The role of polycyclic frameworks in modulating P2X7 receptor function

Article abstract of DOI:10.1016/j.tet.2017.10.075

Herein we describe our recent attempts to target the P2X₇ receptor for potential treatment of neurological disorders. This work focusses on different polycycles including carborane, adamantane or cubane, joined by either a cyanoguanidine or an amide linker to phenyl or isoquinoline moieties. We have demonstrated the superiority of the adamantyl moiety over other polycycles in terms of synthetic accessibility and biological (cellular) activity. We have also shown that an amide or cyanoguanidine linker can greatly alter the biological activity of compounds. This SAR study provides important insights into the types of functionality required to target the P2X₇ receptor.

Full text of DOI:10.1016/j.tet.2017.10.075

Accepted Manuscript
The role of polycyclic frameworks in modulating P2X receptor function
7
Timothy B. Callis, Tristan A. Reekie, James O'Brien-Brown, Erick C.N. Wong, Eryn
L. Werry, Nabiha Ellias, William T. Jorgensen, John Tsanaktsidis, Louis M. Rendina,
Michael Kassiou
PII:
S0040-4020(17)31121-3
10.1016/j.tet.2017.10.075
TET 29078
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 12 September 2017
Revised Date: 27 October 2017
Accepted Date: 31 October 2017
Please cite this article as: Callis TB, Reekie TA, O'Brien-Brown J, Wong ECN, Werry EL, Ellias N,
Jorgensen WT, Tsanaktsidis J, Rendina LM, Kassiou M, The role of polycyclic frameworks in modulating
P2X receptor function, Tetrahedron (2017), doi: 10.1016/j.tet.2017.10.075.
7
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ACCEPTED MANUSCRIPT
NC
N
N
H
N
H
N
NC
N
NC
N
NC
H2N
PhO
N
+
N
H
PhO
OPh
N
N
N
H
N
H
N
O
NH2
OH
NH
+
N
O
R
N
O
R
O

ACCEPTED MANUSCRIPT
The role of polycyclic frameworks in modulating P2X7 receptor
function.
Timothy B. Callis,a# Tristan A. Reekie,a# James O’Brien-Brown,a Erick C.N. Wong,b Eryn L.
Werry,c Nabiha Ellias,a William T. Jorgensen,a John Tsanaktsidis,d Louis M. Rendina,a
Michael Kassioua*
a School of Chemistry, The University of Sydney, NSW, 2006, Australia
b School of Medical Sciences, The University of Sydney, NSW, 2006, Australia
c Faculty of Health Sciences, The University of Sydney, NSW, 2006, Australia
d CSIRO, Manufacturing, Clayton South, Victoria 3169, Australia
# Joint first authors
.
Prof. Michael Kassiou. Tel.: +612-9351-2745; e-mail: michael.kassiou@sydney.edu.au
1

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Abstract
Herein we describe our recent attempts to target the P2X7 receptor for potential treatment of
neurological disorders. This work focusses on different polycycles including carborane,
adamantyl or cubane, joined by either a cyanoguanidine or an amide linker to phenyl or
isoquinoline moieties. We have demonstrated the superiority of the adamantyl moiety over
other polycycles in terms of synthetic accessibility and biological (cellular) activity. We have
also shown that an amide or cyanoguanidine linker can greatly alter the biological activity of
compounds. This SAR study provides important insights into the types of functionality
required to target the P2X7 receptor.
Keywords
P2X7; polycyclic; structure-activity relationships; isoquinoline; cyanoguanidine
2

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Introduction
The P2X7 receptor (P2X7R) is a member of the purinergic P2X family of ligand-gated cation
channels (P2X1-7).1-4 They are trimeric structures, predominantly expressed on immune cells,
with adenosine 5’-triphosphate (ATP) acting as the endogenous ligand. P2X7 activation by
physiological levels of ATP allows the influx of calcium and sodium ions, and efflux of
potassium ions.1, 4, 5 In the periphery the P2X7R is expressed on monocytes and macrophages,
6
while in the central nervous system (CNS) they are located on microglia.2,
Both
psychological and physiological stress, such as injury or neurodegeneration, releases ATP
into the extracellular environment resulting in prolonged activation of the P2X7R.7, 8 The
prolonged activation of the P2X7R results in the formation of a large pore and eventual
apoptosis, but not before the release of pro-inflammatory cytokines IL-1β and IL-18.2, 7-12
Higher levels of pro-inflammatory cytokines, including IL-1β, have been reported in patients
suffering from Alzheimer’s disease (AD)13 and major depressive disorders (MDD).14 In
several animal models of depression, abolishing P2X7R expression offered an antidepressant
effect.15 Additionally, amyloid-β protein (Aβ), a hallmark of AD pathology, did not cause
release of ATP or IL-1β from microglia derived from P2X7R-deficient mice, while their wild
type counterparts did.16 Given that microglial P2X7R expression is upregulated in many
neurodegenerative conditions such as AD and multiple sclerosis (MS),1, 17 and its role in the
inflammatory response, P2X7R antagonism appears a promising treatment strategy.
In recently reported work, we have used compounds 1–3 as lead molecules to develop hybrid
structures that sought to combine the beneficial pharmacophoric features, pharmacokinetics
and enhanced blood-brain barrier (BBB) penetration to reach an optimal structure (Figure 1).
18, 19 We found the simple cyanoguanidine hybrid 4a to have nanomolar potency at the P2X7R
(IC50 = 100 nM).18 To improve potency, hydrogen bonding interactions were obtained
through the incorporation of a nitrogen atom into the 3-position and a lengthening of the
chain to give 4b (IC50 = 69 nM). Additional conclusions from this work showed that a
methylene linker between the cyanoguanidine and adamantyl moieties was essential for
potency. A follow up study clearly demonstrated the superiority of the cyanoguanidine linker
as the urea, thiourea and guanidine linkages all proved to have significantly reduced P2X7
inhibitory activity (IC50 = 2.4–6.41 µM).19
Exploration of the benzamide series involved an examination of polycyclic alternatives to the
adamantyl moiety.20 Incorporation of the hydrophobic closo-1,2-carborane cluster to afford
compound 5 showed similar potency to the adamantyl lead 1, albeit, with improved metabolic
3

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stability. Its conversion to the nido-7,8-carborane (isolated as the cesium salt) reduced its
potency (371 nM) but improved its log D (4.29 for the closo to 1.44 for the nido-carborane),
enabling the crossing of the BBB to exhibit significant in vivo antidepressant behavior in the
forced-swim test mouse model of depression.20 The study represented the first account of a
carborane molecule possessing CNS-modifying activity, and it warranted further
investigation. The incorporation of the cubane moiety as a polycyclic replacement of the
adamantyl group reduced potency (IC50 = 436 nM) but improved the log D value (3.42).20
Finally, we have recently reported the synthesis of the trifluoroadamantyl compound 7, which
exhibits improved physicochemical properties and metabolic stability with comparable IC50
values (23–43 nM).21
a
Figure 1. P2X7 lead molecules. IC50 value ranges are reported for IL-1β release, calcium
flux, and dye uptake assays. bIC50 values are obtained from dye uptake assays.
The aim of the work presented here is to combine the many results from our previous
research to establish a new class of compounds to act at the P2X7 receptor (Figure 2).
Compound 8 incorporates the functional moieties of lead compound 1 but uses the
cyanoguanidine linker to connect the two, which should improve pharmacokinetic properties,
as previously reported.18 Compounds 9–11 examine the role that different polycycles can
play in targeting the P2X7 receptor, whilst aiming to improve their lipophilic properties.
Isoquinoline-containing compounds have been shown to act as antagonists at the P2X7
receptor, namely compound 12.22, 23 With this property in mind, we sought to synthesize
4

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compounds 12–15 to examine amide and cyanoguanidine linkers between the adamantyl and
isoquinoline groups (12–13 and 14–15, respectively). In conjunction, we also wanted to
examine different hydrogen bonding characteristics of the N-substituent, namely the alcohol
(12 and 14) versus the carboxylic acid (13 and 15), neither of which have been reported
previously.
NC
NC
N
N
Cl
polycycle
N
N
N
N
H
H
H
H
N
9, polycycle = cubane
OMe
10, polycycle = closo-1,2-carborane
11, polycycle = nido-7,8-carborane
8
NC
N
O
NH
NH
N
H
N
R
N
R
O
O
12, R = CH2OH
13, R = CO2H
14, R = CH2OH
15, R = CO2H
Figure 2. Novel structures to target P2X7 receptor.
Results and Discussion
The synthesis of target compounds initially required the preparation of precursor amines
(Scheme 1). The formation of the diazonium of aniline 16 and quenching with water under
Sandmeyer conditions gave phenol 17. Methylation of both the carboxylic acid and phenol
afforded 18 that was then directly amidated with ammonia to give 19. Borane reduction then
afforded benzylic amine 20 in 49% yield over 4 steps. Using 1,1’-carbonyldiimidazole (CDI)
coupling, adamantyl acid 21 was converted to amide 22 which was then reduced to the
corresponding amine and isolated as its hydrochloride salt 23.24 The dimethyl cubane-1.4-
dicarboxylate 24 was mono-saponified to acid 25 which, following Barton decarboxylation,
gave the singularly carboxylated compound 26. Hydrolysis to acid 27 followed by amide
formation afforded compound 28, which could then be reduced, via LiAlH4, to the amine
24-26
29.20,
Though these conditions were analogous to those used for the adamantyl
derivative, yields were lower and the amine was more conveniently isolated as the free base
and used immediately, rather than stored as the salt. The closo-1,2-carborane precursor was
5

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prepared from alkyne 30 and decaborane-acetonitrile adduct to give 31, which was then
reduced and hydrolyzed to afford the amine as its hydrochloride salt 32.27
The synthesis of isoquinoline derivatives began with benzoic acid 33 that was initially
converted to the ester 34. Subsequent chromene formation proved to be capricious. The best
results for this system arose from enamine formation with N,N-dimethylformamide dimethyl
acetal followed by acid catalyzed cyclisation. When HCl in ethanol was used then a mixture
of 35 and 36 resulted. Alternatively, stirring the same enamine with silica gave pure 36 in
good yields. The mixture of compounds could be subjected to reaction with ethanolamine to
form exclusively 37 exclusively. Acetate protection of the alcohol then afforded 38 that could
be reduced under hydrogenation conditions to give the desired aniline 39. By using the pure
36, reaction with glycine methyl ester afforded 40 that, under the same hydrogenation
conditions, gave aniline 41. With these building blocks in hand, we could then focus on the
synthesis of our target molecules.
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Scheme 1. Synthesis of precursors. Reagents and conditions: a) NaNO2, H2SO4/H2O, 0 – 100
ºC, 1.5 h; b) MeI, K2CO3, DMF, 40 ºC, 21 h; c) NH3, H2O, RT, 24 h; d) BH3•THF, THF, –78
ºC – RT, 24 h; e) CDI, THF, RT, 4 h, then NH3 in H2O, 20 h; f) LiAlH4, THF, 0 ºC to reflux,
24 h; g) NaOH, THF, RT, 18 h; h)1) (COCl)2, CH2Cl2, RT, 1 h; 2) mercaptopyridine N-oxide
sodium salt, DMAP, hv, CHCl3, reflux, 1 h; i) NaOH, MeOH, reflux, 1 h; j) CDI, THF, RT, 4
h, then NH3 in H2O, 4 h; k) LiAlH4, THF, 0 ºC to reflux, 20 h; l) decaborane-acetonitrile
adduct, toluene, reflux, 2 h; m)1) NaBH4, 2-propanol, RT, 23 h; 2) HCl (conc.), AcOH, H2O,
100 ºC, 2 h; n) H2SO4, MeOH, reflux, 48 h; o)1) N,N-dimethylformamide dimethyl acetal,
7

ACCEPTED MANUSCRIPT
pyrrolidine, toluene, 140 ºC, 99 h; 2) HCl, EtOH, reflux, 48 h; p)1) N,N-dimethylformamide
dimethyl acetal, DMF, 115 ºC, 17 h; 2) SiO2, 1,4-Dioxane, RT, 24 h; q) ethanolamine, DMF,
reflux, 6 h; r) Ac2O, DMF, reflux, 4 h; s) Pd/C, H2 (1 atm), EtOAc, rt, 12 h; t) glycine methyl
ester hydrochloride, Et3N, toluene, reflux, 24 h.
Synthesis of compound 8 utilized amine 20 and the commercially available compound 42
(Scheme 2). Simply stirring 42 in 2-propanol overnight and collecting the precipitate afforded
the carbamimidate 43 in 68% yield. The addition of the second amine 23 required base and
heating under reflux to afford target compound 8.
NC
NC
Cl
N
Cl
N
Cl
NC
H2N
N
PhO
N
N
H
N
+
a)
b)
H
H
PhO
OPh
68%
67%
OMe
OMe
OMe
20
42
43
8
Scheme 2. Synthesis of compound 8. Reagents and conditions: a) 2-propanol, RT, 18 h; b)
23, 2-propanol, Et3N, reflux 18 h.
By reacting pyridyl amine 44 with 42, the key intermediate 45 was obtained to be used as the
precursor to the polycyclic compounds (Scheme 3). This intermediate could then be reacted
with adamantyl amine 23 to give 4b or cubanyl amine 29 to give 9. Unfortunately,
subsequent attempts at repeating the same process with closo-carborane 32 were not
successful, with no reaction occurring. Considering compound 42 is more electrophilic than
intermediate 45, we reversed the order of addition and reacted the free base of amine 32 with
42 to give carbamimidate 46. We then subjected this compound to cyanoguanidine formation
with amine 44 in 2-propanol. The high temperatures required resulted in decomposition of
materials with none of the target compound isolated. Changing the solvent to the less
nucleophilic acetonitrile was also unsuccessful. Attempting the reaction at 0 ºC under
microwave irradiation resulted in consumption of starting material but without isolation of
10. Unfortunately, with these results the desired closo-carborane cyanoguanidine 10, and the
corresponding nido-analogue 11, could not be synthesized.
8

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Scheme 3. Synthesis of polycyclic analogues. Reagents and conditions: a) 2-propanol, RT,
18 h; b) 23, 2-propanol, Et3N, reflux 18 h; c) 29, 2-propanol, reflux, 18 h; d)1) 32, Na2CO3,
CHCl3/H2O; 2) 42, 2-propanol, RT, 18 h.
Converting carboxylic acid 47 to the corresponding acid chloride and reacting it with aniline
39 gave amide 48, with subsequent deprotection revealing alcohol 12 (Scheme 4). The
carboxylic acid derivative 13 followed a very similar procedure, first with the synthesis of
amide 49 from aniline 41 and carboxylic acid 47, followed by hydrolysis to reveal the acid
functionality. Unfortunately, the corresponding cyanoguanidine formation proved
troublesome. Heating aniline 39 with 42 to give 50 proved to be ineffectual, as was the case
with the ester derivative 41. Multiple conditions were attempted to effect the desired
transformation. This included sealed tube, microwave, deprotonation with NaH and the
addition of AlMe3 as a Lewis acid catalyst. All conditions proved to be ineffectual and as
such, the cyanoguanidine derivatives 14 and 15 could not be synthesized by this method. To
ensure all avenues for synthesis were exhausted, we prepared compound 52 and attempted
9

ACCEPTED MANUSCRIPT
cyanoguanidine formation with amines 39 and 41. Not unexpectedly, due to its reduced
reactivity, this compound failed to react meaning that no route to 14 and 15 could be realized
in our hands.
O
O
NH2
NH
NH
O
a)
b)
+
N
99%
N
58%
N
OH
OAc
OAc
OH
O
O
O
47
39
48
12
O
O
NH2
NH
NH
O
c)
d)
O
O
O
+
N
42%
N
77%
N
OH
OMe
OMe
OH
O
O
O
47
41
49
13
NC
N
PhO
NH
NH2
NC
NH2•HCl
N
+
OAc
N
N
OAc
PhO
OPh
O
23
O
39
42
50
e)
89%
NC
N
NC
N
PhO
NH
NH2
N
H
OPh
NC
O
N
O
+
OMe
N
N
OMe
PhO
OPh
52
O
O
41
42
51
Scheme 4. Synthesis of isoquinoline containing compounds. Reagents and conditions: a)1)
47, SOCl2, reflux, 2 h; 2) 39, Et3N, CH2Cl2, RT, 4 h; b) LiOH, H2O, THF, RT, 18 h; c)1) 47,
SOCl2, reflux, 2 h; 2) 41, Et3N, CH2Cl2, RT, 4 h; d) LiOH, H2O, THF, RT, 18 h; e) 2-
propanol, RT, 18 h.
Synthesized compounds were assayed for their ability to inhibit 2'(3')-O-(4-
benzoylbenzoyl)adenosine-5'-triphosphate- (BzATP)-induced P2X7R activity using a human
monocytic cell line (THP-1) (Table 1). BzATP is a stable and potent P2X7R agonist with the
10

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P2X7R being endogenously expressed by THP-1 cells. The cellular uptake of the fluorescent
dye, YO-PRO-1, was measured as an indicator of pore formation, and therefore P2X7R
activity. Experimental protocols are further detailed in the Experimental Section. In addition
to the newly synthesized compounds, the previously reported cyanoguanidine 4b18 was also
subjected to the same experiments and showed a comparable value to that obtained
previously (69 vs 57 nM). The direct substitution of adamantyl for cubane to give 9 however,
saw a marked reduction in activity. While this was also observed for the amide analogues (1
and 6), the difference here was greater, dropping into the micromolar range (3.47 µM). The
cyanoguanidine 8 showed a reduction in values compared to the amide analogue 1 (162 nM
vs 2-10 nM), which could potentially be the result of a bigger distance between linker and
aromatic portion. Isoquinoline 12 has been subjected to interleukin 1β (IL-1β) release assays
to determine its IC50 to give a value of 1.3 nM,22, 23 but no data for pore formation and dye
uptake has been reported. We have shown a similar IC50 value of 2.9 nM for the same
compound using the dye uptake assay. A greater than 100-fold reduction in IC50 is seen for
the carboxylic acid analogue 13 (331 nM), which suggests that at physiological pH, the
carboxylate anion has a negative impact on the binding interactions.
Table 1. Biological results from pore-formation inhibition.
Compound
Structure
cLogP a
IC50 mean ± S.E.M. (nM) b
NC
N
4b
3.48
57 ± 3
N
H
N
H
N
NC
N
Cl
8
9
5.25
1.30
162 ± 2.6
3467 ± 54
N
H
N
H
OMe
NC
N
N
H
N
H
N
O
NH
NH
12
13
2.53
2.31
2.9 ± 0.2
331 ± 40
N
OH
OH
O
O
O
O
N
a Calculated using ChemDraw Professional 15.
b Values pertain to pore-formation inhibition and are the mean of three to five experiments and uncertainty was determined by the standard error of
the mean (SEM)
11

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Given the high potency observed for isoquinoline compound 12 we wanted to test further
compounds that contain this moiety. Maintaining the alcohol functionality, we incorporated
additional side chains with defined stereocenters (Scheme 5). Starting from compound 36 and
commercially available enantiomerically-pure amines we could obtain compounds 53a-d,
that underwent facile hydrogenation to 54a-d. Attempts to afford the amide through coupling
reagents were unsuccessful. Instead, the formation of the acid chloride of 47, and treating
with half an equivalent of the requisite aniline 54a-d afforded the amide, in addition to the
ester, which could be simply hydrolyzed to give the target compound.
O
NH
NO2
NH2
a)
e)
f)
N
N
N
N
N
N
N
75%
99%
16%
OH
OH
OH
OH
OH
OH
OH
O
O
O
O
O
O
54a
O
O
53a
55a
NH
NO2
NH2
b)
e)
f)
75%
99%
46%
NO2
O
54b
53b
55b
NH
O
NO2
NH2
O
c)
e)
f)
36
90%
100%
50%
N
N
OH
OH
O
O
53c
54c
55c
O
NO2
NH2
NH
d)
e)
f)
92%
100%
26%
N
N
N
OH
OH
OH
O
O
O
53d
54d
55d
Scheme 5. Synthesis of isoquinoline containing compounds. Reagents and conditions: a) (S)-
2-aminopropan-1-ol, Et3N, DMF, 100 ºC, 2 h; b) (R)-2-aminopropan-1-ol, Et3N, DMF, 100
ºC, 2 h; c) (S)-2-amino-3-methylbutan-1-ol, Et3N, DMF, 100 ºC, 2 h; d) (R)-2-amino-3-
methylbutan-1-ol, Et3N, DMF, 100 ºC, 2 h; e) Pd/C, H2 (1 atm), EtOAc, RT, 17 h; f)1) 47,
(COCl)2, CH2Cl2, rt, 1h; 2) 53a, b, c or d, Et3N, CH2Cl2, rt, 18 h; 3) 3 M LiOH, THF/MeOH,
rt, 18 h.
Newly-synthesized compounds 55a-d were then subjected to the same YO-PRO-1 uptake
assay described above (Table 2). Despite seeing comparable results between the dye-uptake
12

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assay and reported IL-1β assay results for compound 12,22, 23 it became apparent that the same
observation was not observed for all isoquinolines. Low nanomolar potency reported for the
methyl analogues 55a and 55b (0.15 and 1 nM, respectively) gave higher results of 107 and
20 nM in the dye-uptake assay, suggesting a disparity between assays. The previously
reported values for the isopropyl side chain where for the racemic mixture of 54c and d,
though with very good potency (0.05 nM). The results obtained for the dye-uptake assay
were about 200-fold less, with values of 98 and 112 nM, even as the enantiomerically-pure
compounds. These results illustrate the challenges associated with comparing results between
different functional assays.
Table 2. Biological results for isoquinoline compounds.
IC50 mean
(nM) b
± S.E.M.
Compound
Structure
IC50 (nM) a
O
NH
12
1.3
2.9 ± 0.2
107 ± 10
20 ± 6
N
N
N
N
OH
OH
OH
OH
O
O
O
O
O
O
O
NH
NH
NH
55a
55b
55c
0.15
1.0
98 ± 12
0.05 (racemic)
O
NH
55d
126 ± 10
N
OH
O
a (IL-1β) release assay.22, 23
b Values pertain to pore-formation inhibition and are the mean of three to five experiments and uncertainty was determined by the standard error of
the mean (SEM)
Conclusions
We have described efforts towards the synthesis of 12 compounds to target the P2X7 receptor
containing adamantyl or cubane functionality. The formation of cyanoguanidines of
carborane and isoquinoline targets proved to be the most challenging and all efforts to
13

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prepare these compounds proved fruitless. We observed that the cubane-containing target had
significantly reduced potency in comparison to its adamantyl analogue, identifying the
important role that this latter functionality plays in this system. Using dye uptake functional
assays, rather than IL-1β assays, we have shown reduced activity for the former over the
latter for isoquinoline compounds. Based on these results, our current work in this area will
be to expand the scope of the aryl portion of the cyanoguanidine and benzamide families and
develop functionality to improve potency. The results of this study will be reported in due
course.
Experimental
General Experimental
1H NMR spectra were recorded at 300 K unless stated otherwise, using a Bruker AVANCE
DRX300 (300 MHz) or AVANCE III 500 Ascend (500.1 MHz) spectrometer. The data is
reported as the chemical shift in parts per million (δ ppm) relative to the deuterated solvent
residual peak, and coupling constants are reported in hertz (J Hz). Low resolution mass
spectra (LRMS) were recorded using electrospray ionization (ESI) atmospheric pressure
chemical ionization (APCI) recorded on a Finnigan LCQ ion trap spectrometer, or by
electron impact gas chromatography mass spectrometry (GC/MS). High resolution mass
spectra were run on a Bruker 7T Apex Qe Fourier Transform Ion Cyclotron resonance mass
spectrometer equipped with an Apollo II ESI/APCI/MALDI Dual source by the Mass
Spectrometry Facility of the School of Chemistry at The University of Sydney. Elemental
analyses were performed by the Chemical Analysis Facility in the Department of Chemistry
and Biomolecular Sciences, Macquarie University, Australia. Flash chromatography was
performed using Grace Davisil silica gel, pore size 60 Å, 230–400 mesh particle size, and
thin-layer chromatography (TLC) was carried out on Merck aluminum backed silica gel 60
F254 plates. IR absorption spectra were recorded on a Bruker ALPHA FT-IR spectrometer as
solid or thin film from ethanol, and the data are reported as vibrational frequencies (cm–1).
Melting points were measured with open capillaries using a Stanford Research Systems
(SRS) MPA160 melting point apparatus with a ramp rate of 5 ºC/min.
Chemistry
2-Chloro-5-hydroxybenzoic acid (17)
Sodium nitrite (90 mL of a 0.5 M aq. solution) was added to an ice cold stirred suspension of
5-amino-2-chlorobenzoic acid (16) (5.01 g, 29.6 mmol) and sulfuric acid (6.00 mL, 0.167
14

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mol) in water (460 mL), maintaining the temperature between 0–5 ºC during addition. After
stirring for 1 h the reaction mixture was diluted with water (900 mL) and subsequently stirred
at reflux for another 30 min. The reaction mixture was then cooled to room temperature, and
extracted into ethyl acetate (3 x 500 mL). The combined organic phases were dried (MgSO4),
filtered and concentrated under reduced pressure to yield 17 as a light brown solid (4.97 g,
98% yield).
m.p. 165-168 ºC; IR (ZnSe): 3311, 2920, 2598, 1676, 1599, 1573, 1474, 1447, 1228, 826 cm–
1
1; H NMR (300 MHz, MeOD): δ 7.26 (1H, d, J = 8.7 Hz), 7.23 (1H, d, J = 3.0 Hz), 6.89
(1H, dd, J = 3.0 and 8.7 Hz) ppm 2xOH signals not observed; 13C NMR (75 MHz, MeOD): δ
169.1, 157.4, 132.7, 132.8, 123.8, 120.7, 116.2 ppm; LRMS (–ESI): 171 ([M–H]–, 100%),
127 ([M–CO2H]–, 36%).
Methyl 2-chloro-5-methoxybenzoate (18)
Methyl iodide (7.0 mL, 112 mmol) was added to a solution of 2-chloro-5-hydroxybenzoic
acid (17) (3.51 g, 20.3 mmol) and anhydrous potassium carbonate (12.4 g, 90.0 mol) in
dimethylformamide (100 mL). The reaction mixture was then warmed to 40 ºC and stirred for
5 h following which additional methyl iodide (5.2 mL, 83.5 mmol) was added and stirring
continued for a further 16 h. After cooling to room temperature, the mixture was diluted with
water (300 mL) and extracted into diethyl ether (3 x 100 mL). The organic extracts were then
washed with aqueous sodium hydroxide (0.1 M, 100 mL) and water (2 x 100 mL) and dried
(MgSO4), filtered and concentrated under reduced pressure to give 18 (3.38 g, 83%) as a light
orange oil.
IR (ZnSe): 2953, 1731, 1598, 1574, 1478, 1433, 1217, 1114, 1055, 1024 cm–1; 1H NMR (300
MHz, d6-DMSO): δ 7.47 (1H, d, J = 9.0 Hz),ꢀ7.31 (1H, d, J = 3.0 Hz), 7.15 (1H, dd, J = 9.0
Hz, J = 3.0 Hz), 3.85 (3H, s), 3.80 (3H, s) ppm; 13C NMR (75 MHz, CDCl3): δ 165.9, 157.9,
131.8, 130.5, 124.8, 118.8, 116.0, 55.6, 2.4 ppm; LRMS (+ESI): 201 ([M+H]+, 100%).
2-Chloro-5-methoxybenzamide (19)
Methyl (2-chloro-5-methoxy)benzoate (18) (1.19 g, 6.47 mmol) was stirred vigorously in
neat aqueous ammonia (28%, 7.8 mL) at room temperature. After 24 h, the resulting mixture
was treated with chloroform and the separated organic phase washed with aqueous
hydrochloric acid (5 M, 55 mL), water (40 mL), brine (40 mL) then dried (MgSO4), filtered
and concentrated under reduced pressure to give 19 as a white powder (0.93 g, 78% yield).
15

ACCEPTED MANUSCRIPT
m.p. 125.3-126.9 ºC; IR (ZnSe): 3372, 3178, 1650, 1480, 1461, 1446, 1239, 1028, 821 cm–1;
1H NMR (300 MHz, MeOD): δ 7.34 (1H, d, J = 8.8 Hz), 7.05 (1H, d, J = 2.9 Hz), 7.00 (1H,
13
dd, J = 2.9 and 8.8 Hz,), 3.81 (3H, s) ppm; C NMR (75 MHz, MeOD): δ 172.1, 159.8,
137.9, 132.0, 122.8, 117.9, 115.2, 56.2 ppm; LRMS (GC-MS): 185 ([M], 60%), 169 ([M-
NH ], 100%).
2
(2-Chloro-5-methoxyphenyl)methanamine (20)
A solution of 2-chloro-5-methoxybenzamide (19) (347 mg, 1.87 mmol) in THF (5 mL) was
added dropwise under an atmosphere of nitrogen to a borane-THF complex solution (1 M, 9
mL) at –78°C. Following addition, the reaction mixture was warmed to room temperature,
and then stirred at reflux for 24 h. After cooling to room temperature, the mixture was
quenched via the slow addition of aqueous hydrochloric acid (6 M, 1.5 mL). Once the
effervescence had ceased, the THF solvent was removed under reduced pressure, the residue
basified with aqueous sodium hydroxide (6 M, 3 mL), and extracted in chloroform (3 x 40
mL). The combined organic extracts were dried (MgSO4), filtered and concentrated under
reduced pressure. The crude product was purified by flash column chromatography (49:1:0.1
v/v, CH2Cl2:MeOH:NH3(aq)) to yield 20 as a yellow oil (249 mg, 78% yield).
1
IR (ZnSe): 3469, 3346, 2954, 1601, 1579, 1482, 1461, 1246, 1025 cm–1; H NMR (300
MHz,ꢀCDCl3): δ 7.25 (1H, d, J = 8.7 Hz), 6.94 (1H, d, J = 3.0 Hz), 6.73 (1H, dd, J = 3.0 and
13
8.7 Hz), 3.88 (2H, s), 3.80 (3H, s) ppm; C NMR (75 MHz, CDCl3): δ 158.6, 141.5, 130.2,
124.5, 114.5, 113.5, 55.6, 44.7 ppm; LRMS (+ ESI): 172/170 ([M+H]+, 42/19%), 157/155
([M–CH3]+, 100/31%).
Methyl 2-methyl-3-nitrobenzoate (34)
A mixture of sulfuric acid (8.3 mL, 0.23 mol) in methanol (330 mL) was added to 2-methyl-
3-nitrobenzoic acid (33) (30.1 g, 0.16 mol). The reaction mixture was heated at reflux for 48
h and the solvent removed under reduced pressure. The residue was dissolved in
dichloromethane (200 mL) and subsequently washed with saturated sodium hydrogen
carbonate solution (2 x 50 mL) and then brine (2 x 50 mL). The combined organic phases
were dried (MgSO4), filtered and concentrated under reduced pressure to yield 34 as a light
beige solid (28.8 g, 89% yield).
1
m.p. 68 ºC; IR (ZnSe): 1727, 1530, 1359, 1288, 1265, 1122 cm–1; H NMR (200 MHz,
CDCl3): δ 7.98ꢀ(1H, dd, J = 1.0 and 7.6 Hz), 7.83 (1H, dd, J = 1.0 and 7.6 Hz), 7.37 (1H, t, J
16

ACCEPTED MANUSCRIPT
= 7.6 Hz), 3.93 (3H, s), 2.61 (3H, s) ppm; 13C NMR (75 MHz, CDCl3): δ 167.0, 152.1, 133.7,
133.5, 133.2, 126.8, 126.5, 52.7, 16.3 ppm; LRMS (+ESI): 219 ([M+Na]+, 86%), 195
([M+H]+, 100%), 182 ([M–CH3]+, 84%), 149 [(M – NO2]+, 86%).
3-Ethoxy-5-nitroisochroman-1-one (35) compound and 5-nitro-1H-isochromen-1-one
(36)
Method i) A mixture of methyl 2-methyl-3-nitrobenzoate (34) (9.58 g; 49.1 mmol), N,N’-
dimethylformamide-dimethylacetal (23.9 g; 201 mmol), and pyrrolidine (4.37 g; 61.5 mmol)
in toluene (100 mL) was stirred under nitrogen at 140 °C for 99 h. The solvent was removed
under reduced pressure to yield crude (E)-methyl 3-nitro-2-(2-pyrrolidin-1-yl)vinyl) benzoate
(13.5 g) as a red oil. This crude enamine was then stirred under reflux in a mixture of
aqueous hydrochloric acid in ethanol (1:19 v/v, 200 mL) for 48 h. The reaction mixture was
then concentrated under reduced pressure and the remaining heterogeneous mixture was
diluted with water (200 mL), extracted in ethyl acetate (3 x 100 mL). The combined organic
phases were washed with sodium hydrogen carbonate (2 x 100 mL), dried (MgSO4), filtered
and concentrated under reduced pressure. The crude residue, which was purified by column
chromatography (3:1 v/v, CH2Cl2:hexane) to give 35 (7.25 g; 30.5 mmol; 62%) as beige
crystals and 36 (1.13 g; 5.93 mmol; 12%) a light beige powder. Co-eluted products could also
be used in subsequent reactions.
Method ii) Methyl 2-methyl-3-nitrobenzoate (34) (19.4 g, 99.4 mmol) and freshly distilled
N,N- dimethylformamide dimethyl acetal (48 mL) in anhydrous DMF (120 mL) were stirred
at 115 °C for 17 h. DMF was removed on a rotary evaporator to give (E)-methyl 2-(2-
(dimethylamino)-vinyl)-3-nitrobenzoate which was then dissolved in 1,4-dioxane (450 mL)
and treated with silica gel (150 g). The mixture was stirred vigorously under a nitrogen
atmosphere at room temperature for 24 h, followed by filtration to remove the silica washing
with acetonitrile. The filtrate was concentrated under reduced pressure to yield a red-brown
solid. The solid was purified by flash chromatography (hexane/ethyl acetate, 9:1 v/v) to give
36 as a beige powder (9.69 g, 51%).
1
36; m.p. 152-154 ºC; IR (ZnSe): 1734, 1626, 1523, 1385, 1316, 1256, 1224, 1064 cm–1; H
NMR (300 MHz, CDCl3): δ 8.60 (1H, d, J = 8.1 Hz), 8.46 (1H, d, J = 8.1 Hz), 7.65 (1H, t, J
13
= 8.1ꢀHz), 7.42 (1H, d, J = 6.3 Hz), 7.34 (1H, d, J = 6.3 Hz) ppm; C NMR (75 MHz,
CDCl3): δ 160.1, 147.8, 144.3, 135.8, 131.3, 130.8, 128.2, 123.8, 101.8 ppm; LRMS
17

ACCEPTED MANUSCRIPT
(+APCI): 192.0 ([M+H]+, 100%).
35; m.p. 95-96 ºC; IR (ZnSe):ꢀ2981, 2939, 1733, 1529, 1386, 1353, 1306, 1262, 1072, 1018
cm–1; 1HꢀNMR (400 MHz, CDCl3): δ 8.43 (1H, dd, J = 1.2 and 8.0 Hz), 8.28 (1H, dd, J = 1.2
and 8.0 Hz), 7.58 (1H, t, J = 8.0 Hz), 5.61 (1H, t, J = 3.6 Hz), 3.99-3.64 (2H, m, J = 2.8, 4.4
13
and 7.2 Hz), 3.56 (2H, t, J = 4.4 Hz), 1.17 (3H, t, J = 7.2 Hz) ppm; C NMR (100 MHz,
CDCl3): δ 162.3, 148.2, 135.2, 132.2, 129.8, 128.0, 127.7, 100.0, 65.5, 31.1, 15.0 ppm;
LRMS (+ESI): 519 ([2M+Na]+, 100%), 479 ([2M+H]+, 18%).
2-(2-Hydroxyethyl)-5-nitroisoquinolin-1(2H)-one (37)
A mixture of 35 and 36 (1.12 g; 4.84 mmol) and ethanolamine (0.65 mL; 10.7 mmol) in
dimethylformamide (10 mL) was stirred at reflux for 6 h. The reaction mixture was diluted
with water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic
phases were washed with aqueous hydrochloric acid (1 M, 60 mL), dried (MgSO4), filtered
and concentrated under reduced pressure to give 37 as an orange powder (1.05 g; 93% yield).
m.p. 130-131 ºC; IR (ZnSe): 3425, 1644, 1521, 1345, 1319, 1204, 1601 cm–1; 1H NMRꢁ(300
MHz, CDCl3): δ 8.72 (1H, d, J = 12.0 Hz), 8.40 (1H, d,ꢀJ = 12.0 Hz), 7.55 (1H, t, J = 12.0
Hz), 7.36 (1H, d, J = 12.0 Hz), 7.30 (1H, d, J = 12.0 Hz), 4.21 (2H, t, J = 9.2 Hz), 4.00 (2H, t,
13
J = 9.2 Hz) ppm; C NMR (75 MHz, CDCl3): δ 161.6, 144.8, 136.4, 134.4, 131.1, 129.6,
128.0, 125.9, 100.8, 61.4, 52.7 ppm; LRMS (+APCI): 469 ([2M+H]+, 100%), 191 ([M–
CH CH OH]–, 35%).
2
2
2-(5-Nitro-1-oxoisoquinolin-2(1H)-yl)ethyl acetate (38)
Acetic anhydride (0.84 mL; 8.89 mmol) was added to a solution of 37 (0.94 g; 4.03 mmol) in
dimethylformamide (9 mL). The mixture was stirred at reflux for 4 h then treated with
aqueous sodium hydroxide (1 M, 50 mL) extracted with ethyl acetate (100 mL, 2 x 50 mL),
dried (MgSO4), filtered and concentrated under reduced pressure to give 38 as a yellow
powder (1.10 g; 99% yield).
m.p. 146-147 ºC; IR (ZnSe): 1739, 1655, 1625, 1523, 1345, 1319, 1237, 1050 cm–1; 1H NMR
(300 MHz, CDCl3): δ 8.76 (1H, d, J =8.0ꢀHz), 8.41 (1H, d, J = 8.0 Hz), 7.57 (1H, t, J = 8.0
Hz), 7.27 (2H, q, J = 8.1 Hz), 4.44 (2H, t, J = 5.1 Hz), 4.27 (2H, t, J = 5.1 Hz), 2.05 (3H, s)
13
ppm; C NMR (75 MHz, CDCl3): δ 170.7, 160.8, 144.8, 135.9, 134.5, 131.1, 129.6, 128.2,
126.0, 100.6, 62.0, 48.8, 20.9 ppm; LRMS (+ESI): 315 ([M+K]+, 22%), 277 ([M+H]+, 58%),
18

ACCEPTED MANUSCRIPT
217 ([M – (OCOCH3)]+, 100%).
2-(5-Amino-1-oxoisoquinolin-2(1H)-yl)ethyl acetate (39)
10% w/w Pd/C (49 mg) was added to a solution of 38 (96 mg; 3.48 mmol) in ethyl acetate
(100 mL) and stirred under an atmosphere of H2 for 12 h. The reaction mixture was filtered
through Celite® washing with ethyl acetate. The filtrate was concentrated under reduced
pressure to yield 39 as a beige solid (806 mg; 94% yield).
m.p. 108-110 ºC; IR (ZnSe) 3437, 3361, 3237, 1736, 1634, 1588, 1228 cm–1;ꢀ1H NMR (300
MHz, CDCl3): δ 7.87 (1H, d, J = 8.1 Hz), 7.29 (1H, t, J = 7.8 Hz), 7.03 (1H, d, J = 7.8 Hz),
6.95 (1H, d, J = 7.8 Hz), 6.43 (1H, d, J = 7.5 Hz), 4.41 (2H, t, J = 5.3 Hz), 4.23 (2H, t, J = 5.3
13
Hz), 2.04 (3H, s) ppm; C NMR (75 MHz, CDCl3): δ 170.8, 162.3, 141.4, 131.0, 127.6,
127.1, 125.3, 118.6, 117.5, 99.9, 62.4, 48.4, 20.9 ppm; LRMS (+ESI) 269 ([M+Na]+, 39%),
247 ([M+H]+, 100%), 187 ([M–OAc]+, 83%).
Methyl 2-(5-nitro-1-oxoisoquinolin-2(1H)-yl)acetate (40)
A mixture of 36 (1.04 g; 5.43 mmol), glycine methyl ester hydrochloride (1.56 g; 12.5 mmol)
and triethylamine (1.99 g; 19.7 mmol) in toluene (50 mL) was stirred at reflux for 24 h under
Dean-Stark conditions. The solvent was removed under reduced pressure and the residue
dissolved in chloroform (50 mL), washed with aqueous hydrochloric acid (0.5 M; 2 x 50
mL). The combined aqueous phases were then re-extracted in chloroform (3 x 50 mL). The
combined organic phases were dried (MgSO4), filtered and concentrated under reduced
pressure and the crude residue was purified by column chromatography (1:1 v/v, ethyl
acetate: hexane) to give 40 as a yellow powder (268 mg; 19% yield).
m.p. 195-197 ºC; IR (ZnSe) 2927, 1747, 1656, 1627, 1523, 1419, 1346, 1319, 1214, 1179
cm–1; 1H NMR (300 MHz, CDCl3): δ 8.74 (1H, d, J = 8.1 Hz), 8.42 (1H, d, J = 8.1 Hz), 7.58
(1H, t, J = 8.1 Hz), 7.34 (1H, d, J = 7.8 Hz), 7.20 (1H, d, J = 7.8 Hz), 4.74 (2H, s), 3.80 (3H,
s) ppm; 13C NMR (75 MHz, CDCl3): δ 168.0, 160.8, 144.9, 135.4, 134.5, 131.2, 129.8, 128.0,
126.2, 101.2, 53.0, 50.3 ppm; LRMS (+APCI): 263 ([M+H]+, 33%), 231 ([M–OCH3]+,
100%), 203 ([M–CO2CH3]+, 46%).
Methyl 2-(5-amino-1-oxoisoquinolin-2(1H)-yl)acetate (41)
10% w/w Pd/C (9 mg) was added to a solution of 40 (68 mg; 0.260 mmol) in ethyl acetate
(10 mL) and stirred under an atmosphere of H2 for 12 h. The reaction mixture was filtered
through Celite® washing with ethyl acetate. The filtrate was concentrated under reduced
19

ACCEPTED MANUSCRIPT
pressure and purified by column chromatography (4:1 v/v, ethyl acetate:hexane) to yield 41
as a white solid (45 mg; 75% yield).
m.p. 199-200 ºC; IR (ZnSe) 3357, 3241, 1745, 1614, 1592, 1213 cm–1; 1H NMR (200 MHz,ꢀ
d6-acetone): δ 7.62 (1H, d, J = 8.0 Hz), 7.27 (1H, d, J =ꢀ7.4 Hz), 7.22 (1H, t, J = 7.8 Hz),
13
7.00 (1H, d, J = 7.8 Hz), 6.73 (1H, d, J = 8.0 Hz), 4.75 (2H, s), 3.71 (3H, s); C NMR (75
MHz, d6-acetone): δ 133.1, 131.6, 128.0, 122.7, 122.0, 116.8, 116.6, 100.8, 52.4, 50.6, 2
peaks were unresolved; LRMS (+APCI) 232.87 ([M+H]+, 100%).
Phenyl N-(2-chloro-5-methoxybenzyl)-N'-cyanocarbamimidate (43)
Amine 20 (142 mg, 0.826 mmol) in 2-propanol (3.5 mL) was treated with
diphenylcyanocarbonimidate (42), (197 mg, 0.826 mmol) and the homogeneous solution was
stirred at room temperature for 18 h. The resulting precipitate was collected by filtration,
washed with diethyl ether (10 mL) and dried under vacuum to give 43 as a white solid (177
mg, 68%).
m.p. 156-157.0 ºC; IR 3180, 2927, 2192, 1638, 1484, 1459, 1428, 1207, 1027 cm–1; 1H NMR
(300 MHz, CDCl3): δ 7.39 (2H, t, Jꢀ= 7.5 Hz), 7.33-7.28 (2H, m), 7.14ꢀ(1H, m), 7.04 (1H, d,
J = 8.4 Hz), 6.95 (1H, s), 6.83 (1H, dd, J = 2.4 and 8.4 Hz), 4.66 (2H, d, J = 5.7 Hz), 3.80
13
(3H, s) ppm; C NMR (75 MHz, CDCl3): δ 163.9, 158.7, 151, 134.8, 130.7, 129.7, 126.9,
124.8, 121.5, 115.5, 115.3, 114.9, 55.8, 44.5, ppm; LRMS (+APCI) 316/318 ([M+H]+,
100/30%).
1-((-Adamantan-1-yl)methyl)-3-(2-chloro-5-methoxybenzyl)-2-cyanoguanidine (8)
Compound 43 (131 mg, 0.414 mmol) in 2-propanol (3 mL) was treated with adamantane-1-
methylamine hydrochloride (23)18 (86 mg, 0.426 mmol) and triethylamine (60 µL, 0.433
mmol) and stirred at reflux overnight. The solution was concentrated under reduced pressure
and the resulting residue suspended in diethyl ether and filtered to give 8 as a white powder
(107 mg, 67% yield).
m.p. 173.6-175.0°C, IR (ZnSe): 3300, 3271, 2903, 2847, 2164, 1584, 1475, 1451, 1426,
1
1365; H NMR (300 MHz, CDCl3): δꢁ7.27 (1H, d, J = 8.7 Hz), 6.97 (1H, d, J = 3.0ꢀHz),
6.80 (1H, dd, J = 3.0 and 8.7 Hz), 5.85 (1H, s), 4.99 (1H, s), 4.47 (2H, s), 3.79 (3H, s), 2.84
13
(2H, d, J = 5.4 Hz), 1.94 (3H, s), 1.71-1.54 (6H, m), 1.38 (6H, s) ppm; C NMR (75 MHz,
CDCl3): δ 160.4, 159.0, 135.2, 130.6, 124.3, 118.4, 117.6, 115.4, 55.8, 53.9, 43.5, 40.1, 36.8,
20

ACCEPTED MANUSCRIPT
33.9, 28.2 ppm; HRMS (+ESI) Calc. for C21H2735ClN4O [M+Na]+: 409.1771, found:
409.1765; LRMS (+APCI): 387 ([M+H]+, 100%).
Phenyl N'-cyano-N-(pyridin-3-ylmethyl)carbamimidate (45)
Compound 42 (1.02 g, 4.28 mmol) in 2-propanol (16 mL) was treated with 3-
(aminomethyl)pyridine (44) (468 mg, 4.33 mmol) and the homogeneous solution was stirred
at room temperature for 18 h. The resulting precipitate was collected by filtration, washed
with diethyl ether (10 mL) and dried under vacuum to give 45 as a white solid (1.01 g, 94%).
1
m.p. 132.-133. ºC; IR (ZnSe): 3234, 3047, 2191, 1619, 1483, 1456, 1427, 1204 cm–1; H
NMR (300 MHz, CDCl3): δ 8.63 (1H, s),ꢀ8.57 (1H, dd, J = 1.2 and 4.5 Hz), 7.73 (1H, d, J =
7.5 Hz), 7.38 (2H, t, J = 7.5 Hz), 7.33-7.27 (2H, m), 6.99 (2H, d, J = 7.5 Hz), 4.54 (2H, d, J =
6.0 Hz) ppm; 13C NMR (75 MHz, CDCl3): δ 164.0, 151.0, 149.5, 135.9, 132.6, 129.7, 126.9,
123.8 (2 overlapping signals), 121.5, 115.7, 43.7 ppm; LRMS (+ ESI): 275 ([M+Na]+, 99%),
253 ([M+H]+, 100%).
1-((-Adamantan-1-yl)methyl)-2-cyano-3-(pyridin-3-ylmethyl)guanidine (4b)
Compound 45 (302 mg, 1.20 mmol) in 2-propanol (7 mL) was treated with adamantane-1-
methylamine hydrochloride (23)18 (261 mg, 1.29 mmol) and triethylamine (180 µL, 1.30
mmol) and stirred at reflux for 18 h. The solution was concentrated under reduced pressure
and the resulting residue suspended in diethyl ether and filtered to give 4b as a white powder
(107 mg, 67% yield). Data matched that previously reported.18
1-((Cuban-1-yl)methyl)-2-cyano-3-(pyridin-3-ylmethyl)guanidine (9)
Compound 45 (59 mg, 0.236 mmol) in 2-propanol (2 mL) was treated with cubane-1-
methylamine (29)20 (31 mg, 0.232 mmol) and stirred at reflux for 18 h. The solution was
concentrated under reduced pressure and the resulting residue suspended in diethyl ether and
filtered to give 9 as a white powder (64 mg, 95% yield).
m.p. 179-180 °C, IR (ZnSe): 3258, 2976, 2164, 1577, 1478, 1425, 1357 cm–1; 1H NMR (300
MHz, d6-DMSO): δ 8.48 (1H, m), 7.60ꢀ(1H, m), 7.37 (1H, dd, J = 2.2 and 4.8 Hz), 7.11 (1H,
t, J = 5.9 Hz), 4.36 (2H, d, J = 6.0 Hz), 3.95-3.94 (1H, m), 3.77 (6H, m), 3.38 (2H, d, J = 6.1
13
Hz) ppm; C NMR (75 MHz, DMSO): δ 159.6, 148.6, 148.2, 134.8, 134.4, 123.4, 117.8,
56.9, 47.4, 46.9, 43.3, 42.7, 41.9 ppm; HRMS (+ESI) Calc. for C17H11N5 [M+Na ]+:
314.3395, found: 314.1376; LRMS (+ESI): 292 ([M+H]+, 100%).
21

ACCEPTED MANUSCRIPT
N-(O-closo-Carboranyl)methyl)-N’-cyano-O-phenylisourea (46)
A suspension of o-closo-carboranylmethylamine hydrochloride (32)27 (37 mg, 0.175 mmol)
in chloroform (10 mL) was washed with aqueous saturated sodium carbonate (2 x 10 mL),
dried (MgSO4), filtered and concentrated under reduced pressure to give the free base
(colorless oil, 25 mg, 0.145 mmol). This was dissolved in 2-propanol (1 mL) and treated with
42 (36 mg, 0.151 mmol) and stirred at room temperature for 18 h. The resulting precipitate
was collected by filtration, washed with diethyl ether (10 mL) and dried under vacuum to
give 46 as a white solid (31 mg, 56%).
m.p. 237.0-237.5 °C, IR (ZnSe) 3201, 3048, 2582, 2197, 1634, 1456, 1417, 1354, 1207 cm–1,
1H NMR (500 MHz, CDCl3): δ 7.44 (2H, t, J =ꢀ7.5 Hz), 7.35-7.32 (1H, m), 7.10 (2H, d, J =
8.0 Hz), 4.06 (2H, d, J = 7.0 Hz), 3.96 (1H, s), 2.38-2.17 (10H, m) ppm, NH signal not
13
observed; C NMR (75 MHz, d6-acetone): δ 163.9, 153.9, 130.5, 127.7, 122.4, 114.1, 75.7,
62.4, 47.6 ppm; 11B NMR (500 MHz, CDCl3) –0.07, –3.28, –8.25, –10.45, –11.41 ppm;
LRMS (+ APCI) 316/317/318/319/320 ([M+H]+, 25/100%), 199 ([M-B10H10]+, 20%).
2-(5-(2-((Adamantan-1-yl)acetamido)-1-oxoisoquinolin-2(1H)-yl)ethyl acetate (48)
1-Adamantane acetic acid (47) (151 mg, 0.779 mmol) in thionyl chloride (1.5 mL) was
heated at reflux for 2 h then concentrated under reduced pressure to yield 1-adamantane
acetyl chloride. This was dissolved in dichloromethane (0.8 mL) and added dropwise to a
solution of 39 (128 mg, 0.518 mmol) and triethylamine (130 µL, 0.932 mmol) in
dichloromethane (6 mL) and stirred at room temperature for 4 h. The mixture was diluted
with dichloromethane (10 mL) and washed with saturated sodium carbonate (10 mL) and
water (10 mL). The combined organic phases were dried (MgSO4), filtered and concentrated
under reduced pressure to give 48 as a white powder (218 mg; 99% yield).
1
m.p. 158-161 °C; IR (ZnSe) 3256, 2901, 2848, 1742, 1684, 1624, 1051 cm–1; H NMR (300
MHz, CDCl3): δ 8.21ꢀ(1H, d, J = 8.0 Hz), 7.91 (1H, d, J = 7.8 Hz), 7.41 (1H, t, J = 8.0 Hz),
7.02 (1H, d, J = 7.6 Hz), 6.40 (1H, d, J = 7.6 Hz), 4.35 (2H, t, J = 5.2 Hz), 4.17 (2H, t, J = 5.2
Hz), 2.11 (3H, s), 1.98 (3H, s), 1.69 (6H, m), 1.61 (6H, m) ppm; 13C NMR (75 MHz, CDCl3):
δ 170.8, 170.0, 161.9, 136.5, 132.5, 132.3, 131.9, 131.1, 128.3, 127.1, 125.6, 62.3, 52.5, 48.5,
42.9, 36.8, 33.4, 28.8, 20.9 ppm; LRMS (+APCI) 423 ([M+H]+, 23%), 382 ([M– Ac]+,
100%), 363 ([M–OAc]+, 86%).
22

ACCEPTED MANUSCRIPT
2-((Adamantan-1-yl)-N-(2-(2-hydroxyethyl)-1-oxo-1,2-dihydroisoquinolin-5-
yl)acetamide (12)
A solution of compound 48 (103 mg; 0.244 mmol) in tetrahydrofuran (2 mL) was treated
with aqueous lithium hydroxide (3 M, 0.32 mL) and stirred at room temperature for 18 h. The
reaction mixture was acidified with aqueous hydrochloric acid (1 M, 4.8 mL) and extracted
with dichloromethane (3 x 10 mL). The combined organic phases were dried (MgSO4),
filtered and concentrated under reduced pressure and the residue was purified by column
chromatography (3:1 v/v, ethyl acetate: hexane) to yield compound 12 as a white solid (54
mg; 58% yield).
m.p. 230-231.5 ºC; IR (ZnSe) 3343, 3263, 2899, 2847, 1650, 1598, 1560 cm–1; 1H NMR (300
MHz, d6-acetone): δ 8.03 (1H,ꢀd, J = 8.1 Hz), 7.91 (1H, d, J = 7.5 Hz), 7.33 (1H, t, J = 8.0
Hz), 7.26 (1H, d, J = 7.8 Hz), 6.57 (1H, d, J = 7.5 Hz), 3.98 (2H, t, J = 5.3 Hz), 3.74 (2H, t, J
13
= 5.3 Hz), 2.15 (2H, s), 1.93 (3H, s), 1.67 (6H, s), 1.61-1.58 (6H, m) ppm; C NMR (75
MHz, DMSO): δ 169.6, 160.8, 133.7, 132.9, 131.3, 127.8, 126.3, 126.0, 123.7, 99.6, 58.8,
51.0, 50.1, 42.1, 36.4, 32.7, 28.1; HRMS (+ESI): Calc. C23H28N2O3 [M+Na]+: 403.1998,
found 403.1991, Calc. C23H28N2O3 [M+H]+: 381.2178, found 381.2173; LRMS (+APCI) 381
([M+H]+, 100%), 363 ([M–OH]+, 27%).
Methyl 2-(5-(2-(-adamantan-1-yl)acetamido)-1-oxoisoquinolin-2(1H)-yl)acetate (49)
1-Adamantane acetic acid (47) (86 mg, 0.44 mmol) in thionyl chloride (0.9 mL) was heated
at reflux for 2 h then concentrated under reduced pressure to yield 1-adamantane acetyl
chloride. This was dissolved in dichloromethane (0.6 mL) and added dropwise to a solution
of 41 (86 mg, 0.37 mmol) and triethylamine (130 µL, 0.93 mmol) in dichloromethane (5 mL)
and stirred at room temperature for 4 h. The mixture was diluted with dichloromethane (10
mL) and washed with saturated sodium carbonate (10 mL) and water (10 mL). The combined
organic phases were dried (MgSO4), filtered and concentrated under reduced pressure to give
49 as a white powder (77 mg; 42% yield).
m.p. 218-220 °C; IR (ZnSe): 3247, 2905, 2848, 1752, 1654, 1623, 1143 cm–1; 1H NMR (300
MHz, CDCl3): δ 8.21 (1H, d, J = 8.1 Hz), 7.92 (1H, d, J = 7.5 Hz), 7.44 (1H, t, J = 8.1 Hz),
6.98 (1H, d, J = 7.5 Hz, CH), 6.45 (1H, d, J = 7.5 Hz), 4.69 (2H, s), 3.78 (3H, s), 2.20 (2H,
13
s), 1.74 (3H, s), 1.68 (6H, m), 1.65 (6H, m) ppm; C NMR (75 MHz, CDCl3): δ 170.1,
168.7, 162.0, 139.0, 132.0, 131.2, 128.6, 127.2, 126.7, 125.6, 100.8, 52.8, 50.3, 48.6, 36.8,
33.5, 28.8 ppm; LRMS (+APCI): 409 ([M+H]+, 85%), 377 ([M-OMe]+, 100%), 349 ([M–
23

ACCEPTED MANUSCRIPT
CO Me]+, 55%).
2
2-(5-(2-(Adamantan-1-yl)acetamido)-1-oxoisoquinolin-2(1H)-yl)acetic acid (13)
A solution of compound 49 (23 mg; 0.056 mmol) in tetrahydrofuran (0.6 mL) was treated
with aqueous lithium hydroxide (3 M, 32 µL) and stirred at room temperature for 18 h. The
reaction mixture was acidified with aqueous hydrochloric acid (1 M, 0.45 mL) and extracted
into dichloromethane (3 x 5 mL). The combined organic phases were dried (MgSO4), filtered
and concentrated under reduced pressure and the residue was purified by column
chromatography (3:1 v/v, ethyl acetate: hexane) to yield compound 13 as a white solid (17
mg; 77% yield).
m.p. 258-259 ºC; IR (ZnSe) 2902, 2848, 1714, 1651, 1529, 1201 cm–1; 1HꢀNMR (300 MHz,
MeOD): δ 8.21 (1H, d, J = 7.8 Hz), 7.79 (1H, dd, J = 1.2 and 7.8 Hz), 7.52 (1H, t, J = 7.8
Hz), 7.36 (1H, d, J = 7.8 Hz), 6.75 (1H, d, J = 7.5 Hz), 4.77 (2H, s), 2.24 (2H, s), 2.03 (3H,
s,), 1.79-1.79 (6H, m), 1.74-1.66 (6H, m); 13C NMR (75 MHz, MeOD): δ 173.3, 171.3,
163.8, 135.9, 134.3, 133.9, 131.1, 127.8, 127.6, 126.4, 102.9, 51.9, 51.3, 43.9, 37.9, 34.3,
30.2; HRMS (+ESI) Calc. C23H26N2O4 [M+Na]+: 417.1785, found 417.1783; LRMS (+APCI)
395 ([M+H]+, 100%), 377 ([M–OH]+, 34%), 349 ([M–CO H]+, 21%).
2
Phenyl N-((adamantan-1-yl)methyl)-N'-cyanocarbamimidate (52)
Compound 42 (424 mg, 1.78 mmol) in 2-propanol (40 mL) was treated with adamantane-1-
methylamine hydrochloride (23)18 (357 mg, 1.77 mmol) and triethylamine (300 µL, 2.16
mmol) and the homogeneous solution was stirred at room temperature for 18 h. The resulting
precipitate was collected by filtration, washed with diethyl ether (10 mL) and dried under
vacuum to give 52 as a white solid (275 mg, 50%). Data matched that previously reported.18
(S)-2-(1-Hydroxypropan-2-yl)-5-nitroisoquinolin-1(2H)-one (53a)
Compound 36 (1.01 g, 5.28 mmol) and trimethylamine (5 mL, 35 mmol) in DMF (10 mL)
was treated with (S)-2-aminopropan-1-ol (583 mg, 7.76 mmol) in DMF (10 mL). The
resulting solution was heated at 100 ºC for 2 h, then diluted with water (50 mL) and extracted
with ethyl acetate (3 x 50 mL). The combined organics were washed with water (2 x 50 mL),
aqueous hydrochloric acid (1 M, 2 x 50 mL), and brine (1 x 50 mL), and then dried (MgSO4),
filtered and concentrated under reduced pressure to yield compound 53a as a brown
crystalline solid (985 mg; 75% yield).
24

ACCEPTED MANUSCRIPT
1
m.p. 164-166 ºC; IR (ZnSe) 3373, 1641, 1624, 1597, 1521, 1318 cm–1; H NMR (300 MHz,
d6-DMSO): δ 8.61 (1H, d, J = 7.9 Hz), 8.45 (1H, dd, J = 7.9 Hz, J = 1.0 Hz), 7.75 (1H, d, J =
8.0 Hz), 7.65 (1H, app t, J = 8.0 Hz), 7.06 (1H, d, J = 8.0 Hz), 5.09-4.98 (2H, m), 3.73-3.59
13
(2H, m), 1.31 (3H, d, J = 7.0 Hz) ppm; C NMR (75 MHz, DMSO) δ 160.8, 145.1, 134.8,
134.7, 130.6, 130.2, 128.1, 126.8, 99.8, 63.7, 53.5, 16.7 ppm; LRMS (–ESI) 247 ([M–H]–,
100%).
(R)-2-(1-Hydroxypropan-2-yl)-5-nitroisoquinolin-1(2H)-one (53b)
Compound 36 (1.02 g, 5.28 mmol) and trimethylamine (5 mL, 35 mmol) in DMF (10 mL)
was treated with (R)-2-aminopropan-1-ol (590 mg, 7.86 mmol) in DMF (10 mL). The
resulting solution was heated at 100 ºC for 2 h, then diluted with water (50 mL) and extracted
with ethyl acetate (3 x 50 mL). The combined organics were washed with water (2 x 50 mL),
aqueous hydrochloric acid (1 M, 2 x 50 mL), and brine (1 x 50 mL), and then dried (MgSO4),
filtered and concentrated under reduced pressure to yield compound 53b as a brown
crystalline solid (991 mg; 75% yield).
1
m.p. 166-167 ºC; IR (ZnSe) 3379, 1642, 1623, 1598, 1521, 1318 cm–1; H NMR (300 MHz,
d6-DMSO): δ 8.61 (1H, d, J = 8.0 Hz), 8.45 (1H, dd, J = 1.2 and 8.0 Hz), 7.75 (1H, d, J = 8.0
Hz), 7.65 (1H, app t, J = 8.0 Hz), 7.05 (1H, d, J = 7.9 Hz), 5.09-4.98 (2H, m), 3.73-3.59 (2H,
13
m), 1.31 (3H, d, J = 7.0 Hz) ppm; C NMR (75 MHz, DMSO): δ 160.7, 145.1, 134.80,
134.75, 130.6, 130.2, 128.1, 126.8, 99.8, 63.7, 53.5, 16.7 ppm; LRMS (–ESI) 247 ([M–H]–,
100%).
(S)-2-(1-Hydroxy-3-methylbutan-2-yl)-5-nitroisoquinolin-1(2H)-one (53c)
Compound 36 (1.00 g, 5.38 mmol) and trimethylamine (5 mL, 35 mmol) in DMF (10 mL)
was treated with (S)-2-amino-3-methylbutan-1-ol (845 mg, 8.19 mmol) in DMF (10 mL). The
resulting solution was heated at 100 ºC for 2 h, then diluted with water (50 mL) and extracted
with ethyl acetate (3 x 50 mL). The combined organics were washed with water (2 x 50 mL),
aqueous hydrochloric acid (1 M, 2 x 50 mL), and brine (1 x 50 mL), and then dried (MgSO4),
filtered and concentrated under reduced pressure to yield compound 53c as an orange
crystalline solid (1.31 g; 90% yield).
1
m.p. 100-102 ºC; IR (ZnSe) 3410, 2966, 1642, 1624, 1522, 1321 cm–1; H NMR (500 MHz,
d6-DMSO): δ 8.63 (1H, d, J = 7.8 Hz), 8.46 (1H, dd, J = 1.5 and 8.0 Hz),ꢀ7.74 (1H, d, J = 8.0
Hz), 7.66 (1H, app t, J = 7.8 Hz), 7.07 (1H, d, J = 8.0 Hz), 4.90 (1H, app t, J = 5.3 Hz), 4.64
25

ACCEPTED MANUSCRIPT
(1H, br s), 3.87-3.82 (1H, m), 3.76-3.72 (1H, m), 2.16-2.15 (1H, m), 1.04 (3H, d, J = 6.5 Hz),
13
0.70 (3H, d, J = 6.5 Hz) ppm; C NMR (125 MHz, DMSO): δ 160.4, 144.2, 134.5, 134.0,
129.7, 129.3, 127.2, 125.9, 98.7, 60.3, 28.0, 19.8, 19.4 ppm (1 signal not resolved); HRMS
(+ESI): Calc. C14H16N2O4 [M+Na]+: 299.1008, found 299.1004; LRMS (–ESI) 275 ([M–H]–,
100%).
(R)-2-(1-Hydroxy-3-methylbutan-2-yl)-5-nitroisoquinolin-1(2H)-one (53d)
Compound 36 (992 mg, 5.38 mmol) and trimethylamine (5 mL, 35 mmol) in DMF (10 mL)
was treated with (R)-2-amino-3-methylbutan-1-ol (868 mg, 8.19 mmol) in DMF (10 mL).
The resulting solution was heated at 100 ºC for 2 h, then diluted with water (50 mL) and
extracted with ethyl acetate (3 x 50 mL). The combined organics were washed with water (2
x 50 mL), aqueous hydrochloric acid (1 M, 2 x 50 mL), and brine (1 x 50 mL), and then dried
(MgSO4), filtered and concentrated under reduced pressure to yield compound 53d as an
orange crystalline solid (1.32 g; 92% yield).
1
m.p. 103-105 ºC; IR (ZnSe) 3426, 2967, 1642, 1624, 1522, 1320 cm–1; H NMR (500 MHz,
d6-DMSO): δ 8.63 (1H, d, J = 8.0 Hz), 8.46 (1H, dd, J = 1.2 and 8.0 Hz),ꢀ7.74 (1H, d, J = 8.0
Hz), 7.66 (1H, app t, J = 8.0 Hz), 7.07 (1H, d, J = 7.9 Hz), 4.90 (1H, app t, J = 5.1 Hz), 4.64
(1H, br s), 3.87-3.82 (1H, m), 3.76-3.72 (1H, m), 2.16-2.15 (1H, m), 1.04 (3H, d, J = 6.5 Hz),
13
0.70 (3H, d, J = 6.7 Hz) ppm; C NMR (75 MHz, DMSO): δ 160.4, 144.2, 134.6, 134.0,
129.7, 129.3, 127.1, 125.9, 98.7, 60.3, 28.0, 19.8, 19.4 ppm (1 signal not resolved); LRMS (–
ESI) 275 ([M–H]–, 100%).
(S)-5-Amino-2-(1-hydroxypropan-2-yl)isoquinolin-1(2H)-one (54a)
10% w/w Pd/C (100 mg) was added to a solution of 53a (734 mg; 2.96 mmol) in ethyl acetate
(100 mL) and stirred under and atmosphere of H2 for 18 h. The reaction mixture was filtered
through Celite® washing with ethyl acetate. The filtrate was concentrated under reduced
pressure to yield 54a as a pale yellow solid (637 mg; 99% yield).
1
m.p. 144-146 ºC; IR (ZnSe) 3351, 3245, 2981, 2933, 2875, 1704, 1560 cm–1; H NMR (300
MHz, d6-DMSO): δ 7.48 (1H, d, J = 7.7 Hz), 7.33 (1H, d, J = 7.8 Hz), 7.17 (1H, app t, J =
7.8 Hz), 6.88ꢀ(1H, dd, J = 1.1 and 7.7 Hz), 6.79 (1H, d, J = 7.8 Hz), 5.63 (2H, br s), 5.13-
5.01 (1H, m), 4.93 (1H, t, J = 5.45 Hz), 3.72-3.57 (2H, m), 1.29 (3H, d, J = 7.0 Hz) ppm; 13C
NMR (75 MHz, DMSO): δ 161.3, 144.1, 127.0, 126.6, 126.5, 123.5, 114.7, 114.6, 100.3,
63.3, 51.6, 16.1 ppm; LRMS (+APCI) 219 ([M+H]+, 100%).
26

ACCEPTED MANUSCRIPT
(R)-5-Amino-2-(1-hydroxypropan-2-yl)isoquinolin-1(2H)-one (54b)
10% w/w Pd/C (100 mg) was added to a solution of 53a (864 mg; 3.48 mmol) in ethyl acetate
(100 mL) and stirred under and atmosphere of H2 for 18 h. The reaction mixture was filtered
through Celite® washing with ethyl acetate. The filtrate was concentrated under reduced
pressure to yield 54b as a pale yellow solid (7.49 mg; 99% yield).
m.p. 147-149 ºC; IR (ZnSe) 3352, 3247, 2981, 2928, 2877, 1705, 1563 cm–1; 1H NMR (300
MHz, d6-DMSO): δ 7.48 (1H, d, J = 7.7 Hz), 7.33 (1H, d, J = 7.8 Hz), 7.17 (1H, app t, J =
7.8 Hz), 6.88ꢀ(1H, dd, J = 1.1 and 7.7 Hz), 6.79 (1H, d, J = 7.8 Hz), 5.63 (2H, br s), 5.13-
5.01 (1H, m), 4.93 (1H, t, J = 5.45 Hz), 3.72-3.57 (2H, m), 1.29 (3H, d, J = 7.0 Hz) ppm; 13C
NMR (75 MHz, DMSO): δ 161.3, 144.1, 127.0, 126.6, 126.5, 123.5, 114.7, 114.6, 100.3,
63.3, 51.6, 16.1 ppm; LRMS (+APCI) 219 ([M+H]+, 100%).
(S)-5-Amino-2-(1-hydroxy-3-methylbutan-2-yl)isoquinolin-1(2H)-one (54c)
10% w/w Pd/C (100 mg) was added to a solution of 53c (1.10 mg; 3.97 mmol) in ethyl
acetate (100 mL) and stirred under and atmosphere of H2 for 18 h. The reaction mixture was
filtered through Celite® washing with ethyl acetate. The filtrate was concentrated under
reduced pressure to yield 54c as a pale yellow solid (1.00 mg; 100% yield).
1
m.p. 138-140 ºC; IR (ZnSe) 3351, 3251, 2964, 1706, 1580, 1562 cm–1; H NMR (300 MHz,
d6-DMSO): δ 7.45 (1H, d, J = 7.8 Hz), 7.30 (1H, d, J = 7.8ꢀHz), 7.15 (1H, app t, J = 7.8 Hz),
6.85 (1H, d, J = 7.7 Hz),ꢀ6.74 (1H, d, J = 7.7 Hz), 5.60 (2H, br s), 4.80 (1H, app t, J = 4.9
Hz), 4.64 (1H, br s), 3.87-3.79 (1H, m), 3.73-3.67 (1H, m), 2.18-2.11 (1H, m), 1.03 (3H, d, J
= 6.5 Hz), 0.67 (3H, d, J = 6.7 Hz) ppm; 13C NMR (75 MHz, DMSO): δ 161.8, 144.9, 128.5,
127.7, 127.4, 124.3, 115.6, 115.5, 100.8, 62.5, 61.7, 28.9, 20.8, 20.3 ppm; LRMS (+APCI)
247 ([M+H]+, 100%).
(R)-5-Amino-2-(1-hydroxy-3-methylbutan-2-yl)isoquinolin-1(2H)-one (54d)
10% w/w Pd/C (100 mg) was added to a solution of 53d (1.13 mg; 4.10 mmol) in ethyl
acetate (100 mL) and stirred under and atmosphere of H2 for 18 h. The reaction mixture was
filtered through Celite® washing with ethyl acetate. The filtrate was concentrated under
reduced pressure to yield 54d as a pale yellow solid (1.02 mg; 100% yield).
1
m.p. 137-141 ºC; IR (ZnSe) 3351, 3251, 2964, 1706, 1580, 1562 cm–1; H NMR (300 MHz,
d6-DMSO): δ 7.45 (1H, d, J = 7.8 Hz), 7.30 (1H, d, J = 7.8ꢀHz), 7.15 (1H, app t, J = 7.8 Hz),
27

ACCEPTED MANUSCRIPT
6.85 (1H, d, J = 7.7 Hz),ꢀ6.74 (1H, d, J = 7.7 Hz), 5.60 (2H, br s), 4.80 (1H, app t, J = 4.9
Hz), 4.64 (1H, br s), 3.87-3.79 (1H, m), 3.73-3.67 (1H, m), 2.18-2.11 (1H, m), 1.03 (3H, d, J
= 6.5 Hz), 0.67 (3H, d, J = 6.7 Hz) ppm; 13C NMR (75 MHz, DMSO): δ 161.8, 144.9, 128.5,
127.7, 127.4, 124.3, 115.6, 115.5, 100.8, 62.5, 61.7, 28.9, 20.8, 20.3 ppm; LRMS (+APCI)
247 ([M+H]+, 100%).
2-(Adamantan-1-yl)-N-(2-((S)-1-hydroxypropan-2-yl)-1-oxo-1,2-dihydroisoquinolin-5-
yl)acetamide (55a)
1-Adamantaneacetic acid (47) (470 mg, 2.4 mmol) in dichloromethane (2 mL) was treated
with oxalyl chloride (500 µL, 5.8 mmol), followed by anhydrous DMF (2 drops) and the
mixture was stirred at room temperature for 1 h then concentrated under reduced pressure.
The acid chloride was dissolved in dichloromethane (1 mL) and added dropwise to a solution
of triethylamine (320 µL, 4.3 mmol) and isoquinolinone 54a (104 mg, 0.48 mol) in
dichloromethane (2 mL) then stirred at room temperature for 16 h before being concentrated
under reduced pressure. The crude mixture was diluted with THF (3 mL) and methanol (2
mL) then treated with aqueous lithium hydroxide (3 M, 2 mL) and stirred at room
temperature for 17 h. The mixture was concentrated under reduced pressure diluted with
ethyl acetate (25 mL) and aqueous hydrochloric acid (1 M, 10 mL). The organic fraction was
washed with hydrochloric acid (1 M, 10 mL), then sodium hydroxide (1 M, 2 x 10 mL),
water (10 mL), and brine (15 mL). The organic layer was dried (MgSO4), filtered and
concentrated under reduced pressure. The crude product was recrystallized from ethyl acetate
to yield 55a as a white crystalline solid (30 mg, 16%).
1
m.p. 228-229 ºC; IR (ZnSe) 3275, 2902, 1651, 1595, 1529 cm–1; H NMR (300 MHz, d6-
DMSO): δ 8.14 (1H, d, J = 8.0 Hz), 7.81 (1H, d, J = 7.6 Hz), 7.52 (1H, s), 7.35ꢀ(1H, app t, J
= 7.9 Hz), 7.14 (1H, d, J = 7.7 Hz), 6.39 (1H, d, J = 7.7 Hz), 5.12-5.06 (1H, m), 3.92-3.77
13
(2H, m), 2.20 (3H, s), 2.01 (3H, s), 1.74-1.64 (12H, m), 1.40 (3H, d, J = 7.1 Hz) ppm; C
NMR (75 MHz, CD3OD): δ 173.3, 163.8, 133.7, 133.6, 130.8, 129.9, 127.9, 127.6, 126.6,
103.0, 65.0, 54.3, 51.9, 43.9, 37.9, 34.3, 30.2, 16.2 ppm; HRMS (+ESI): Calc. C24H30N2O3
[M+Na]+: 417.2154, found 417.2150; LRMS (+ESI) 395 ([M+H]+, 100%).
2-(Adamantan-1-yl)-N-(2-((R)-1-hydroxypropan-2-yl)-1-oxo-1,2-dihydroisoquinolin-5-
yl)acetamide (55b)
1-Adamantaneacetic acid (47) (470 mg, 2.4 mmol) in dichloromethane (2 mL) was treated
with oxalyl chloride (500 µL, 5.8 mmol), followed by anhydrous DMF (2 drops) and the
28