Structure-activity relationships for the anaesthetic and analgaesic properties of aromatic ring-substituted ketamine esters

Article abstract of DOI:10.3390/molecules25122950

A series of benzene ring substituted ketamine N-alkyl esters were prepared from the corresponding substituted norketamines. Few of the latter have been reported since they have not been generally accessible via known routes. We report a new general route to many of these norketamines via the Neber (oxime to α-aminoketone) rearrangement of readily available substituted 2-phenycyclohexanones. We explored the use of the substituents Cl, Me, OMe, CF₃, and OCF₃, with a wide range of lipophilic and electronic properties, at all available benzene ring positions. The 2- and 3-substituted compounds were generally more active than 4-substituted compounds. The most generally acceptable substituent was Cl, while the powerful electron-withdrawing substituents CF₃ and OCF₃ provided fewer effective analogues.

Full text of DOI:10.3390/molecules25122950

molecules
Article
Structure-Activity Relationships for the Anaesthetic
and Analgaesic Properties of Aromatic
Ring-Substituted Ketamine Esters
Ivaylo V. Dimitrov 1, Martyn G. Harvey 2, Logan J. Voss 2, James W. Sleigh 2,
Michael J. Bickerdike 3 and William A. Denny 1,
*
1
Auckland Cancer Society Research Centre, School of Medical Sciences, Auckland 1142, New Zealand;
i.dimitrov@auckland.ac.nz
Waikato Clinical School, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand;
Martyn.Harvey@waikatodhb.health.nz (M.G.H.); Logan.Voss@waikatodhb.health.nz (L.J.V.);
Jamie.Sleigh@waikatodhb.health.nz (J.W.S.)
2
3
Kea Therapeutics Ltd, Level 20, Auckland 1010, New Zealand; Mike@keatx.com
Correspondence: b.denny@auckland.ac.nz
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editors: Larry Wakelin and Sandra Gemma
Received: 3 June 2020; Accepted: 23 June 2020; Published: 26 June 2020
Abstract: A series of benzene ring substituted ketamine N-alkyl esters were prepared from the
corresponding substituted norketamines. Few of the latter have been reported since they have
not been generally accessible via known routes. We report a new general route to many of these
norketamines via the Neber (oxime to α-aminoketone) rearrangement of readily available substituted
2-phenycyclohexanones. We explored the use of the substituents Cl, Me, OMe, CF3, and OCF3, with a
wide range of lipophilic and electronic properties, at all available benzene ring positions. The 2- and
3-substituted compounds were generally more active than 4-substituted compounds. The most
generally acceptable substituent was Cl, while the powerful electron-withdrawing substituents CF3
and OCF3 provided fewer effective analogues.
Keywords: ketamine; esters; anaesthesia; short-acting; structure-activity relationship
1. Introduction
Racemic ketamine (
1, Figure 1) is an effective and widely used anaesthetic/analgaesic [1],
and tiletamine (2) is a thiophene analogue widely used as an anaesthetic in veterinary medicine [2].
O
O
Cl
S
HN
HN
2
Tiletamine ( )
Ketamine (1)
Figure 1. Ketamine, tiletamine.
The sedative and analgaesic effects of
antagonism of the calcium channel pore of the N-methyl-D-aspartate (NMDA) receptor [
although this has recently been called into question [ ]. Compared to opioid-type pain-relieving
drugs, ketamine has the major advantages of no immediate respiratory depression or hyperalgesic
effects, and an absence of longer-term effects such as increased tolerance [ ]. The primary drawback
of is its substantial psychotogenic effects, which have recently been attributed to its blockade of
1
have commonly been attributed to its non-competitive
3,4],
5
6
1
Molecules 2020, 25, 2950; doi:10.3390/molecules25122950
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2950
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GluN2C-containing NMDA receptors [
7]. These detrimental properties are exacerbated by its relatively
long elimination half-life, which means that patients can be exposed to prolonged hallucinogenic
events as levels of drug slowly decline. To control these effects ketamine is frequently co-administered
with respiratory depressant hypnotic drugs like midazolam or propofol, but these can markedly reduce
its clinical safety [
8
]. In an alternative approach, we have recently shown, in a rat infusion model [
9],
that alkyl ester derivatives of ketamine (e.g. 5a
,
5b) are effective short-term anaesthetics/analgaesics.
They minimise psychotomimetic side effects during recovery by undergoing very rapid metabolism
by tissue esterases to the corresponding, much more polar, and inactive acids [10]. Ester side chains
i
(CH2)2CO2 Pr and (CH2)4CO2Me were particularly suitable [9,11].
We now extend these structure-activity studies to include analogues with Cl, Me, OMe, CF3 and
OCF3 substituted at each available position on the benzene ring, together with the unsubstituted ring
and 2-F variants (Table 1). Such substituents can potentially greatly affect drug binding to target proteins
through the lipophilic, electronic, and steric changes that they have on the molecule. They collectively
cover a wide range of lipophilic and electronic properties while keeping the steric effect broadly
similar [12]. For further comparison we also included the corresponding esters (20a, 20b) of the
veterinary thiophene analogue tiletamine.
Table 1. Biological and physiochemical data for esters of ring-substituted ketamine analogues.
Behavioural
dysfunction
No
X
n
R
Sedation
LORR a
Analgaesia
potency c
Rat
NMDA
Score g
(SEM)
RORR b
cTFL d
(sec)
t21
e
(min)
Duration h
(SEM)
IC50
(mg/kg) (SEM)
(mg/kg) (SEM)
(µM)f
(sec) (SEM)
1
3a
3b
2-Cl
H
H
2-F
2-F
3
3
3
3
2
3
3
3
3
1
2
2
2
1
2
3
3
2
2
1
2
1
2
2
3
3
3
1
2
2
1
Me
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
21 (4)
>200 (0)
40 (5)
59 (10)
35 (4)
33 (10)
34 (12)
>200 (0)
37 (2)
>200 (0)
34 (9)
64 (12)
154 (42)
62 (0)
>200 (0)
67 (21)
51 (6)
1075 (89)
N/Ai
28
>200 (0)
45 (5)
62 (6)
40 (5)
37 (11)
44 (17)
>200 (0)
43 (3)
>200 (0)
49 (11)
64 (12)
156 (45)
62 (0)
671
0
0
1297
11
1384
374
393
91
313
2332
652
1864
80
119
332
54
44.4
0.7
>1000
9.9
249
3.1
134
3.2
428
3.4
172
>1000
290
128
184
121
164
13
466
5.2
201
5.6
3 (0)
0.5 (0.7)
1 (0)
1 (0)
3 (1.4)
2 (1)
2 (0)
0 (0)
1 (0)
0 (0)
3 (1)
2 (1)
1 (0)
1 (0)
3 (1)
1 (0)
3 (1)
2 (1)
2 (1)
2 (0)
3 (1.4)
0 (0)
0 (0)
0 (0)
0 (0)
2 (1)
3 (1)
1 (0)
1 (0)
0 (0)
3 (0)
3 (1)
0 (0)
2 (0)
1 (0)
4 (0)
1 (0)
1 (0)
0 (0)
2 (1)
0 (0)
2 (0)
1 (0)
1 (0)
1 (0)
3 (0)
1 (0)
2 (0)
1 (0)
1 (0)
1 (0)
4 (0)
0 (0)
0 (0)
0 (0)
0 (0)
1 (0)
3 (0)
1 (0)
0 (0)
0 (0)
3 (0)
78 (22)
64 (7)
84 (20)
83 (33)
99 (28)
N/A
4a
4b
5aj
5bj
6a
2-Cl
2-Cl
3.4
0.7
2-Me
2-Me
2-OMe
2-OMe
2-CF3
2-CF3
2-OCF3
2-OCF3
3-Cl
6b
7a
7b
85 (14)
N/A
135 (12)
58 (4)
66 (8)
65 (0)
N/A
10 (1)
114 (46)
108 (39)
137 (32)
10 (0)
120 (3)
N/A
N/A
N/A
N/A
15 (9)
82 (40)
10 (0)
75 (11)
N/A
8a
8b#
9a
9b
>200 (0)
58 (11)
61 (4)
50 (5)
43 (6)
10a
10b
11a
11b
12a
12b
13ak
13b#
14a
14b
15a
15b
16a
16b
17a
17b
3-Cl
3-Me
3-Me
3-OMe
3-OMe
3-CF3
3-CF3
3-OCF3
3-OCF3
4-Cl
38 (4)
39 (6)
79 (0)
50 (8)
63
104
284
210
208
8
274
444
491
576
650
0
83 (0)
58 (6)
>200 (0)
>200 (0)
>200 (0)
>200 (0)
99 (43)
89 (40)
47 (0)
50 (6)
>200 (0)
Seizure
>200 (0)
>200 (0)
>200 (0)
>200 (0)
>200 (0)
100 (23)
52 (0)
229
172
211
57
129
101
161
23
4-Cl
4-Me
4-Me
4-OMe
4-OMe
55 (9)
>200 (0)
167
551
396
94.1

Molecules 2020, 25, 2950
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Table 1. Cont.
Behavioural
dysfunction
No
X
n
R
Sedation
LORR a
Analgaesia
potency c
Rat
NMDA
Score g
RORR b
cTFL d
(sec)
t21
e
(min)
Duration h
IC50
(mg/kg) (SEM)
(mg/kg) (SEM)
(µM)f
(SEM)
(sec) (SEM)
(SEM)
18a#
18b#
19a
19b
20a
4-CF3
4-CF3
4-OCF3
4-OCF3
thioph
thioph
1
2
1
1
3
2
A
B
A
B
A
B
>200 (0)
>200 (0)
>200 (0)
>200 (0)
>200 (0)
52 (6)
N/A
N/A
N/A
N/A
N/A
10 (2)
>200 (0)
>200 (0)
>200 (0)
>200 (0)
>200 (0)
74 (7)
412
37
166
194
362
196
438
470
314
597
157
8.9
0 (0)
0 (0)
0 (0)
0 (0)
1 (1)
4 (0)
0 (0)
0 (0)
0 (0)
0 (0)
1 (0)
1 (0)
20b
a
b
LORR: minimal dose (mg/kg) for loss of righting reflex (measure of sedative potency); RORR: time (sec) for
return of righting reflex after infusion stopped; c minimal dose (mg/kg) for loss of pedal withdrawal reflex (measure
of analgaesic potency); d cTFL (composite tail flick latency, a measure of analgaesic magnitude/duration); e Drug
half-life in rats (see ref 10 for method); f NMDA; IC50
(µM) for inhibition of the N-methyl-D-aspartate receptor;
conducted by Eurofins Eurofins Panlabs Taiwan; g Severity of behavioural dysfunction on recovery (see biology
methods); h Duration of behavioural dysfunction (see biology methods); i N/A; animals failed to demonstrate LORR
(sedation)/PWR of 1 (analgaesia) [9].
2. Chemistry and Biology
2.1. Chemistry
The synthesis of the compounds of Table 1 from the corresponding norketamines is straightforward
as we have previously demonstrated [9]. (Scheme 1).
i
Scheme 1. Synthesis of ketamine alkyl esters; Reagents and conditions: (i) Br(CH2)2CO2 Pr, K2CO3,
KI, MeCN, reflux 12–48h; (ii) Br(CH2)4CO2Me, KI, MeCN, reflux 12–48h.
However, few analogues of norketamine with substituents other than a 2-Cl in the aromatic ring
have been reported; only the unsubstituted compound 21 [13] and the 4-Cl (22) and 4-Br (23) [14]
analogues. The 3-OMe (25) and 3-OH (26) derivatives have also been characterised, but only as
metabolites of methoxetamine (24) [15] (Figure 2).
O
O
O
MeO
XO
HN
methoxetamine (
NH2
: X=OMe
NH2
X=H
X
25
26: X=OH
21:
24
)
22: X=Cl
23
: X=Br
Figure 2. Known norketamine analogues.

Molecules 2020, 25, 2950
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We initially sought to prepare the required new substituted norketamines by the published method
for norketamine itself [13] that we had used previously [ ], but this was not successful, probably due
9
to the lower nucleophilicity of ammonia compared with methylamine in that process. The use of more
nucleophilic precursor reagents (N-methylhydrazine, 4-methoxybenzylamine) was also not successful.
We therefore developed a new general route to many of these required norketamines, via the Neber
(oxime to α-aminoketone) rearrangement [16] of substituted 2-phenycyclohexanones 27a–27p via the
hydrazines 28a–28p and hydrazinium salts 29a
30b,c,e–i, k–p (Scheme 2).
–29p to give the required norketamines 21, 22 and
Scheme 2. Synthesis of ring-substituted norketamines 21, 22, 30b,c, e–i, k–p. R = :
, 4-Cl; , 2-Me; , 3-Me; , 4-Me; , 2-OMe; , 3-OMe; , 4-OMe; , 2-CF3; , 3-CF3,
, 3-OCF3; p, 4-OCF3; Reagents and conditions: (i) NH2NMe2, EtOH, reflux 18 h; (ii) (a) MeI, MeCN,
a
, H;
b, 2-F;
c, 3-Cl;
d
e
f
g
h
i
j
k
l
m, 4-CF3; n
, 2-OCF3;
o
40 ◦C for 2 h then 70 ◦C for 3 h; (iii) NaOEt, EtOH, reflux, 1 h.
The known 4-methoxynorketamine analogue 30j could not be prepared by the Neber
rearrangement, presumably because of the powerful electron-donating and/or inductive effects
of this substituent para to the reaction centre, and was prepared instead by the method described by
Sato et al [17].
In a further exploration of the nature of the aromatic ring in these esters, we also prepared the
thiophene-based tiletamine ester analogues 20a and 20b. Tiletamine itself (
2: Figure 1) is a well-known
veterinary animal anaesthetic [ ], considered to have a similar mechanism of action to ketamine.
2
The required nortiletamine was prepared by the method of Sato et al. [17] (Scheme 3).
Scheme 3. Synthesis of nortiletamine; Reagents and Conditions: (i) BzCl, pyridine, n-hexane-CH2Cl2
(10:1), 4 h r.t., then TFAA, CH2Cl2, r.t. 12 h, 89%; (ii) thiophene, n-BuLi, MeAlCl, THF, r.t. 30 min,
then 32, reflux, 3 h, 66%; (iii) NaOH (5% in MeOH), r.t. 12 h; (iv) Jones reagent, 84%.
2.2. Biology
The compounds were evaluated for their ability to anaesthetise rats when administered by
continuous intravenous infusion, as reported previously [9]. Compounds were administered to initially
deliver 20 mg/kg/min (weight-adjusted flow) to achieve a pedal withdrawal reflex score (PWR = 1),
then titrated to maintain loss of righting reflex (LORR) for 10 min. Three rats were used in each study,
with each group of rats also acting as their own ketamine control. Data were collected on the total
dose of drug (mg/kg), to achieve LORR and a PWR = 1, and on the time (in seconds, from cessation of
the infusion) to recovery of righting reflex (RORR) (recovery from the hypnotic anaesthesia effect).
Given the complexity of the experimental protocol, the total dose for LORR (Table 1) is very consistent,
with ranges of only 1.5-fold within each group. The consistency of the post-sedation recovery times are

Molecules 2020, 25, 2950
5 of 14
expectedly lower, with ranges of about 2.5-fold. Average data for LORR and RORR are given in Table 1.
During recovery the rats were monitored for multiple signs of behavioural dysfunction (see biology
section for details) and the sum of these scores (from 0 to 4, with 0 being no effect and 4 being severe
dysfunction) is given in Table 2. IC50 values for inhibition of the NMDA receptor were conducted by
Eurofins PanLabs, Taiwan.
3. Results and Discussion
Ketamine has long been of interest for its multiple biological activities and its potential as a
non-opioid anaesthetic. While a large number of side-chain analogues are known, relatively few
with benzene ring substituents have been reported. The new route that we report here by the Neber
rearrangement [16] of readily available substituted 2-phenycyclohexanones gives access to a wide range
of benzene-substituted ketamines. We compare two sets of N-alkyl esters as short-acting anaesthetics,
exploring Cl, Me, OMe, CF3, and OCF3 benzene ring substituents.
Table 1 gives structural and biological data for ketamine (
1
), two previously-reported [
9
,
,
10] ester
4b 19b)
analogues (5a 5b), two series of esters of novel benzene ring-substituted analogues (4a
,
–19a
–
and two similar esters (20a, 20b) of the thiophene-based analogue tiletamine (2, Figure 1) [12].
It has often been stated, [ ] and equally disputed [ ], that the sedative analgaesic and
3
,4
5
psychotomimetic properties of ketamine are due to interaction with/inhibition of the NMDA receptor.
The data acquired in this study for ketamine (1) and the ketamine esters (3a–19a, 3b–19b) provide an
opportunity to test these claims.
Of the 34 compounds studied (compound 17b was omitted due to its seizure-inducing effect),
12 showed no sedative or analgaesic activity at doses up to 200 mg/kg. All but one of these compounds
(9b) also showed no psychotomimetic properties, as judged by the behavioural dysfunction test.
They also showed much weaker inhibition of the NMDA receptor than ketamine (IC50 0.7 µM),
with IC50s ranging from 57 to >1000 (average IC50 350 µM).
In contrast, the 22 actively sedative compounds (including ketamine and the previously reported
ester analogues 5a and 5b) had a wide range of IC50s for inhibition of NMDA (from 0.7 to >1000 M) but
did have a much lower average IC50 (167 M). All but compounds 5a and 17a also showed analgaesic
µ
µ
activity (most at potencies <60 mg/kg). This is broadly consistent with some relationships between
these properties and NMDA inhibition. The majority of the active compounds were also much less
psychotomimetic than ketamine (behavioural dysfunction score 3), but this may be at least in part due
to the much faster recovery times for the esters (the main reason for this work). The shorter-chain ester
analogue 20a of tiletamine (2) was not active, but the longer-chain analogue 20b was also an effective
and relatively potent anaesthetics and analgaesics. However, 20b generated severe dysfunction on
awakening (score 4).
In terms of sedative activity structure-activity relationships for the benzene ring substituents,
the active (CH2)4CO2Me series compounds were on average about 2.5-fold more potent than the
i
corresponding (CH2)2CO2 Pr shorter-chain series, but the ring substituent effects were broadly similar
across both series. The 2- and 3-substituted compounds were generally more active than 4-substituted
i
compounds. The active anaesthetic compounds with the shorter (CH2)2CO2 Pr chain (4a
–
19a) included
all of the 2-substituted examples except 2-Me (6a), making this overall the favoured position for
substitution. The 3-Cl, 3-Me and 3-OMe compounds (10a 12a) and the 4-Cl and 4-OMe (15a 16a)
–
,
analogues were also active anaesthetics. Overall, the most generally acceptable substituent was
Cl, while the non-polar and powerful electron-withdrawing substituents CF3 and OCF3 were the
least successful. All of the compounds generated very little dysfunction in the rats during recovery
(averaged scores of mostly 0 or 1, of short duration), in contrast to ketamine (average score 3 for a
prolonged period).
Overall, this study has helped to define the SAR for this series of ketamine esters and provide
useful information towards selection of a clinical candidate.

Molecules 2020, 25, 2950
6 of 14
Conclusions
The above results show that the short chain aliphatic ester analogues of ketamine across the range
of different benzene ring substituted compounds broadly retain the parent’s desirable anaesthetic and
analgaesic properties, yet are sufficiently rapidly metabolised to minimise the drawbacks of ketamine
in this capacity. The structure activity relationships for the esters were not straightforward, the results
suggest the (CH2)4CO2Me series compounds were on average more active than the corresponding
i
(CH2)2CO2 Pr shorter-chain series. The 2- and 3-substituted compounds were generally more active
than 4-substituted compounds.
4. Experimental
4.1. Chemistry
All reagents and solvents were obtained from commercial suppliers and were used without
further purification. Reactions requiring anhydrous conditions were performed under nitrogen
atmospheres. Reactions were monitored by thin layer chromatography (TLC) on preloaded silica
gel F254 plates (Merck, Darmstadt, Germany). with a UV indicator. Column chromatography was
performed with Merck 230–400 mesh silica gel. 1H and 13C NMR spectra were obtained with a Bruker
Avance 400 spectrometer (Bruker, Zuerich, Switzerland) at 400 MHz for 1H and 101 MHz for 13C spectra.
Spectra were obtained in CDCl3 or (CD3)2SO. The chemical shifts are reported in parts per million
(δ) downfield using tetramethylsilane (SiMe4) as internal standard. Spin multiplicities are given as s
(singlet), d (doublet), dd (double doublet), br (broad), m (multiplet), and q (quartet). Coupling constants
(J values) were measured in hertz (Hz). All LC/MS data were gathered by direct injection of methanolic
solutions into a Surveyor MSQ mass spectrometer using an atmospheric pressure chemical ionisation
(APCI) with a corona voltage of 50 V and a source temperature of 400 ◦C. High-resolution electrospray
ionisation (HRESIMS) mass spectra were determined on a Bruker micrOTOFQ II mass spectrometer
(Bruker, Switzerland). Final products were analysed by reverse-phase HPLC (Alltima C18 5
150 mm 3.2 mm; Alltech Associated, Inc., Deerfield, IL, USA) using an Agilent HP1100 equipped with
a diode array detector. The mobile phase was 80% MeCN/20% H2O (v/v) in 45 mM HCO2NH4 at pH 3.5
and 0.5 mL/min. The purity was determined by monitoring at 272 nm and was 95% for final products
µm column,
×
≥
unless otherwise stated. DCM refers to dichloromethane, DMF refers to N,N-dimethylformamide,
EtOAc refers to ethyl acetate, EtOH refers to ethanol.
4.2. Synthesis of Ring-Substituted Norketamine Analogues. (Scheme 2)
2-Amino-2-(4-chlorophenyl)cyclohexan-1-one (22). A solution of 2-(4-chlorophenyl)cyclohexan-1-one
(
27d) (3.0 g, 14.4 mmol) and unsym.-dimethylhydrazine (3.46 g, 58.0 mmol), in EtOH (20 mL) was heated
to 96 ◦C in a sealed tube for 12 h. The reaction mixture was cooled to room temperature, filtered and the
solvent evaporated. The residue was purified by column chromatography on silica gel. Elution with
EtOAc/hexanes (0–40%) gave 2-(2-(4-chlorophenyl)cyclohexylidene)-1,1-dimethylhydrazine (28d) (3.2 g,
90%) as a pale yellow oil. 1HNMR (CDCl3)
δ 7.30–7.25 (m, 2H), 7.22–7.19 (m, 2H), 2.88–2.64 (dt,
J = 13.96 Hz, 4.56 Hz, 1H), 2.47 (s, 6 H), 2.36–2.26 (m, 1H), 2.20–2.10 (m, 1 H), 2.00–1.92 (m, 1H),
1.82–1.72 (m 1H), 1.70–1.48 (m, 4H); MS m/z 251.20 (MH+).
A solution of 28d (3.2 g, 12.8 mmol) and MeI (2.20 g, 15.4 mmol), in MeCN (20 mL) was heated in a
sealed tube to 40 ◦C for 2 h, followed by heating to 70 ◦C for 3 h. The reaction mixture was cooled
to room temperature, diluted with Et2O (60 mL) and left overnight in the fridge for the product
to crystallise out. The solid was filtered and dried under high vacuum to yield the desired salt
2-(2-(4-chlorophenyl)cyclohexylidene)-1,1,1-trimethylhydrazinium iodide (29d) (4.99 g, 99%) as pale
1
cream solid. HNMR (MeOD)
δ 7.32–7.29 (m, 2H), 7.25–7.23 (m, 2H), 3.82–3.79 (q, J = 4.8 Hz, 1H),
3.46 (s, 9 H), 3.20–3.10 (m, 1H), 2.78–2.68 (m, 1H) 2.30–2.08 (m, 3H), 2.20–1.78 (m, 3H); MS m/z 251.20
((MH-MeI)+).

Molecules 2020, 25, 2950
7 of 14
Sodium (0.33g, 14.5 mmol), was washed with hexane, dried, cut into small pieces and placed in EtOH
(40 mL) at r.t. The solution was stirred for approximately 20 min, until the sodium disappeared.
The quaternary salt 29d (5 g, 12 mmol) was added to the above solution and then it was refluxed for
1 h. The solution was cooled on ice and quenched with HCl (4 M, 40 mL). The ethanol was removed
under reduced pressure, the residue was diluted with water (20 mL) and neutralised with NaOH
(2 M) solution until pH 7. The aqueous layer was extracted with dichloromethane, MgSO4 dried and
concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting
with EtOAc/hexanes (30–100%) to obtain 2-amino-2-(4-chlorophenyl)cyclohexan-1-one (22) (1.87 g,
1
70%) as a pale yellow oil. HNMR (CDCl3)
δ
7.37–7.33 (m, 2H), 7.22–7.18 (m, 2H), 2.82–2.76 (m, 1H),
2.52–2.44 (m, 1H), 2.40–2.32 (m, 1H), 2.20–1.94 (m, 1H), 1.82–1.62 (m, 4H); MS m/z 224.20 (MH+).
Similarly were prepared:
2-Amino-2-phenylcyclohexan-1-one (21). Similar reaction of 2-phenylcyclohexan-1-one (27a) (2.43 g,
13.9 mmol) and unsym.-dimethylhydrazine gave 1,1-dimethyl-2-(2-phenylcyclohexylidene)hydrazine
1
(
28a) (2.62 g, 87%). HNMR (CDCl3)
δ
7.30–7.29 (m, 3H), 7.21–7.19 (m, 2H), 3.01–2.95(dt, J = 13.84 Hz
,
4.32 Hz, 1H), 2.50 (s, 6H), 2.33–2.32 (m, 1H), 2.07–1.91 (m, 2H), 1.81–1.70 (m, 2H), 1.69–1.60 (m, 1H),
1.59–1.52 (m, 2H); MS m/z 217.30 (MH+). Reaction of 28a (2.62 g, 12.1 mmol) and methyl iodide
as above gave 1,1,1-trimethyl-2-(2-phenylcyclohexylidene)hydrazin-1-ium iodide (29a) (3.0 g, 70%).
1HNMR (MeOD)
δ 7.35–7.30 (m, 2H), 7.26–7.20 (m, 3H), 3.82–3.78 (dd, J = 9.49 Hz, 4.64 Hz, 1H), 3.47
(s, 9H), 3.05–2.89 (m, 1H), 2.82–2.76 (m, 1H), 2.36–2.30 (m, 1H), 2.18–2.15 (m, 1H), 2.08–2.03 (m, 1H),
1.97–1.79 (m, 3 H); MS m/z 217.2 ((MH-MeI)+). Reaction of 29a (2.87 g, 8.0 mmol) with Na/EtOH as
1
above then gave 21 (0.90 g, 60%). HNMR (CDCl3)
δ 7.40–7.36 (m, 2H), 7.31–7.24 (m, 3H), 2.88–2.84 (m,
1H), 2.45–2.38 (m, 2H), 2.00–1.98 (m, 1H), 1.79–1.72 (m, 4H); MS m/z 190.20 (MH+).
2-Amino-2-(2-fluorophenyl)cyclohexan-1-one (30b). Similar reaction of 2-(2-fluorophenyl)cyclohexan
-1-one (27b) (0.77 g, 4.0 mmol) and unsym.-dimethylhydrazine gave 2-(2-(2-fluorophenyl)cyclohexylidene)
-1,1-dimethylhydrazine (28b) (0.72g, 82%) 1HNMR (CDCl3)
δ 7.26–7.15 (m, 2H), 7.08–7.00 (td,
J = 6.24 Hz, 1.24 Hz, 1 H), 6.98–6.95 (m, 1H), 3.86–3.74 (m, 1H), 3.10–3.04 (dt, J = 13.76 Hz,
4.40 Hz), 2.32 (s, 6 H), 2.28–2.19 (m, 2H), 2.08–1.98 (m, 2H), 1.90–1.78 (m, 2H), 1.68–1.56 (m,
2H); MS m/z 235.20 (MH+). Reaction of (28b) (0.66 g, 2.80 mmol) and methyl iodide as above
gave 2-(2-(2-fluorophenyl)cyclohexylidene)-1,1,1-trimethylhydrazin-1-ium iodide (29b) (0.97 g, 94%)
1HNMR (MeOD)
δ 7.31–7.24 (m, 2H), 7.15–7.11 (td, J = 7.56 Hz, 1.20 Hz, 1H), 7.07–7.02 (m, 1H),
4.01–3.97 (t, J = 8.52 Hz, 1H), 3.42 (s, 9H), 2.71–2.63 (m, 1H), 2.23–2.20 (m, 3H), 2.04–2.00 (m, 1H),
1.84–1.79 (m, 3H); MS m/z 235.30((MH-MeI)+). Reaction of 29b (0.97 g, 2.60 mmol) with Na/EtOH
as above then gave 30b (0.37 g, 70%). 1HNMR (CDCl3)
δ 7.53–7.48 (td, J = 7.80 Hz, 1.72 Hz, 1H),
7.33–7.28 (m, 1H), 7.22–7.18 (td, J = 7.68 Hz, 1.32 Hz, 1H), 7.07–7.02 (dd, J = 8.16 Hz, 1.24 Hz, 1H),
2.80–2.75 (m,1 H), 2.57–2.52 (m, 1H), 2.48–2.43 (m, 1H), 2.00–1.98 (m, 1H), 1.83–1.65 (m, 4H); MS m/z
208.20 (MH+).
2-Amino-2-(3-chlorophenyl)cyclohexan-1-one (30c). Similar reaction of 2-(3-chlorophenyl)cyclohexan
-1-one (27c) (3.0 g, 14.4 mmol) and unsym.-dimethylhydrazine gave 2-(2-(3-chlorophenyl)cyclohexylidene)
-1,1-dimethylhydrazine (28c) (3.2 g, 90%) as pale yellow oil. 1HNMR (CDCl3)
δ 7.30–7.25
(m, 2H), 7.22–7.19 (m, 2H), 2.88–2.64 (dt, J = 13.96 Hz, 4.56 Hz, 1H), 2.47 (s, 6 H),
2.36–2.26 (m, 1H), 2.20–2.10 (m, 1 H), 2.00–1.92 (m, 1H), 1.82–1.72 (m 1H), 1.70–1.48 (m, 4H);
MS m/z 251.20 (MH+). Reaction of 28c (3.2 g, 12.8 mmol) and methyl iodide as above gave
2-(2-(3-chlorophenyl)cyclohexylidene)-1,1,1-trimethylhydrazinium iodide (29c) (4.99 g, 99%) as a
solid. 1HNMR (MeOD)
δ 7.29–7.27 (m, 2H), 7.25–7.24 (m, 1H), 7.19–7.17 (m, 1H), 3.84–3.79 (q,
J = 5.12 Hz, 1H), 3.46 (s, 9H), 3.20–3.15 (m, 1H), 2.70–2.69 (m, 1 H), 2.22–2.12 (m, 3H), 1.98–1.96 (m, 1H),
1.84–1.82 (m, 2H); MS m/z 251.20 ((MH-MeI)+). Treatment of 29c (3.8 g, 9.6 mmol) with Na/EtOH as
1
above then gave 30c 1.8 g, 84%) as an yellow oil. H NMR (CDCl3) δ 7.29–7.28 (m, 2 H), 2.27–7.26 (m,

Molecules 2020, 25, 2950
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1 H), 7.14–7.11 (dt, J = 7.36 Hz, 1.68 Hz, 1H), 2.82–2.78 (m, 1H), 2.52–2.48 (m, 1H), 2.40–2.36 (m, 1H),
2.04–2.00 (m, 1H), 1.80–1.72 (m, 4H); MS m/z 224.20 (MH)+.
2-Amino-2-(o-tolyl)cyclohexan-1-one (30e). Similar reaction of 2-(o-tolyl)cyclohexan-1-one 27e (3.0 g,
16.0 mmol) and unsym.-dimethylhydrazine gave 1,1-dimethyl-2-(2-(o-tolyl)cyclohexylidene)hydrazine
(
28e) (3.65 mg, 99%). 1HNMR (CDCl3)
δ 7.25–7.17 (m, 1H), 7.16–7.13 (m, 1H), 7.12–7.08 (m, 1H),
3.16–3.11 (dt, J = 10.4 Hz, 4.44 Hz, 1H), 2.34 (s, 6H), 2.26 (s, 3H), 2.12–2.02 (m, 2H), 2.00–1.82 (m,
3 H), 1.68–1.58 (m, 3H); MS m/z 231.30 (MH)+. Reaction of 28e (3.65 g, 16.0 mmol) and methyl iodide
as above gave 1,1,1-trimethyl-2-(2-(o-tolyl)cyclohexylidene)hydrazin-1-ium iodide (29e) (5.1 g, 86%).
1HNMR (MeOD)
δ 7.20–7.18 (m, 1H), 7.16–7.08 (m, 3H), 3.95–3.91 (m, 1H), 3.39 (s, 9H), 2.74–2.66 (m,
1H), 2.26–2.21 (m, 3H), 2.24 (s, 3H), 2.10–1.98 (m, 1H), 1.92–1.78 (m, 2H); MS m/z 231.30 ((MH-MeI)+).
1
Reaction of 29e (5.10 g, 13.7 mmol) with Na/EtOH as above then gave 30e (2.0 g, 72%). HNMR (CDCl3)
δ
7.56–7.54 (dd, J = 8.76 Hz, 1.12 Hz, 1H), 7.27–7.18 (m, 3H), 2.92–2.88 (m, 1H), 2.52–2.36 (m, 1H), 2.17
(s, 3H), 2.04–1.93 (m, 2H), 1.76–1.72 (m, 4H); MS m/z 204.20 (MH)+.
2-Amino-2-(m-tolyl)cyclohexan-1-one (30f). Similar reaction of 2-(m-tolyl)cyclohexan-1-one (27f) (0.89 g,
4.70 mmol) and unsym.-dimethylhydrazine gave 1,1-dimethyl-2-(2-(m-tolyl)cyclohexylidene)hydrazine
(
28f) (0.9 g, 90%). 1HNMR (CDCl3)
δ 7.23–7.16 (m, 1H), 7.12–7.08 (m, 1H), 7.04–6.98 (m, 2H),
3.02–2.96 (dt, J = 9.52 Hz, 4.21 Hz, 1H), 2.52 (s, 6H), 2.48 (s, 3H), 2.08–1.88 (m, 2H), 1.86–1.50
(m, 6H); m/z 231.3 (MH+). Reaction of 28f (0.9 g, 3.90 mmol) and methyl iodide as above gave
1
1,1,1-trimethyl-2-(2-(m-tolyl)cyclohexylidene)hydrazin-1-ium iodide (29f) (1 g, 70%). HNMR (MeOD)
δ
7.21–7.17 (t, J = 7.64 Hz, 1H), 7.07–7.04 (t, J = 6.92 Hz, 3H), 3.78–3.74 (dd, J = 9.24 Hz, 4.6 Hz, 1H), 3.48
(s, 9H), 3.02–2.96 (m, 1H), 2.86–2.74 (m, 1H), 2.40–2.30 (m, 1H), 2.36 (s, 3H), 2.18–2.00 (m, 2H), 2.00–1.76
(m, 3H); MS m/z 231.20 ((MH-MeI)+). Reaction of 29f (0.83 g, 2.23 mmol) with Na/EtOH as above then
1
gave 30f (0.3 g, 67%). HNMR (CDCl3)
δ 7.28–7.24 (t, J = 8.3 Hz, 1H), 7.11–7.09 (m, 1H), 7.07–7.05 (m,
2H), 2.90–2.82 (m, 1H), 2.48–2.40 (m, 2H), 2.34 (s, 3H), 2.04–1.98 (m, 1H), 1.82–1.62 (m, 4H); MS m/z
204.2 (MH)+.
2-Amino-2-(p-tolyl)cyclohexan-1-one (30g). Similar reaction of 2-(p-tolyl)cyclohexan-1-one (27g) (3 g,
16.0 mmol) and unsym.-dimethylhydrazine gave 1,1-dimethyl-2-(2-(p-tolyl)cyclohexylidene)hydrazine
1
(
28g) (3.07 g, 83%). H NMR (CDCl3)
δ
7.18–7.16 (m, 2H), 7.14–7.10 (m, 2H), 2.99–2.94 (dt, J = 9.8 Hz
,
4.28 Hz, 1H), 2.50 (s, 6H), 2.30 (s, 3H), 2.08–1.89 (m, 2H), 1.82–1.60 (m, 4H), 1.60–1.49 (m, 2 H);
MS m/z 231.20 (MH)+. Reaction of 28g (3.07 g, 13.3 mmol) and methyl iodide as above gave
1
2-(2-(p-tolyl)cyclohexylidene)-1,1,1-trimethylhydrazinium iodide (29g) (3.72 g, 75%). H NMR (MeOD)
δ
7.24–7.16 (m, 1H), 7.15–7.13 (m, 3H), 3.77–3.74 (m, 1H), 3.48 (s, 9H), 3.02–2.94 (m, 1H), 2.84–2.74 (m,
1H), 2.40–2.32 (m, 1H), 2.30 (s, 3H), 2.19–2.08 (m, 1H), 2.06–1.98 (m, 1H), 1.94–1.84 (m, 2H), 1.86–1.76
(m, 1H); MS m/z 231.20 ((MH-MeI)+). Reaction of 29g (3.72 g, 10.0 mmol) with Na/EtOH as above then
1
gave 30g (1 g, 50%). HNMR (CDCl3)
δ 7.20–7.18 (m, 2 H), 7.14–7.12 (m, 2H), 2.84 (br s, 1H), 2.35–2.30
(m, 2H), 2.34 (s, 3H), 2.20–2.00 (m, 1H), 1.80–1.70 (m, 4H); MS m/z 204.20 (MH)+.
2-Amino-2-(2-methoxyphenyl)cyclohexan-1-one (30h) Similar reaction of 2-(2-methoxyphenyl)
.
cyclohexan-1-one (27h) (3.0 g, 14.7mmol) and unsym.-dimethylhydrazine gave 2-(2-(2-methoxyphenyl)
1
cyclohexylidene)-1,1-dimethylhydrazine (28h) (3.0 g, 83%). HNMR (CDCl3)
δ 7.19–7.14 (m, 2H),
6.92–6.82 (m, 2H), 3.77 (s, 3H), 3.00–2.94 (m, 1H), 2.58–2.38 (m, 1H), 2.38 (s, 4H), 2.22–2.12 (m, 1H), 2.02–1.98
(m, 2H), 1.82–1.70 (m, 2H), 1.70–1.58 (m, 2H); MS m/z 247.20 (MH+). Reaction of 28h (3.0 g, 12.2 mmol)
and methyl iodide as above gave 2-(2-(2-methoxyphenyl)cyclohexylidene)-1,1,1-trimethylhydrazin-1-ium
1
iodide (29h) (4.31 g, 91%). HNMR (MeOD)
δ
7.24–7.17 (m, 2H), 6.94–6.88 (m, 2H), 4.01–3.97 (m, 1H), 3.82
(s, 3H), 3.38 (s, 9H), 2.70–2.62 (m, 2H), 2.22–2.14 (m, 2H), 2.08–2.00 (m, 2H), 1.90–1.74 (m, 2H); MS m/z
247.20 ((MH-MeI)+). Reaction of 29h (1.00 g, 2.60 mmol) with Na/EtOH as above then gave 30h (0.30 g,
1
53%). HNMR (CDCl3)
δ
7.54–7.52 (dd, J = 7.76 Hz, 1.6 Hz, 1H), 7.32–7.27 (td, J = 7.48 Hz, 1.6 Hz, 1H),
7.06–7.01 (td, J = 7.6 Hz, 1.16 Hz, 1H), 6.90–6.87 (dd, J = 8.29 Hz, 0.92 Hz, 1H), 3.73 (s, 3H), 2.40–2.31 (m,
2H), 1.99–1.92 (m, 2H), 1.76–1.58 (m, 4H); MS m/z 220.20 (MH+).

Molecules 2020, 25, 2950
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2-Amino-2-(3-methoxyphenyl)cyclohexan-1-one (30i). Similar reaction of 2-(3-methoxyphenyl)
cyclohexan-1-one 27i (1.0 g, 4.90 mmol) and unsym.-dimethylhydrazine gave
2-(2-(3-methoxyphenyl)cyclohexylidene)-1,1-dimethylhydrazine (28i) (1.2 g, 100%).
1HNMR
(CDCl3) 7.26–7.22 (m, 1H), 6.90–6.81 (m, 1H), 6.78–6.68 (m, 2H), 3.76 (s, 3H), 3.04–2.98 (dt, J = 13.81
(
)
δ
Hz, 4.17 Hz, 1 H), 2.49 (s, 6H), 2.06–1.88 (m, 2H), 1.82- 1.70 (m, 2H), 1.70–1.62 (m, 2H), 1.60–1.50
(m, 2 H); MS m/z 247.20 (MH+). Reaction of 28i (1.2 g, 4.88 mmol) and methyl iodide as above gave
1
2-(2-(3-methoxyphenyl)cyclohexylidene)-1,1-dimethylhydrazine (29i) (1.20 g, 63%). HNMR (MeOD)
δ
7.26–7.21 (td, J = 9.12 Hz, 1.52 Hz, 1 H), 6.85–6.79 (m, 3H), 3.78 (s, 3H), 3.49 (s, 9H), 3.04–2.98 (m, 1H),
2.83–2.77 (m, 1H), 2.70–2.58 (m, 1H), 2.40–2.32 (m, 1H), 2.20–2.10 (m, 1H), 2.10–2.00 (m, 1H), 2.00–1.80
(m, 3H); MS m/z 247.20 ((MH-MeI)+). Reaction of 29i (0.67 g, 1.72 mmol) with Na/EtOH as above then
gave 30i (0.20 g, 54%).1H NMR (CDCl3) 1HNMR (CDCl3)
δ 7.31–7.26 (m, 1H), 6.84–6.82 (m, 3H), 3.79 (s,
3H), 2.83–2.81 (m, 1H), 2.44–2.40 (m, 2H), 2.04–1.95 (m, 2H), 1.76–1.71 (m, 3H); MS m/z 220.20 (MH+).
2-Amino-2-(2-(trifluoromethyl)phenyl)cyclohexan-1-one (30k). Similar reaction of 2-(2-(trifluoromethyl)
phenyl)cyclohexan-1-one (27k) (1.28 g, 5.30 mmol) and unsym.-dimethylhydrazine gave
1,1-dimethyl-2-(2-(2-(trifluoromethyl)phenyl)cyclohexylidene)hydrazine (28k) (1.20, 80%) 1HNMR
(CDCl3)
δ 7.60–7.58 (d, J = 7.92 Hz, 1H), 7.46–7.42 (m, 2H), 7.29–7.27 (m, 1H), 3.82–3.78 (dd,
J = 12.25 Hz, 4.36 Hz, 1H), 3.44–3.39 (m, 1H), 2.24 (s, 6H), 2.10–1.80 (m, 5H), 1.72–1.48 (m, 3H);
MS m/z 285.20 (MH+). Reaction of 28k (1.00 g, 3.50 mmol) and methyl iodide as above gave
1,1,1-trimethyl-2-(2-(2-(trifluoromethyl)phenyl)cyclohexylidene)hydrazin-1-ium iodide (29k) (1.44 g,
96%) 1HNMR (MeOD)
δ 7.67–7.65 (d, J = 7.88 Hz, 1H), 7.62–7.52 (m, 2H), 7.44–7.40 (m, 1H), 4.09–4.05
(dd, J = 7.88 Hz, 4.36 Hz, 1H), 3.38 (s, 9H), 2.68–2.54 (m, 1H), 2.38–2.22 (m, 2H), 2.10–1.96 (m, 2H),
1.90–1.82 (m, 2H), 1.76–1.62 (m, 1H); MS m/z 285.20((MH-MeI)+). Reaction of 29k (1.44 g, 3.40 mmol)
1
with Na/EtOH as above then gave 30k (0.62 g, 72%). HNMR (CDCl3)
δ 7.98–7.96 (d, J = 8.05 Hz, 1H),
7.71–7.68 (dd, J = 7.88 Hz, 1.2 Hz, 1H), 7.61–7.57 (td, J = 7.52 Hz, 0.68 Hz, 1H), 7.52–7.42 (t, J = 7.64 Hz,
1H), 2.76–2.68 (m, 1H), 2.57–2.42 (m, 1H), 1.98–1.92 (m, 3H), 1.87–1.77 (m, 5H); MS m/z 258.20 (MH+).
2-Amino-2-(3-(trifluoromethyl)phenyl)cyclohexan-1-one (30l). Similar reaction of 2-(3-(trifluoromethyl)
phenyl)cyclohexan-1-one (27l) (1.28 g, 5.30 mmol) and unsym.-dimethylhydrazine gave
1,1-dimethyl-2-(2-(3-(trifluoromethyl)phenyl)cyclohexylidene)hydrazine (28l) (1.2 g, 71%) 1HNMR
(CDCl3)
δ 7.54–7.50 (m,1H), 7.49–7.37 (m, 3H), 3.70–3.67 (t, J = 5.2Hz, 1H), 2.47 (s,
6H), 2.33–2.20 (m, 2H), 2.08–1.98 (m, 1H), 1.94–1.66 (m, 3H), 1.56–1.44 (m, 2H); MS m/z
285.20 (MH+). Reaction of 28l (1.07 g, 2.80 mmol) and methyl iodide as above gave
1,1,1-trimethyl-2-(2-(3-(trifluoromethyl)phenyl)cyclohexylidene)hydrazin-1-ium iodide (29l) (1.40 g,
86%) 1HNMR (MeOD)
δ 7.55–7.52 (m, 4H), 3.94–3.90 (m, 1H), 3.44 (s, 9H), 2.73–2.69 (m, 1H), 2.53–2.50
(m, 1H), 2.24–2.18 (m, 3H), 2.03–1.98 (m, 1H), 1.88–1.82 (m, 2H); MS m/z 285.20((MH-MeI)+). Reaction of
1
29l (1.40 g, 3.30 mmol) with Na/EtOH as above then gave 30l (0.52 g, 62%). HNMR (CDCl3)
δ
7.58–7.55
(m, 1H), 7.52–7.49 (t, J = 7.72 Hz, 2H), 7.45–7.43 (m, 1H), 2.88–2.82 (m, 1H), 2.58–2.50 (m, 1H), 2.38–2.30
(m, 1H), 2.06–1.98 (m, 1H), 1.92–1.80 (m, 3H), 1.80–1.70 (m, 1H); MS m/z 258.20 (MH+).
2-Amino-2-(4-(trifluoromethyl)phenyl)cyclohexan-1-one (30m). Similar reaction of 2-(4-(trifluoromethyl)
phenyl)cyclohexan-1-one (27m) (1.28 g, 5.30 mmol) and unsym.-dimethylhydrazine gave
1,1-dimethyl-2-(2-(4-(trifluoromethyl)phenyl)cyclohexylidene)hydrazine (28m) (1.0 g, 76%) 1HNMR
(CDCl3)
δ 7.58–7.54 (m, 2H), 7.40–7.37 (d, J = 8.56 Hz, 2H), 3.70–3.67 (t, J = 5.16 Hz, 1H), 2.86–2.78
(m, 1H), 2.48 (s, 6H), 2.38–2.28 (m, 1H), 2.28–2.18 (m, 1H), 2.20–1.96 (m, 1H), 1.82–1.60 (m, 4H);
MS m/z 285.20 (MH+). Reaction of 28m (1.00 g, 3.50 mmol) and methyl iodide as above gave
1,1,1-trimethyl-2-(2-(4-(trifluoromethyl)phenyl)cyclohexylidene)hydrazin-1-ium iodide (29m) (1.22,
82%) 1HNMR (MeOD)
δ 7.65–7.59 (d, J = 13.36 Hz, 2H), 7.46–7.44 (d, J = 8.44 Hz, 2H), 4.84 (s, 9H),
3.92–3.88 (q, J = 4.88 Hz, 1H), 2.74–2.69 (m, 1H), 2.53–2.49 (m, 1H), 2.22–1.97 (m, 3H), 1.93–1.88 (m,
1H), 1.88–1.84 (m, 2H); MS m/z 285.20((MH-MeI)+). Reaction of 29m (1.22 g, 2.80 mmol) with Na/EtOH
1
as above then gave 30m (0.48 g, 66%). HNMR (CDCl3)
δ
7.65–7.63 (d, J = 8.25 Hz, 2H), 7.41–7.39 (d,

Molecules 2020, 25, 2950
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J = 8.20 Hz, 2H), 2.88–2.80 (m, 1H), 2.58–2.50 (m, 1H), 2.40–2.32 (m, 1H), 2.04–1.98 (m, 1H), 1.88–1.72
(m, 4H); MS m/z 258.20 (MH+).
2-Amino-2-(2-(trifluoromethoxy)phenyl)cyclohexan-1-one (30n). Similar reaction 2-(2-(trifluoromethoxy)
phenyl)cyclohexan-1-one (27n) (1.11 g, 5.30 mmol) and unsym.-dimethylhydrazine gave
2-(2,2-dimethylhydrazono)-1-(2-(trifluoromethoxy)phenyl)cyclohexan-1-amine (28n) (1.0 g, 78%)
1HNMR (CDCl3)
δ
7.32–7.29 (m, 1H), 7.26–7.18 (m, 3H), 3.79–3.76 (dd,
J = 12.40
Hz, 4.16 Hz, 1H), 2.27 (s, 6H), 2.04–1.82 (m, 5H), 1.72–1.52 (m, 3H); MS m/z
301.20 (MH+). Reaction of 28n (1.00 g, 3.30 mmol) and methyl iodide as above gave
2-(2-amino-2-(2-(trifluoromethoxy)phenyl)cyclohexylidene)-1,1,1-trimethylhydrazin-1-ium iodide
(
29n) (1.35g, 92%) 1HNMR (MeOD)
δ 7.44–7.42 (m, 1H), 7.36–7.32 (m, 2H), 7.28–7.26 (m, 1H), 4.07–4.03
(m, 1H), 3.40 (s, 9H), 2.69–2.66 (m, 1H), 2.26–2.19 (m, 3H), 2.06–2.03 (m, 2H), 1.84–1.79 (m, 2H); MS m/z
301.20((MH-MeI)+). Reaction of 29n (1.35 g, 3.05 mmol) with Na/EtOH as above then gave 30n (0.42 g,
1
52%). HNMR (CDCl3)
δ
7.69–7.67 (m, 1H), 7.39–7.34 (m, 2H), 7.29–7.26 (m, 1H), 2.77–2.71 (m, 1H),
2.58–2.52 (m, 1H), 2.45–2.38 (m, 1H), 1.82–1.77 (m, 1H), 1.82–1.62 (m, 4H); MS m/z 274.20 (MH+).
2-Amino-2-(3-(trifluoromethoxy)phenyl)cyclohexan-1-one (30o). Similar reaction of 2-(3-(trifluoromethoxy)
phenyl)cyclohexan-1-one (27o) (1.62 g, 5.40 mmol) and unsym.-dimethylhydrazine gave
2-(2,2-dimethylhydrazono)-1-(3-(trifluoromethoxy)phenyl)cyclohexan-1-amine (28o) (2.0 g, 84%)
1HNMR (CDCl3)
δ 7.37–7.31 (m, 1H), 7.22–7.18 (m, 1H), 7.14–7.11 (m, 1H), 7.08–7.04 (m, 1H), 3.68–3.64
(t, J = 5.0 Hz, 1H), 2.48 (s, 6H), 2.34–2.28 (m, 1H), 2.20–2.10 (m, 1H), 2.06–1.96 (m, 1H), 1.94–1.88 (m,
1H), 1.80–1.50 (m, 4H); MS m/z 301.20 (MH+). Reaction of 28o (1.62 g, 5.40 mmol) and methyl iodide as
above gave 2-(2-amino-2-(3-(trifluoromethoxy)phenyl)cyclohexylidene)-1,1,1-trimethylhydrazin-1-ium
iodide (29o) (2.00 g, 84%) 1HNMR (MeOD)
δ 7.40–7.38 (t, J = 7.97 Hz, 1H), 7.30–7.26 (m, 1H), 7.22–7.18
(m, 1H), 7.17–7.11 (m, 1H), 3.89–3.85 (t, J = 7.73 Hz, 1H), 3.44 (s, 9H)HH, 2.52–2.44 (m, 1H), 2.24–2.14
(m, 2H), 2.04–1.96 (m, 2H), 1.92–1.78 (m, 2H), 1.70–1.60 (m, 1H); MS m/z 301.20((MH-MeI)+). Reaction
1
of 29o (2.00 g, 4.50 mmol) with Na/EtOH as above then gave 30o (0.60 g, 50%). HNMR (CDCl3)
δ
7.43–7.41 (td, J = 7.93 Hz, 0.96 Hz, 1H), 7.18–7.16 (m, 3H), 2.82–2.76 (m, 1H), 2.54–2.48 (m, 1H), 2.40–2.32
(m, 1H), 2.04–1.98 (m, 1H), 1.90–1.70 (m, 4H); MS m/z 274.20 (MH+).
2-Amino-2-(4-(trifluoromethoxy)phenyl)cyclohexan-1-one (30p). Similar reaction of 2-(4-(trifluoromethoxy)
phenyl)cyclohexan-1-one (27p) (2.24 g, 8.68 mmol) and unsym.-dimethylhydrazine gave
2-(2,2-dimethylhydrazono)-1-(4-(trifluoromethoxy)phenyl)cyclohexan-1-amine (28p) (2.0 g, 76%)
1HNMR (CDCl3)
δ 7.30–7.27 (m, 2H), 7.17–7.13 (m, 2H), 3.67–3.64 (t, J = 7.13 Hz, 1H),
2.89–2.58 (m, 1H), 2.45 (s, 6H), 2.32–2.14 (m, 2H), 2.02–1.94 (m, 1H) 1.80–1.58 (m, 4H);
MS m/z 301.20 (MH+). Reaction of 28p (1.82 g, 6.10 mmol) and methyl iodide as above
gave 2-(2-amino-2-(4-(trifluoromethoxy)phenyl)cyclohexylidene)-1,1,1-trimethylhydrazin-1-ium iodide
(
29p) (2.62 g, 98%) 1HNMR (MeOD)
δ 7.36–7.34 (m, 2H), 7.22–7.20 (d, J = 7.92 Hz, 2H), 3.89–3.85 (m,
1H), 3.46 (s, 9H), 2.78–2.70 (m, 1H), 2.58–2.49 (m, 1H), 2.30–2.10 (m, 3H), 2.00–1.80 (m, 3H); MS m/z
301.20((MH-MeI)+). Reaction of 29p (2.62 g, 5.90 mmol) with Na/EtOH as above then gave 30p (0.80 g,
1
50%). HNMR (CDCl3)
δ
7.32–7.29 (m, 2H), 7.23–7.21 (m, 2H), 2.84–2.78 (m, 1H), 2.52–2.49 (m, 1H),
2.40–2.34 (m, 1H), 2.06–1.98 (m, 1H), 1.88–1.62 (m, 4H); MS m/z 274.20 (MH+).
4.3. Synthesis of Nortiletamine (35) (Scheme 3).
N-(Benzoyloxy)-N-(cyclohex-1-en-1-yl)-2,2,2-trifluoroacetamide (32). To a solution of cyclohexanone
oxime (31) (5.5 g, 48.6 mmol) in n-hexane:DCM (160 mL, 10:1) was added pyridine (3.84 g, 48.6 mmol)
and benzoyl chloride (6.8 g, 48.6 mmol) dropwise at room temperature. The mixture was stirred at room
temperature for 4 h and then diluted with water (100 mL). The organic layer was washed with water
(3
×
70 mL), dried (MgSO4) and concentrated in vacuo to afford the corresponding O-benzoyloxime
ether (10.17 g, 96%), which was used in the subsequent reaction without purification. A solution
of this in DCM (70 mL) was treated with TFAA (35 mL) dropwise at 0 ◦C. The mixture was stirred

Molecules 2020, 25, 2950
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at room temperature for 12 h and then concentrated in vacuo. The residue was purified by flash
1
chromatography (hexane-EtOAc, 50:1) to give enamide 32 (13 g, 89%). HNMR (CDCl3)
δ 8.12–8.08 (d,
J = 7.5 Hz, 2H), 7.66–7.59 (m, 1H), 7.51–7.44 (m, 2H), 6.36–6.20 (br m, 1H), 2.37–2.21 (m, 4H), 1.79–1.71
(m, 2H), 1.66–1.58 (m, 2H); MS m/z 314.10 (MH+).
2-(Thiophen-2-yl)-2-(2,2,2-trifluoroacetamido)cyclohexyl benzoate (33). A solution of thiophene
(2.67 g, 31.9 mmol) in THF (10 mL), was treated with n-BuLi (2M in cyclohexane, 15.95 mL, 31.9 mmol)
dropwise at –78 ◦C. The mixture was stirred at 0 ◦C for 30 min and then Et2AlCl (1M in hexane, 31.9 mL,
31.9 mmol) was added. The mixture was then stirred for additional 30 min at room temperature.
The resulting diethyl(thiophene)aluminium reagent was used directly in the subsequent reaction.
A solution of enamide 32 (5 g, 15.9 mmol) in THF (60 mL) was added dropwise to the above-generated
aluminium reagent at room temperature. The reaction was stirred under reflux for 3 h and quenched
with 1.3 M aq. Rochelle’s salt (150 mL). The aqueous layer was DCM extracted (3x 100 mL) and
the combined organic layers were washed with water (100mL). The residue was purified by flash
1
chromatography (hexane-EtOAc, 5:1) to give 33 (4.14 g, 66%). HNMR (CDCl3)
δ 7.95–7.93 (dm,
J = 8.5 Hz, 2H), 7.61–7.57 (tm, J = 7.5 Hz, 1H), 7.48–7.44 (tm, J = 7.0 Hz, 2H), 7.16–7.14 (dd, J = 5.0 Hz,
1.5 Hz, 1H), 7.00–6.98 (dd, J = 3.5 Hz, 1.5 Hz, 2H), 6.89–6.87 (dd, J = 5.0 Hz, 3.5 Hz, 1H), 5.29–5.26 (dd,
J = 10.5 Hz, 4.0 Hz, 1H), 3.32–3.28 (dm, J = 14.5 Hz, 1H), 2.16–2.04 (m, 2H), 1.89–1.67 (m, 3H), 1.62–1.39
(m, 2H); MS m/z 396.1 (M-H+).
2-Amino-2-(thiophen-2-yl)cyclohexan-1-ol (34). The O-benzoyl ester 33 (4.14 g, 10.4 mmol) was
stirred with 5% NaOH in MeOH (200 mL) at room temperature for 16 h. The solvent was then removed
in vacuo, the residue was dissolved in DCM (100 mL) and washed with water (3
×
200 mL). The residue
was purified with flash chromatography eluting with EtOAc (100%) to yield amino alcohol 34 (1.8 g,
1
88%). HNMR (CDCl3)
δ
7.21–7.19 (dm, J = 5.0 Hz, 1H), 7.00–6.98 (m, 2H), 3.87–3.83 (dd, J = 9.5 Hz,
4 Hz, 1 H), 2.00–1.92 (m, 1H), 1.86–1.73 (m, 2H), 1.70–1.32 (m, 5H); MS m/z 198.2 (MH+).
2-Amino-2-(thiophen-2-yl)cyclohexan-1-one (nortiletamine) (35). A solution of amino alcohol 34
(0.79 g, 4.0 mmol) in acetone (180 mL) was treated slowly with Jones reagent (2.5 M, 4.0 mmol, 1.6 mL) at
room temperature. The reaction was stirred at room temperature for 30 min, filtered and concentrated in
vacuo. The residue was diluted with water and neutralised with 2.0 M aq. NaOH solution. The aqueous
layer was extracted with Et2O (3x 60 mL), dried with MgSO4, concentrated in vacuo and purified
1
with flash chromatography EtOAc (100%) to yield 35 (0.66 g, 84%). HNMR (CDCl3)
δ
7.29–7.28 (dd,
J = 5.0 Hz, 1.0 Hz, 1H), 6.97–6.96 (dd, J = 5.0 Hz, 3.5 Hz, 1H), 6.82–6.81 (dd, J = 3.5 Hz, 1.0 Hz, 1H),
2.74–2.66 (m, 1H), 2.66–2.48 (m, 2H), 2.10–1.90 (m, 2H), 1.90–1.80 (m, 2H), 1.78–1.62 (m, 1H); MS m/z
196.2 (MH+).
4.4. Synthesis of Ketamine Esters (Example) (Scheme 1).
Methyl 5-((1-(4-chlorophenyl)-2-oxocyclohexyl)amino)pentanoate (15b). A solution of 22 (0.9 g,
4.03 mmol), methyl 5-bromovalerate (1.02 g, 5.2 mmol), KI (0.23 g, 1.4 mmol), K2CO3 (1.67 g, 12.0 mmol)
in MeCN (20 mL) was heated to a 112 ◦C in sealed tube for 20 h. The reaction mixture was cooled
to room temperature, filtered and the solvent evaporated. The residue was purified by column
chromatography on silica gel eluting with EtOAc/hexanes (30–60%) to obtain 15b (1 g, 74%) as pale
yellow oil. This was dissolved in Et2O (10 mL) and cooled to 0 ◦C, HCl in Et2O (2M, 4.45 mmol) were
added dropwise. The solvent was evaporated under reduced pressure, the residue was taken up in
EtOAc (5 mL) and sonicated at room temperature for 2 min. The white precipitate was diluted with
EtOAc (5 mL), filtered washed with EtOAc and dried under vacuum to give 15b as the solid HCl
1
salt. HNMR (CDCl3)
δ 7.34–7.28 (m, 2H), 7.19–7.16 (m, 2H), 3.64 (s, 3 H), 2.82–2.72 (m, 1H), 2.48–2.38
(m, 1H), 2.38–2.32 (m, 1H), 2.28–2.18 (m, 3H), 2.08–1.92 (m, 4H), 1.88–1.78 (m, 2H), 1.78–1.64 (m, 2H),
1.48–1.32 (m, 2H); 13C (CDCl3)
210.99, 174.29, 138.32, 133.47, 129.09, 128.67, 69.59, 51.74, 41.90, 39.79,
δ

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36.74, 33.88, 30.20, 27.70, 22.76, 22.38; MS m/z 338.20 (MH+). Calculated for C18H24ClNO3 (MH+)
338.15175, found 338.15170.
The other compounds of Table S1 were prepared similarly. See Supplementary Information
for details.
4.5. Biology
4.5.1. Animals
All animal experiments were conducted at the Ruakura Research Centre, Hamilton, New Zealand,
using experimental protocols reviewed and approved by the Ruakura Animal Ethics Committee
(ethics ref 12604/13786). Adult female Sprague-Dawley rats of approximately 250–350 g were evaluated
in both anaesthetic and analgaesic study protocols. All study agents were delivered by tail vein cannula
connected via minibore extension tubing to mechanised infusion pump.
4.5.2. Anaesthetic Assessment Protocol
Following acquisition of baseline physiologic parameters (heart rate, respiratory rate, PWR, and
righting reflex (RR)) ketamine or an experimental compound at 10mg/mL were commenced at a
rate (weight-adjusted) to deliver 20 mg/kg/min. Dose to loss of righting reflex (LORR see below),
and subsequent pedal withdrawal score of 1 (PWR see below) were recorded. Following attaining a
PWR of 1, infusion rate was reduced to 6.7 mg/kg/min, then titrated in an up-and-down fashion as
required to maintain both dorsal recumbency, and a PWR = 1, to 10 min before cessation. Each study
used three rats, with each group of rats also acting as their own ketamine control. Prior odds/evens
randomisation determined the order of study drug administration was determined by with a recovery
interval of at least three hours afforded between experiments. Records of PWR and RR were made
at one-minute intervals throughout, from cessation of infusion to return of righting reflex (RORR),
and from cessation of infusion to the animals displaying independent locomotion (walk).
Loss of Righting Reflex (LORR): This is primarily used to assess anaesthetic hypnotic effect.
Righting reflex is judged absent when the rat fails to right from a position of dorsal recumbency to
a position of sternal recumbency on three attempts performed in rapid succession. Dose to LORR is
termed effective potency.
Pedal Withdrawal Reflex (PWR) scoring: Nociceptive testing in animals was conducted via 1 s
application of constant pressure (firm digital pressure) over the forepaw of the animal. Pedal withdrawal
reflex testing is primarily used to assess analgaesic effect, and responses are graded accordingly: 0,
absent; 1, flicker; 2, moderate withdrawal; 3, fast withdrawal; 4, Fast withdrawal with cry/preceding
apnoea (modified from [18]).
Behavioural dysfunction scoring: This was undertaken according to the following table.
Observations were made over a one minute interval every five minutes from cessation of infusion until
return of normal behaviours (total score = 0). A score of 1 was accorded for any positive behavioural
aberration for each of four categories (maximal score 4) during wake-up.
Duration of any behavioural aberration was recorded from RORR as follows: score 0 = nil;
score1 = 0–120 s; score 2 = 121–300 s; score 3 = 301–600 s; score 4 = 600+ s.
Analgaesic assessment protocol: Animal preparation was in accord with the anaesthetic assessment
protocol above. Three rats were used in each study. Following venous cannulation, animals underwent
infusion of ketamine or experimental compound at 20 mg/kg over a ten minute interval. A tail flick
analgaesia meter (Colombus Instruments, Colombus, Ohio) was then used to determine thermal pain
sensitivity. Radiant heat was applied using a shutter-controlled lamp as a heat source focused on a
spot located 6–8 cm from the tip of the tail. The intensity of the beam was set at a level producing basal
latency times between 2 and 4 s. To prevent thermal tissue injury the cut off time as set at 10 s. A digital
response time indicator with a resolution of 0.1s measured the time from initiation of stimulus until
tail withdrawal (the flick; TFL).

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Table 2. Behavioural dysfunction score (score 1 for any behavioural aberration in each category to
maximum score 4).
(1) General
Exophthalmos
Increased respiration
Decreased respiration
(2) Motor
Hunched posturing
Subdued
Increased/decreased body tone
Increased/decreased locomotor activity
(3) Reactivity
Increased irritability on gentle handling
Decreased irritability on gentle handling
Rearing
(4) Stereotypical behaviours
Head weaving
Ataxia/decreased co-ordination
Splayed hind legs
The TFL response following infusion of control and study drugs was calculated as a percentage of
the maximum possible effect (MPE) such that:
%MPE = [TFL (post-drug) - TFL (pre-drug)/10 s - TFL (pre-drug)] × 100%
(1)
TFL latency was recorded at 5 min intervals from cessation of study drug infusion (time zero) to
60 min. Individual %MPE-time curves were constructed for each animal, and the area under the curve
(AUC) adopted as a composite measure of induced analgaesia (cTFL).
Supplementary Materials: The following are available online, Syntheses and characterisation of the compounds
of Table S1.
Author Contributions: Conceptualisation, supervision, formal analysis, original draft preparation, W.A.D. and
J.W.S.; investigation, methodology, I.V.D., M.G.H., L.J.V.; funding acquisition, project administration, resources,
M.J.B. All authors have read and agreed to the published version of the manuscript.
Acknowledgments: The authors thank the Return on Science Initiative from Auckland UniServices Ltd and the
NZ Ministry of Business, Innovation and Enterprise for funding. We thank Ric Broadhurst and Bobby Smith of
the Ruakura Research Centre for assistance in the performance of animal procedures.
Conflicts of Interest: Authors declare no conflict of interest
Abbreviations
DCM
Dichloromethane
LORR
NMDA
PWR
Loss of righting reflex
N-methyl-D-aspartate
Pedal withdrawal reflex score
Trifluoroacetic acid anhydride.
TFAA
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