Ketamine esters and amides as short-acting anaesthetics: Structure-activity relationships for the side-chain

Article abstract of DOI:10.1016/j.bmc.2019.02.010

N-Aliphatic ester analogues of the non-opioid ketamine (1) retain effective anaesthetic/analgesic properties while minimising ketamine's psychomimetic side-effects. We show that the anaesthetic/analgesic properties of these ester analogues depend critically on the length (from 2 to 4 carbons), polarity and steric cross-section of the aliphatic linker chain. More stable amide and ethylsulfone analogues generally showed weaker anaesthetic/analgesic activity. There was no correlation between the anaesthetic/analgesic properties of the compounds and their binding affinities for the N-methyl-D-aspartate (NMDA) receptor.

Full text of DOI:10.1016/j.bmc.2019.02.010

Accepted Manuscript
Ketamine esters and amides as short-acting anaesthetics: structure-activity re-
lationships for the side-chain
Ivaylo V. Dimitrov, Martyn G. Harvey, Logan J. Voss, James W. Sleigh,
Michael J. Bickerdike, William A. Denny
PII:
S0968-0896(18)32129-1
https://doi.org/10.1016/j.bmc.2019.02.010
BMC 14746
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 December 2018
4 February 2019
4 February 2019
Please cite this article as: Dimitrov, I.V., Harvey, M.G., Voss, L.J., Sleigh, J.W., Bickerdike, M.J., Denny, W.A.,
Ketamine esters and amides as short-acting anaesthetics: structure-activity relationships for the side-chain,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.02.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ketamine esters and amides as short-acting anaesthetics: structure-activity
relationships for the side-chain
Ivaylo V. Dimitrova, Martyn G. Harveyb, Logan J. Vossb, James W. Sleighb, Michael J.
Bickerdikec, William A. Dennya
aAuckland Cancer Society Research Centre, School of Medical Sciences, University of
b
Auckland, New Zealand, Waikato Clinical School, University of Auckland, New Zealand
cKea Therapeutics Ltd, Auckland, New Zealand.
Abstract
N-Aliphatic ester analogues of the non-opioid ketamine (1) retain effective
anaesthetic/analgesic properties while minimising ketamine’s psychomimetic side-effects.
We show that the anaesthetic/analgesic properties of these ester analogues depend critically
on the length (from 2-4 carbons), polarity and steric cross-section of the aliphatic linker
chain. More stable amide and ethylsulfone analogues generally showed weaker
anaesthetic/analgesic activity. There was no correlation between the anaesthetic/analgesic
properties of the compounds and their binding affinities for the N-methyl-D-aspartate
(NMDA) receptor.
Key words
Ketamine; esters; anaesthesia; short-acting; structure-activity relationship
Abbreviations
DCC, N,N'-Dicyclohexylcarbodiimide; DCM, dichloromethane; DMAP, 4-
Dimethylaminopyridine; LDA, Lithium diisopropylamide; LRR, loss of righting reflex;
NMDA, N-methyl-D-aspartate; PWR, pedal withdrawal reflex score; TFAA, trifluoroacetic
acid anhydride.
Corresponding Author
Ph (+649) 923 6156
Email i.dimitrov@auckland.ac.nz (I. V. Dimitrov)
1. Introduction
Ketamine is a dissociative anaesthetic, which finds widespread application in the treatment of
battlefield injuries, emergency departments and for use in children. Ketamine’s lack of
cardiovascular depression associated with other anaesthetics (especially opioids) offers
significant advantages. It is however plagued by the feeling of detachment from the
environment and the user and the near-death experiences it produces on recovery. It can also
cause hallucinations. These side effects have limited its potential use. In recent times,
however ketamine has enjoyed a resurgence due in large part to the impact it has had in

treating chronic depression and the rapid onset of its antidepressant effect. This in turn has
led to increase in ketamine related publications in recent years. There has been a number of
ketamine related analogues synthesised over the years, they have all proved less potent than
the parent compound, however reduction of the hallucinogenic side effects have been
achieved in certain cases.1
We have previously shown in a rat intravenous infusion model that aliphatic ester analogues
of the non-opioid anaesthetic/analgesic ketamine (1) retain these desirable therapeutic
activities,2 while at the same time minimising its psychomimetic side-effects during recovery
by undergoing very rapid metabolism to the much more polar and inactive acids,3 resulting in
a 10-fold shorter period for recovery compared with that of 1. Structure-activity relationships
for these alkyl-linked esters N(CH2)nCO2R (e.g., compounds 4, 6) showed that their potency
as sedatives was not very dependent upon the N-alkylester chain length (from 2-4 carbons),
but dropped away more quickly with the size of the ester R group.
In the present paper we extend these structure-activity relationships for the side chain by
further variations (Table 1) to the length and nature of the spacer alkyl chain and to the nature
of the terminating group.
O
Cl
HN
1
ketamine
2. Results and discussion
2.1. Chemistry
The compounds of Table 1 were prepared from racemic norketamine4 (23), by reaction with
the appropriate alkyl bromides in acetonitrile under reflux, in the presence of K2CO3 and KI,
for the appropriate time (between 12-168 h) (Scheme 1). The methylaluminium amide and
dimethylaluminium amide reagents5 for the preparation of amides 19 and 20 were generated
by reacting trimethylaluminium with methylamine or dimethylamine hydrochloride at room
temperature for an hour. Subsequent addition of the known2 norketamine ester 24 and reflux
overnight afforded the desired amides (Scheme 2).
Scheme 1. General synthesis of the compounds of Table 1
O
O
Cl
Cl
O
O
Br
alkyl
i
+
X
alkyl
HN
n
NH2
X
n
X = O, NH
2 3 5 7-
compounds , , ,
18 21 22
of Table 1
23
,
,
Reagents and conditions: (i) alkyl bromide, K2CO3, KI, MeCN, heat (4 -168 h)

Scheme 2. Synthesis of compounds 19, 20 of Table 1
O
O
Cl
Cl
R
N
i
HN
19
OEt
HN
24
O
O
: R = H
20
: R = Me
Reagents and conditions: (i) Al reagent, toluene, reflux, 15 h, 72% for 19 and 71% for 20
Many of the required alkyl bromides were commercially available or prepared by literature
methods; others were prepared by the routes shown in Scheme 3. Phenyl 3-bromopropanoate
(26) for the preparation of 5 was prepared6 by reaction of 3-bromopropionic acid (25) with
phenol in the presence of TFAA (Scheme 3A). Methoxy ethyl 5-bromopentanoate (28) for
the preparation of 12 was prepared by reacting 5-bromovaleric acid (27) with 2-
methoxyethanol in the presence of DCC and DMAP (Scheme 3B). Ethyl 5-bromo-2-
methylpentanoate (31) for the preparation of 9 was prepared by reacting diethyl
methylmalonate (29) with 1,3-dibromopropane in the presence of NaH to give diester (30),
which was then treated with conc. HBr under reflux (Scheme 3C).6 Similarly methyl 5-
bromo-2,2-dimethylpentanoate (38) was prepared by reacting methyl isobutyrate (37) with
1,3-dibromopropane in the presence of LDA (Scheme 3F).
Methyl 2-(2-bromoethoxy)acetate (34) for the preparation of 11 was synthesized by reacting
2-(benzyloxy)ethanol (32) with methyl bromoacetate in the presence of NaH to yield ether
33. Benzyl deprotection of this by hydrogenation (Pd/C) gave an alcohol, which in turn
underwent an Appel reaction7 (CBr4/PPh3) to give 34 (Scheme 3D). Ethyl 3-(2-
bromoethoxy)propanoate (36) for the preparation of 13 was synthesized by similarly reacting
32 with ethyl acrylate in the presence of Na metal to give ether 35. Benzyl group deprotection
(Pd/C/H2) of this ether yielded an alcohol, which in turn underwent an Appel reaction to give
36 (Scheme 3E).8
Scheme 3. Preparation of required bromides

O
O
i
Br
OH
Br
O
A
26
25
O
O
ii
O
B
C
Br
OH
Br
O
28
27
O
O
O
O
iii
iv
Br
OEt
Br
OEt
EtO
OEt
29
30
31
O
O
OEt
O
O
v
vi
OH
OH
O
D
E
BnO
BnO
Br
OMe
O
BnO
BnO
OMe
34
32
33
vii
viii
O
OEt
O
OEt
Br
32
O
O
36
35
O
ix
F
OMe
Br
OMe
37
38
Reagents and conditions: (i) phenol, TFAA, 25oC, 12 h, 74%; (ii) 2-methoxyethanol, DCC, DMAP,
DCM, 25 oC, 48 h, 84%; (iii) NaH, 1,3-dibromopropane, THF, 0-15 oC, 20 h, 87%; (iv) HBr (48%),
reflux, 7 h, then 25 oC. 15 h, 80%; (v) methyl bromoacetate, NaH, THF, 0-25 oC, 12 h, 55%; (vi)
Pd/C, H2, EtOAc, 12 h, then CBr4, PPh3, THF, 25 oC, 3 h, 62% over 2 steps; (vii) ethyl acrylate, Na,
THF, 25 oC, 20 h, 60%; (viii) Pd/C, H2, EtOAc, 12 h, then CBr4, PPh3, THF, 25 oC, 3 h, 64% over 2
steps; (ix) 1,3-dibromopropane, LDA, THF, -78-0 oC, 4 h, 86%.
2.2. Biology
Table 1 gives both structural and biological data for ketamine (1) itself and two previously-
reported esters2,3 (4, 6), along with 20 new analogues. Compounds 2 and 3 are examples of
the shortest possible (1-atom) chain, and both were inactive at the highest dose tested (200
mg/kg). This contrasts with the results for the previously-reported2 (CH2)2- and (CH2)4-linked
compounds 4 and 6, which had analgesic potencies close to that of 1, and defines two atoms
as the minimum length spacer. There is a marked drop in calculated pKa in going from a
(CH2)2 to a CH2 link (from 4.35 to 3.17) but it is not known if this is the reason for the loss of
activity. Compounds 7 and 8 explore longer chain lengths (5 and 6 atoms respectively). Both
were inactive, suggesting an optimal upper size limit to the spacer chain as well.
Table 1. Structural and biological data for new ketamine ester and amide analogues

O
Cl
HN
R
N
n
R
LORRa
mg/kg
PWRb
mg/kg
RORRc NMDAd pKae
sec
IC50
(µM)
0.7
1f
Ester chain length
23
28
1075
7.49
2
3
4f
5
6f
7
8
1
1
2
2
4
5
6
CH2CO2Me
>200
>200
33
>200
34
>200
>200
37
>200
44
NAg
NA
83
NA
99
308
45
134
73
3
3.17
3.67
4.35
5.02
6.29
6.47
6.57
i
CH2CO2 Pr
i
(CH2)2CO2 Pr
(CH2)2CO2Ph
(CH2)4CO2Me
(CH2)5CO2Me
(CH2)6CO2Me
>200
>200
>200
>200
NA
NA
44
51
Ester chain hindrance
9
10
4
4
(CH2)3CHMeCO2Et
(CH2)3C(Me)2CO2Me
50
52
55
>200
260
580
107
125
6.42
6.36
Ester chain polarity
11
12
13
4
4
5
(CH2)2OCH2CO2Me
(CH2)4CO2(CH2)2OMe
(CH2)2O(CH2)2CO2Et
>200
>200
>200
>200
>200
>200
NA
NA
NA
13
19
87
5.03
6.38
5.34
Ester replacement
14
15
16
17
18
19
20
21
22
2
2
2
2
2
4
4
4
2
(CH2)2CONH2
>200
58
110
112
82
83
>200
67
>200
>200
113
NA
85
623
237
82
125
NA
556
NA
500
73
416
935
58
77
>1000
41
5.45
5.14
5.42
5.33
5.45
6.37
6.42
9.56
4.20
(CH2)2CONHMe
(CH2)2CONHEt
(CH2)2CONHiPr
(CH2)2CONMe2
(CH2)4CONHMe
(CH2)4CONMe2
(CH2)4CONpiperidine
(CH2)2SO2Et
139
>200
>200
>200
130
>200
>200
>1000
Footnotes for Table 1
aLORR: minimal dose for loss of righting reflex; bPWR: minimal dose for loss of pedal
withdrawal reflex; cRORR: time for return of righting reflex after infusion stopped; dNMDA:
Binding affinity (IC50) at the NMDA glutamate ion channel using [3H]MK-801 as the
competitive radioligand (see text for details); epKa of amine, calculated using
ACD/PhysChem Suite v12; f Data from ref. 1; gNA: not attained.
Compounds 9 and 10 are methyl-substituted analogues of 6 and explore the effect of steric
bulk around the ester. Both compounds had slightly lower sedative potency than 6, but longer

recovery times (roughly 2.5 and 6 times than that of 6), consistent with increasing resistance
to enzymic hydrolysis expected with greater steric bulk close to the ester.9
Compounds 11-13 investigate the effect of inserting a polar ether link in the side chain
(11,13) or in the ester group (12) of compounds that are essentially analogues of 6 (4-atom
linker chain). Replacement of CH2 by an O atom lowers logP by only about half a unit
(calculated) but irrespective of its placement abolishes the excellent sedative/analgesic
properties of 6.
Compounds 14-21 explore the replacement of the ester unit with similar but more stable
amide groups). Of the five (CH2)2-linked amides (14-18), the primary amide 14 did not show
activity due to insolubilisation. The other four secondary/tertiary amides (15-18) showed
sedative activity, but were 2-3 fold less potent than ester 6. Comparisons between esters 4
and 6 and the direct corresponding amides 17 and 19 showed the amides to be anaesthetic and
analgesic, but with lesser potency and longer offsets than their counterpart esters.
Interestingly, while the (CH2)4-linked tertiary amide 20 was inactive, the corresponding
piperidide 21 showed significant anaesthetic potency, but with a long offset (556 sec).
Finally, compound 22 explored the use of a sulfone as a stable ester analogue but this very
polar compound was completely inactive.
In Table 1 we also present the binding affinities of these compounds for the N-methyl-D-
aspartate (NMDA) glutamate ion channel, determined by a radioligand binding assay using
Wistar rat brain preparations.10 Many of the pharmacological effects of ketamine
(anaesthetic, analgesic, psychomimetic, anti-depressant), have previously been linked to this
property13,14 since ketamine has high binding affinity for the NMDA receptor, with its Ki
reported in the literature to be 1.9 µM.15 However, our data clearly suggests that NMDA
potency does not correlate at all with the anaesthetic or analgesic activity of the compounds
discussed here. While ketamine (1) had a minimal dose for loss of righting reflex of 23 mg/kg
and an IC50 for NMDA inhibition of 0.7 nM, the most active anaesthetic analogues in Table 1
(compounds 4, 6, 9, 10) had NMDA affinities ranging from 3-580 nM; the same range as that
for the most inactive compounds 2, 5, 7, 8, 11-14 (i.e. 13-560 nM).
2.3. Conclusions
Compounds 2-8 of Table 1 define the acceptable length of the N-ester spacer chain to be from
4-6 atoms. Compounds 9 and 10 suggest that more bulk around the carbonyl carbon does
result in slower hydrolysis (longer recovery times) but does not markedly affect anaesthetic
activity. In contrast, compounds 11 and 13 show that polarity in the spacer chain abolishes
activity, even when the chain is in the allowable length range. Compounds 14-21 explore the
use of more stable aliphatic amide groups; the bulk of these were active but were
considerably less potent, with the best being the piperidide 21. There was no relationship
between anaesthetic/antinociceptive activity and NMDA channel affinity for these
compounds.

3. Experimental
3.1. Chemistry
All reagents and solvents were obtained from commercial suppliers and used without further
purification unless otherwise stated. Reactions requiring anhydrous conditions were
performed under nitrogen atmospheres. Reactions were monitored by thin layer
chromatography (TLC) on preloaded silica gel F254 plates (Sigma-Aldrich) with a UV
1
indicator. Column chromatography was performed with Merck 230-400 mesh silica gel. H
and 13C NMR spectra were obtained with a Bruker Avance 400 spectrometer 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 ionization (APCI)
with a corona voltage of 50 V and a source temperature of 400 oC. High-resolution
electrospray ionization (HRESIMS) mass spectra were determined on a Bruker micrOTOFQ
II mass spectrometer. Final products were analyzed by reverse-phase HPLC (Alltima C18 5
μm column, 150 mm × 3.2 mm; Alltech Associated, Inc., Deerfield, IL) 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 unless otherwise stated. DCM refers
to dichloromethane, DMF refers to N,N-dimethylformamide, EtOAc refers to ethyl acetate,
EtOH refers to ethanol.
3.1.1. Methyl (1-(2-chlorophenyl)-2-oxocyclohexyl)glycinate hydrochloride (2) (Scheme
1). A solution of racemic norketamine (23) (0.4 g, 1.78 mmol), methyl 2-bromoacetate (0.32
g, 2.14 mmol), KI (0.089g, 0.54 mmol), K2CO3 (0.74 g, 5.35 mmol) in MeCN (10 mL) was
heated to a 100 oC in 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 eluting with EtOAc/hexanes (0-35%) to give 2 (0.42 g, 80%) as
pale yellow oil. This was dissolved in Et2O (5 mL), cooled to 0 oC, and HCl in Et2O (2M,
2.11 mmol) was added dropwise. The solvent was evaporated under reduced pressure, the
residue was taken up in EtOAc (1 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 2; 1HNMR (CDCl3) δ 7.55-7.52 (dd, J= 7.84 Hz, 1.72 Hz, 1H), 7.38-
7.35 (dd, J= 7.76 Hz, 1.48 Hz, 1H), 7.33-7.28 (td, J= 7.44 Hz, 1.52 Hz, 1H), 7.26-7.21 (td, J=
9.36 Hz, 1.76 Hz, 1H), 3.66 (s, 3H), 3.33-3.28 (d J=17.64 Hz, 1H), 3.15-3.11 (d, d J=17.65
Hz, 1H), 2.74 (br. 1H), 2.66-2.58 (m, 2H), 2.49-2.45 (m, 1H), 2.01-1.74 (m 5H); 13C (CDCl3)
δ 208.20, 172.88, 138.65, 133.58, 131.32, 129.04, 128.97, 127.00, 69.04, 51.99, 44.76, 39.28,
39.25, 26.95, 21.66; MS m/z 296.20 (MH+). Calculated for C15H19ClNO3 (MH+) 296.1048,
found 296.1045.

3.1.2. Isopropyl (1-(2-chlorophenyl)-2-oxocyclohexyl)glycinate hydrochloride (3). Similar
reaction of 23 (0.2 g, 0.89 mmol) and isopropyl 2-bromoacetate (2.67 g, 14.9 mmol), KI
(0.05 g, 0.31 mmol) K2CO3 (0.43 g, 3.13 mmol) in MeCN (7 mL) at 120 oC in a sealed tube
for 168 h, followed by chromatographic purification on silica gel, eluting with
EtOAc/hexanes (0-15%), gave a pale yellow oil (0.08 g, 27%), which was converted to 3
with HCl in Et2O as above. 1HNMR (CDCl3) δ 7.55-7.5 2 (dd, J= 7.84 Hz, 1.72 Hz, 1H),
7.38-7.35 (dd, J= 7.76 Hz, 1.44 Hz, 1H), 7.33-7.28 (td, J= 7.44 Hz, 1.48 Hz, 1H), 7.26-7.21
(td, J= 9.36 Hz, 1.76 Hz, 1H), 5.14-5.05 (q, J=6.28 Hz, 1H), 3.29-3.24 (d, J=17.69 Hz, 1H),
3.11-3.07 (d, J=17.69 Hz, 1H), 2.67-2.56 (m, 2H), 2.52-2.48 (m, 1H), 1.99-1.77 (m, 5H),
1.21-1.20 (d, J=6.16 Hz, 6H); 13C (CDCl3) δ 208.25, 172.19, 139.09, 133.62, 131.40, 129.12,
128.96, 127.05, 68.97, 68.57, 45.26, 39.37, 39.29, 26.81, 22.01, 21.96; MS m/z 324.20 (MH+)
Calculated for C17H22ClNO3 (MH+) 324.1362, found 324.1362.
3.1.3. Phenyl 3-((1-(2-chlorophenyl)-2-oxocyclohexyl)amino)propanoate hydrochloride
(5). Similar reaction of 23 (0.3 g, 1.34 mmol) and phenyl 3-bromopropanoate (26) (1.84 g,
8.07 mmol) with K2CO3/KI in MeCN at 100 oC for 48 h, followed by chromatography on
silica gel (EtOAc/hexanes) gave a pale yellow oil (0.35 g, 70%), which was converted to 5 as
above. 1HNMR (CDCl3) δ 7.61-7.58 (dd, J= 7.6 Hz, 1.6 Hz, 1H), 7.40-7.36 (m, 3 H), 7.33-
7.29 (td, J= 9.2 Hz, 1.6 Hz, 1H), 7.26-7.21 (m, 3H), 7.09-7.07 (m, 2H), 2.75-2.64 (m, 4 H),
2.5-2.47 (m, 3H), 2.0-1.9 (m 2 H), 1.79-1.57 (m, 3H); 13C (CDCl3) δ 208.33, 171.81, 150.74,
138.67, 133.82, 131.43, 129.66, 129.18, 128.89, 126.98, 126.12, 121.76, 69.77, 39.58, 39.19,
38.26, 35.40, 27.36, 21.91; MS m/z 372.10 (MH+) Calculated for C21H23ClNO3 (MH+)
372.1361, found 372.1352.
3.1.4. Methyl 5-((1-(2-chlorophenyl)-2-oxocyclohexyl)amino)hexanoate hydrochloride
(7). Similar reaction of 23 (0.35 g, 1.56 mmol) and methyl 6-bromohexanoate (0.49 g, 2.4
mmol) with K2CO3/KI in MeCN at 120 oC for 12 h, gave a pale yellow oil (0.40 g, 73%),
which was purified and converted to 7 as above. 1HNMR (CDCl3) δ 7.55-7.52 (dd, J= 7.84
Hz, 1.72 Hz, 1H), 7.38-7.35 (dd, J= 7.76 Hz, 1.48 Hz, 1H), 7.33-7.28 (td, J= 7.44 Hz, 1.52
Hz, 1H), 7.26-7.21 (td, J= 9.36 Hz, 1.76 Hz, 1H), 3.65 (s, 3H), 2.75-2.71 (m, 1H), 2.53-2.43
(m 2H), 2.28-2.25 (t, J= 7.36 Hz, 2H), 2.06-1.98 (m, 3H), 1.81-1.76 (m, 3H), 1.65-1.39 (m,
5H), 1.39-1.13 (m, 3H); 13C (CDCl3) δ 209.09, 174.31, 138.69, 133.83, 131.32, 129.35,
128.76, 126.82, 70.01, 51.62, 42.28, 39.64, 39.22, 34.13, 30.45, 27.97, 26.87, 24.91 22.03;
MS m/z 352.20 (MH+) Calculated for C19H27ClNO3 (MH+) 352.1674, found 352.1686
3.1.5. Ethyl 7-((1-(2-chlorophenyl)-2-oxocyclohexyl)amino)heptanoate hydrochloride
(8). Similar reaction of 23 (0.35 g, 1.56 mmol) and ethyl 7-bromoheptanoate (0.56 g, 2.4
mmol) with K2CO3/KI in MeCN at 90 oC for 48 h gave a pale yellow oil (0.55 g, 91%) which
was purified and converted to 8 as above. 1HNMR (CDCl3) δ 7.55-7.52 (dd, J= 7.84 Hz, 1.72
Hz, 1H), 7.38-7.35 (dd, J= 7.76 Hz, 1.48 Hz, 1H), 7.33-7.28 (td, J= 7.44 Hz, 1.52 Hz, 1H),
7.26-7.21 (td, J= 9.36 Hz, 1.76 Hz, 1H), 4.14-4.08 (m, 2H), 2.76-2.67 (M, 1H), 2.52-2.41 (m,
2H), 2.34-2.28 (m, 1H), 2.27-2.23 (t, J=7.64 Hz, 2H), 2.05-1.35 (m, 14 H), 1.25-1.22 (t,
J=7.16 Hz, 3H); 13C (CDCl3) δ 208.99, 173.98, 138.83, 133.82, 131.32, 129.36, 128.74,

126.80, 69.98, 60.58, 42.45, 39.63, 39.18, 34.47, 30.63, 29.14, 27.87, 27.02, 25.06, 22.04,
14.38; MS m/z 380.20 (MH+) Calculated for C21H31ClNO3 (MH+) 380.1987, found 380.1919
3.1.6. Ethyl 5-((1-(2-chlorophenyl)-2-oxocyclohexyl)amino)-2-methylpentanoate
hydrochloride (9). Similar reaction of 23 (0.35 g, 1.56 mmol), ethyl 5-bromo-2-
methylpentanoate (31) (0.55 g, 2.4 mmol) with K2CO3/KI in MeCN at 95 oC for 12 h gave a
pale yellow oil (0.45 g, 79 %), which was purified and converted to 9as above. 1HNMR
(CDCl3) δ 7.55-7.52 (dd, J= 7.84 Hz, 1.72 Hz, 1H), 7.38-7.35 (dd, J= 7.76 Hz, 1.48 Hz, 1H),
7.33-7.28 (td, J= 7.44 Hz, 1.52 Hz, 1H), 7.26-7.21 (td, J= 9.36 Hz, 1.76 Hz, 1H), 4.13-4.07
(q, J=7.12 Hz), 2.74-2.71 (m, 1), 2.54-2.29 (m, 4 H), 2.15-1.93 (m, 3H), 1.92-1.82 (m, 1H),
1.82-1.72 (m 3H), 1.68-1.37 (m, 3H), 1.28-1.19 (t, J= 7.08 Hz, 3H), 1.12-0.98 (d, J= 3.96
Hz, 3H); 13C (CDCl3) δ 208.94, 176.80, 138.65, 133.76, 131.27, 129.28, 128.72, 126.76,
69.94, 60.26, 42.31, 39.57, 39.48 39.44, 39.18, 34.74, 31.37, 28.47, 22.15, 17.20; MS m/z
366.20 (MH+) Calculated for C20H29ClNO3 (MH+) 366.1830, found 366.1827
3.1.7. Ethyl 5-((1-(2-chlorophenyl)-2-oxocyclohexyl)amino)-2,2-dimethylpentanoate
hydrochloride (10). Similar reaction of 23 (0.35 g, 1.56 mmol) and methyl 5-bromo-2,2-
dimethylpentanoate (38) (0.55 g, 2.4 mmol) with K2CO3/KI in MeCN at 95 oC for 12 h gave
a pale yellow oil (0.49 g, 86 %), which was purified and converted to 10 as above. 1HNMR
(CDCl3) δ 7.55-7.52 (dd, J= 7.84 Hz, 1.72 Hz, 1H), 7.38-7.35 (dd, J= 7.76 Hz, 1.48 Hz, 1H),
7.33-7.28 (td, J= 7.44 Hz, 1.52 Hz, 1H), 7.26-7.21 (td, J= 9.36 Hz, 1.76 Hz, 1H), 3.64 (s,
3H), 2.79-2.69 (m, 1H), 2.56-2.40 (m. 2H), 2.32-2.22 (m 1H), 2.04-1.92 (m, 3H), 1.91-1.82
(m, 1H), 1.82-1.70 (m, 3H), 1.52-1.44 (m, 2H), 1.42-1.32 (m, 1H), 1.14 (s, 6H); 13C (CDCl3)
δ 209.16, 178.56, 138.66, 133.88, 131.36, 129.98, 129.37, 126.83, 70.05, 51.88, 43.05, 42.31,
39.68, 39.31, 38.39, 28.07, 26.44, 25.35, 22.06; MS m/z 366.20 (MH+) Calculated for
C20H29ClNO3 (MH+) 366.1830, found 366.1842
3.1.8. Methyl 2-(2-((1-(2-chlorophenyl)-2-oxocyclohexyl)amino)ethoxy)acetate
hydrochloride (11). Similar reaction of 23 (0.35 g, 1.56 mmol) and methyl 2-(2-
bromoethoxy)acetate (34) (0.61 g, 3.1 mmol) with K2CO3/KI in MeCN at 110 oC for 48 h
gave a pale yellow oil (0.37 g, 70 %), which was purified and converted to 11 as above.
1HNMR (CDCl3) δ 7.55-7.52 (dd, J= 7.84 Hz, 1.72 Hz, 1H), 7.38-7.35 (dd, J= 7.76 Hz, 1.48
Hz, 1H), 7.33-7.28 (td, J= 7.44 Hz, 1.52 Hz, 1H), 7.26-7.21 (td, J= 9.36 Hz, 1.76 Hz, 1H),
4.10 (s, 2H), 3.75 (s, 3H), 3.64-3.60 (m, 2H), 2.72-2.62 (m, 1H), 2.60-2.43 (m, 3H), 2.40-
2.31 (m, 1H), 2.20-1.88 (m, 2H), 1.88-1.82 (m, 3H); 13C (CDCl3) δ 208.06, 171.11, 138.97,
133.61, 131.24, 129.23, 128.69, 126.8, 71.84, 69.35, 68.36, 51.94, 42.16 39.41, 39.06, 27.07,
21.71; MS m/z 340.10 (MH+) Calculated for C17H23ClNO4 (MH+) 340.1310, found 340.1313
3.1.9. 2-Methoxyethyl 5-((1-(2-chlorophenyl)-2-oxocyclohexyl)amino) pentanoate
hydrochloride (12). Similar reaction of 23 (0.35 g, 1.56 mmol) and 2-methoxyethyl 5-
bromopentanoate (28) (0.75 g, 3.1 mmol) with K2CO3/KI at 100 oC for 48 h gave a pale
yellow oil (0.40 g, 67%), which was converted to 12 as above. 1HNMR (CDCl3) δ 7.55-7.52
(dd, J= 7.84 Hz, 1.72 Hz, 1H), 7.38-7.35 (dd, J= 7.76 Hz, 1.48 Hz, 1H), 7.33-7.28 (td, J=
7.44 Hz, 1.52 Hz, 1H), 7.26-7.21 (td, J= 9.36 Hz, 1.76 Hz, 1H), 4.22-4.18 (m, 2H), 3.58-3.54

(m, 2H), 3.38 (s, 3H), 2.77-2.69 (m, 1H), 2.53-2.40 (m, 2H), 2.34-2.29 (m, 3H), 2.10-1.95
(m, 2H), 1.93-1.83 (m, 1H), 1.82-1.72 (m, 3H), 1.64-1.60 (m, 2H), 1.5-1.40 (m, 2H); 13C
(CDCl3) δ 209.09, 173.77, 138.68, 133.85, 131.36, 129.34, 128.80, 126.84, 70.66, 70.01,
63.50, 59.17, 42.11, 39.64, 39.26, 34.08, 30.28, 27.99, 22.72, 22.04; MS m/z 382.20 (MH+)
Calculated for C20H29ClNO4 (MH+) 382.1780, found 382.1774
3.1.10. Ethyl 3-(2-((1-(2-chlorophenyl)-2-oxocyclohexyl)amino)ethoxy)propanoate
hydrochloride (13). Similar reaction of 23 (0.18 g, 0.81 mmol) and ethyl 3-(2-
bromoethoxy)propanoate (36) (0.36 g, 1.61 mmol) with K2CO3/KI in MeCN at 100 oC for 20
h gave a pale yellow oil (0.17 g, 60 %), which was purified and converted to 13 as above.
1HNMR (CDCl3) δ 7.55-7.52 (dd, J= 7.84 Hz, 1.72 Hz, 1H), 7.38-7.35 (dd, J= 7.76 Hz, 1.48
Hz, 1H), 7.33-7.28 (td, J= 7.44 Hz, 1.52 Hz, 1H), 7.26-7.21 (td, J= 9.36 Hz, 1.76 Hz, 1H),
4.19-4.11 (1, J=7.16 Hz, 2H), 3.74-3.66 (m, 2H), 3.60-3.46 (m, 2H), 2.69-2.42 (m, 6H), 2.34-
2.26 (m, 1H), 2.2-1.88 (m, 2H), 1.84-1.72 (m, 3H), 1.29-1.23 (t, J=7.13 Hz, 3H); 13C
(CDCl3) δ 207.94, 171.81, 139.19, 133.64, 131.28, 129.24, 128.68, 126.84, 70.76, 69.31,
66.30, 60.68, 42.09, 39.44, 28.98, 35.29, 26.97, 21.73, 14.41; MS m/z 368.10 (MH+)
Calculated for C19H27ClNO4 (MH+) 368.1623, found 368.1635.
3.1.11. 3-((1-(2-Chlorophenyl)-2-oxocyclohexyl)amino) propanamide hydrochloride
(14). Similar reaction of 23 (0.30 g, 1.35 mmol) and 3-bromopropanamide (0.51 g, 3.33
mmol) with K2CO3/KI in MeCN at 120 oC for 168 h, followed by purification by column
chromatography on silica gel, gave a pale yellow oil (0.20 g, 52%), which was converted to
14 as above. 1HNMR (CDCl3) δ 8.24 (br, 1H), 7.40-7.25 (m, 2H), 6.81 (br, 2H), 6.27-6.20
(dd, J=10.16 Hz, 17.08 Hz, 1H), 6.07-6.02 (dd, J= 17.12Hz, 2.28 Hz, 1H), 3.31-3.26 (q,
J=7.0 Hz, 2H), 2.27-2.23 (m, 4H), 2.21- 2.18 (m, 2H), 1.89-1.72 (m, 4H); 13C (CDCl3) δ
205.16, 172.97, 140.02, 131.48, 130.48, 129.05, 128.43, 126.85, 124.78, 68.39, 56.47, 35.18,
34.89, 30.70, 25.01, 20.57; MS m/z 295.20 (MH+) Calculated for C15H20ClN2O2 (MH+)
295.1208, found 295.1210
3.1.12. 3-((1-(2-Chlorophenyl)-2-oxocyclohexyl)amino)-N-methylpropanamide
hydrochloride (15). Similar reaction of 23 (0.30 g, 1.35 mmol) and 3-bromo-N-
methylpropanamide (0.56 g, 3.34 mmol) with K2CO3/KI in MeCN at 120 oC for 168 h,
followed by purification as above, gave a pale yellow oil, gave a pale yellow oil (0.20 g,
50%) which was converted to 15 as above. 1HNMR (CDCl3) δ 7.55-7.52 (dd, J= 7.84 Hz,
1.72 Hz, 1H), 7.38-7.35 (dd, J= 7.76 Hz, 1.48 Hz, 1H), 7.33-7.28 (td, J= 7.44 Hz, 1.52 Hz,
1H), 7.26-7.21 (td, J= 9.36 Hz, 1.76 Hz, 1H), 3.01-2.92 (m, 1H), 2.69-2.62 (d, J=4.81 Hz,
3H), 2.52-2.46 (m, 2H), 2.36-2.26 (m, 3H), 2.10-1.90 (m 4H), 1.84-1.72 (m 2H); 13C (CDCl3)
δ 210.84, 173.30, 136.99, 134.20, 131.64, 129.69, 129.50, 127.16, 70.46, 39.83, 39.55, 38.03,
36.09, 29.72, 26.02, 22.33; MS m/z 309.20 (MH+) Calculated for C16H22ClN2O2 (MH+)
309.1364, found 309.1361.
3.1.13. 3-((1-(2-Chlorophenyl)-2-oxocyclohexyl)amino)-N,N-dimethylpropanamide
hydrochloride (16). Similar reaction of 23 (0.30 g, 1.35 mmol) and 3-bromo-N,N-

dimethylpropanamide (0.48g, 2.70 mmol) with K2CO3/KI in MeCN at 100 oC for 48 h,
followed by purification as above, gave a pale yellow oil (0.30 g, 70%) which was converted
to 16 as above. 1HNMR (CDCl3) δ 7.55-7.52 (dd, J= 7.84 Hz, 1.72 Hz, 1H), 7.38-7.35 (dd,
J= 7.76 Hz, 1.48 Hz, 1H), 7.33-7.28 (td, J= 7.44 Hz, 1.52 Hz, 1H), 7.26-7.21 (td, J= 9.36 Hz,
1.76 Hz, 1H), 2.99 (s, 3H), 2.95 (s, 3H), 2.66-2.59 (m, 2H), 2.54-2.46 (m, 4H), 2.41-2.38 (m,
1H), 1.98-1.88 (m, 2H), 1.81-1.77 (m, 3H); 13C (CDCl3) δ 207.50, 172.35, 139.57, 133.62,
131.28, 129.08, 128.58, 126.84, 69.44, 39.49, 38.82, 38.48, 37.34, 35.53, 33.98, 26.36, 21.78;
MS m/z 323.20 (MH+) Calculated for C17H24ClN2O2 (MH+) 323.1521 found 323.1515
3.1.14. 3-((1-(2-Chlorophenyl)-2-oxocyclohexyl)amino)-N-ethylpropanamide
hydrochloride (17). Similar reaction of 23 (0.35 g, 1.56 mmol) and 3-bromo-N-
ethylpropanamide (0.56 g, 3.31 mmol) with K2CO3/KI in MeCN at 100 oC for 12 h, followed
by purification as above, gave a pale yellow oil (0.36 g, 72%), which was converted to 17 as
above. 1HNMR (CDCl3) δ 7.55-7.52 (dd, J= 7.84 Hz, 1.72 Hz, 1H), 7.38-7.35 (dd, J= 7.76
Hz, 1.48 Hz, 1H), 7.33-7.28 (td, J= 7.44 Hz, 1.52 Hz, 1H), 7.26-7.21 (td, J= 9.36 Hz, 1.76
Hz, 1H), 7.14 (br, 1H), 3.23-3.13 (m, 2H), 2.95-2.89 (m, 1H), 2.65-2.59 (m, 1H), 2.55-2.45
(m, 2H), 2.38-2.25 (m, 4H), 1.86-1.62 (m, 4H), 1.07-1.03(t, J= 7.28 Hz, 3H); 13C (CDCl3) δ
210.45, 172.46, 137.38, 134.16, 131.61, 129.56, 129.33, 127.11, 70.33, 39.81, 39.52, 38.26,
36.36, 34.17, 29.33, 22.26, 15.05; MS m/z 323.20 (MH+) Calculated for C17H24ClN2O2
(MH+) 323.1521, found 323.1509
3.1.15. 3-((1-(2-Chlorophenyl)-2-oxocyclohexyl)amino)-N-isopropylpropanamide
hydrochloride (18). Similar reaction of 23 (0.20 g, 0.89 mmol) with 3-bromo-N-
isopropylpropanamide (0.34 g, 1.80 mmol) with K2CO3/KI in MeCN at 110 oC for 48 h,
followed by purification as above, gave a white solid (0.20 g, 67%) that was converted to 18
as above. 1HNMR (CDCl3) δ 7.55-7.52 (dd, J= 7.84 Hz, 1.72 Hz, 1H), 7.38-7.35 (dd, J= 7.76
Hz, 1.48 Hz, 1H), 7.33-7.28 (td, J= 7.44 Hz, 1.52 Hz, 1H), 7.26-7.21 (td, J= 9.36 Hz, 1.76
Hz, 1H), 6.74 (br, 1H), 4.04-3.99 (m, 1H), 2.96-2.82 (m, 1H), 2.60-2.42 (m, 3H), 2.41-2.32
(m, 1H), 2.30-2.22 (m, 2H), 2.08-1.99 (m, 1H), 1.90-1.54 (m, 4H), 1.12-1.11 (d, J= 6.57 Hz,
3H), 1.06-1.05 (d, J= 6.60 Hz, 3H); 13C (CDCl3) δ 210.12, 171.66, 137.69, 134.12, 131.62,
129.45, 129.22, 127.09, 70.26, 41.14, 39.84, 39.54, 38.53, 36.64, 28.98, 22.94, 22.85, 22.20;
MS m/z 337.70 (MH+) Calculated for C18H26ClN2O2 (MH+) 337.1677 found 337.1676.
3.1.16. 5-((1-(2-Chlorophenyl)-2-oxocyclohexyl)amino)-N-methylpentanamide
hydrochloride (19) (Scheme 2). Trimethylaluminium (4 mmol, 2M in hexanes) was slowly
added to a suspension of methylamine hydrochloride (0.27g, 4.0 mmol) in toluene (4 mL) at -
5 oC, and the resulting solution was warmed to room temperature and stirred for 2 h until no
more gas evolved. A solution of ethyl 5-((1-(2-chlorophenyl)-2-
oxocyclohexyl)amino)pentanoate (24) (0.34g, 0.98 mmol) in toluene (10 mL) was added to
the above aluminium reagent and refluxed overnight. The reaction mixture was quenched
with water and extracted with ethyl acetate and the organic layers were dried with magnesium
sulphate. Column chromatography EtOAc/Hexanes (60-100%) afforded a pale yellow oil
(0.24g, 72%), which was converted to 19 as above. 1HNMR (CDCl3) δ 7.55-7.52 (dd, J= 7.84
Hz, 1.72 Hz, 1H), 7.38-7.35 (dd, J= 7.76 Hz, 1.48 Hz, 1H), 7.33-7.28 (td, J= 7.44 Hz, 1.52

Hz, 1H), 7.26-7.21 (td, J= 9.36 Hz, 1.76 Hz, 1H), 5.68 (br, 1H), 2.81-2.80 (d, J=3.77 Hz,
3H), 2.54-2.42 (m, 2H), 2.37-2.35 (m, 2H), 2.18-2.09 (m, 2H), 2.06-1.99 (m, 2H), 1.92-1.84
(m, 2H), 1.82-1.72 (m, 3H), 1.68-1.58 (m, 3H), 1.52-1.39 (m, 2H); 13C (CDCl3) δ 209.54,
173.75, 138.33, 133.92, 132.12, 130.04, 129.50, 126.92, 70.22, 42.02, 39.74, 39.34, 36.36,
30.11, 28.30,26.48, 23.86, 22.08; MS m/z 337.20 (MH+) Calculated for C18H25ClN2O2 (MH+)
337.1677 found 337.1676.
3.1.17. 5-((1-(2-Chlorophenyl)-2-oxocyclohexyl)amino)-N,N-dimethylpentanamide
hydrochloride (20) (Scheme 2). Trimethylaluminium (4 mmol, 2M in hexanes) was slowly
added to a suspension of dimethylamine hydrochloride (0.33g, 4.1 mmol) in toluene (4 mL)
at -5 oC and the resulting solution was warmed to room temperature and stirred for 2 h until
no more gas evolved. A solution of ethyl 5-((1-(2-chlorophenyl)-2-
oxocyclohexyl)amino)pentanoate (24) (0.32g, 0.96 mmol) in toluene (10 mL) was added to
the above formed aluminium reagent and refluxed overnight. The reaction mixture was
worked up as for the preparation of 19 to give a pale yellow oil (0.22g, 71%), which was
converted as above to 20. 1HNMR (CDCl3) δ 7.59-7.53 (dd, J= 7.77Hz, 1.72 Hz, 1H), 7.41-
7.32 (m, 2H), 7.28-7.25 (td, J= 7.57 Hz, 1.76 Hz, 1H), 2.92-2.89 (d, J=14.0 Hz, 3H), 2.80-
2.78 (d, J= 9.56 Hz, 3H), 2.40-2.28 (m, 2H), 2.27-2.18 (m, 3H), 1.94-1.82 (m, 4H), 1.74-1.68
(m, 2H), 1.66-1.38 (m, 5H); 13C (CDCl3) δ 205.40, 171.87, 140.36, 132.42, 130.53, 129.36,
128.92, 126.88, 68.20, 42.14, 38.42, 38.10, 36.72, 34.77, 32.13, 29.67, 25.21, 22.44, 20.77;
MS m/z 351.20 (MH+) Calculated for C19H27ClN2O2 (MH+) 351.18338 found 351.18272.
3.1.18. 2-(2-Chlorophenyl)-2-((3-(piperidin-1-yl)propyl)amino)cyclohexan-1-one
hydrochloride (21). Similar reaction as in Scheme 1 of 23 (0.15 g, 0.67 mmol) and 1-(3-
chloropropyl)piperidine hydrochloride (0.80 g, 4.04 mmol) with K2CO3/KI in MeCN at 110
oC for 12 h, followed by purification as above, gave a pale yellow oil (0.12 g, 52%) which
was converted to 21 as above. 1HNMR (CDCl3) δ 7.71-7.65 (dd, J= 8.0 Hz, 1.56 Hz, 1H),
7.42-7.34 (m, 2H), 7.31-7.24 (td, J=7.24 Hz, 1.6 Hz, 1H), 3.15-3.05 (m, 2H), 3.00-2.85 (m,
3H), 2.51-2.45 (m, 3H), 2.45-2.35 (m, 2H), 2.10-1.90 (m, 7H), 1.80-1.70 (m, 4H), 1.72-1.65
(m, 3H); 13C (CDCl3) δ 211.15, 136.71, 134.09, 131.29, 130.10, 129.50, 127.66, 70.96, 57.20,
53.76, 53.64, 53.42, 40.91, 40.86, 40.33, 29.64, 23.96, 23.38, 22.48, 22.11; MS m/z 349.20
(MH+) Calculated for C20H29ClN2O (MH+) 349.2041 found 349.2043
3.1.19. 2-(2-Chlorophenyl)-2-((2-(ethylsulfonyl)ethyl)amino)cyclohexan-1-one
hydrochloride (22).Similar reaction of 23 (0.44 g, 1.97 mmol) and 1-bromo-2-
(ethylsulfonyl)ethane14 (0.67 g, 3.30 mmol) with K2CO3/KI in MeCN at 120 oC for 168 h,
followed by purification as above, gave a pale yellow oil (0.46 g, 70 %) which was converted
to 22 as above. 1HNMR (CDCl3) δ 7.94-7.92 (m, 1H), 7.60-7.56 (m, 3H), 3.20-3.02 (m, 5H),
2.81-2.72 (m, 1H), 2.29-2.19 (m, 2H), 2.05-1.82 (m, 6H), 1.36-1.28 (t, J= 7.49 Hz, 3H); 13C
(CDCl3) δ 209.28, 135.22, 133.64, 133.20, 132.08, 131.99, 129.94, 69.06, 47.98, 47.42,
39.52, 37.94, 23.20, 22.39, 21.04, 6.77; MS m/z 344.10 (MH+) Calculated for C16H23ClNO3S
(MH+) 344.1082 found 344.1078.

3.2. Biological activity
3.2.1. General
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 13420). Following acquisition of baseline physiologic parameters
(heart rate, respiratory rate, PWR, and documentation of intact righting reflex (RR)) adult
female Sprague-Dawley rats (age 119 to 168 days; weight 230 to 320g) were put under non-
traumatic restraint and the marginal tail vein cannulated. Ketamine or an experimental
compound at 10mg/ml was administered by automated syringe driver connected via a
minibore extension tube adequately secured to the animal’s tail. Infusions were commenced
at a rate (weight-adjusted) to deliver 20 mg/kg/min initially (continued until loss of righting
reflex (LORR) and subsequent pedal withdrawal reflex score PWR=1), then were reduced to
a rate of 6.7 mg/kg/min. Infusion rate was then titrated in an up-and-down fashion to
maintain dorsal recumbency and a PWR=1 to 10 minutes before cessation. Three rats were
used in each study, with each group of rats also acting as their own ketamine control. The
order of study drug administration was determined by prior odds/evens randomisation with a
recovery interval of at least four hours afforded between experiments. PWR and RR were
recorded at 1 minute intervals throughout. The times from cessation of infusion to return of
righting reflex (RORR), and from cessation of infusion to the animals displaying independent
locomotion (walk) were recorded.
4.2.2. Pedal Withdrawal Reflex (PWR) scoring: Nociceptive testing in animals was
conducted via 1 second application of constant pressure (firm digital pressure) over the
forepaw of the animal. Pedal withdrawal reflex testing is primarily used to assess analgesic
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 ref 16).
4.2.3. 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.
4.2.4. Determination of NMDA inhibition: Determinations of NMDA inhibition were
carried out by Eurofins Panlabs Taiwan, Ltd. Pharmacology Laboratories 158 Li-Teh Road,
Peitou Taipei, Taiwan 11259, in Wistar rat brain (minus cerebellum) preparations with MK-
801 {[5R,10S]-[+]-5-methyl-10,11- dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine} as
ligand, by radioimmunoassay, in 5 mM Tris-acetate buffer (pH 7.4) for 3 h at 25°C).
Acknowledgements

The authors thank the Biopharma Thematic Research Initiative, University of Auckland, and
Kea Therapeutics Ltd, Auckland for funding.
References
1. Dimitrov, I.; Jose, J.; Denny, W.A., Synthesis, 2018, 50, 4201-4215.
2. Jose, J.; Gamage, S.A.; Harvey, M.G.; Voss, L.J.; Sleigh, J.W.; Denny, W.A., Bioorg.
Med. Chem., 2013, 21, 5098-5106.
3. Harvey, M.G.; Voss, L.J.; Jose, J.; Gamage, S.A.; Pruijn, F.B.; Liyanage, S.; Denny, W.A.;
Sleigh, J.W., Anesthesia & Analgesia, 2015, 121, 925-933.
4. Buchwald, P., Mini-Rev. Med. Chem. 2001, 1, 101-111.
5. Hong, S., Davisson, J., J. Pharm. Sci. 1982, 71, 912-914.
6. Levin, J., Turos, E., Weinreb, S., Synth. Commun., 1982, 12, 989-993.
7. Dyke, C., Bryson, T., Tetrahedron Lett., 2001, 42, 3959-3961.
8. Ng, P., Laing, B., Balasundarum, G., Pingle, M., Friedman, A., Bergstrom, D.,
Bioconjugate Chem., 2010, 1545-1553
9. Evans, R., Baiga, T., Bock, M., Downes, M., Embler, E., Fan, W., Keana, F., Kluge A.,
Noel, J., Patane, M., WO 2014/165827
10. NMDA radioligand binding assays were carried out at Panlabs Taiwan Ltd Pharmacology
Laboratories, 158 Li-Teh Road, Peitou Taipei, Taiwan 11259.
11. Anis, A., Berry, S., Burton, N., Lodge, D., Br. J. Pharmacol. 1983, 79, 565-575.
12. Mion, G., Villevieille, T., CNS Neurosci. Therap. 2013, 19, 370-380.
13. Fagg, G.E., NeuroScience Lett. 1987, 76, 221-227
14. Zanos, P., Moaddel, R., Morris, P., et al., Nature 2016, 533, 481-486.
15. Caplar, V., Sunjic, V., Kafjez, F., Journal of Heterocyclic Chemistry 1974, 11(6), 1055-
1060
16. Buitrago, S., Martin, T.E., Tetens-Woodring, J., Belicha-Villanueva, A., Wilding, G.E. J.
Amer. Assoc. Lab. Animal Sci. 2008, 47, 11-17.