Supporting Information

Validating Eatons Hypothesis: Cubane as a Benzene Bioisostere

Benjamin A. Chalmers, Hui Xing, Sevan Houston, Charlotte Clark, Sussan Ghassabian,
Andy Kuo, Benjamin Cao, Andrea Reitsma, Cody-Ellen P. Murray, Jeanette E. Stok,
Glen M. Boyle, Carly J. Pierce, Stuart W. Littler, David A. Winkler, Paul V. Bernhardt,
Cielo Pasay, James J. De Voss, James McCarthy, Peter G. Parsons, Gimme H. Walter,
Maree T. Smith, Helen M. Cooper, Susan K. Nilsson, John Tsanaktsidis,* G. Paul Savage,* and
Craig M. Williams*

anie_201510675_sm_miscellaneous_information.pdf
Author Contributions: C.M.W., G.P.S. and J.T. conceived the project. C.M.W., B.A.C., H.X. and S.D.H.
chose the cubane targets. B.A.C., H.X. and S.D.H. undertook the synthetic preparation of all cubane
analogues and obtained the respective characterisation data. C.M.W. and B.A.C. wrote the paper with
assistance from all authors. C.E.J.C. and H.M.C. designed the leteprinim study and analysed results. S.G. and
M.T.S undertook the Phase I and II leteprinim metabolism study and analysed the results. A.K. and M.T.S.
designed the benzocaine study and analysed results. B.C., A.R., D.W., and S.K.N. designed the SAHA
mouse study and analysed results. C.-E.M. and G.H.W. designed the diflubenzuron study and analysed
results. J.S. and J.D.V. designed the t-butylbenzene metabolism study and analysed results. G.M.B., C.J.P.,
and P.G.P. designed the SAHA cell line study and analysed results. S.W.L. performed the LogP analysis.
P.V.B. performed the X-ray crystallographic structure determination for compounds 4 and 12. C.P. and J.Mc.
designed the scabies study and analysed results.
 Table of Contents
Supplementary Materials Part 1: Synthesis ..................................................................................S3
General Experimental ........................................................................................................................S3
log P Measurements ........................................................................................................................... S6
SAHA (3) and SUBACUBE (4).................................................................................................... S6
Cubane (1), Benzene (2), Benzocaine (7), Cubocaine (8), Benzyl benzoate (9), Cubyl cubates
(10a–c), Diflubenzuron (11) and Diflucuburon (12) .................................................................... S6
Leteprinim (5) and Letepricube (6) ............................................................................................. S12
log P Summary ............................................................................................................................ S14
Experimental Procedures and Characterisation Data ....................................................................... S15
SUBACUBE (4) ..........................................................................................................................S15
Letepricube (6) ............................................................................................................................ S18
Cubocaine (8) .............................................................................................................................. S21
Cubyl cubates (10a–c) ................................................................................................................. S23
Diflucuburon (12) ........................................................................................................................ S25
1-(tert-Butyl)cubane (S24) .......................................................................................................... S27
Supplementary Materials Part 2: Biological Studies .................................................................. S28
SUBACUBE (4) ............................................................................................................................... S28
Letepricube (6) ................................................................................................................................. S32
Cubocaine (8) ................................................................................................................................... S34
Cubyl cubates (10a-c) ...................................................................................................................... S36
Diflucuburon (12) ............................................................................................................................ S37
Metabolism....................................................................................................................................... S41
tert-Butylcubane .......................................................................................................................... S41
Leteprinim (5) and Letepricube (6) ............................................................................................. S42
Supplementary Materials Part 3: Crystallographic Data .......................................................... S47
SUBACUBE (4) ............................................................................................................................... S47
Diflucuburon (12) ............................................................................................................................ S49
Supplementary Materials Part 4: 1H and 13C Spectra................................................................ S51
Supplementary Materials Part 5: References ............................................................................. S84

S2
Supplementary Materials Part 1: Synthesis

General Experimental

Abbreviations
°C degrees Celsius
Δ heat/reflux
Δ chemical shift
µm microliter
µm micrometer
Boc tert-Butyl carbonate
br broad
calcd calculated
CDCl3 deuterated chloroform
CSIRO Commonwealth Scientific and Industrial Research Organisation
D2 O deuterium oxide
Da Dalton(s)
decomp decomposition
DCM dichloromethane
DMF N,N-dimethylformamide
DMAP N,N-dimethyl-4-aminopyridine
DMSO-d6 deuterated dimethyl sulfoxide
DMSO dimethyl sulfoxide
d doublet
EI electron ionisation
et al et alii / et aliae (and others)
EtOAc ethyl acetate
ESI electrospray ionisation
eV electron volts
GCMS gas chromatography mass spectrometry
g gram
HCl hydrochloric acid

S3
h hour(s)
HRMS high resolution mass spectrometry
hυ photoirradiation
Hz Hertz
IC50 50% maximal inhibitory concentration
in vacuo in a vacuum
in vitro within the glass
in vivo within the living
J coupling constant
LC-MS/MS liquid chromatography-mass spectrometry/mass spectrometry
Methanol-d4 deuterated methanol
MHz mega-hertz
min minute(s)
mL millilitre(s)
mmol millimole(s)
mm milimetre
m.p. melting point
m/z mass to charge ratio
m multiplet
NADPH nicotinamide adenine dinucleotide phosphate
NGF nerve growth factor
nmol nanomole(s)
NMR nuclear magnetic resonance
PBS phosphate buffered saline
post hoc after this
ppm parts per million
q quartet
rh relative humidity
rpm revolutions per minute
SAHA suberoylanilide hydroxamic acid

S4
SE/SEM standard error/standard error of the mean
s singlet
THF tetrahydrofuran
TLC thin layer chromatography
t tertiary
t triplet
UDPGA glucuronosyltransferase
UV ultraviolet
wt weight

NMR Spectroscopy
NMR spectra were recorded under standard conditions (unless stated otherwise) using Brooker
AVANCE 500, 400 and 300 MHz spectrometers and were referenced with residual monoprotic
solvent peaks (e.g. CDCl3, C6D6 etc.) [29]. Samples run in D2O were referenced using a dioxane
standard (1H = δ 3.75 ppm, 13C = 67.2 ppm). Coupling constants (J) are quoted to the nearest 0.1
Hz.

Mass Spectrometry
High resolution mass spectra were recorded using a Bruker MicroTOF-Q (quadrupole-Time of
Flight) with a Bruker ESI source.

Melting Points
Melting points were determined using a Digimelt MPA 160 melting point apparatus and are
reported uncorrected.

Chromatography
Flash column chromatography was run using Merck silica gel 60 (230–400 mesh). Fractions were
initially visualised using UV irradiation and subsequently by heating TLC plates exposed to either
ceric ammonium molybdate (Goofy’s stain) or 10 % aqueous potassium permanganate or similar.
TLC was performed with Merck silica gel plates, precoated with silica gel 60 F254 (0.2 mm).

Experimental Procedures
All reactions were magnetically stirred and, where required, were performed in oven-dried
glassware under a positive pressure of dry argon. Dry solvents were distilled prior to use based on
the methods described by Amarego and Chai [30]. Commercially available chemicals were used
without further purification unless specified otherwise.

Known Pharmaceutical and Agrochemical Compounds

SAHA (3), leteprinim (6) and diflubenzuron (11) were purchased from commercial sources as
indicated (vide infra). Benzocaine (7) was synthesised following previously reported literature [31].
Benzyl benzoate (9) was synthesised using a modified Schotten-Baumann procedure.

S5
log P Measurements

SAHA (3) and SUBACUBE (4)

Octanol/water partition coefficients (log P) were indirectly determined by means of reverse-phase
HPLC. All measurements were performed using a Waters 600 liquid chromatographic pump system
fitted with a 5 µm octadecylsilane (Alltima C18) column (4.6 x 150 mm) kept at 32 °C in a constant
temperature column heater. Peaks were detected using a Waters 2487 UV detector at wavelengths
254 nm and 214 nm, and recorded on a Waters Millennium Data Management System. Generally,
10 µL of each sample as a solution in methanol (1–2 mg/mL) was injected using a Waters 717
Autosampler. The mobile phase consisted of 72% methanol / 28% 0.005 M phosphate buffer
adjusted to pH 7.6, and containing 1.0 g/L sodium dodecyl sulphate and 0.1 M sodium perchlorate.
Flow rate was 0.8 mL/min.

log P Estimation:
The chromatographic capacity factor, k', of each compound was calculated by the formula:
k' = (t - to)/to
where t is the retention time of the compound and to is the retention time of an unretained substance,
determined in this case by injection of a solution of uracil.
The relationship between octanol/water partition coefficients and HPLC retention behaviour in this
system was established by the determination of k' values of 108 reference compounds of varying
functionality and structure type, and plotting the log P values of these compounds (obtained from
the literature) versus the logarithm of their k' values (log k') obtained from the HPLC system. The
log P’s of acidic and basic compounds are determined in their uncharged form, i.e., acidic
compounds the mobile phase at pH 2.2, and at pH 7.6 for basic compounds. The log P’s of all other
compounds are usually determined at pH 4.6, but can be measured at any pH.
Two linear relationships between these two parameters were obtained:
log P = 2.766 log k' + 1.74
for hydrocarbons, halides, heterocycles and proton acceptor compounds,
and log P = 1.975 log k' + 1.89
for amphiprotic compounds,
allowing a simple estimation of log P values of compounds from their measured k' values.

Cubane (1), Benzene (2), Benzocaine (7), Cubocaine (8), Benzyl benzoate (9), Cubyl cubates
(10a–c), Diflubenzuron (11) and Diflucuburon (12)
The HPLC measurements were performed using a system comprising a Waters 600 pump controller,
Waters 717 autosampler and Waters 2996 PDA detector with data processing and control by Waters
Empower software. Chromatography was performed on a 150 x 4.6 mm Alltima C18 column kept
at a constant 30 °C by a column oven with mobile phase comprised of 72% methanol containing
28% 0.05 M phosphate buffer and 0.1% sodium dodecyl sulphate and 0.1% sodium perchlorate
adjusted to either pH 2.2 or pH 4.6 or pH 7.6 as necessary at 1.0 mL / min flow rate. Generally, 10
µL of each sample as a solution in methanol (1–2 mg/mL) was injected.

log P Estimation:
The chromatographic capacity factor, k', of each compound was calculated by the formula:
k' = (t - to)/to
where t is the retention time of the compound and to is the retention time of an unretained substance,
determined in this case by injection of a solution of uracil.
The relationship between octanol/water partition coefficients and HPLC retention behaviour in this
system was established by the determination of k' values of 30 reference compounds of varying
functionality and structure type, and plotting the log P values of these compounds (obtained from
the literature) versus the logarithm of their k' values (log k') obtained from the HPLC system. The
log P’s of acidic and basic compounds are determined in their uncharged form, i.e., acidic
S6
compounds the mobile phase at pH 2.2, and at pH 7.6 for basic compounds. The log P’s of all other
compounds are usually determined at pH 4.6, but can be measured at any pH.
Linear relationships were established between these two parameters for each class of compounds
allowing a simple estimation of log P values of compounds from their measured k' values:

S7
Cubane (1) and benzene (2)

Compound tR k' log k' log Poct log PHPLC CLog P

Benzene (1) 5.19 1.95 0.29 2.13 2.09 2.14
Toluene 7.71 3.38 0.53 2.69 2.74 2.79
Ethyl-benzene 11.04 5.27 0.72 3.15 3.27 3.32
Diphenyl 16.84 8.57 0.93 3.89 3.84 4.03
Naphthalene 10.68 5.07 0.70 3.30 3.22 3.32
Table S1: Chromatographic data (measured at pH 4.6, t0(Uracil) = 1.76), log Poct (literature), log
PHPLC (calculated) and Clog P (ChemDraw®) for hydrocarbon reference compounds used to
calculate log PHPLC values for cubane (1) and benzene (2).

log Poct vs log k'

4.50
4.00 y = 2.7298x + 1.2967
3.50 R² = 0.9848
3.00
log Poct

2.50
2.00
1.50
1.00
0.50
0.00
0.00 0.20 0.40 0.60 0.80 1.00
log k'

Fig. S1: Linear relationship between log Poct and log k’ (log PHPLC = 2.7298 log k’ + 1.2967) used
to calculate log PHPLC values for cubane (1) and benzene (2).

S8
Benzyl benzoate (9) and cubyl cubates (10a-c)

Compound tR k' log k' log Poct log PHPLC CLog P

Ethyl benzoate 6.06 2.44 0.39 2.64 2.74 2.64
Phenyl benzoate 9.74 4.53 0.66 3.59 3.49 3.62
Ethyl cinnamate 8.32 3.73 0.57 2.99 3.25 3.09
Diphenyl ether 14.52 7.25 0.86 4.21 4.06 4.24
Acetophenone 3.26 0.85 -0.07 1.58 1.47 1.58
Table S2: Chromatographic data (measured at pH 4.6, t0(Uracil) = 1.76), log Poct (literature), log
PHPLC (calculated) and Clog P (ChemDraw®) for H acceptor reference compounds used to calculate
log PHPLC values for benzyl benzoate (9) and cubyl cubates (10a-c).

log Poct vs log k'

4.50
4.00 y = 2.7842x + 1.6618
3.50 R² = 0.9684
3.00
log Poct

2.50
2.00
1.50
1.00
0.50
0.00
-0.20 0.00 0.20 0.40 0.60 0.80 1.00
log k'

Fig. S2: Linear relationship between log Poct and log k’ (log PHPLC = 2.7842 log k’ + 1.6618) used
to calculate log PHPLC values for benzyl benzoate (9) and cubyl cubates (10a-c).

S9
Benzocaine (7) and cubocaine (8)

Compound tR k' log k' log Poct log PHPLC CLog P

m-Toluidine 2.96 0.68 -0.17 1.4 1.24 1.56
3,5-Dimethylaniline 3.75 1.13 0.05 1.7 1.80 2.21
N,N-Dimethylaniline 6.34 2.60 0.42 2.31 2.73 2.34
Diphenylamine 8.42 3.78 0.58 3.5 3.14 3.62
Table S3: Chromatographic data (measured at pH 7.6, t0(Uracil) = 1.76), log Poct (literature), log
PHPLC (calculated) and Clog P (ChemDraw®) for aniline reference compounds used to calculate log
PHPLC values for benzocaine (7) and cubocaine (8).

log Poct vs log k'

4
3.5
y = 2.5609x + 1.6639
3 R² = 0.8697
2.5
log Poct

2
1.5
1
0.5
0
-0.40 -0.20 0.00 0.20 0.40 0.60 0.80
log k'

Fig. S3: Linear relationship between log Poct and log k’ (log PHPLC = 2.5609 log k’ + 1.6639) used
to calculate log PHPLC values for benzocaine (7) and cubocaine (8).

S10
Diflubenzuron (11) and diflucuburon (12)

Compound tR k' log k' log Poct log PHPLC CLog P

Chlorobenzene 7.39 3.20 0.50 2.84 2.86 2.86
Bromobenzene 8.36 3.75 0.57 2.99 3.04 3.00
Fluorobenzene 5.02 1.85 0.27 2.27 2.24 2.28
p-Dichlorobenzene 11.19 5.36 0.73 3.50 3.45 3.57
Table S4: Chromatographic data (measured at pH 4.6, t0(Uracil) = 1.76), log Poct (literature), log
PHPLC (calculated) and Clog P (ChemDraw®) for aromatic halide reference compounds used to
calculate log PHPLC values for diflubenzuron (11) and diflucuburon (12).

log Poct vs log k'

4.00
3.50 y = 2.626x + 1.5373
R² = 0.9912
3.00
2.50
log Poct

2.00
1.50
1.00
0.50
0.00
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80
log k'

Fig. S4: Linear relationship between log Poct and log k’ (log PHPLC = 2.626 log k’ + 1.5373) used to
calculate log PHPLC values for diflubenzuron (11) and diflucuburon (12).

S11
Leteprinim (5) and Letepricube (6)
The HPLC measurements were performed using a system comprising a Waters 600 pump controller,
Waters 717 autosampler and Waters 2996 PDA detector with data processing and control by Waters
Empower software. Chromatography was performed on a 150 x 4.6 mm Alltima C18 column kept
at a constant 30 °C by a column oven with mobile phase comprised of 50% methanol containing
50% 0.05 M phosphate buffer and 0.1% sodium dodecyl sulphate and 0.1% sodium perchlorate
adjusted to pH 2.2 at 1.0 mL / min flow rate. Generally, 10 µL of each sample as a solution in the
mobile phase (1–2 mg/mL) was injected.

log P Estimation:
The chromatographic capacity factor, k', of each compound was calculated by the formula:
k' = (t - to)/to
where t is the retention time of the compound and to is the retention time of an unretained substance,
determined in this case by injection of a solution of uracil.
The relationship between octanol/water partition coefficients and HPLC retention behaviour in this
system was established by the determination of k' values of 5 reference compounds of varying
functionality and structure type, and plotting the log P values of these compounds (obtained from
the literature) versus the logarithm of their k' values (log k') obtained from the HPLC system. The
log P’s of acidic and basic compounds are determined in their uncharged form, i.e., acidic
compounds the mobile phase at pH 2.2, and at pH 7.6 for basic compounds. The log P’s of all other
compounds are usually determined at pH 4.6, but can be measured at any pH.
A linear relationship was established between these two parameters allowing a simple estimation of
log P values of compounds from their measured k’ values:

S12
Leteprinim (5) and letepricube (6)

Compound tR k' log k' log Poct log PHPLC CLog P

Benzene (1) 13.72 6.80 0.83 2.13 2.29 2.14
Toluene 27.63 14.70 1.17 2.69 2.65 2.79
Ethyl benzoate 22.66 11.88 1.07 2.64 2.55 2.64
Benzoic acid 5.71 2.24 0.35 1.87 1.76 1.88
Indole-2-carboxylic acid 8.68 3.93 0.59 1.95 2.03 2.3
Table S5: Chromatographic data (measured at pH 2.2, t0(Uracil) = 1.76), log Poct (literature), log
PHPLC (calculated) and Clog P (ChemDraw®) for reference compounds used to calculate log PHPLC
values for leteprinim (5) and letepricube (6).

log Poct vs log k'

3
y = 1.0922x + 1.3779
2.5
R² = 0.9132
2
log Poct

1.5

0.5

0
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40
log k'

Fig. S5: Linear relationship between log Poct and log k’ (log PHPLC = 1.0922 log k’ + 1.3779) used
to calculate log PHPLC values for leteprinim (5) and letepricube (6).

S13
log P Summary

Compound tR k' log k' log PHPLC

Benzene (1) 5.19 1.95 0.29 2.09
Cubane (2) 19.21 9.91 1.00 4.02
SAHA (3) 2.61 0.35 -0.45 0.99
SUBACUBE (4) 3.18 0.65 -0.19 1.52
Leteprinim (5) 3.42 0.94 -0.03 1.35
Letepricube (6) 2.53 0.44 -0.36 0.99
Benzocaine (7) 2.88 0.64 -0.20 1.16
Cubocaine (8) 2.95 0.68 -0.17 1.23
Benzyl benzoate (9) 12.62 6.17 0.79 3.86
Cubyl cubate (10a) 41.36 22.50 1.35 5.43
Cubyl benzoate (10b) 31.98 17.17 1.23 5.10
Benzyl cuboate (10c) 16.30 8.26 0.92 4.22
Diflubenzuron (11) 10.25 4.82 0.68 3.33
Diflucuburon (12) 8.85 4.03 0.61 3.13
Table S6: Chromatographic data and log PHPLC values (measured and calculated using the
experimental parameters outlined in above sections) for benzene (1), cubane (2), known
pharmaceutical and agrochemical compounds (3, 5, 7, 9, 11) and their cubane analogues (4, 6, 8,
10a-c, 12).

S14
Experimental Procedures and Characterisation Data

SUBACUBE (4)

Scheme S1: Synthesis of SUBACUBE (4).

4-Methoxycarbonylcubane-1-carboxylic acid (14)

Following the procedure of Eaton et al. [32]: dimethyl cubane-1,4-dicarboxylate 13 (6.079 g, 27.60
mmol) was suspended in THF (250 mL). A solution of sodium hydroxide (1.220 g, 30.50 mmol) in
methanol (14 mL) was added dropwise and the solution was left to stir for 16 h. The THF was
removed in vacuo and the residual solid was suspended in water (200 mL), and washed with DCM
(3 x 100 mL). The aqueous phase was acidified to pH 2 with aqueous hydrochloric acid (10 M) and
extracted with DCM (3 x 100 mL). The combined organic phases were dried over magnesium
sulfate and concentrated to give 14 (5.385 g, 95%) as a white solid. 1H-NMR (300 MHz, CDCl3): δ
4.28 (s, 6H), 3.73 (s, 3H).

Methyl cubane-1-carboxylate (S1)

Following the procedure of Ko et al. [33]: carboxylic acid 14 (9.654 g, 46.82 mmol) was dissolved in
anhydrous DCM (500 mL) under an argon atmosphere. Oxalyl chloride (4.81 mL, 56.08 mmol) and
anhydrous DMF (0.3 mL) were added and the solution was left to stir for 1 h. The DCM was
removed in vacuo and the residual brown oil was exposed to high vacuum for 1 h. Separately,
freshly ground 2-mercaptopyridine N-oxide sodium salt 15 (10.639 g, 71.33 mmol) and DMAP (59
mg, 0.48 mmol) were suspended in anhydrous chloroform[34] (500 mL) under an argon atmosphere

S15
and heated to reflux whilst under irradiation from a 500-W tungsten lamp. The nascent acid chloride
was suspended in anhydrous chloroform (500 mL) under an argon atmosphere and added slowly
over 1 h to the refluxing mixture. After 4 h reflux the suspension was washed with water (3 x 500
mL), dried over magnesium sulfate and concentrated to give a brown oil. Purification by column
chromatography (petroleum ether:EtOAc 9:1 v/v) gave S1 (6.820 g, 90%) as a white sweet smelling
solid. 1H-NMR (300 MHz, CDCl3): δ 4.26–4.24 (m, 3H), 4.04–3.99 (m, 4H), 3.70 (s, 3H).

Cubanecarboxylic acid (16)

Adapted from the procedure of Eaton et al. [32]: ester S1 (6.600 g, 0.041 mmol) was suspended in
THF (200 mL). A solution of sodium hydroxide (2.004 g, 50.10 mmol) in methanol (12 mL) was
added dropwise and the solution was left to stir for 16 h. The THF was removed in vacuo and the
residual solid was suspended in water (200 mL) and extracted with DCM (3 x 100 mL). The
aqueous phase was acidified to pH 2 with aqueous hydrochloric acid (10 M) and extracted again
with DCM (3 x 100 mL). The combined organic phases were dried over magnesium sulfate and
concentrated to give 16 (5.324 g, 88%) as a yellow solid. 1H-NMR (400 MHz, CDCl3): δ 4.33–4.29
(m, 3H), 4.07–4.00 (m, 4H).

t-Butyl cubanylcarbamate (S2)

Adapted from the procedure of Shioiri et al. [35]: carboxylic acid 16 (0.500 g, 3.37 mmol), freshly
distilled diphenylphosphoryl azide (0.73 mL, 3.39 mmol) and anhydrous triethylamine (0.47 mL,
3.37 mmol) were suspended in anhydrous t-butyl alcohol (10 mL) under an argon atmosphere. The
mixture was heated to reflux for 24 h. On cooling and concentration in vacuo the residual material
was purified by column chromatography (hexanes:EtOAc 3:2 v/v) to give S2 (0.490 g, 67%) as an
off-white solid. 1H-NMR (400 MHz, CDCl3): δ 5.04 (br, s, 1H), 4.06 (m, 3H), 3.95 (br, m, 1H),
3.88 (br, s, 3H), 1.46 (s, 9H).

Aminocubane hydrochloride (17)

Adapted from the procedure of Eaton et al. [36]: acetyl chloride (1 mL, 14mmol) was added
dropwise to a stirred solution of anhydrous methanol (2 mL) at -30 °C under an argon atmosphere
and allowed to stir for 15 min. Solid S2 (100 mg, 0.456 mmol) was added and the solution was
allowed to stir for 1 h, and then allowed to warm to room temperature. Concentration in vacuo gave
a brown solid which was washed with a solution of diethyl ether:acetone (4:1 v/v) and filtered to
give S7 as a white solid (60 mg, 90%). 1H-NMR (300 MHz, D2O): δ 4.12 (m, 3H), 3.91 (m, 4H).

8-Ethoxy-8-oxooctanoic acid (S4)

Adapted from the procedure of Grolla et al. [37]: to a solution of 1-ethyl 8-methyloctanedioate S3
(12.66 mL, 0.054 mol) in THF (25 mL) was added 2.5 M ethanolic KOH (21.5 mL, 0.054 mol) and
the solution stirred at 0 °C for 18 h. The solution was then stirred for a further 6 h at room
temperature. The solvent was removed in vacuo and water (200 mL) was added. The solution was
extracted with diethyl ether (4 x 50 mL) before being acidified to pH 2–3 with aqueous
S16
hydrochloric acid (10 M). The acidified aqueous phase was extracted with DCM (4 x 100 mL). The
organic layer was dried over magnesium sulfate, filtered and the concentrated in vacuo to yield (8.6
g, 79%) of low melting solid. The solid was purified by Kugelrohr distillation to give (6 g, 55%) of
S4 as an amorphous white solid. 1H-NMR (300 MHz, CDCl3): δ 4.10 (q, J = 7.1 Hz, 2H), 2.30 (dt, J
= 17.5, 7.5 Hz, 4H), 1.62 (m, 4H), 1.34 (m, 4H), 1.23 (t, J = 7.1 Hz, 3H).

Ethyl 8-(cubylamino)-8-oxooctanoate (S5)

Octanoic acid S4 (1.500 g, 7.55 mmol) was dissolved in anhydrous DCM (5 mL) at 0 °C under an
argon atmosphere. Oxalyl chloride (0.75 mL, 8.26 mmol) and anhydrous DMF (1 drop) were added
and the solution was left to stir for 2 h. The DCM was removed in vacuo to give ethyl 8-chloro-8-
oxooctanoate (18). The nascent acid chloride (240 mg, 1.09 mmol) was dissolved in anhydrous
DCM (20 mL) under an argon atmosphere to give a 0.055 M solution. Hydrochloric acid salt 17 (31
mg, 0.19 mmol was suspended in anhydrous DCM (20 mL) under an argon atmosphere and cooled
to -30 °C. A solution of ethyl 8-chloro-8-oxooctanoate (3.69 mL, 0.20 mmol) in anhydrous DCM
was added and the solution was stirred for 5 min. Anhydrous triethylamine (0.11 mL, 0.80 mmol)
was added dropwise and the solution was allowed to stir for 15 min. A precipitate formed and the
solution was allowed to warm to room temperature before concentration in vacuo. The residue was
purified by column chromatography (hexanes:EtOAc 4:1 v/v) to give S5 (51 mg, 85%) as a white
solid. m.p. 100–102 °C; 1H-NMR (400 MHz, CDCl3): δ 5.93 (br, s, 1H), 4.10 (m, 5H), 3.91 (m,
4H), 2.26 (t, J = 7.5 Hz, 2H), 2.15 (t, J = 7.5 Hz, 2H), 1.60 (m, 4H), 1.32 (m, 4H), 1.23 (t, J = 7.1
Hz, 3H); 13C-NMR (100 MHz, CDCl3): δ 173.79, 172.28, 66.61, 60.19, 52.58, 47.81, 42.92, 52.58,
47.81, 42.92, 36.18, 34.25, 28.82, 28.78, 25.24, 24.75, 14.24; HRMS-ESI calcd for C18H25NO3Na
([M+Na]+): 326.1732, found 326.1729.

SUBACUBE (4)

Adapted from the procedure of Gediya et al. [38]: a solution of KOH (260 mg, 3.89 mmol) in
methanol (2 mL) was added slowly to a stirred solution of hydroxylamine hydrochloride (138 mg,
1.98 mmol) in methanol (2 mL) at 0 °C. The solution was stirred for 30 min. Amide S5 (60 mg,
0.198 mmol) in methanol (3 mL) was added dropwise at 0 °C and the solution was allowed to stir
overnight. The solvent was removed in vacuo and water (10 mL) was added followed by slow
addition of acetic acid until pH 6–7. The resultant solid precipitate was filtered, dried and
recrystallised from methanol to obtain 4 (35 mg, 62%) as a white solid. m.p. 165–167 °C (decomp);
1
H-NMR (300 MHz, DMSO-d6): δ 10.30 (s, 1H), 8.63 (s, 1H), 8.40 (s, 1H), 4.00 (m, 3H), 3.86 (m,
4H), 2.05 (t, J = 7.4 Hz, 2H), 1.91 (dd, J = 9.6, 5.1 Hz, 2H), 1.45 (m, 4H), 1.22 (m, 4H); 13C-NMR
(100 MHz, DMSO-d6) δ: 171.38, 168.97, 65.92, 51.82, 46.69, 41.88, 34.96, 32.13, 28.23, 24.86;
HRMS-ESI calcd for C16H22N2O3Na ([M+Na]+): 313.1528, found 313.1523; log P: 1.52.

S17
Letepricube (6)

Scheme S2: Synthesis of Letepricube (6).

1-Methyl-4-(t-butoxycarbonylamino)cubylcarboxylate (S6)

Adapted from the procedure of Shioiri et al. [35]: carboxylic acid 14 (2.000 g, 9.70 mmol) was
dissolved in anhydrous t-butyl alcohol (40 mL) and stirred under an argon atmosphere. Anhydrous
triethylamine (1.35 mL, 9.68 mmol) was added and the solution was allowed to stir until clear.
Freshly distilled diphenylphosphoryl azide (2.1 mL, 9.74 mmol) was then added and the solution
was heated to reflux for 2 days. On cooling the solvent was removed in vacuo and the residual
material was purified via column chromatography (EtOAc) to give S6 (1.770 g, 66%) as a white
solid. m.p. 166–168 °C; 1H-NMR (300 MHz, CDCl3): δ 4.07 (m, 6H), 3.68 (s, 3H), 1.43 (s, 9H);
13
C-NMR (125 MHz, CDCl3): δ 203.0, 172.6, 66.2, 55.9, 51.4, 50.2, 50.2, 44.5, 28.2; HRMS-ESI
calcd for C15H19NO4Na ([M+Na]+): 300.1206, found 300.1214.

4-(Methoxycarbonyl)cubaneammonium chloride (S7)

Adapted from the procedure of Eaton et al. [36]: acetyl chloride (15 mL, 0.21 mol) was added slowly
to anhydrous methanol (30 mL) and stirred vigorously at -30 °C under an argon atmosphere. After
15 min S6 (1.500 g, 5.41 mmol) was added. The solution was stirred for 1 h then allowed to warm
to room temperature. The solvent was removed in vacuo to give a green/yellow solid which was
S18
washed with diethyl ether:acetone (4:1 v/v) and filtered to give S7 (0.943 g, 82%) as an off-white
solid. 1H-NMR (300 MHz, D2O): δ 4.14 (s, 6H), 3.64 (s, 3H).

Methyl 4-acrylamidocubanecarboxylate (20)

Cubaneammonium chloride S7 (1.500 g, 7.02 mmol) and acryloyl chloride (S8) (0.63 mL, 7.79
mmol) were stirred in anhydrous DCM (40 mL) at -30 °C under an argon atmosphere. Anhydrous
triethylamine (3.87 mL, 27.75 mmol) in anhydrous DCM (11 mL) was added dropwise via syringe
pump over 30 min. The solution was stirred for a further 20 min before the solvent was removed in
vacuo. The residual material was purified by column chromatography (EtOAc) to give 20 (1.070 g,
66%) as a white solid. m.p. 184–186 °C (decomp); 1H-NMR (300 MHz, CDCl3): δ 6.30–6.26 (dd, J
= 17.0, 1.3 Hz, 1H), 6.08 (m, 2H), 5.66–5.64 (dd, J = 10.1, 1.4 Hz, 1H), 4.16 (m, 6H), 3.69 (s, 3H);
13
C-NMR (100MHz, CDCl3): δ 172.65, 164.74, 130.01, 127.00, 66.60, 55.76, 51.52, 50.23, 45.09;
HRMS-ESI calcd for C13H13NO3Na ([M+Na]+): 254.0793, found 254.0795.

Methyl 4-[3-(6-amino-9H-purin-9-yl)propanamido]cubanecarboxylate (S10)

Adapted from the procedure of Lei at al. [39]: adenine (S9) (321 mg, 2.38 mmol) was suspended in
anhydrous methanol (40 mL) under an argon atmosphere. Sodium methoxide in methanol (25%
w/w, 0.32 mL) was added and the solution was allowed to stir for 30 min until all adenine had
dissolved. Amide 20 (500 mg, 2.16 mmol) was added and the solution was heated at reflux
overnight. The solvent was removed in vacuo the resultant precipitate was purified by column
chromatography (chloroform:methanol 9:1 v/v) to give S10 (278 mg, 35%) and recovered 20 (300
mg, 60%). m.p. >250 °C; 1H-NMR (500 MHz, DMSO-d6): δ 8.13 (s, 1H), 7.96 (s, 1H), 7.82 (s, 1H),
4.43 (t, J = 6.6 Hz, 2H), 3.98–3.91 (m, 6H), 3.60 (s, 3H), 2.71 (t, J = 6.6 Hz, 2H); 13C-NMR (75
MHz, DMSO-d6): δ 171.68, 169.00, 155.90, 152.33, 149.36, 140.91, 118.68, 65.91, 54.94., 51.16,
49.42, 44.15, 34.86, 30.68; HRMS-ESI calcd for C18H18N6O3Na ([M+Na]+): 389.1338, found
389.1346.

Methyl 4-[3-(6-oxo-5H-purin-9(6H)-yl)propanamido]cubanecarboxylate (S11)

Adapted from the procedure of Lei at al. [39]: amide S10 (168 mg, 0.46 mmol) was dissolved in
glacial acetic acid (1.5 mL). The solution was stirred for 15 min before a solution of sodium nitrite
(970 mg, 14.06 mmol) was added and the solution was heated at reflux overnight. After
concentration in vacuo the residual material was purified by column chromatography
(chloroform:methanol 9:1 v/v) to give S11 (87 mg, 51%) as a white solid. m.p. 225–227 °C
(decomp); 1H-NMR (300 MHz, DMSO-d6): δ 12.26 (s, 1H), 8.71 (s, 1H), 8.03 (s, 1H), 7.95 (s, 1H),
4.34 (t, J = 6.5 Hz, 2H), 3.98 (dd, J = 16.8, 4.3 Hz, 6H), 3.61 (s, 3H), 2.68 (t, J = 6.5 Hz, 2H); 13C-
NMR (100 MHz, DMSO-d6): δ 174.39, 172.99, 171.65, 146.58, 142.39, 65.9, 55.0, 51.2, 49.4, 44.2,
35.1; HRMS-ESI calcd for C19H18N6O3 ([M+H]+): 368.1353; found 368.1359.

S19
4-[3-[6-Oxo-1H-purin-9(6H)-yl]propanamido]cubanecarboxylic acid (S12)

Amide S11 (6 mg, 0.016 mmol) was suspended in water (1 mL). Sodium hydroxide (1 mg, 0.025
mmol) was added and the solution was stirred overnight. Aqueous hydrochloric acid (10 M) was
added until pH 1 whereupon a precipitate was formed. The solvent was removed in vacuo and the
solid was washed with water to give S12 (5 mg, 90%) as an amorphous solid. m.p. 220–222 °C
(decomp); 1H-NMR (400 MHz, DMSO-d6): δ 8.06 (s, 1H), 7.83 (s, 1H), 4.45 (t, J = 6.5 Hz, 2H),
3.96 (m, 6H), 2.75 (t, J = 6.5 Hz, 2H); 13C-NMR (100 MHz, DMSO-d6): δ 175.33, 168.80, 164.44,
151.42, 149.21, 137.41, 124.15, 65.96, 59.44, 48.57, 44.45, 35.35; HRMS-ESI calcd for
C17H15N5O4K ([M+K]+): 392.0756; found 392.0761.

Letepricube (6)

Amide S12 (10 mg, 0.028 mmol) was suspended in water (1 mL). To this was added aqueous
potassium hydroxide (0.1000 M, 283 µL, 0.028 mmol) and the solution was stirred for 5 min. The
water was removed in vacuo to give 6 (11 mg, 100%) as a white solid. m.p. 215–217 °C (decomp);
1
H-NMR (400 MHz, D2O): δ 8.21 (s, 1H), 8.10 (s, 1H), 4.56 (t, J = 6.3 Hz, 2H), 3.99–3.96 (m, 3H),
3.90–3.88 (m, 3H), 2.82 (t, J = 6.3 Hz, 2H); 13C-NMR (100 MHz, D2O): δ 181.5, 172.4, 159.1,
149.4, 146.3, 142.8, 123.8, 66.3, 58.6, 49.6, 45.5, 41.5, 36.7; HRMS-ESI calcd for C17H15N5O4
([M-K]-): 352.1051; found 352.1050; log P: 0.99.

S20
Cubocaine (8)

Scheme S3: Synthesis of Cubocaine (8).

Diethyl cubane-1,4-dicarboxylate (S13)

Dimethyl cubane-1,4-dicarboxylate 13 (8.000 g, 36.32 mmol) was suspended in ethanol (300 mL)
with aqueous hydrochloric acid (10 M, 1 mL). The solution was heated to reflux for 4 h, at which
point the ethanol was removed in vacuo. The residual white solid was partitioned between water
(300 mL) and diethyl ether (300 mL). After separation, the aqueous phase was extracted with
diethyl ether (2 x 300 mL), dried over magnesium sulfate and concentrated in vacuo to give S13
(8.741 g, 97%) as an off white solid. 1H-NMR (300 MHz, CDCl3): δ 4.22 (s, 6H), 4.16 (q, J = 7.2
Hz, 4H), 1.27 (t, J = 7.2 Hz, 6H).

4-(Ethoxycarbonyl)cubane-1-carboxylic acid (S14)

Adapted from the procedure of Eaton et al. [32]: bis(ethyl) ester S13 (4.049 g, 16.44 mmol) was
suspended in THF (150 mL). A solution of sodium hydroxide (768 mg, 19.20 mmol) in ethanol (10
mL) was added dropwise and the solution was left to stir for 15 h. The THF was removed in vacuo
and the residual solid was suspended in water (150 mL) and washed with DCM (3 x 100 mL). The
aqueous phase was acidified to pH 2 with aqueous hydrochloric acid (10 M) and extracted with
DCM (3 x 100 mL). The combined organic phases were dried over magnesium sulfate and
concentrated to give S14 (3.145 g, 87%) as a white solid. m.p. 113.8–115.4 °C; 1H-NMR (400
MHz, CDCl3): δ 4.28–4.24 (m, 6H), 4.16 (q, J = 8.0 Hz, 2H), 1.26 (t, J = 8.0 Hz, 3H); 13C-NMR
(100 MHz, CDCl3): δ 177.5, 171.6, 60.5, 56.0, 55.5, 47.1, 47.0, 14.3; HRMS-ESI calcd for
C12H13O4 ([M+H]+): 219.0663, found 219.0656.

4-Ethoxycarbonylcubane Boc-protected amide (S15)

Adapted from the procedure of Shioiri et al. [35]: carboxylic acid S14 (1.018 g, 4.62 mmol),
diphenylphosphoryl azide (1.08 mL, 5.00 mmol) and anhydrous triethylamine (0.70 mL, 5.00
S21
mmol) were suspended in anhydrous t-butyl alcohol (5 mL) under an argon atmosphere. The
mixture was left to stir at room temperature for 30 min before heating to reflux for 1 h. On cooling
and concentration in vacuo the residual material was purified by column chromatography
(petroleum ether:EtOAc 5:1 v/v) to give S15 (1.047 g, 78%) as a white solid. m.p. 123.7–126.0 °C;
1
H-NMR (400 MHz, CDCl3): δ 4.15 (q, J = 8.0 Hz, 2H), 4.08 (br s, 6H), 1.45 (s, 9H), 1.26 (t, J =
8.0 Hz, 3H); 13C-NMR (75 MHz, CDCl3): δ 172.4, 79.9, 66.3, 60.2, 56.1, 50.2, 44.4, 28.3, 14.3;
HRMS-ESI calcd for C16H21NO4Na ([M+Na]+): 314.1363, found 314.1368.

Cubocaine (8)

Adapted from the procedure of Eaton et al. [36]: carbamate S15 (100 mg, 0.34 mmol) was suspended
in anhydrous ethanol (2.5 mL) under an argon atmosphere and cooled to -78 °C. Acetyl chloride
(1.0 mL, 14.01 mmol) was added and the solution was allowed to warm to room temperature and
left to stir for 4 h. The ethanol was removed in vacuo and the crude mixture was filtered with cold
diethyl ether:acetone (4:1 v/v) to give 8 (45 mg, 58%) as a white solid. m.p. 169.5–170.5 °C; 1H-
NMR (400 MHz, D2O): δ 4.24 (s, 6H), 4.19 (q, J = 8.0 Hz, 2H), 1.27 (t, J = 8.0 Hz, 3H); 13C-NMR
(100 MHz, D2O): δ 175.3, 64.8, 62.6, 56.9, 48.4, 48.3, 45.7, 45.6, 13.9; HRMS-ESI calcd for
C11H14NO2 ([M-Cl]+): 192.1019, found 192.1028; log P: 1.23.

S22
Cubyl cubates (10a–c)

R1 R2 Product Yield

85%

87%

86%

Scheme S4: Synthesis of Cubyl cubates (10a-c).

Cubylcarbinol (19)
Cubylcarbinol (19) was synthesised from cubanecarboxylic acid (16) following the procedure of
Priefer, Farrell and Harp.[40]

General method
Carboxylic acid (R1, 1.00 mmol) was dissolved in anhydrous DCM (2 mL) under an argon
atmosphere. Oxalyl chloride (0.10 mL, 1.20 mmol and anhydrous DMF (1 drop) were added and
the solution was left to stir for 30 min before concentration in vacuo. The nascent acid chloride was
suspended in DCM (2 mL) under an argon atmosphere, to which was added a solution of alcohol
(R2, 1.00 mmol) and 4-N,N-dimethylaminopyridine (1.10 mmol) in anhydrous DCM (2 mL). The
solution was stirred at room temperature for 1 h before the addition of aqueous sodium bicarbonate
(10%). The solution was then extracted with DCM (3 x 5 mL) and the combined organic phases
were dried over magnesium sulate. After concentration in vacuo, the crude material was purified by
column chromatography (petroleum ether:EtOAc 17:3 v/v) to give the pure ester. Yields and
spectroscopic data are reported below.

(Cubanyl)methyl cubanecarboxylate (10a)

White solid (225 mg, 85%). m.p.: 98–99 °C; 1H-NMR (300 MHz, CDCl3): δ 3.88 (m, 6H), 3.99 (m,
5H), 4.21 (m, 3H), 4.24 (s, 2H); 13C-NMR (75 MHz, CDCl3): δ 44.55, 45.18, 47.40, 47.82, 48.56,
49.56, 55.88, 56.00, 64.57, 172.73; HRMS-ESI calcd for C18H16O2Na ([M+Na]+): 192.1019, found
192.1028; log P: 5.43.

(Cubanyl)methyl benzoate (10b)

S23
Colourless oil (207 mg, 87%). 1H-NMR (300 MHz, CDCl3): δ 3.95 (m, 6H), 4.03 (m, 1H), 4.46 (s,
2H), 7.45 (m, 2H), 7.57 (m, 1H), 8.05 (m, 2H); 13C-NMR (75 MHz, CDCl3): δ 44.64, 47.43, 48.64,
55.90, 65.53, 128.31, 129.59, 130.54, 132.79, 166.87; HRMS-ESI calcd for C16H14O2Na
([M+Na+]): 261.0891, found 261.0886; log P: 5.10.

Benzyl cubanecarboxylate (10c)

Colourless oil (205 mg, 86%). 1H-NMR (300 MHz, CDCl3): δ 3.99 (m, 4H), 4.25 (m, 3H), 5.13 (s,
2H), 7.32 (m, 5H); 13C-NMR (75 MHz, CDCl3): δ 45.12, 47.81, 49.47, 55.69, 65.78, 128.04,
128.06, 128.49, 136.33, 172.20; HRMS-ESI calcd for C16H14O2Na ([M+Na+]): 261.0891, found
261.0886; log P: 4.22.

S24
Diflucuburon (12)

Scheme S5: Synthesis of Diflucuburon (12).

Methyl 4-chloro-cubane-1-carboxylate (21)

Following the procedure of Kuduva et al. [41]: carboxylic acid 14 (1.000 g, 4.80 mmol) was
dissolved in anhydrous DCM (15 mL), under an argon atmosphere. Oxalyl chloride (0.46 mL, 5.80
mmol) and anhydrous DMF (1 drop) were added and the solution was left to stir for 1 h before
concentration in vacuo. The nascent acid chloride was dissolved in anhydrous carbon tetrachloride
(20 mL) and added dropwise to a stirred solution of 2-mercaptopyridine N-oxide sodium salt 15
(1.100 g, 7.30 mmol) and 4-N,N-dimethylaminopyridine (5 mg, 0.048 mmol) in anhydrous carbon
tetrachloride (20 mL). The solution was then irradiated using a 500W Tungsten lamp. After 2 h the
solvent was removed in vacuo and the residue was purified by column chromatography
(hexanes:EtOAc 20:1 v/v) to give 21 (500 mg, 52%). 1H-NMR (300 MHz, CDCl3): δ 4.23–4.18 (m,
3H), 4.16–4.12 (m, 3H), 3.69 (s, 3H).

4-chloro-cubane-1-carboxylic acid (S16)

Following the procedure of Kuduva et al. [41]: ester 21 (200 mg, 1.02 mmol) was dissolved in
methanolic sodium hydroxide solution (5.0 mL, 0.51 M, 2.55 mmol) and heated at reflux for 1 h.
On cooling the reaction mixture was concentrated in vacuo. Water was added and the mixture was
extracted with DCM (3 x 15 mL). The aqueous phase was acidified to pH 1–2 with aqueous
hydrochloric acid (1 M), and then extracted with DCM (3 x 15 mL). The combined organic phases
were dried over sodium sulfate and concentrated to give S16 (160 mg, 90%) as yellow solid. 1H-
NMR (300 MHz, CDCl3): δ 4.27–4.23 (m, 3H), 4.20–4.15 (m, 3H).

Diflucuburon (12)

Adapted from the procedure of Shioiri et al. [35]: carboxylic acid S16 (1.000 g, 5.48 mmol),
diphenylphosphoryl azide (1.17 mL, 5.42 mmol) and anhydrous triethylamine (0.90 mL, 6.45
mmol) were suspended in anhydrous toluene (10 mL) under an argon atmosphere. The mixture was
S25
left to stir at room temperature for 30 min before heating to 90 °C for 2 h. This hot mixture
containing the nascent isocyanate S17 was slowly added over 10 min to a refluxing solution of 2,6-
diflurobenzamide S18 (950 mg, 6.05 mmol) in anhydrous toluene (20 mL) under an argon
atmosphere. After addition the reaction mixture was stirred at reflux for 12 h. On cooling a
precipitate formed, which was filtered and washed with toluene and acetone to give 12 as a white
solid (924 mg, 50%). m.p. 230–231 °C (decomp); 1H-NMR (400 MHz, CDCl3): δ 9.27 (br s, 1H),
8.81 (s, 1H), 7.53–7.45 (m, 1H), 7.04–6.99 (m, 2H), 4.13–4.11 (m, 3H), 4.04–4.01 (m, 3H); 13C-
NMR (100 MHz, CDCl3): δ 161.7, 161.2, 161.2, 158.7, 158.6, 151.2, 133.5, 133.4, 133.3, 112.5,
112.4, 112.4, 112.3, 112.2, 112.2, 72.9, 66.6, 51.8, 48.8; HRMS-ESI calcd for C16H11ClF2N2O2Na
([M+Na]+): 261.0891, found 261.0886; log P: 3.13.

S26
1-(tert-Butyl)cubane (S19)

Adapted from the procedure of Eaton et al. [42]: 1,4-Diiodocubane[43] (0.500 g, 1.41 mmol) in THF
(40 mL) was added dropwise to a stirred solution of t-butyl lithium (1.2 M solution in hexanes, 24
mL, 28 mmol) in THF (20 mL) at -100 °C (internal thermometer, liquid nitrogen/ethanol). The
solution was stirred for 30 min, quenched with methanol (5 mL), warmed to 0 °C and water (20
mL) was added. The resultant solution was then extracted with pentane (80 mL, 2 x 20 mL), dried
with magnesium sulfate, filtered and concentrated via distillation using a 20 cm Vigreux column.
The resultant liquid was diluted with pentane (5 mL), filtered and purified by preparatory gas
chromatography (injection: 200 °C; column: 165 °C; retention: 5:00 – 6:25 min) to give a colourless
liquid S24 (10 mg, 2.8%). 1H-NMR (300 MHz, Methanol-d4): δ 3.97 (m, 1 H), 3.76 (m, 6 H), 0.75
(s, 9H); 13C-NMR (75 MHz, Methanol -d4): δ 49.89, 46.91, 44.09, 31.21, 22.68; HRMS-EI calcd for
C12H17 [(M+H)·]: 161.1330; found 161.9904; calcd for C11H13 [(M-CH3)·]: 145.1017; found
145.1012.

S27
Supplementary Materials Part 2: Biological Studies

SUBACUBE (4)

Inhibition of cancer cell growth in culture (performed by the group led by Prof. Peter Parsons at the
QIMR Berghofer Medical Research Institute):

Materials and Methods

SAHA was purchased from Sigma-Aldrich (Catalogue #10009929). SUBACUBE (4) was
synthesized as detailed in Supplementary Materials Part 1. Both compounds were dissolved in
DMSO. MM96L and MCF7 were human tumor cell lines derived from melanoma and breast cancer
respectively. NFF were early passage neonatal foreskin fibroblasts.

Sulforhodamine B assay for inhibition of growth of cells cultured as monolayers

Cells were seeded at 5,000 per microtitre well (96-well plate) in 10% FCS-RPMI 1640 culture
medium, treated, and allowed to grow until the controls were nearly confluent (5-6 days). Culture
media was removed; the wells were then washed twice with phosphate buffered solution (PBS),
fixed with ethanol for a minimum of 5 min. and washed with water. Sulforhodamine B (SRB)
solution (50 µL of 0.4% in 1% acetic acid) was added and the mixture left at room temperature for a
minimum of 15 min. The plate was washed rapidly with tap water and then twice with 0.1-1%
acetic acid, the liquid being removed by tapping each time. After addition of 100 µL/well of 10
mM Tris base (unbuffered, pH > 9), plates were left for a minimum of 5 min, then the absorbance
was read at 564 nm on an ELISA reader with SOFTmax® PRO Version 3.1.2, with a 3 second prior
shaking operation. Data were exported as text files and analysed via an Excel spreadsheet. After
subtraction of a blank (i.e. wells with no cells, A564 typically ~0.04), growth inhibition was
calculated as a percentage of the untreated control and plotted against dose [44]. Non-linear
regression was performed using GraphPad Prism version 6.05 for Windows (GraphPad Software,
La Jolla California USA).

Results
SAHA (3) was observed to inhibit the growth of both tumour cell lines with an IC50 of
approximately 0.03 µg/mL (Fig. S6). The healthy cells (NFF) however, were observed to not be
affected to the same extent. SUBACUBE (4) gave similar results (Fig. S7).

S28
 SAHA
120
M M 96L
100 M C F7
NFF
80
% C o n tr o l

60

40

20

0
1 0 -6 1 0 -5 1 0 -4 1 0 -3 1 0 -2 1 0 -1 10 0 10 1 10 2

C o n c e n tr a tio n (  g /m l)

Fig. S6: Inhibition of cell growth by 3.

SUBACUBE
120
M M 96L
100 M C F7
NFF
80
% C o n tr o l

60

40

20

0
1 0 -6 1 0 -5 1 0 -4 1 0 -3 1 0 -2 1 0 -1 10 0 10 1 10 2
C o n c e n tra tio n (m g /m l)

Fig. S7: Inhibition of cell growth by 4.

S29
Mouse xenograft (performed by the group led by A/Prof. Susan Nilsson at CSIRO Manufacturing
Flagship and Australian Regenerative Medicine Institute, Monash University):

Materials and Methods

NODSCIDIL2Rγ−/− (NSG) mice were bred at Monash Animal Research Platform (MARP), Monash
University, Clayton, Australia. Female mice were 6 to 12 weeks old for experiments. All
experiments were approved by the MARP ethics committee.

Cells and Xenograft Model

MyLa 2059 cells [45] were maintained in RPMI 1640 (Gibco, Life Technologies, Mulgrave,
Victoria, Australia) supplemented with 10% fetal bovine serum (FBS)(SAFC, Sigma-Aldrich Pty
Ltd, Sydney, Australia) and 2 mM L-Glutamine (Gibco, Life Technologies, Mulgrave, Victoria,
Australia) with 10% CO2 at 37 oC. Solid tumours were generated using 100,000 MyLa 2059 cells
injected subcutaneously (s.c.) into the hind right flank of NSG mice. Cells were in log growth phase
when transplanted. Tumours were detected by palpation at 13-16 days post transplantation. Therapy
was commenced when individual tumours reached 20-50 mm [46]. Tumours were measured daily
with calipers, (INSIZE Mini Digital Caliper Series IIII, Cheektowaga, NY, USA) and tumour
volume was calculated by (a x b x c) x pi/6. When tumours reached 500 mm [46] animals were
terminated.

Treatment
SAHA (3) (ChemiTek, Indianapolois, IN, USA) (102 mg, 0.386 mmol) in methanol (20 mL) was
treated with 0.2 M sodium hydroxide (1.93 mL, 0.386 mmol) for 1 h at room temperature. The
solution was concentrated in vacuo, diluted with water, filtered through a 0.45 m syringe filter unit
and lyophilized to give SAHA sodium salt (107 mg, 97%) as a fluffy colourless powder. The same
procedure was used to obtain SUBACUBE sodium salt. For in vivo studies, SAHA and
SUBACUBE salts were dissolved in freshly prepared 10% (w/v) hydroxypropyl-beta-cyclodextrin
(HPCD)/normal saline to a final concentration of 6 mg/mL. A dose of 60 mg/kg of SAHA and
SUBACUBE salts was delivered to tumour engrafted NSG mice via intraperitoneal (i.p.) injection
(10 mL/g mouse wt) once a day. A vehicle only group received equivalent volumes of 10%
HPbCD/saline.

Tumour growth and modelling

Tumours typically undergo an early exponential growth phase, followed by a pseudo exponential
slowing when the growth of large tumours becomes increasingly nutrient limited. This behaviour
can be described by a number of double exponential functions, the most common one being the
Gompertz function [46].
S(t) = S(0)exp[(1-exp(-t))/]
where S(t) is the tumour size at time t, and  and  are positive constants. This equation may be
transformed to give a linear relationship:
ln[lnS(max) –ln(S(t)] = ln(/) –t
where S(max) is the maximum size of the tumour at sacrifice. When the double logarithm function
on the left hand side of the above equation is plotted against time, the intercept is ln(/), and the
slope is , the specific growth rate of the tumour. According to the theory, the rate is independent of
whether area or volume of the tumour is used to estimate its size.
Considerable variability was detected between individual animals but the slopes were usually linear,
suggesting these data are well described by the model. The specific growth rates (slopes of
Gompertz graph) for individual mice were averaged.

S30
Results
According to the Gompertz theory, the greater the growth rate of a tumour, the greater the slope of
the line will be. The specific tumour growth rates (slopes of fitted lines in Fig. 3A for cubane-
treated and control animals, averaged over all animals for each treatment, showed equivalent
reduced tumour growth rates for SUBACUBE (4) and SAHA (3) treated animals. Both treatments
demonstrated efficacy, with the tumour growth rates reduced to 63% of that of the control
(untreated) animals.

S31
Letepricube (6)

Biological studies concerned with leteprinim (5) and letepricube (6) (performed by the group led by
A/Prof. Helen Cooper at the Queensland Brain Institute):

Materials and Methods

Adherent PC12 cells were grown in RPMI 1640 (Gibco, 11875-093) + 10% heat-inactivated horse
serum (HI-HS, Sigma, H1270) + 5% HI-fetal calf serum (FCS, SAFC Biosciences, 12003C) + 1%
Penicillin-Streptomycin (Invitrogen, 15140122). For differentiation, PC12 cells were plated at a
density of 2 x 103 cells per well in 24-well plates on #1 round glass coverslips coated with 0.015%
poly-L-ornithine (Sigma, P4957). Cells were grown in growth medium for 1 h, before the medium
was replaced with RPMI 1640 + 1% HI-FCS +1% Penicillin-Streptomycin, containing NGF (50
ng/ml Biosensis, 105-01925) and commercial leteprinim (5) (100 µM) (Toronto Research
Chemicals Inc., L329900, MW 365.39) or letepricube (6) (100µM, MW 391.42) (was synthesized
as detailed in Supplementary Materials Part 1. Compounds were made up to 10 mM stock solutions
in 0.1 M PBS, pH 7.4. Differentiation medium was replaced every 48 h. Four independent
experiments were performed, with triplicates for each condition. Cells were fixed after 6 days in
vitro with 4% paraformaldehyde in PBS.
Cells were prepared for immunohistochemistry as follows: cells were blocked with 2% HI-goat
serum (GS, Sigma, G9023) + 2% HI-FCS + 0.25% Triton-X 100 (Sigma, X100) in 1 x PBS for 2 h
at room temperature. Cells were then incubated with mouse anti-neuronal-specific βIII-tubulin
(TUJ1, 1:2000, Covance, MMS-425P) in blocking solution overnight at 4 °C. After three PBS
washes, cells were incubated with goat anti-mouse Alexa 488 (1:500, Invitrogen, A1101) and 4’-6-
diamidino-2-phenylindole (DAPI) nuclear stain (1:1000, Sigma, D9542) for 1 h at room
temperature. After three PBS washes, the cells were mounted with ProLong GOLD (Molecular
Probes, P36934).

Figure 3b: Percentage of differentiated cells

Images used for the quantification of neurite growth were captured using a Zeiss AxioCam high-
resolution monochrome camera on a Zeiss Axio Imager upright microscope using a 20X Plan-
APOCHROMAT objective and Zen software. Five regions per coverslip (fifteen images per
condition per experiment) were captured. Regions were randomly selected based on detection of
DAPI-positive nuclei. Image analysis was performed using the Cell Counter plugin in NeuronJ
software. Cells were classed as either undifferentiated (no processes or processes < 20 µm in
length) or differentiated (at least one process > 20 µm in length). All data were collected with the
investigator blind to the experimental condition. Statistical analysis was performed using GraphPad
Prism 6. Data were compared using ordinary one-way analysis of variance (ANOVA) followed by
the Dunnett’s multiple comparisons test (p<0.0001 ***).

Results
The length of the longest neurite (Fig. S8) per neuron was measured using a modified ImageJ
plugin, NeuronJ. These data were extracted in MATLAB, transferred to an Excel spreadsheet and
compared in GraphPad Prism. The data were taken from the data set used in Fig. 3B (n=4
experiments, from >20 cells per condition).
NGF-induced neurite growth was not enhanced by leteprinum or letepricube. The length of the
longest neurite per neuron was significantly increased in the presence of NGF alone, NGF +
Leteprinum or NGF + Letepricube. There was no difference in neurite length in the presence of
NGF + Leteprinum or NGF + Letepricube compared to NGF alone. Ordinary one-way ANOVA,
Sidak’s multiple comparisons test; p<0.0001 ****.

S32
Conclusion
Both leteprinim (5) and letepricube (6) effect the initial phase of neuronal differentiation (i.e.
neurite outgrowth, Fig. 3B but not subsequent neurite growth (Fig. S8).

Fig. S8: Length of longest neurite.

S33
Cubocaine (8)

Antinociception evaluation (performed by the group led by Prof. Maree Smith at the Centre for
Integrated Preclinical Drug Development):

Materials and Methods

Experimental Animals
All animal care and experimental procedures complied with the Australia Code of Practice for the
Care and Use of Animals for Scientific Purposes (8th edition, 2013). Ethics approval was obtained
from the Animal Ethics Committee of The University of Queensland (Brisbane, Australia).
Male Sprague-Dawley (SD) rats were purchased from the Animal Resources Centre (Perth, WA,
Australia). Rats weighed 180–200 g upon arrival and they were housed in a purpose-built Physical
Containment Level 2 (PC2) animal holding facility in groups of three to four in individually
ventilated cages (BioZone, Thorne Hill, Ramsgate, Kent, UK). Rat chow (Specialty Feeds, Glen
Forrest, WA, Australia) and tap water were available ad libitum throughout the housing period. Rats
were maintained in cages that contained recycled paper bedding material (FibreCycle Pty Ltd,
Yatala, QLD, Australia) and environmental enrichment comprised Kimwipes (Kimberly-Clark
Professional, Milsons Point, NSW, Australia), a rodent hutch (red Perspex hutch) and Rat
Chewsticks (Able Scientific, Welshpool, WA, Australia) in each cage. The animal holding facility
had a 12 h/12 h light/dark cycle and a mean (±SEM) room temperature of 23 (±3) °C. Animals were
acclimatised for at least three days in the animal holding facility prior to initiation of any
experimentation.

Test Compound Administration

Stock solutions of each of benzocaine (7) and cubocaine (8) were prepared at a concentration of 10
ug/µL using sterile H2O for injection as vehicle and these were stored refrigerated and protected
from light until required. Stock solutions were assigned a one week expiry date from the date of
preparation. Working solutions were prepared freshly on the day of experimentation from the stock
solution (10 µg/µL) and using H2O for injection as diluent. Both 7 and 8 were synthesized as
detailed in Supplementary Materials Part 1.
Rats were anaesthetized briefly with 3% isoflurane (Abbott Australasia Pty Ltd, Botany, NSW,
Australia) delivered in oxygen to facilitate intraplantar (i.pl.) drug administration. The i.pl. injection
volumes were fixed at 10 µL and injections were made using a 50 μL Hamilton syringe (SGE
Analytical Science Pty Ltd, Ringwood, VIC, Australia) with a 25G x 5/8 needle (Terumo
Corporation, Shibuya-ku, Tokyo, Japan). Each rat received a maximum of two doses with at least 2
days between each dose. Each hindpaw was injected once only.

Rat model of acute pain: Noxious thermal stimuli to the hindpaws

The Hargreaves’ apparatus was used to apply acute noxious thermal (heat) stimuli (Ugo Basile,
Comerio, Italy) to the hindpaws of normal rats. One the day of experimentation, awake freely-
moving rats were acclimatised to the Hargreaves holding chamber for at least 15 min prior to
measurement of the time to withdraw the hindpaws in response to an applied noxious heat stimulus.
For individual rats, the heat source of the Hargreaves apparatus was placed beneath the plexiglass
surface of the holding chamber and aimed at the mid-plantar region of one hindpaw. Baseline paw
thermal thresholds (PTTs) were in the range 10 to 15 s. A cut-off time of 30 s was used to prevent
tissue damage. For each Hargreaves baselining (pre-dosing; 0 min) session, the PTTs for both
hindpaws were measured up to six times with each successive reading separated by a minimum of a
5 min interval. The closest three readings were averaged and used as the baseline PTT value.
Following dose administration, hindpaw PTTs were measured at the following post-dosing times
30, 45, 60, 75, 90, 120 and 180 min.

S34
Results and Data Analysis
For individual rats, delta (Δ) PTT values were calculated by subtracting the pre-dosing PTT value
from the corresponding post-dosing PTT values and any negative Δ PTT values were arbitrarily
assigned a value of 0. For each rat, the extent and duration of antinociception (pain relief) was
determined by using trapezoidal integration to estimate the area under the ∆PTT versus time curve
(Δ PTT AUC values) using GraphPad Prism (version 6.04; GraphPad Software, San Diego, CA,
USA).
The i.pl. bolus doses of benzocaine (7) at 10 and 20 µg, and cubocaine (8) at 8.86 and 17.72 µg
were converted to nmoles. As the i.pl. bolus doses of benzocaine were larger than those of
cubocaine, the ∆PTT AUC values for benzocaine were normalized by multiplying each value by a
scaling factor of 0.7846 (Table S7) to give dose-normalized ∆PTT AUC values.
Analysis of variance (ANOVA) with a post Dunnett’s Multiple Comparison test was performed on
the mean (±SEM) normalized-ΔPTT AUC values for groups of SD-rats administered single i.pl.
bolus doses of cubocaine or the positive control item, benzocaine relative to animals administered
vehicle (H2O for injection). Microsoft Excel (version 14.0.7128.5000; Microsoft Corporation,
Redmond, WA, USA) and GraphPad Prism were used for all data and statistical analysis. The
statistical significance criterion was p ≤ 0.05.

Test Items Amount (µg) Molecular Weight (g) nmole Normalization Factor
Benzocaine-HCl 10 201.65 49.59 0.7846
20 201.65 99.18 0.7846
Cubocaine-HCl 8.86 227.69 38.91 Not Applicable
17.72 227.69 77.82 Not Applicable
Table S7: Summary of the calculations for the normalisation factors.

S35
Cubyl cubates (10a-c)

Acaricidal activity of scabies mites (performed by the group led by Prof. James McCarthy at the
QIMR Berghofer Medical Research Institute):

Materials and Methods

Scabies mites
Sarcoptes scabiei var suis mites were collected from scabies infected pigs maintained at the Centre
for Advanced Animal Science (CAAS), University of Queensland, Gatton Campus, Australia. Pigs
were kept on an optimised immunosuppression regimen to ensure a steady supply of scabies mites
for acaridical activity studies [47].

Bioassay method
Cubyl cubates 10a, 10b, and 10c (synthesized as detailed in Supplementary Materials Part 1 were
diluted to 50 mM concentrations with mineral oil. With the exception of 10c, 10a and 10b, required
heating at 60 °C for 10 min to completely dissolve. Thirty-five microliters of each test compound
was spread evenly across the surface of a 30 mm x 615 mm plastic petri dish (Proscitech, QLD,
Australia) using a microtip. Live mites were placed directly into the petri dishes to allow direct
contact with the test compounds and controls. Benzyl benzoate (9) (25 mM) was used as the
positive control acaricide, while mineral oil alone was used as the negative control. Mites were
incubated in a 28 °C humidified incubator and observed microscopically within the first 30 min,
hourly thereafter, up to 8 h, and again at the completion of the assay (after 24 h). Mortality was
recorded by microscopic verification of absence of leg movement and gut peristalsis when touched
with a probe. The contact bioassays to test the cubyl cubates were performed in duplicate dishes
with ten mites per dish. Duplicate assays were performed independently twice so the total sample
size or n=40 mites per compound tested.

Results
The in-vitro survival of scabies mites after 24 h of exposure to cubyl cubates 10a-c is shown above
(Fig. S9). The highest killing effect was observed with 10c with only 45% of mites remaining alive
after 24 h of exposure to the compound. Compound 10b showed only moderate acaricidal activity
killing 32.5% of mites after 24 h of exposure. The least acaricidal activity was observed with 10a
killing only 25% of mites after 24 h of exposure. All mites died within 5 min of exposure to benzyl
benzoate (9), the positive control acaricide used while all mites remained alive in mineral oil
(negative control) after 24 h of exposure. Benzyl benzoate (9) is a quick acting acaricide with
acaricidal effects usually observed within a few minutes of contact of scabies mites in-vitro. In
contrast, 10c manifested a slower activity against scabies mites with dead mites observed only after
24 h of exposure.
M it e s u r v iv a l in c u b y l c u b a t e s ( 5 0 m M )

100 10a
10b
P e r c e n t s u r v iv a l

10c
B e n z y l B e n z o a te ( 9 )

50 M in e r a l o il

0
0 10 20 30
T im e (h o u r)

Fig. S9: Mite survival in cubyl cubates.

S36
Diflucuburon (12)

Tribolium castaneum (rust-red flour beetle) evaluation (performed by the group led by Prof.
Gimme Walter at the School of Biological Sciences):

Introduction
Tribolium has often been used in investigations of the mode of action of insecticides, including
insect growth regulators (IGRs) and benzoylphenylureas (BPUs) such as diflubenzuron (11) [48], an
inhibitor of chitin formation [49]. Diflubenzuron (11) is considered most effective on the larval
stages of arthropods and results in abortive moulting [50].
We tested the mortality imposed on T. castaneum larvae by diflubenzuron (11) at different doses
relative to that caused by the cubane analogue (12). We tested different laboratory strains (with
different levels of resistance to phosphine, simply because they were readily available) and also
included a field strain.

Materials and Methods

Rearing T. castaneum
Three hundred T. castaneum adults were placed in a whole-wheat flour + 20 g torula yeast mixture
(400 g) at 25 °C and ~70% rh overnight. The following day the adults were sieved from the
medium, which was retained so as to culture the larvae that hatched from the eggs deposited in it.
After three weeks these larvae were ~6 mm long, so were close to pupation, and were used in
experiments.

Experimental methods
The treatment doses and application methods for testing chitin inhibition in insects using IGR’s are
numerous and varied [51]. We used the following method, which is fairly representative.
Controls comprised a Petri dish containing 10 late instar larvae in a 10 g organic whole wheat flour
medium enriched with 5% torula yeast. The diflubenzuron (11) and cubane analogue (12)
treatments were conducted in the same manner as the controls, with the appropriate weight of the
relevant compound (see below) added to the flour medium and mixed thoroughly, 30 seconds
clockwise then 30 seconds anticlockwise, before addition of the larvae. All treatments were held at
25 °C and 70% rh for 10 days. The larvae were sieved from the medium to record mortality. Data
were analysed by means of ANOVA’s and Tukey’s tests were used for post hoc comparisons.
Tests were conducted on two strains that have been cultured in the laboratory for some time, one
susceptible to the fumigant phosphine, the other resistant to it [52]. The third strain tested comprised
first generation offspring from field-collected beetles (Dalby, Queensland).

Preliminary comparison - diflubenzuron (11) and cubane analogue (12)

A preliminary test was conducted on the susceptible laboratory strain. Slightly different molecular
weights of the test compounds were used. Three replicates of each molecular weight treatment level
were run, together with three blank controls.

Comparison with equal molecular weights of (11) and (12)

The efficacy of the cubane analogue (12) and diflubenzuron (11) at equal molecular doses were
tested on all three strains of T. castaneum (see above). Three replicates of each dose level were run
for each of the strains, plus three blank controls.

Results
Fig. S10 shows that the cubane analogue (12) resulted in significantly higher levels of larval
mortality in T. castaneum than did diflubenzuron (11). Control mortality was uniformly low in this
test, and this resulted in a consistently clear pattern of mortality across compounds and across
S37
treatments. This result is consistent with the results presented in Fig. 3D, even though lower
molecular weights of the cubane analogue (12) than diflubenzuron (11) were used in tests.

Fig. S10: Mean (±SE) mortality of larval T. castaneum (phosphine resistant strain) caused by the
cubane analogue 12 (white) and diflubenzuron (11) (black) at different doses. Superscripts above
bars indicate significant differences across test substances (and control) and across doses of
particular test substances.

S38
Figures S11 and S12 illustrate the levels of larval mortality caused by equal molecular dose
treatments of the cubane analogue (12) and diflubenzuron (11) on, respectively, the phosphine
susceptible laboratory strain and field strain of T. castaneum. The general pattern is the same as that
recorded in Fig. 3D and S10, but with greater variance.

Fig. S11: Mean (±SE) larval mortality of phosphine resistant T. castaneum caused by the cubane
analogue 12 (white) and diflubenzuron (11) (black) (with equal molecular weight doses in each
treatment). Superscripts above bars indicate significant differences across test substances (and
control) and across doses of particular test substances.

Fig. S12: Mean (±SE) larval mortality of field strain larval T. castaneum caused by the cubane
analogue 12 (white) and diflubenzuron (11) (black) (with equal molecular weight doses in each
treatment). Superscripts above bars indicate significant differences across test substances (and
control) and across doses of particular test substances.

S39
The additional trials we ran (Fig. S10–S12) produced similar results to those presented in Fig. 3D.
That is, the cubane analogue (12) consistently killed more T. castaneum larvae at the higher dose
treatments than did diflubenzuron (11), and it did so across the two independent experiments. The
results, though repeatable, manifested different levels of variance across experiments, which is
particularly evident in the control treatments. A median lethal dose (LD50) for the cubane analogue
(12) would be about 2.25 mg per 10 larvae, based on the results obtained from the laboratory
insects.
Larvae from the field showed the lowest response to treatment (Fig. S12), with a maximum mean
mortality of ~50%, and none of the treatments imposed mortality that was significantly higher than
that recorded in the control tests. The culture medium could have imposed stresses on insects
adapted to field conditions and led to an increase in the variance in recorded mortality. However,
the consistency of this response by field-collected beetles should be checked.

S40
Metabolism

tert-Butylcubane
P450cam catalysed oxidation of tert-butylcubane (performed by the group led by Prof. James De
Voss at the School of Chemistry and Molecular Biosciences, UQ):

Materials and Methods

P450cam was expressed and purified according to previously published protocols [53]. The
dissociation constant (Kd) and spin state change were determined using established procedures [53a].
Tert-butylcubane appears to have a tighter binding constant (Kd) with P450cam (6.1  0.4 mM) than
the corresponding tert-butylbenzene with P450cam (639  71 mM).

P450cam catalysed oxidation was performed in the following manner: P450cam (1 mM),
putidaredoxin reductase (2 mM), putidaredoxin (8 mM), NADH (3 mM) and tert-butylcubane (5
mM) in 50 mM Tris.HCl and 100 mM KCl, pH 7.4 was incubated for approximately 40 min at
room temperature with stirring. The reaction products and remaining substrate were recovered by
employing a solid-phase extraction cartridge (SPE; Phenomenex, strata-X reverse phase absorbant,
30 mg/mL). The ethyl acetate extract was analysed by GCMS (Zebron ZB-5MS, Phenomenex; 50
°C for 2 min, 50–250 °C at 16 °C/min).

GCMS analysis revealed that the P450cam catalysed oxidation of tert-butylcubane gave a product
with a molecular ion consistent with hydroxylation of the substrate (7.8 min; m/z (EI, 70 eV) 57
(29), 79 (18), 91 (100), 105 (19), 119 (65), 133 (19), 161 (71), 176 (M+.,16)). Following acetylation
of the reaction mixture a new peak (9.2 min) was observed with a GCMS fragmentation pattern
similar to other cubane compounds (m/z (EI, 70 eV) 43 (100), 55 (26), 91 (45), 105 (16), 119 (29),
133 (12), 161 (27), 175 (1), 203 (1)).

S41
Leteprinim (5) and Letepricube (6)
Phase 1 and 2 leteprinim (5) and letepricube (6) (performed by the group led by Prof. Maree Smith
at the Centre for Integrated Preclinical Drug Development, UQ):

Materials and Methods

Leteprinim (5) was purchased from Toronto Research Chemicals (ON, Canada). Human liver
microsomes (HLM) were purchased from BD Gentest (North Ryde, Sydney, Australia). β-
Nicotinamide adenine dinucleotide 2´-phosphate (NADPH), magnesium chloride, alamethicin and
uridine 5´-diphosphoglucuronic acid (UDPGA) were purchased from Sigma Aldrich (Castle Hill,
Sydney, Australia).
Detection of the analytes of interest using liquid chromatography-tandem mass spectrometry (LC-
MS/MS) was achieved in positive ESI mode using a QTrap 5500 instrument (AB Sciex, Concord,
ON, Canada). The highest abundance product ions were selected. The mass transitions for
leteprinim and its cubane analogue were 328.3→192.1 and 354.3→137.1, respectively. Paracetamol
and paracetamol glucuronide were used as the positive control for the Phase 2 drug metabolism
experiments and their mass transitions were 152.1→110.0 and 327.9→152.1, respectively.
The LC system comprised a Symbiosis Pharma System (SPARK Holland System (SPARK Holland,
Emmen, The Netherlands) with a pair of binary LC pumps, a Reliance autosampler (SPARK
Holland, Emmen, The Netherlands) and a Mistral column oven (SPARK Holland, Emmen, The
Netherland). An X-Terra® MS C18 column (2.1 mm x 150 mm, 5 µm) from Waters (Sydney,
Australia) and a Phenomenex security guard column (Phenomenex, Sydney, Australia) were used
for chromatographic separation of leteprinim (5) and its cubane analogue [letepricube (6)]. The
mobile phase comprised solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in
acetonitrile); the mobile phase flow rate was 0.4 mL/min and the analytes of interests were eluted
using gradient elution. The initial mobile phase composition was 90% solvent A and 10% solvent B
and this was maintained for 1 min. Thereafter the percentage of solvent B was increased to 90% at
2.5–2.7 min. This was then decreased to 10% after 22 seconds and this was maintained until the end
of the chromatographic run (5 min). The column oven temperature was 40 oC and 10 µl of each
sample was injected sequentially into the instrument.
The same chromatography conditions were used for quantification of Phase 2 metabolites except
that solvent A comprised 0.2% formic acid in water and solvent B was acetonitrile. The mobile
phase gradient commenced at 20% solvent B which was maintained for 1 min. Thereafter, the
percentage of solvent B in the mobile phase was increased to 80% over a 35 second period and this
was maintained for another 0.5 min. The percentage of solvent B in the mobile phase was then
decreased to 20% over a 10 second period and this was maintained until the end of the
chromatographic run (4.5 min).
The LC-MS/MS data were processed using Analyst software version 1.6, AB Sciex (Concord, ON,
Canada).

Metabolic Stability
Phase 1 metabolism
Aliquots of 25 µM solutions of 5 and 6 dissolved in methanol (10 µL) were added to the incubation
tubes and evaporated to dryness. Aliquots of phosphate buffer (100 mM, pH=7.4, 150 µL) and
HLM (1.5 mg/mL, 50 µl) were added to the reaction tubes and they were maintained in a shaking
water bath at 37 °C for 5 min. Aliquots of NADPH (5 mM, 50 µL) were added to each incubation
tube to initiate the reaction and samples were collected at 0, 1, 2, 3 and 4 h for leteprinim (5) and at
0, 15, 30, 1 and 2 h for letepricube (6). The final concentrations of each of 5 and 6, HLM and
NADPH in the incubation medium (250 µl) were 1 µM, 0.3 mg/mL and 1 mM, respectively. Each
experiment was performed in triplicate.
Reaction was terminated in the incubate samples by addition of 250 µL aliquots of ice-cold
acetonitrile. Samples were vortex-mixed and centrifuged for 5 min at 14,000 rpm. Aliquots of
supernatant (100 uL) were mixed with water (200 uL) and injected directly into the LC-MS/MS
S42
system. The concentrations of both 5 and 6 were quantified in separate incubate samples (each in
triplicate) using LC-MS/MS.

Phase 2 metabolism
Aliquots of 6.25 mM solutions of 5 and 6 dissolved in methanol (20 µL) were added to the
incubation tubes and evaporated to dryness. Aliquots of 5 mM magnesium chloride solution in
phosphate buffer 100 mM, pH=7.4 (50 µL) were added to the reaction tubes followed by addition of
HLM (1 mg/mL in phosphate buffer 100 mM, pH=7.4, 75 µL) which was pre-mixed with 67 µg/mL
alamethicin in phosphate buffer 100 mM, pH=7.4, 75 µl) and kept on ice for 15 min. The reaction
was pre-incubated in a shaking water bath at 37 °C for 5 min. Then, an aliquot of UDPGA (25 mM,
50 µL) was added to each tube (each in triplicate) to initiate the reaction and samples were collected
at 0, 0.25, 0.5, 1, 2 h thereafter. The final concentrations of 5 and 6, HLM, UDPGA, magnesium
chloride and alamethicin in the incubation medium (250 µL) were 500 µM, 0.3 mg/mL, 5 mM, 1
mM, and 20 µg/mL, respectively.
Reaction was terminated in the incubate samples by the addition of a mixture of ice cold acetonitrile
(375 µl) and 0.1% formic acid in water (375 µL). Samples were vortex-mixed and centrifuged for 5
min at 14,000 rpm. The supernatants were injected directly into the LC-MS/MS system. A negative
control (no UDPGA) and positive control (paracetamol 500 µM) were used in parallel incubations
with the study samples.
The presence of putative Phase 2 metabolites of 5 and 6 were investigated using LC-MS Q1 scan,
precursor ion scan, neutral loss, product ion scan and selected reaction monitoring modes.

Results
There were no significant changes in the concentrations of either 5 and 6 during the course of
incubation with human liver microsomes under conditions favouring Phase 1 metabolism. These
findings show that neither compound undergoes significant Phase 1 metabolism when assessed
using human liver microsomes (Fig. S13).
C o n c n e t r a t io n (  M )

1 .0

L e te p rin im
C u b a n e a n a lo g u e
0 .5

0 30 60 90 120
T im e ( m in )

Fig. S13: The mean (±SEM) concentration of leteprinim (5) and its cubane analogue [letepricube
(6)] remained unaltered by Phase 1 metabolic reactions in human liver microsomes during a 2 h
experimental period. Data are from three independent experiments.

S43
Under conditions favouring Phase 2 metabolism in human liver microsomes, paracetamol (positive
control) was metabolized to paracetamol glucuronide thereby confirming the suitability of the
reaction conditions for monitoring possible Phase 2 metabolism of the two compounds of interest
(Fig. S14). Following incubation of 5 and 6 with human liver microsomes under the same reaction
conditions, glucuronide metabolites were not detected.

Fig. S14: Paracetamol glucuronide formed after 2 h incubation with human liver microsomes under
conditions favouring Phase 2 drug metabolism.

S44
Mass spectrometry investigation
Neutral loss
Both Q1 and Q3 were scanned for a preselected mass shift between them. In positive mode,
glucuronidation produces an m/z shift of 176. The mass shift of +176 between paracetamol and
paracetamol glucuronide is shown in Fig. S15.

Fig. S15: Neutral loss experiment (top) and its products in the EPI (Enhanced Product Ion) sane
mode (bottom).

S45
Precursor ion scan
The masses of both leteprinim and its cubane derivative were used as the products by the Q3 mass
filter and Q1 was scanned for possible glucuronide conjugates. Again no ions that matched the
putative glucuronide metabolites were detected.

Conclusion
Our findings collectively show no significant Phase 1 or 2 metabolism of either 5 and 6 over 2 h in
human liver microsomes. The lack of in vitro metabolism of leteprinim by human liver microsomes
mirrors previous work by Grundma et al [54].

S46
Supplementary Materials Part 3: Crystallographic Data

SUBACUBE (4)

Fig. S16: ORTEP molecular structure of 4 (CCDC1418507, 30% ellipsoids).

Fig. S17: Crystal packing of 4.

S47
______________________________________________________________________________
Identification code 4
Empirical formula C16H22N2O3
Formula weight 290.36
Temperature 293(2) K
Wavelength 0.71073 Å
Crystal system Triclinic
Space group P1
Unit cell dimensions a = 5.2841(17) Å α = 99.06(4)°.
b = 7.616(3) Å β = 97.15(3)°.
c = 17.736(7) Å γ = 90.22(3)°.
Volume 699.2(5) Å3
Z 2
Density (calculated) 1.379 Mg/m3
Absorption coefficient 0.096 mm-1
F(000) 312
Crystal size 0.4 x 0.05 x 0.05 mm3
Theta range for data collection 3.12 to 25.00°.
Index ranges -6<=h<=6, -9<=k<=9, -21<=l<=21
Reflections collected 2916
Independent reflections 2916 [R(int) = 0.0000]
Completeness to theta = 25.00° 99.8%
Absorption correction Semi-empirical from equivalents
Max. and min. transmission 1 and 0.59575
Refinement method Full-matrix least-squares on F2
Data / restraints / parameters 2916 / 0 / 192
Goodness-of-fit on F2 0.961
Final R indices [I>2sigma(I)] R1 = 0.0855, wR2 = 0.1922
R indices (all data) R1 = 0.1492, wR2 = 0.2171
Extinction coefficient 0.010(5)
Largest diff. peak and hole 0.382 and -0.297 e.Å-3
Table S8: Crystal data and structure refinement for 4.

S48
Diflucuburon (12)

Fig. S18: ORTEP molecular structure of 12 (CCDC1418506, 30% ellipsoids).

Fig. S19: Crystal packing of 12.

S49
______________________________________________________________________________
Identification code 12
Empirical formula C16 H11 Cl F2 N2 O2
Formula weight 336.72
Temperature 190(2) K
Wavelength 1.54180 Å
Crystal system Monoclinic
Space group I 2/a
Unit cell dimensions a = 20.0309(7) Å = 90°.
b = 5.7090(2) Å = 107.414(4)°.
c = 26.3097(9) Å = 90°.
Volume 2870.79(17) Å3
Z 8
Density (calculated) 1.558 Mg/m3
Absorption coefficient 2.685 mm-1
F(000) 1376
Crystal size 0.1 x 0.1 x 0.05 mm3
Theta range for data collection 3.52 to 62.50°.
Index ranges -23<=h<=21, -5<=k<=6, -28<=l<=30
Reflections collected 5717
Independent reflections 2287 [R(int) = 0.0322]
Completeness to theta = 62.50° 99.6 %
Absorption correction Semi-empirical from equivalents
Max. and min. transmission 1 and 0.88289
Refinement method Full-matrix least-squares on F2
Data / restraints / parameters 2287 / 0 / 208
Goodness-of-fit on F2 1.056
Final R indices [I>2sigma(I)] R1 = 0.0377, wR2 = 0.0997
R indices (all data) R1 = 0.0426, wR2 = 0.1045
Largest diff. peak and hole 0.206 and -0.330 e.Å-3
Table S9: Crystal data and structure refinement for 12.

S50
Supplementary Materials Part 4: 1H and 13C Spectra

S51
 5.93

4.10
3.91

2.26
2.15

1.60
1.32
1.23
Ethyl 8-(cubanylamino)-8-oxooctanoate (S5)

1
H, 400 MHz, CDCl3

10 9 8 7 6 5 4 3 2 1 0 ppm
0.939

5.415
4.000

2.225
2.067

4.481
4.325
3.430
S52
 173.79
172.28

66.61
60.19

52.58
47.81
42.92
36.18
34.25
28.82
28.78
25.24
24.75
14.24
Ethyl 8-(cubanylamino)-8-oxooctanoate (S5)

13
C, 100 MHz, CDCl3

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
S53
 1

14
13
SUBACUBE (4)

H, 400 MHz, DMSO-d6

12
11
0.760 10.30

10
9
0.775 8.63
0.950 8.40

8
7

S54
6
5

2.958 4.00
4

4.000 3.88
3.84
3

1.958 2.05
2

1.599 1.91
4.156 1.45
4.219 1.22
1
0
ppm
 171.38
168.97

65.92

51.82
46.69
41.88
34.96
32.13
28.23
24.86
SUBACUBE (4)

13
C DEPT-135, 100 MHz, DMSO-d6
13
C, 100 MHz, DMSO-d6

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
S55
 4.07

3.68

1.43
1-Methyl-4-(t-butoxycarbonylamino)cubane carboxylate (S6)

1
H, 300 MHz, CDCl3

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
6.535

3.432

9.166
S56
 172.85

66.42

56.08
51.63
50.28
44.69

28.44
1-Methyl-4-(t-butoxycarbonylamino)cubane carboxylate (S6)

13
C DEPT-135, 100MHz, CDCl3
13
C, 100 MHz, CDCl3

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
S57
 6.30
6.26
6.08
5.66
5.64

4.16

3.69
Methyl 4-acrylamidocubane carboxylate (20)

1
H, 300 MHz, CDCl3

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
0.976
1.763

0.947

5.947

3.000

S58
 172.65

164.74

130.01
127.00

66.60

55.76
51.52
50.23
45.09
Methyl 4-acrylamidocubane carboxylate (20)

13
C DEPT-135, 75 MHz, CDCl3
13
C, 75 MHz, CDCl3

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
S59
 8.73

8.31
8.04
7.96

4.34
4.01
4.00
3.96
3.95
3.61

2.68
Methyl 4-[3-(6-amino-9H-purin-9-yl)propanamido]cubane carboxylate (S10)

1
H, 400 MHz, DMSO-d6

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
1.063

1.403
1.113
0.984

2.311

6.000

3.191

2.253
S60
 171.71
168.83

156.69

148.27
145.51

123.91

79.18

65.92

54.97
51.20
49.44
44.18

35.10
Methyl 4-[3-(6-amino-9H-purin-9-yl)propanamido]cubane carboxylate (S10)

13
C, 100 MHz, DMSO-d6

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
S61
 12.26

8.71

8.03
7.95

4.33
4.00
3.94
3.60

2.67
Methyl 4-{3-[6-oxo-1H-purin-9(6H)-yl]propanamido}cubane carboxylate (S11)

1
H, 400 MHz, DMSO-d6

14 13 12 11 10 9 8 7 6 5 4 3 2 1 ppm
0.790

0.984

1.033
0.948

2.000
5.936
3.000

2.067
S62
 172.10
171.67
168.79

156.62

148.23
145.43
140.34

123.88

65.90

54.95
51.16
49.41
44.15

35.07
Methyl 4-{3-[6-oxo-1H-purin-9(6H)-yl]propanamido}cubane carboxylate (S11)

13
C DEPT-135, 100 MHz, DMSO-d6
13
C, 100 MHz, DMSO-d6

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
S63
 8.65

7.88
7.63

4.22

3.74
3.67

2.61
4-{3-[6-Oxo-1H-purin-9(6H)-yl]propanamido}cubane carboxylic acid (S12)

1
H, 400 MHz, DMSO-d6

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
1.000

0.878
0.954

2.186

3.355
3.481

2.200
S64
 175.33

168.80
164.44

151.42
149.21

137.41

124.15

65.96

59.45

48.57
44.45

35.35
4-{3-[6-Oxo-1H-purin-9(6H)-yl]propanamido}cubane carboxylic acid (S12)

13
C DEPT-135, 100 MHz, DMSO-d6
13
C, 100 MHz, DMSO-d6

190 180 170 160 150 140 130 120 110 100S65 90 80 70 60 50 40 30 20 10 ppm
Letepricube (6)

1
H, 400 MHz, D2O

S66
Letepricube (6)

13
C, 100 MHz, D2O

S67
4-(Ethoxycarbonyl)cubane-1-carboxylic acid
(S14)

1
H, 400 MHz, CDCl3

S68
4-(Ethoxycarbonyl)cubane-1-carboxylic acid (S14)

13
C, 100 MHz, CDCl3

S69
4-Ethoxycarbonylcubane Boc-protected amide (S15)

1
H, 400 MHz, CDCl3

S70
4-Ethoxycarbonylcubane Boc-protected amide (S15)

13
C, 75 MHz, CDCl3

S71
Cubocaine (8)

1
H, 400 MHz, D2O

S72
Cubocaine (8)

13
C, 100 MHz, D2O

S73
 4.24
4.21
3.99
3.88
(Cubanyl)methylcubanecarboxylate (10a)

1
H, 300 MHz, CDCl3

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
2.015
3.000
4.990
5.995

S74
 172.73

64.57
56.00
55.88
49.56
48.56
47.82
47.40
45.18
44.55
(Cubanyl)methylcubanecarboxylate (10a)

13
C, 75 MHz, CDCl3

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
S75
 8.05

7.57
7.45

4.46
4.03
3.95
(Cubanyl)methyl benzoate (10b)

1
H, 300 MHz, CDCl3

10 9 8 7 6 5 4 3 2 1 0 ppm
1.970

1.000
2.045

2.068
1.049
5.952

S76
(Cubanyl)methyl benzoate (10b)

13
C, 75 MHz, CDCl3

S77
 7.32

5.13

4.25
3.99
Benzyl cubanecarboxylate (10c)

1
H, 300 MHz, CDCl3

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
5.031

2.033

2.926
4.000

S78
 172.20

136.33
128.49
128.06
128.04

65.78

55.69
49.47
47.81
45.19
Benzyl cubanecarboxylate (10c)

13
C, 75 MHz, CDCl3

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
S79
Diflucuburon (12)

1
H, 400 MHz, CDCl3

S80
Diflucuburon (12)

13
C, 100 MHz, CDCl3

S81
 3.97
3.78
3.73

0.75
1-(tert-Butyl)cubane (S19)

1
H, 400 MHz, Methanol-d4

10 9 8 7 6 5 4 3 2 1 0 ppm
0.877
2.929
2.878

9.000
S82
 49.89
46.91
44.09

31.21
26.22
22.68
1-(tert-Butyl)cubane (S19)

13
C, 100 MHz, Methanol-d4

S83
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
Supplementary Materials Part 5: References

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S85