) d 192.8, 158.8, 156.6, 151.4, 151.1, 119.2, 117.5, 52.8,
(s, 3H), 2.31 (dd, J ¼ 17.1, 1.2 Hz, 1H), 1.39 (s, 3H), 1.15 (s, 3H); 13C
48.6, 39.7, 29.7, 25.4, 13.7; HRMS (ESI) calcd for [C14H15BrN4O þ H]þ
NMR (100 MHz, CDCl3) d 192.0, 151.5, 151.2, 149.0, 145.2, 117.1, 116.7,
335.0507, found 335.0522.
50.8, 48.3, 40.4, 29.5, 25.2, 13.4; HRMS (ESI) calcd for
2-Hydrazinylpyrazine (31). To 35% hydrazine hydrate (4.48 g,
[C15H16BrN3O þ H]þ 334.0555, found 334.0562.
16

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
2-Hydrazinyl-3-methylpyridine (37). A solution of 2-bromo-3-
methyl pyridine (1.54 g, 8.98 mmol) and 80% hydrazine hydrate
(74.5 mmol, 4.72 g) in EtOH (3 mL) was stirred at 105 ꢁC for 40 h.
After evaporating EtOH, the resulting slurry was filtered and
washed with diisopropyl ether (9 mL). The solid was collected,
dissolved in CH2Cl2, dried over anhydrous Na2SO4 and evaporated
calcd for [C16H19N3O þ H]þ 270.1606, found 270.1607.
7-Bromo-3,6,6-trimethyl-1-(5-methylpyridin-2-yl)-6,7-
dihydro-1H-indazol-4(5H)-one (43). Prepared from compound 42
(50 mg, 0.18 mmol) according to the general procedure for
bromination. Yield: 81% (105 mg); yellowish oil. 1H NMR (400 MHz,
CDCl3)
d
8.32 (dd, J ¼ 1.6, 0.7 Hz, 1H), 7.87 (d, J ¼ 8.4 Hz, 1H), 7.65
to give compound 37 as
a
white solid (394 mg, 36%).
(dd, J ¼ 8.4, 2.3 Hz, 1H), 6.36 (d, J ¼ 1.3 Hz, 1H), 2.86 (dd, J ¼ 16.8,
0.8 Hz, 1H), 2.54 (s, 3H), 2.39 (s, 3H), 2.26 (dd, J ¼ 16.8, 1.4 Hz, 1H),
Mp ¼ 122e130 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.08 (dd, J ¼ 5.0,
0.9 Hz, 1H), 7.25 (m, 1H), 6.65 (dd, J ¼ 7.1, 5.1 Hz, 1H), 5.51 (br s, 1H),
1.39 (s, 3H), 1.26 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 192.9, 150.5,
4.03 (br s, 2H), 2.08 (s, 3H),; 13C NMR (100 MHz, CDCl3)
144.9, 137.1, 116.9, 114.4, 16.2.
d
158.4,
149.8, 149.2, 147.9, 139.3, 131.8, 116.4, 114.9, 52.9, 48.7, 39.7, 29.7,
25.4, 18.0, 13.5; HRMS (ESI) calcd for [C16H18BrN3O þ H]þ 348.0711,
found 348.0707.
3,6,6-Trimethyl-1-(3-methylpyridin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (38). Prepared from compound 37 (213 mg,
1.73 mmol) and 2-acetyl-5,5-dimethy1-cyclohexane-1,3-dione (1,
300 mg, 1.65 mmo1) according to the general procedure for the
formation of 4,5,6,7-tetrahydroindazoles. Yield: 30% (133 mg); pale
2-Hydrazinyl-3-(trifluoromethyl)pyridine (44). A solution of
2-bromo-3-(trifluoromethyl)pyridine (500 mg, 2.21 mmol) and
80% hydrazine hydrate (22.1 mmol, 1.38 g) in EtOH (0.8 mL) was
stirred at 105 ꢁC for 29 h. After EtOH was removed by evaporation,
H2O (2.5 mL) was added to the residue. The resulting slurry was
filtered, washed with H2O (0.4 mL) and diisopropyl ether (0.5 mL).
The filtered solid was dried under vacuum to afford compound 44
as a pale yellow solid (419 mg, 53%). 1H NMR (400 MHz, DMSO‑d6)
yellow solid. Mp ¼ 100e103 ꢁC; 1H NMR (400 MHz, CDCl3)
d 8.40 (d,
J ¼ 4.7 Hz, 1H), 7.73 (d, J ¼ 7.7 Hz, 1H), 7.31 (dd, J ¼ 7.6, 4.7 Hz, 1H),
2.71 (s, 2H), 2.54 (s, 3H), 2.39 (s, 2H), 2.33 (s, 3H), 1.11 (s, 6H); 13C
NMR (100 MHz, CDCl3)
d 193.6, 151.0, 149.9, 149.4, 146.3, 140.7,
130.1, 124.1, 116.2, 52.6, 36.4, 35.7, 28.4, 17.9, 13.4; HRMS (ESI) calcd
d
11.90 (br s,1H), 8.32 (dd, J ¼ 4.5,1.6 Hz,1H), 8.10 (dd, J ¼ 7.9,1.6 Hz,
for [C16H19N3O þ H]þ 270.1606, found 270.1601.
1H), 6.94 (dd, J ¼ 7.9, 4.6 Hz,1H), 5.53 (br s, 2H); 13C NMR (100 MHz,
7-Bromo-3,6,6-trimethyl-1-(3-methylpyridin-2-yl)-6,7-
DMSO‑d6) d 152.3, 148.4, 148.1, 129.5, 113.7, 106.1; HRMS (ESI) calcd
dihydro-1H-indazol-4(5H)-one
(39)
and
5-Bromo-3,6,6-
for [C6H6F3N3 þ H]þ 178.0592, found 178.0599.
trimethyl-1-(3-methylpyridin-2-yl)-6,7-dihydro-1H-indazol-
4(5H)-one (40). Two compounds (39 and 40) were obtained from
compound 38 (50 mg, 0.18 mmol) according to the general pro-
cedure for bromination. Yield for 39: 39% (25 mg), yield for 40: 17%
(11 mg); white solids. Compound 39: Mp ¼ 150e160 ꢁC; 1H NMR
3,6,6-Trimethyl-1-(3-(trifluoromethyl)pyridin-2-yl)-6,7-
dihydro-1H-indazol-4(5H)-one (45). Prepared from compound 44
(380 mg, 1.78 mmol) and 2-acetyl-5,5-dimethy1-cyclohexane-1,3-
dione (1, 324 mg, 1.78 mmo1) according to the general procedure
for the formation of 4,5,6,7-tetrahydroindazoles. Yield: 22%
(128 mg); white solid. Mp ¼ 82e90 ꢁC; 1H NMR (400 MHz, CDCl3)
(400 MHz, CDCl3)
d
8.41 (dd, J ¼ 4.6, 1.0 Hz, 1H), 7.76 (dd, J ¼ 7.6,
0.7 Hz, 1H), 7.36 (dd, J ¼ 7.6, 4.8 Hz, 1H), 5.36 (d, J ¼ 0.8 Hz, 1H), 2.80
d
8.76 (dd, J ¼ 4.8,1.4 Hz,1H), 8.23 (dd, J ¼ 8.0,1.6 Hz,1H), 7.59 (ddd,
(d, J ¼ 16.6 Hz,1H), 2.54 (s, 3H), 2.35 (s, 3H), 2.27 (dd, J ¼ 16.7,1.0 Hz,
J ¼ 7.9, 4.8, 0.7 Hz, 1H), 2.68 (s, 2H), 2.54 (s, 3H), 2.40 (s, 2H), 1.10 (s,
1H), 1.32 (s, 3H), 1.31 (s, 3H); 13C NMR (100 MHz, CDCl3)
d
192.4,
3H); 13C NMR (100 MHz, CDCl3)
d
193.5, 151.8, 151.7, 150.1, 137.5,
150.0, 149.6, 149.0, 146.3, 141.1, 130.6, 124.5, 114.9, 49.8, 48.6, 39.6,
29.5, 25.5, 18.1, 13.3; HRMS (ESI) calcd for [C16H18BrN3O þ H]þ
348.0711, found 348.0703. Compound 40: Mp ¼ 135e149 ꢁC; 1H
124.1, 123.5, 120.8, 116.8, 116.1, 52.6, 36.1, 35.8, 28.3, 13.3; HRMS
(ESI) calcd for [C16H16F3N3O þ H]þ 324.1324, found 324.1318.
7-Bromo-3,6,6-trimethyl-1-(3-(trifluoromethyl)pyridin-2-
yl)-6,7-dihydro-1H-indazol-4(5H)-one (46) and 5-Bromo-3,6,6-
trimethyl-1-(3-(trifluoromethyl)pyridin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (47). Two compounds (46 and 47) were ob-
tained from compound 45 (50 mg, 0.15 mmol) according to the
general procedure for bromination. Yield for 46: 11% (6.9 mg), Yield
for 47: 10% (6.4 mg); pale yellow solids. Compound 46:
NMR (400 MHz, CDCl3)
d
8.41 (dd, J ¼ 4.7, 1.0 Hz, 1H), 7.73 (dd,
J ¼ 7.6, 0.8 Hz, 1H), 7.33 (dd, J ¼ 7.6, 4.8 Hz, 1H), 4.14 (s, 1H), 3.02 (d,
J ¼ 17.3 Hz, 1H), 2.60 (d, J ¼ 17.5 Hz, 1H), 2.56 (s, 3H), 2.33 (s, 3H),
1.28 (s, 3H), 1.21 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 186.8, 150.7,
149.7, 149.4, 146.4, 140.9, 130.0, 124.2, 113.2, 62.5, 38.9, 33.0, 27.9,
24.7, 18.0, 13.4; HRMS (ESI) calcd for [C16H18BrN3O þ H]þ 348.0711,
found 348.0709.
mp ¼ 140e152 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.77 (dd, J ¼ 4.8,
2-Hydrazinyl-5-methylpyridine (41). A solution of 2-bromo-5-
methyl pyridine (1.00 g, 5.81 mmol) and 80% hydrazine hydrate
(3.64 g, 58.1 mmol) in EtOH (2 mL) was stirred at 105 ꢁC for 5 days.
After evaporating EtOH, the residue was extracted with 10% iso-
propyl alcohol/CH2Cl2 (4 ꢃ 5 mL). The combined organic layer was
washed with H2O (2.5 mL) and the water layer was extracted with
CH2Cl2 (5 mL). The combined organic layer was dried over anhy-
drous Na2SO4 and evaporated to give compound 41 as a red oil
1.5 Hz,1H), 8.26 (dd, J ¼ 7.9,1.6 Hz,1H), 7.60 (ddd, J ¼ 7.9, 4.8, 0.5 Hz,
1H), 5.39 (d, J ¼ 1.1 Hz, 1H), 2.82 (dd, J ¼ 16.7, 0.7 Hz, 1H), 2.54 (s,
3H), 2.29 (dd, J ¼ 16.7, 1.2 Hz, 1H), 1.32 (s, 3H), 1.30 (s, 3H); 13C NMR
(100 MHz, CDCl3)
d 192.4, 151.6, 150.2, 149.8, 138.1, 124.1, 123.4,
122.6, 120.7, 115.9, 49.1, 48.6, 39.7, 29.4, 25.5, 13.3; HRMS (ESI) calcd
for [C16H15BrF3N3O þ H]þ 402.0429, found 402.0422. Compound
47: mp ¼ 148e161 ꢁC; 1H NMR (400 MHz, CDCl3)
8.78 (dd, J ¼ 4.8,
d
1.5 Hz, 1H), 8.24 (dd, J ¼ 8.0, 1.7 Hz, 1H), 7.60 (ddd, J ¼ 7.9, 4.8,
0.7 Hz, 1H), 4.14 (s, 1H), 3.03 (d, J ¼ 17.2 Hz, 1H), 2.55 (s, 3H), 2.49 (d,
J ¼ 17.2 Hz, 1H), 1.28 (s, 3H), 1.19 (s, 3H); 13C NMR (100 MHz, CDCl3)
(563 mg, 79%). 1H NMR (400 MHz, CDCl3)
d
7.96 (dd, J ¼ 1.3, 0.7 Hz,
1H), 7.32 (dd, J ¼ 8.3, 2.1 Hz,1H), 6.63 (d, J ¼ 8.4 Hz,1H), 5.68 (s, 1H),
3.73 (s, 2H), 2.21 (s, 3H); 13C NMR (100 MHz, CDCl3)
139.2, 123.4, 107.3, 17.4.
d
159.0, 145.8,
d 186.7, 151.9, 151.4, 150.1, 137.6, 124.3, 123.5, 120.8, 113.9, 62.1, 39.0,
35.5, 32.8, 27.9, 24.4, 13.3; HRMS (ESI) calcd for
3,6,6-Trimethyl-1-(5-methylpyridin-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (42). Prepared from compound 41 (355 mg,
2.88 mmol) and 2-acetyl-5,5-dimethy1-cyclohexane-1,3-dione (1,
500 mg, 2.74 mmo1) according to the general procedure for the
formation of 4,5,6,7-tetrahydroindazoles. Yield: 83% (616 mg);
[C16H15BrF3N3O þ H]þ 402.0429, found 402.0448.
1-(5-Chloropyridin-2-yl)-3,6,6-trimethyl-6,7-dihydro-1H-
indazol-4(5H)-one
(48).
Prepared
from
5-chloro-2-
hydrazinopyridine (300 mg, 2.09 mmol) and 2-acetyl-5,5-
dimethy1-cyclohexane-1,3-dione (1, 363 mg, 1.99 mmo1) accord-
ing to the general procedure for the formation of 4,5,6,7-
tetrahydroindazoles. Yield: 83% (478 mg); pale orange solid.
white solid. Mp ¼ 122e129 ꢁC; 1H NMR (400 MHz, CDCl3)
d 8.27
(dd, J ¼ 1.6, 0.7 Hz,1H), 7.80 (d, J ¼ 8.4 Hz,1H), 7.64 (ddd, J ¼ 8.4, 2.3,
0.6 Hz,1H), 3.25 (s, 2H), 2.54 (s, 3H), 2.39 (s, 2H), 2.38 (s, 3H), 1.13 (s,
Mp ¼ 144e151 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.39 (dd, J ¼ 2.5,
6H); 13C NMR (100 MHz, CDCl3)
d
194.1, 150.6, 150.3, 149.9, 147.7,
0.5 Hz,1H), 7.93 (dd, J ¼ 8.8, 0.5 Hz,1H), 7.79 (dd, J ¼ 8.8, 2.5 Hz,1H),
139.1, 131.4, 117.7, 115.2, 52.5, 38.8, 35.4, 28.6, 17.9, 13.5; HRMS (ESI)
3.26 (s, 2H), 2.54 (s, 3H), 2.39 (s, 2H), 1.14 (s, 6H); 13C NMR
17

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
(100 MHz, CDCl3)
d
194.0, 150.9, 150.7, 150.5, 146.3, 138.3, 129.4,
13.5; HRMS (ESI) calcd for [C17H17N3OS þ H]þ 312.1171, found
312.1172.
118.2, 116.4, 52.4, 39.0, 35.4, 28.6, 13.5; HRMS (ESI) calcd for
[C15H16ClN3O þ H]þ 290.1060, found 290.1055.
1-(Benzo[d]thiazol-2-yl)-7-bromo-3,6,6-trimethyl-6,7-
dihydro-1H-indazol-4(5H)-one (55). Prepared from compound 54
(150 mg, 0.48 mmol) according to the general procedure for
bromination. Yield: 37% (47 mg); white solid. Mp ¼ 206e218 ꢁC; 1H
7-Bromo-1-(5-chloropyridin-2-yl)-3,6,6-trimethyl-6,7-
dihydro-1H-indazol-4(5H)-one (49). Prepared from compound 48
(150 mg, 0.52 mmol) according to the general procedure for
bromination. Yield: 60% (77 mg); white solid. Mp ¼ 117e123 ꢁC; 1H
NMR (400 MHz, CDCl3)
d
8.01 (d, J ¼ 8.2 Hz, 1H), 7.87 (d, J ¼ 8.0 Hz,
NMR (400 MHz, CDCl3)
d
8.46 (d, J ¼ 2.5 Hz, 1H), 7.98 (d, J ¼ 8.8 Hz,
1H), 7.52 (ddd, J ¼ 8.0, 7.4, 1.2 Hz, 1H), 7.41 (m, 1H), 6.16 (d,
J ¼ 1.2 Hz, 1H), 2.92 (dd, J ¼ 16.8, 0.7 Hz, 1H), 2.56 (s, 3H), 2.31 (dd,
J ¼ 16.9, 1.3 Hz, 1H), 1.45 (s, 3H), 1.27 (s, 3H); 13C NMR (100 MHz,
1H), 7.82 (dd, J ¼ 8.8, 2.6 Hz, 1H), 6.26 (d, J ¼ 1.1 Hz, 1H), 2.86 (d,
J ¼ 16.9 Hz, 1H), 2.53 (s, 3H), 2.27 (dd, J ¼ 16.8, 1.2 Hz, 1H), 1.39 (s,
3H), 1.25 (s, 3H); 13C NMR (100 MHz, CDCl3)
d
192.8, 150.7, 150.4,
CDCl3) d 192.4, 159.3, 151.8, 151.3, 149.9, 132.8, 126.7, 125.4, 123.1,
149.6, 146.5, 138.5, 129.9, 116.8, 116.2, 52.7, 48.7, 39.7, 29.6, 25.4,
13.5; HRMS (ESI) calcd for [C15H15BrClN3O þ H]þ 368.0165, found
368.0174.
121.5, 117.3, 50.7, 48.9, 39.8, 29.5, 25.3, 13.4; HRMS (ESI) calcd for
[C17H16BrN3OS þ H]þ 390.0276, found 390.0290.
3-(3,6,6-Trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-
yl)propanenitrile (56). Prepared from cyanoethylhydrazine
(426 mg, 5.00 mmol) and 2-acetyl-5,5-dimethy1-cyclohexane-1,3-
dione (1, 844 mg, 4.63 mmo1) according to the general procedure
for the formation of 4,5,6,7-tetrahydroindazoles. Yield: 61%
(657 mg); pale yellow solid. Mp ¼ 57e63 ꢁC; 1H NMR (400 MHz,
1-(6-Chloropyridazin-3-yl)-3,6,6-trimethyl-6,7-dihydro-1H-
indazol-4(5H)-one
(50).
Prepared
from
3-chloro-6-
hydrazinopyridazine (300 mg, 2.08 mmol) and 2-acetyl-5,5-
dimethy1-cyclohexane-1,3-dione (1, 360 mg, 1.98 mmo1) accord-
ing to the general procedure for the formation of 4,5,6,7-
tetrahydroindazoles. Yield: 73% (420 mg); pale yellow solid.
CDCl3)
d
4.25 (t, J ¼ 6.3 Hz, 2H), 2.95 (t, J ¼ 6.3 Hz, 2H), 2.72 (s, 2H),
Mp ¼ 140e153 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.22 (d, J ¼ 9.2 Hz,
2.46 (s, 3H), 2.35 (s, 2H), 1.14 (s, 6H); 13C NMR (100 MHz, CDCl3)
1H), 7.65 (d, J ¼ 9.2 Hz, 1H), 3.39 (s, 2H), 2.54 (s, 3H), 2.42 (s, 2H),
d 193.0, 150.0, 149.9, 116.9, 115.8, 52.4, 44.2, 35.7, 35.2, 28.5, 28.2,
1.15 (s, 6H); 13C NMR (100 MHz, CDCl3)
d
194.0, 155.4, 154.6, 151.7,
19.0, 13.3; HRMS (ESI) calcd for [C13H17N3O þ H]þ 232.1450, found
130.5, 122.4, 119.0, 52.4, 39.2, 35.4, 28.5, 13.5; HRMS (ESI) calcd for
232.1460.
[C14H15ClN4O þ H]þ 291.1013, found 291.1007.
3-(7-Bromo-3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-
indazol-1-yl)propanenitrile (57). Prepared from compound 56
(300 mg, 1.30 mmol) according to the general procedure for
bromination. Yield: 51% (205 mg); white solid. Mp ¼ 94e106 ꢁC; 1H
7-Bromo-1-(6-chloropyridazin-3-yl)-3,6,6-trimethyl-6,7-
dihydro-1H-indazol-4(5H)-one (51). Prepared from compound 50
(150 mg, 0.52 mmol) according to the general procedure for
bromination. Yield: 23% (30 mg); white solid. Mp ¼ 179e192 ꢁC; 1H
NMR (400 MHz, CDCl3)
d
5.01 (d, J ¼ 1.1 Hz, 1H), 4.36 (m, 2H), 3.11
NMR (400 MHz, CDCl3)
d
8.24 (d, J ¼ 9.2 Hz, 1H), 7.68 (d, J ¼ 9.2 Hz,
(m, 1H), 2.94 (m, 1H), 2.76 (dd, J ¼ 16.7, 0.9 Hz,1H), 2.46 (s, 3H), 2.23
1H), 6.30 (d, J ¼ 1.2 Hz, 1H), 2.88 (dd, J ¼ 16.8, 0.7 Hz, 1H), 2.54 (s,
(dd, J ¼ 16.7,1.2 Hz,1H),1.36 (s, 3H),1.22 (s, 3H); 13C NMR (100 MHz,
3H), 2.31 (dd, J ¼ 16.9, 1.3 Hz, 1H), 1.39 (s, 3H), 1.26 (s, 3H); 13C NMR
CDCl3)
d 191.8, 149.9, 148.6, 116.8, 114.4, 48.8, 48.1, 44.5, 39.6, 29.7,
(100 MHz, CDCl3)
d
192.7, 155.3, 155.0, 151.6, 150.6, 130.7, 122.3,
25.5, 18.7, 13.3; HRMS (ESI) calcd for [C13H16BrN3O þ H]þ 310.0555,
117.7, 52.1, 48.6, 39.7, 29.5, 25.3, 13.5; HRMS (ESI) calcd for
found 310.0557.
[C14H14BrClN4O þ H]þ 369.0118, found 369.0108.
3,6,6-Trimethyl-1-(pyrimidin-4-yl)-6,7-dihydro-1H-indazol-
4(5H)-one (58). Prepared from 4-hydrazinopyrimidine (300 mg,
2.72 mmol) and 2-acetyl-5,5-dimethy1-cyclohexane-1,3-dione (1,
460 mg, 2.52 mmo1) according to the general procedure for the
formation of 4,5,6,7-tetrahydroindazoles. Yield: 92% (592 mg);
3,6,6-Trimethyl-1-(thiazol-2-yl)-6,7-dihydro-1H-indazol-
4(5H)-one (52). Prepared from 2-hydrazinothiazole (440 mg,
2.41 mmol) and 2-acetyl-5,5-dimethy1-cyclohexane-1,3-dione (1,
300 mg, 2.61 mmo1) according to the general procedure for the
formation of 4,5,6,7-tetrahydroindazoles. Yield: 84% (529 mg); pale
white solid. Mp ¼ 145e148 ꢁC; 1H NMR (400 MHz, CDCl3)
d 9.05 (d,
yellow solid. Mp ¼ 126e131 ꢁC; 1H NMR (400 MHz, CDCl3)
d
7.59 (d,
J ¼ 1.0 Hz, 1H), 8.78 (d, J ¼ 5.6 Hz, 1H), 7.97 (dd, J ¼ 5.6, 1.2 Hz, 1H),
J ¼ 3.5 Hz, 1H), 7.13 (d, J ¼ 3.5 Hz, 1H), 3.28 (s, 2H), 2.53 (s, 3H), 2.41
3.40 (s, 2H), 2.54 (s, 3H), 2.41 (s, 2H), 1.17 (s, 6H); 13C NMR
(s, 2H), 1.17 (s, 6H); 13C NMR (100 MHz, CDCl3)
d
193.6, 161.5, 151.4,
(100 MHz, CDCl3) d 193.9,158.7,158.6,158.0,152.1,151.7,119.2,111.2,
150.2, 140.4, 118.2, 116.2, 52.6, 37.6, 35.4, 28.6, 13.4; HRMS (ESI)
52.4, 39.4, 35.4, 28.6, 13.6; HRMS (ESI) calcd for [C14H16N4O þ H]þ
calcd for [C13H15N3OS þ H]þ 262.1014, found 262.1016.
257.1402, found 257.1391.
7-Bromo-3,6,6-trimethyl-1-(thiazol-2-yl)-6,7-dihydro-1H-
indazol-4(5H)-one (53)Prepared from compound 52 (150 mg,
0.52 mmol) according to the general procedure for bromination.
Yield: 51% (66 mg); white solid. Mp ¼ 167e174 ꢁC; 1H NMR
7-Bromo-3,6,6-trimethyl-1-(pyrimidin-4-yl)-6,7-dihydro-
1H-indazol-4(5H)-one (59). Prepared from compound 58 (200 mg,
0.780 mmol) according to the general procedure for bromination.
Yield: 61% (160 mg); white solid. Mp ¼ 156e162 ꢁC; 1H NMR
(400 MHz, CDCl3)
d
7.66 (d, J ¼ 3.5 Hz, 1H), 7.18 (d, J ¼ 3.5 Hz, 1H),
(400 MHz, CDCl3)
d
9.13 (d, J ¼ 1.1 Hz, 1H), 8.82 (d, J ¼ 5.6 Hz, 1H),
6.03 (d, J ¼ 1.2 Hz, 1H), 2.89 (dd, J ¼ 16.9, 0.8 Hz, 1H), 2.53 (s, 3H),
8.00 (dd, J ¼ 5.7, 1.2 Hz, 1H), 6.34 (d, J ¼ 1.2 Hz, 1H), 2.88 (d,
2.28 (dd, J ¼ 16.9, 1.4 Hz, 1H), 1.40 (s, 3H), 1.23 (s, 3H); 13C NMR
J ¼ 16.9 Hz, 1H), 2.54 (s, 3H), 2.30 (dd, J ¼ 16.7, 1.4 Hz, 1H), 1.42 (s,
(100 MHz, CDCl3)
d
192.3, 160.5, 151.3, 149.0, 140.8, 116.6, 50.8, 48.8,
3H), 1.25 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 192.6, 159.0, 158.3,
39.7, 29.5, 25.3, 13.3; HRMS (ESI) calcd for [C13H14BrN3OS þ H]þ
158.1, 151.6, 151.0, 117.8, 111.2, 52.4, 48.7, 39.7, 29.6, 25.3, 13.6; HRMS
(ESI) calcd for [C14H15BrN4O þ H]þ 335.0507, found 335.0515.
3,6,6-Trimethyl-1-(5-(trifluoromethyl)pyridin-2-yl)-6,7-
340.0119, found 340.0109.
1-(Benzo[d]thiazol-2-yl)-3,6,6-trimethyl-6,7-dihydro-1H-
indazol-4(5H)-one (54). Prepared from 2-hydrazinobenzothiazole
(350 mg, 2.12 mmol) and 2-acetyl-5,5-dimethy1-cyclohexane-1,3-
dione (1, 357 mg, 1.96 mmo1) according to the general procedure
for the formation of 4,5,6,7-tetrahydroindazoles. Yield: 97%
(594 mg); white solid. Mp ¼ 234e240 ꢁC; 1H NMR (400 MHz,
dihydro-1H-indazol-4(5H)-one (60). Prepared from 2-hydrazinyl-
5-(trifluoromethyl)pyridine (300 mg, 1.69 mmol) and 2-acetyl-5,5-
dimethy1-cyclohexane-1,3-dione (1, 285 mg,1.57 mmo1) according
to the general procedure for the formation of 4,5,6,7-
tetrahydroindazoles. Yield: 98% (495 mg); white solid.
CDCl3)
d
7.93 (d, J ¼ 8.1 Hz, 1H), 7.85 (d, J ¼ 8.0 Hz, 1H), 7.49 (ddd,
Mp ¼ 117e121 ꢁC; 1H NMR (400 MHz, CDCl3)
d 8.72 (br s, 1H), 8.13
J ¼ 8.0, 7.4, 1.3 Hz, 1H), 7.38 (m, 1H), 3.41 (s, 2H), 2.55 (s, 3H), 2.44 (s,
2H), 1.21 (s, 6H); 13C NMR (100 MHz, CDCl3)
193.7, 160.3, 151.8,
151.2, 132.8, 126.5, 125.1, 122.7, 121.5, 118.9, 52.7, 37.9, 35.5, 28.6,
(d, J ¼ 8.7 Hz, 1H), 8.05 (dd, J ¼ 8.8, 2.3 Hz, 1H), 3.34 (s, 2H), 2.55 (s,
d
3H), 2.41 (s, 2H), 1.16 (s, 6H); 13C NMR (100 MHz, CDCl3)
151.5, 151.1, 145.1, 135.8, 124.7, 124.4, 124.0, 118.7, 114.9, 52.4, 39.3,
d 194.0,
18

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
35.4, 28.6, 13.6; HRMS (ESI) calcd for [C16H16F3N3O þ H]þ 324.1324,
found 324.1317.
1H), 2.55 (s, 3H), 2.31 (d, J ¼ 16.9 Hz, 1H), 1.42 (s, 3H), 1.26 (s, 3H);
13C NMR (100 MHz, CDCl3)
d 192.6, 155.6, 151.8, 150.9, 144.4, 142.1,
7-Bromo-3,6,6-trimethyl-1-(5-(trifluoromethyl)pyridin-2-
yl)-6,7-dihydro-1H-indazol-4(5H)-one (61). Prepared from com-
pound 60 (120 mg, 0.371 mmol) according to the general procedure
for bromination. Yield: 66% (99 mg); semi solid. 1H NMR (400 MHz,
134.1, 117.9, 115.1, 52.6, 48.6, 39.7, 29.6, 25.4, 13.6; HRMS (ESI) calcd
for [C15H15BrN4O3 þ H]þ 379.0406, found 379.0420.
1-(5-Aminopyridin-2-yl)-7-bromo-3,6,6-trimethyl-6,7-
dihydro-1H-indazol-4(5H)-one (67). To a solution of compound
66 (60 mg, 0.158 mmol) in EtOH (1 mL) was added SnCl2$2H2O
(179 mg, 0.791 mmol). The reaction mixture was stirred at 70 ꢁC for
15 min. After cooling, the reaction mixture was poured into icy
water (13 mL) and neutralized with 5% NaHCO3 (2.8 mL). The
aqueous solution was extracted with CH2Cl2 (3 ꢃ 15 mL). The
combined organic layer was washed with brine (20 mL) and dried
over anhydrous Na2SO4. The residue was purified by preparative
TLC (silica gel, EtOAc:hexanes 2:1) to afford compound 67 as a pale
yellow solid (34 mg, 62%). Mp ¼ 163e173 ꢁC; 1H NMR (400 MHz,
CDCl3)
d
8.80 (br s, 1H), 8.18 (d, J ¼ 8.7 Hz, 1H), 8.07 (dd, J ¼ 8.7,
2.3 Hz, 1H), 6.31 (d, J ¼ 1.2 Hz, 1H), 2.88 (d, J ¼ 16.9 Hz, 1H), 2.55 (s,
3H), 2.29 (dd, J ¼ 16.9, 1.3 Hz, 1H), 1.41 (s, 3H), 1.25 (s, 3H); 13C NMR
(100 MHz, CDCl3)
d 192.7, 151.0, 150.3, 145.3, 136.0, 124.8, 124.6,
124.4, 117.3, 114.9, 52.7, 48.7, 39.7, 29.6, 25.4, 13.5; HRMS (ESI) calcd
for [C16H15BrF3N3O þ H]þ 402.0429, found 402.0423.
2-Hydrazinyl-6-methoxypyridine (62). A mixture of 2-bromo-
6-methoxypyridine (3.00 g, 16.0 mmol) and 80% hydrazine hydrate
(160 mmol, 9.98 g) was stirred at 120 ꢁC for 3.5 h. Excess hydrazine
was removed under reduced pressure and then H2O (3 mL) was
added to the residue. The aqueous mixture was extracted with
EtOAc (4 ꢃ 8 mL). The combined organic layer was washed with
saturated NaHCO3 (5 mL), dried over anhydrous Na2SO4 and
evaporated. The residue was distilled under vacuum (12 mbar) at
100 ꢁC to afford compound 62 as a dark brown oil (909 mg, 41%). 1H
CDCl3)
d
7.94 (d, J ¼ 2.7 Hz, 1H), 7.73 (d, J ¼ 8.8 Hz, 1H), 7.14 (dd,
J ¼ 8.7, 2.9 Hz, 1H), 6.22 (s, 1H), 3.83 (br s, 2H), 2.85 (d, J ¼ 16.9 Hz,
1H), 2.53 (s, 3H), 2.25 (dd, J ¼ 16.8, 1.1 Hz, 1H), 1.37 (s, 3H), 1.25 (s,
3H); 13C NMR (100 MHz, CDCl3)
d 192.8, 149.4, 148.3, 144.5, 141.6,
134.2,124.2, 116.4, 115.8, 52.7, 48.7, 39.7, 29.7, 25.4, 13.4; HRMS (ESI)
calcd for [C15H17BrN4O þ H]þ 349.0664, found 349.0649.
NMR (400 MHz, CDCl3)
d
7.42 (t, J ¼ 7.9 Hz, 1H), 6.23 (d, J ¼ 7.9 Hz,
3,6,6-Trimethyl-6,7-dihydro-1H-indazol-4(5H)-one (68).
A
1H), 6.12 (dd, J ¼ 8.0, 0.4 Hz,1H), 5.69 (br s,1H), 3.87 (s, 3H), 3.79 (br
solution of 80% hydrazine hydrate (32.4 mmol, 2.06 g) and 2-acetyl-
5,5-dimethyl-cyclohexanes-1,3-dione (1, 2.00 g, 11.0 mmo1) in
EtOH (25 mL) was stirred at room temperature for 4 h. EtOH was
then removed by evaporation. To the residue was added CH2Cl2
(30 mL), and then the organic solution was washed with H2O
(20 mL) and brine (20 mL) and dried over anhydrous Na2SO4. After
evaporation, the residue was purified by silica column chroma-
tography (MeOH:CH2Cl2 5:95) to afford compound 68 as a pale
yellow solid (1.71 g, 88%). Mp ¼ 93e107 ꢁC; 1H NMR (400 MHz,
s, 2H); 13C NMR (100 MHz, CDCl3)
53.3.
d 163.6, 160.4, 140.2, 98.9, 97.7,
1-(6-Methoxypyridin-2-yl)-3,6,6-trimethyl-6,7-dihydro-1H-
indazol-4(5H)-one (63). Prepared from compound 62 (300 mg,
2.16 mmol) and 2-acetyl-5,5-dimethy1-cyclohexane-1,3-dione (1,
364 mg, 2.00 mmo1) according to the general procedure for the
formation of 4,5,6,7-tetrahydroindazoles. Yield: 54% (310 mg); light
orange solid. Mp ¼ 108e115 ꢁC; 1H NMR (400 MHz, CDCl3)
d 7.71 (t,
J ¼ 7.9 Hz,1H), 7.50 (d, J ¼ 7.8 Hz,1H), 6.68 (d, J ¼ 8.1 Hz,1H), 3.96 (s,
CDCl3)
(100 MHz, CDCl3)
d
2.71 (s, 2H), 2.56 (s, 3H), 2.37 (s, 2H), 1.11 (s, 6H); 13C NMR
3H), 3.33 (s, 2H), 2.54 (s, 3H), 2.40 (s, 2H), 1.14 (s, 6H); 13C NMR
d 194.6, 154.2, 145.0, 114.6, 53.0, 36.4, 35.6, 28.4,
(100 MHz, CDCl3)
d
193.9, 162.8, 150.4, 150.2, 150.1, 140.9, 117.8,
12.0; HRMS (ESI) calcd for [C10H14N2O þ H]þ 179.1184, found
108.3, 107.2, 53.9, 52.4, 39.3, 35.3, 28.7, 13.5; HRMS (ESI) calcd for
179.1185.
[C16H19N3O3 þ H]þ 286.1556, found 286.1548.
3,6,6-Trimethyl-1-((4-nitrophenyl)sulfonyl)-6,7-dihydro-1H-
indazol-4(5H)-one (69). To a solution of compound 68 (500 mg,
2.81 mmol) and triethylamine (1.17 mL, 8.42 mmol) in CH2Cl2
(12 mL) was added a solution of 4-nitrobenzenesulfonyl chloride
(933 mg, 4.21 mmol) in CH2Cl2 (12 mL). The mixture was stirred at
room temperature for 5 h CH2Cl2 (100 mL) was then added and the
mixture was washed with saturated NaHCO3 (2 ꢃ 60 mL). The
organic layer was dried over anhydrous Na2SO4 and evaporated
under reduced pressure. The residue was purified by silica column
chromatography (EtOAc:hexanes 1:3) to afford compound 69 as a
pale yellow solid (766 mg, 75%). Mp ¼ 150e164 ꢁC; 1H NMR
7-Bromo-1-(6-methoxypyridin-2-yl)-3,6,6-trimethyl-6,7-
dihydro-1H-indazol-4(5H)-one (64). Prepared from compound 63
(150 mg, 0.526 mmol) according to the general procedure for
bromination. Yield: 72% (137 mg); white solid. Mp ¼ 138e144 ꢁC;
1H NMR (400 MHz, CDCl3)
d
7.73 (t, J ¼ 8.0 Hz, 1H), 7.60 (d,
J ¼ 7.8 Hz, 1H), 6.72 (d, J ¼ 8.1 Hz, 1H), 6.31 (s, 1H), 4.04 (s, 3H), 2.88
(d, J ¼ 16.9 Hz, 1H), 2.54 (s, 3H), 2.28 (d, J ¼ 16.9 Hz, 1H), 1.38 (s, 3H),
1.24 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 192.7, 163.0, 150.3, 150.2,
149.1, 140.9, 116.5, 108.9, 107.1, 53.9, 53.1, 48.5, 39.7, 29.9, 25.5, 13.5;
HRMS (ESI) calcd for [C16H18BrN3O2 þ H]þ 364.0661, found
364.0652.
(400 MHz, CDCl3)
d
8.41 (d, J ¼ 8.9 Hz, 2H), 8.22 (d, J ¼ 8.9 Hz, 2H),
3,6,6-Trimethyl-1-(5-nitropyridin-2-yl)-6,7-dihydro-1H-
3.11 (s, 2H), 2.42 (s, 3H), 2.36 (s, 2H),1.14 (s, 6H); 13C NMR (100 MHz,
indazol-4(5H)-one (65).
Prepared from 2-hydrazinyl-5-
CDCl3) d 192.9,153.9,153.8,151.2,142.6,129.5,124.7,118.8, 52.1, 37.1,
nitropyridine (1.00 g, 6.49 mmol) and 2-acetyl-5,5-dimethy1-
cyclohexanes-1,3-dione (1, 1.10 g, 6.01 mmo1) according to the
general procedure for the formation of 4,5,6,7-tetrahydroindazoles.
Yield: 25% (448 mg); pale yellow solid. Mp ¼ 174e181 ꢁC; 1H NMR
35.5, 28.4, 13.7; HRMS (ESI) calcd for [C16H17N3O5S þ H]þ 364.0967,
found 364.0976.
7-Bromo-3,6,6-trimethyl-1-((4-nitrophenyl)sulfonyl)-6,7-
dihydro-1H-indazol-4(5H)-one (70). Prepared from compound 69
(180 mg, 0.599 mmol) according to the general procedure for
bromination. Yield: 56% (103 mg); white solid. Mp ¼ 146e150 ꢁC;
(400 MHz, CDCl3)
d
9.30 (dd, J ¼ 2.7, 0.5 Hz, 1H), 8.60 (dd, J ¼ 9.1,
2.7 Hz, 1H), 8.20 (dd, J ¼ 9.1, 0.5 Hz, 1H), 3.37 (s, 2H), 2.55 (s, 3H),
2.42 (s, 2H), 1.17 (s, 6H); 13C NMR (100 MHz, CDCl3)
d
193.9, 156.0,
1H NMR (400 MHz, CDCl3)
d
8.43 (d, J ¼ 1.3 Hz, 1H), 5.63 (d,
152.1, 151.9, 144.3, 141.8, 133.9, 119.3, 115.0, 52.3, 39.5, 35.4, 28.6,
13.6; HRMS (ESI) calcd for [C15H16N4O3 þ H]þ 301.1301, found
301.1295.
J ¼ 1.1 Hz, 1H), 2.77 (dd, J ¼ 17.1, 0.6 Hz, 1H), 2.44 (s, 3H), 2.28 (dd,
J ¼ 17.1, 1.2 Hz, 1H), 1.39 (s, 3H), 1.20 (s, 3H); 13C NMR (100 MHz,
CDCl3) d 191.8,153.6,152.7,151.4,141.7,130.7,124.4,116.8, 49.4, 48.2,
7-Bromo-3,6,6-trimethyl-1-(5-nitropyridin-2-yl)-6,7-
dihydro-1H-indazol-4(5H)-one (66). Prepared from compound 65
(180 mg, 0.599 mmol) according to the general procedure for
bromination. Yield: 70% (158 mg); white solid. Mp ¼ 119e122 ꢁC;
40.0, 29.4, 25.1, 13.7; HRMS (ESI) calcd for [C15H18BrN5O3S þ H]þ
442.0072, found 442.0067.
Methyl
3-((6-(7-Bromo-3,6,6-trimethyl-4-oxo-4,5,6,7-
tetrahydro-1H-indazol-1-yl)pyridin-3-yl)amino)-3-
oxopropanoate (71). To a solution of compound 67 (70 mg,
0.20 mmol) and monomethyl malonate (23 mL, 0.22 mmol) in
1H NMR (400 MHz, CDCl3)
d
9.38 (d, J ¼ 2.5 Hz, 1H), 8.62 (dd, J ¼ 9.1,
2.6 Hz, 1H), 8.25 (d, J ¼ 9.1 Hz, 1H), 6.27 (s, 1H), 2.88 (d, J ¼ 16.9 Hz,
19

J.C. Lee, K.H. Hong, A. Becker et al.
European Journal of Medicinal Chemistry 214 (2021) 113232
CH2Cl2 (2 mL) were added EDC$HCl (46 mg, 0.24 mmol), HOBT (14%
calcd for [C17H19BrN4O2 þ H]þ 391.0770, found 391.0785.
3,6,6-Trimethyl-1-(naphthalen-2-yl)-6,7-dihydro-1H-inda-
H2O, 38 mg, 0.24 mmol) and N,N-diisopropylethylamine (42
mL,
0.24 mmol). The solution was stirred at room temperature for 24 h.
After adding CH2Cl2 (8 mL), the mixture was washed with saturated
NH4Cl (2 ꢃ 8 mL), saturated NaHCO3 (8 mL) and brine (8 mL). The
organic layer was dried over anhydrous Na2SO4 and evaporated
under reduced pressure. The residue was purified by preparative
TLC (silica gel, MeOH:CH2Cl2 5:95) to afford compound 71 as a
zol-4(5H)-one (75).
A
mixture of 2-acetyl-5,5-dimethy1-
cyclohexanes-1,3-dione (1, 200 mg, 1.10 mmo1), naphthalen-2-
ylhydrazine hydrochloride (224 mg, 1.15 mmo1) and pyridine
(133 m
L, 1.65 mmol) in ethanol (7 m1) was stirred at 80 ꢁC for 2 h.
The solvent was then removed by evaporation. CH2Cl2 (18 mL) was
then added and the solution was washed with saturated NaHCO3
(6 mL) and saturated NH4Cl (9 mL). The organic layer was dried over
anhydrous Na2SO4 and evaporated under reduced pressure. The
residue was purified by silica column chromatography (EtOA-
c:hexanes 1:3) to afford compound 75 as a light red solid (264 mg,
yellowish semisolid (62 mg, 69%). 1H NMR (400 MHz, CDCl3)
d 9.58
(s, 1H), 8.69 (d, J ¼ 2.5 Hz, 1H), 8.19 (dd, J ¼ 8.9, 2.6 Hz, 1H), 7.97 (d,
J ¼ 8.9 Hz, 1H), 6.31 (d, J ¼ 1.1 Hz, 1H), 3.84 (s, 3H), 3.55 (s, 2H), 2.86
(d, J ¼ 16.8 Hz,1H), 2.54 (s, 3H), 2.26 (dd, J ¼ 16.8,1.3 Hz,1H),1.39 (s,
3H), 1.26 (s, 3H); 13C NMR (100 MHz, CDCl3)
d
192.9, 170.4, 163.2,
79%). Mp ¼ 127e131 ꢁC; 1H NMR (400 MHz, CDCl3)
d 7.97 (d,
150.0, 149.2, 148.6, 139.1, 133.0, 130.0, 116.5, 115.5, 52.9, 52.7, 48.7,
40.8, 39.7, 29.6, 25.4, 13.5; HRMS (ESI) calcd for
[C19H21BrN4O4 þ H]þ 449.0824, found 449.0839.
J ¼ 8.8 Hz, 1H), 7.91 (m, J ¼ 4.2 Hz, 3H), 7.65 (dd, J ¼ 8.7, 2.1 Hz, 1H),
7.56 (m, 2H), 2.86 (s, 2H), 2.59 (s, 3H), 2.43 (s, 2H), 1.11 (s, 6H); 13
NMR (100 MHz, CDCl3) 193.4, 150.0, 149.2, 136.1, 133.1, 132.4,
C
d
N-(6-(7-Bromo-3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-
129.5, 128.1, 127.9, 127.2, 126.8, 122.1, 121.8, 117.0, 52.5, 37.3, 35.9,
28.4, 13.4; HRMS (ESI) calcd for [C20H20N2O þ H]þ 305.1654, found
305.1665.
indazol-1-yl)pyridin-3-yl)sulfamoylamide (72).
A solution of
compound 67 (70 mg, 0.20 mmol) and sulfamoyl chloride (26 mg,
0.22 mmol) in dimethylacetamide (0.5 mL) was stirred at room
temperature for 20 h. After adding EtOAc (5 mL), the solution was
washed with brine (2 ꢃ 5 mL), dried over anhydrous Na2SO4 and
evaporated under reduced pressure. The residue was purified by
preparative TLC (silica gel, MeOH:CH2Cl2 5:95) to afford compound
72 as a white solid (76 mg, 89%). Mp ¼ 148e160 ꢁC; 1H NMR
7-Bromo-3,6,6-trimethyl-1-(naphthalen-2-yl)-6,7-dihydro-
1H-indazol-4(5H)-one (76). Prepared from compound 75 (100 mg,
0.329 mmol) according to the general procedure for bromination.
Yield: 68% (86 mg); pale yellow solid. Mp ¼ 65e75 ꢁC; 1H NMR
(400 MHz, CDCl3)
d
8.18 (d, J ¼ 2.0 Hz, 1H), 8.01 (d, J ¼ 8.8 Hz, 1H),
7.94 (m, 2H), 7.74 (dd, J ¼ 8.7, 2.2 Hz, 1H), 7.60 (m, 2H), 4.93 (d,
J ¼ 1.2 Hz, 1H), 2.90 (dd, J ¼ 17.1, 0.7 Hz, 1H), 2.59 (s, 3H), 2.29 (dd,
J ¼ 17.1, 1.3 Hz, 1H), 1.33 (s, 3H), 1.17 (s, 3H); 13C NMR (100 MHz,
(400 MHz, DMSO‑d6)
d
10.07 (s, 1H), 8.35 (d, J ¼ 2.6 Hz, 1H), 7.87 (d,
J ¼ 8.9 Hz, 1H), 7.76 (dd, J ¼ 8.9, 2.6 Hz, 1H), 7.40 (s, 2H), 6.30 (s, 1H),
2.67 (d, J ¼ 16.9 Hz, 1H), 2.42 (s, 3H), 2.25 (d, J ¼ 16.9 Hz, 1H), 1.31 (s,
CDCl3)
d 192.2, 150.1, 149.1, 135.5, 133.1, 132.9, 129.8, 128.4, 127.9,
3H), 1.17 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d
191.7, 148.6, 148.3,
127.3, 127.2, 123.0, 122.6, 115.4, 50.8, 48.5, 40.3, 29.5, 25.2, 13.4;
HRMS (ESI) calcd for [C20H19BrN2O
383.0777.
146.0,137.0,135.3,127.9,115.6,115.5, 59.7, 53.2, 48.1, 28.8, 24.4,13.1;
HRMS (ESI) calcd for [C15H18BrN5O3S þ H]þ 428.0392, found
428.0409.
þ
H]þ 383.0759, found
N-(6-(7-Bromo-3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-
indazol-1-yl)pyridin-3-yl)methanesulfonamide (73). To a solu-
Declaration of competing interest
tion of compound 67 (70 mg, 0.20 mmol) and pyridine (23
0.28 mmol) in CH2Cl2 (2 mL) was added methanesulfonyl chloride
(22 L, 0.28 mmol). The mixture was stirred at room temperature
mL,
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
m
for 23 h CH2Cl2 (7 mL) was then added and the solution was washed
with H2O (5 mL) and brine (5 mL). The organic layer was dried over
anhydrous Na2SO4 and evaporated under reduced pressure. The
residue was purified by preparative TLC (silica gel, EtOAc:hexanes
3:1) to afford compound 73 as a white solid (76 mg, 88%).
Acknowledgments
We gratefully acknowledge financial support from the Contra-
ception Research Branch of the Eunice Kennedy Shriver National
Institute of Child Health and Human Development (NICHD)
through contract HHSN275200503400C. We thank Drs. H. K. Kim,
Diana Blithe, and Min Lee from NICHD for their support of this
project. Funding came from the NIH (US).
Mp ¼ 173e178 ꢁC; 1H NMR (400 MHz, CDCl3)
d
8.40 (d, J ¼ 2.6 Hz,
1H), 8.04 (d, J ¼ 8.9 Hz,1H), 7.82 (dd, J ¼ 8.9, 2.8 Hz,1H), 6.91 (s, 1H),
6.26 (d, J ¼ 1.0 Hz, 1H), 3.11 (s, 3H), 2.87 (d, J ¼ 16.9 Hz, 1H), 2.54 (s,
3H), 2.29 (dd, J ¼ 16.9, 1.3 Hz, 1H), 1.40 (s, 3H), 1.25 (s, 3H); 13C NMR
(100 MHz, CDCl3)
d 192.9, 150.4, 149.9, 149.5, 140.3, 131.8, 131.4,
116.7, 116.1, 52.7, 48.7, 40.2, 39.7, 29.6, 25.4, 13.5; HRMS (ESI) calcd
Appendix A. Supplementary data
for [C16H19BrN4O3S þ H]þ 427.0473, found 427.0457.
N-(6-(7-Bromo-3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-
indazol-1-yl)pyridin-3-yl)acetamide (74). To a solution of com-
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113232.
pound 67 (70 mg, 0.20 mmol) and triethylamine (42
in CH2Cl2 (1.5 mL) was added acetyl chloride (21
m
L, 0.30 mmol)
mL, 0.30 mmol).
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dried over anhydrous Na2SO4 and evaporated under reduced
pressure. The residue was purified by preparative TLC (silica gel,
EtOAc:hexanes 3:1) to afford compound 74 as a white solid (72 mg,
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