Synthesis and identifications of potential metabolites as biomarkers of the synthetic cannabinoid AKB-48

Article abstract of DOI:10.1016/j.tet.2018.04.026

AKB-48 belongs to the family of synthetic cannabinoids. It has strong binding affinity to CB₁ receptor and is psychoactive. It is banned in many countries including USA, Japan, Germany, New Zealand, Singapore and China etc. But the difficulty in detecting the parent compound in urine samples highlights the importance of studies of its metabolites. Here we report the synthesis of 19 potential metabolites of AKB-48, among which, compounds 2, 9, 10, 30 and 31, together with the commercially available substance 5 were identified as metabolites of AKB-48 by comparison with one authentic human urine sample and human liver microsomal data. Compounds 10 and 30 could be of use as biomarkers in detecting AKB-48 in human urine samples.

Full text of DOI:10.1016/j.tet.2018.04.026

Tetrahedron 74 (2018) 2905e2913
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and identifications of potential metabolites as biomarkers of
the synthetic cannabinoid AKB-48
,
,
, c
Jakob Wallgren a, Svante Vikingsson b c, Anna Åstrand b c, Martin Josefsson a
,
Henrik Green , Johan Dahlen , Xiongyu Wu *, Peter Konradsson a
Department of Physics, Chemistry and Biology, Linkoping University, Sweden
Division of Drug Research, Department of Medical and Health Sciences, Faculty of Health Sciences, Linkoping University, Sweden
Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linkoping University, Sweden
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a b s t r a c t
Article history:
AKB-48 belongs to the family of synthetic cannabinoids. It has strong binding affinity to CB1 receptor and
is psychoactive. It is banned in many countries including USA, Japan, Germany, New Zealand, Singapore
and China etc. But the difficulty in detecting the parent compound in urine samples highlights the
importance of studies of its metabolites. Here we report the synthesis of 19 potential metabolites of AKB-
48, among which, compounds 2, 9, 10, 30 and 31, together with the commercially available substance 5
were identified as metabolites of AKB-48 by comparison with one authentic human urine sample and
human liver microsomal data. Compounds 10 and 30 could be of use as biomarkers in detecting AKB-48
in human urine samples.
Received 14 December 2017
Received in revised form
6 April 2018
Accepted 10 April 2018
Available online 22 April 2018
Keywords:
Metabolite
Biomarker
© 2018 Elsevier Ltd. All rights reserved.
AKB-48
Synthetic cannabinoid
Adamantane
1. Introduction
human hepatocytes and high-resolution mass spectrometry.3
Further investigation by Holm et al. found that CYP3A4 mediated
Synthetic cannabinoids have emerged and grown to become a
common substitute to cannabis in recent years. The rate at which
old synthetic cannabinoids are replaced on the market with new
ones as they become classified as harmful substances puts great
pressure on forensic laboratories and governments. Synthetic
cannabinoids are not always detected in routine cannabinoid urine
screening methods; however, their metabolites can be detected
and are therefore important to enable identifications of synthetic
cannabinoid intakes in analysis of urine samples. As a member of
the synthetic cannabinoid family, AKB-48 (Fig. 1) (APINACA) has
stronger binding affinity to CB1 receptor compared to tetrahydro-
cannabinol,1 and is used as an alternative to cannabis. It is banned
in many countries including USA, Japan, Germany, New Zealand,
Singapore and China etc.
the oxidative metabolism of AKB-48.4 The oxidative metabolism
happened mainly on the pentyl chain and the adamantyl moiety
giving mono-, di- or tri-hydroxylated metabolites together with
metabolites as ketones or carboxylic acids. Vikingsson et al. also put
great effort in identification of AKB-48 metabolites using human
liver microsomes and time of flight mass spectrometry.5 Based on
these research groups' findings more knowledge of AKB-48's
metabolism is well described. However, the exact structure of the
metabolites cannot be determined by LC-MS/MS alone. In a previ-
ous study we have synthesized and identified an interesting and
important metabolite of AKB-48, with a secondary alcohol on the
adamantane ring.6 This is uncommon because metabolism of the
adamantyl moiety normally leads to metabolites with a tertiary
alcohol at the bridge-carbon as major.7 Apart from the adamantyl
moiety that is found in several synthetic cannabinoids, such as AB-
001, SDB-001, STS-135; AKB-48 also contains a pentyl chain, which
is an even more common moiety in other synthetic cannabinoids.
There are some commercially available synthetic cannabinoid
metabolites with a hydroxyl group at position 4 or 5 of the pentyl
chain (Fig. 1), but unambiguous structure identification is lacking
for several major metabolites.5 To the best of our knowledge, there
AKB-48 was first reported by a Japanese group in 2012 as an
ingredient in a synthetic cannabis smoking blend.2 In 2013 the
study of its metabolites was carried out by Gandhi et al. using
* Corresponding author.
E-mail address: xiongyu.wu@liu.se (X. Wu).
https://doi.org/10.1016/j.tet.2018.04.026
0040-4020/© 2018 Elsevier Ltd. All rights reserved.

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J. Wallgren et al. / Tetrahedron 74 (2018) 2905e2913
applied to the synthesis of mono- or di-hydroxylations of the pentyl
side chain at different position from compound 7 to give com-
pounds 8, 9 and 10, and from 17 to give 18, 19 and 20, and from
compound 21 to 22, 23, and 24, which are shown in Scheme 2 and
4. The yield of compound 10 was low, and the purification of its
mixture with compound 9 was difficult. It's probably easier to
achieve 10 by reduction of compound 11 with NaBH4 instead.
The ketones 11 and 12 were synthesized by microwave irradi-
ation of the mixture of compound 2 or 15 and 1-chloro-3-
pentanone in THF with t-BuOK as base at 120 ꢀC for one hour
(Scheme 3). Full conversion was not achieved, resulting in low
yields, 24% and 28% respectively. Unfortunately, we were not able to
find compound 11 or 12 in the authentic urine sample. Therefore,
the optimization was not continued.
Fig. 1. Structures and activities of AKB-48 and tetrahydrocannabinol.
The adamantyl moiety of AKB-48 was prone to be oxidized by
CYP3A4.2,4 1-adamantylamine 13 was oxidized instead with
concentrated H2SO4/HNO3 to give compound 14 containing a hy-
droxyl group.8 With 14 as starting material other potential me-
tabolites of AKB-48, compounds 12, 15, 16, 18e20 and 22e24 were
synthesized using similar methods as described above, showed in
Scheme 4. When compound 15, a similar structure to 2 was syn-
thesized, another amide coupling reagent TBTU was used instead,
and a good yield (81%) was achieved compared to that of EDC (27%)
or DCC (31%). Hydroboration-oxidation of 17 gave a mixture of al-
cohols 19 and 20, where the yield of 20 was very low (3%) because
of anti-Markovnikov's rule and therefore there were also diffi-
culties in the purification. Another synthetic route was developed
to achieve 20 with better yield (49%, two steps) by first epoxidation
of 17 with m-CPBA, followed by reduction of the resulting epoxide
with NaBH4 in i-PrOH at 60 ꢀC for 3 h.
Although the tertiary carbons of the adamantyl moiety were
much easier to be oxidized compared to the secondary ones with
concentrated H2SO4/HNO3, it's difficult to say whether it will be the
same in vivo. Compound 25 was used as starting material to syn-
thesize other potential metabolites with a secondary hydroxyl
group. Conditions for a modified Ritter reaction reported in the
literature were used.9 The synthesis of compounds 25e31 was re-
ported in our previous study.5 However, it was not discussed in
detail. But the synthetic route is shown in Scheme 5 for a better
overview.
are not any synthesis and identification studies on the metabolic
position of the pentyl chain of synthetic cannabinoids. To have a
more complete picture of the metabolite profile of AKB-48, we
continued our study of the synthesis and identifications of other
metabolites of AKB-48. By comparison with an authentic urine
sample we can identify its major metabolites, which could even-
tually lead us to discover better biomarkers for AKB-48, or even for
other synthetic cannabinoids containing a pentyl chain or an ada-
mantane moiety, in urine samples.
2. Results and discussion
According to our previous human liver microsome results5 and
the research by Gandhi et al.,3 the oxidative metabolism of AKB-48
gave metabolites in the form of mono- or di-hydroxylations of the
pentyl chain and/or of the adamantyl moiety. Therefore, the syn-
thesis of potential metabolites of AKB-48 was focused on adding
hydroxyl group(s) on its pentyl and/or adamantyl side chains.
Furthermore, the synthetic compounds were used as references in
the analysis of an authentic urine sample.
Compound 2 was synthesized using 3-indazole carboxylic acid 1
and 1-adamantanamine hydrochloride as starting materials with
EDC as the coupling reagent in 48% yield. The yield was unfortu-
nately low because of incomplete conversion.
Compound 32 was also synthesized from compound 19 with
Jones reagent in 59% yield (Scheme 6).
Compounds 4 and 5 are commercially available. They can also be
synthesized using hydroboration-oxidation, which was described
below for the synthesis of compounds 19 and 20. Alkylation of 2
with 5-bromopent-1-ene followed by dihydroxylation with OsO4/
NMO to give compound 6 (Scheme 1). Similar methods were
As described previously,6 compound 30 was shown by LC-MS/
Scheme 1. i) EDC, HOBt, Et3N, DMF, rt, overnight, 48%; ii) 5-bromopent-1-ene, t-BuOK,
DMF/THF (1:5), rt, overnight, 87%; iii) OsO4 (aq), NMO, THF/H2O (3:1), rt, overnight,
66%. Compounds 4 and 5, commercially available.
Scheme 2. i) 1-bromopent-2-ene, t-BuOK in DMF/THF (1:5), rt, overnight, 98%; ii)
OsO4 (aq), NMO, THF/H2O (3:1), rt, overnight, 80%; iii) a) BH3$THF, N2 (g), 0 ꢀC, 2 h. b)
NaOH (aq), H2O2, rt, overnight, 9, 69%; 10, 8%.

J. Wallgren et al. / Tetrahedron 74 (2018) 2905e2913
2907
Scheme 3. i) 1-chloro-3-pentanone, t-BuOK, THF, MW irradiation, 120 ꢀC, 1 h, 11, 24%;
12, 28%.
Scheme 6. Jones reagent, acetone, RT, 59%.
MS to be the major metabolite, hydroxylated at the adamantyl
moiety of AKB-48. In a similar experiment compounds 4, 5, 9 and 10
were analyzed with the urine sample (Fig. 3), indicating that the
major metabolite was 5 with minor contribution of 10 and traces of
9. Small amounts of 4 cannot be ruled out. Compound 2 was
identified unambiguously by MS/MS spectra as a minor metabolite
based on previous results.5 Although 5 was the major identified
mono-hydroxyl metabolite of the pentyl chain, it is unfortunately
less suitable as a biomarker for the detection of AKB-48 intake as it
is also a major metabolite of 5F-AKB-48.5
In summary, Compounds 2, 5, 9, 10, 30 and 31 were identified as
metabolites of AKB-48 in the urine sample and compound 10 and
30 have the potential to be used as biomarkers of AKB-48 intake
with strong intensities and structures uniquely identifying AKB-48
from 5F-AKB-48.
Interestingly, studies of the metabolites of the substances con-
taining an adamantine ring suggest that the tertiary carbon is
normally easier to be oxidized than the secondary carbon unless
the tertiary carbons are substituted, for example: 1-amino-3-
hydroxyadamantane is the only detected metabolite of
amantadine.10e13 Our results highlight the importance of having
access to all the potential metabolites for accurate metabolic study.
Scheme 4. i) Conc. H2SO4, conc. HNO3, 2 h, 10 ꢀC, 74%; ii) TBTU, 1H-indazole-3-
carboxylic acid, Et3N, THF, rt, overnight, 81%; iii) 1-bromopentane, t-BuOK, THF/DMF
(5:1), rt, overnight, 90%; iv) 1-bromo-2-pentene, t-BuOK in THF/DMF (5:1), rt, over-
night, 82%; v) OsO4 (aq), NMO, THF/H2O (3:1), rt, overnight, 82%; vi) a) BH3$THF, N2,
0 ꢀC, 2 h; b) NaOH(aq), H2O2, rt, overnight, 23, 64%; 24, 7%; vii) 5-bromo-1-pentene, t-
BuOK, THF/DMF (5:1), rt, overnight, 79%; viii) OsO4(aq), NMO, THF/H2O (3:1), rt,
overnight, 82%; ix) a) BH3$THF, N2, 0 ꢀC 2 h; b) NaOH(aq), H2O2, rt, overnight, 19, 32%;
20, 3%; x) a) m-CPBA, 66%; b) NaBH4, iPrOH, 60 ꢀC, 3 h, 75%.
3. Conclusion
In conclusion, 19 potential metabolites of AKB-48 have been
synthesized (Fig. 2), and six metabolites were identified by com-
parison with the metabolites in an authentic urine sample (Fig. 4).
Compounds 10 and 30 are of most interest with strong intensities
and structures uniquely identifying AKB-48 from 5F-AKB-48,
therefore they are prime candidates to be used as biomarkers for
detection of AKB-48 intake in routine urine sample analysis. The
developed synthesis methods could be applied to other synthetic
cannabinoids with alkyl adamantyl or pentyl chain moiety. The
findings could also be of use in elucidating the metabolism of novel
synthetic cannabinoids.
4. Experimental section
4.1. General information
TLC was performed using 0.25 mm precoated silica-gel plates
(Merck 60 F254), detection by UV-abs at 254 nm. 1H, 13C and 19F
NMR spectra were recorded on a Varian Mercury 300 MHz instru-
ment (25 ꢀC in CDCl3, MeOH-d4 or acetone-d6). 19F NMR analyses
were compared with an external standard (TFA). HPLC-MS was
performed on a Gilson system (Column: Phenomenex C-18, 5
100 ꢁ 21, 20 mm and Waters X-Bridge C-8 2.5 m, 50 ꢁ 4.6 mm or
C-8 2.5
respectively; using acetonitrile and deionized water with ammo-
nium acetate (10 mM) as mobile phase. Pump: Gilson gradient
pump 322; UV/VIS detector: Gilson 155, detection at 215 nm; MS
mm,
Scheme 5. i) CH3CN, BF3‧Et2O, TFA, 70 ꢀC, 3 h; ii) Conc. HCl, 150 ꢀC MW irradiation 1 h;
iii) TBTU, 1H-indazole-3-carboxylic acid, Et3N, THF, rt, overnight, 30% over 3 steps; iv)
NaBH4, MeOH, rt, 1 h; v) 1-Bromopentane, t-BuOK, DMF/THF (1:5), rt, overnight, 30,
36%; 31, 20%.
m
m
m, 50 ꢁ 4.6 mm for preparative and analytical experiments

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J. Wallgren et al. / Tetrahedron 74 (2018) 2905e2913
Fig. 3. Analysis of reference materials 4, 5, 9, 10, 16, 30, 31 and an authentic urine
sample (red) by LC-MS. A) m/z 382 - > m/z 135 B) 382 - > m/z 151; LC gradient: 26e67%
MeCN in 10 mM ammonium acetate, 100 ꢁ 2.1 mm Cortecs UPLC C18 column (Waters),
7.5 min.
Fig. 2. List of structures of 19 synthesized and two (4 and 5) commercial potential
metabolites of AKB-48.
Fig. 4. Identified synthesized metabolites from authentic urine samples with possible
intake of only AKB-48.
detector: Thermo Finnigan Surveyor MSQ; Gilson Fraction Collector
FC204).Flash chromatography was performed using the following
silica gel: High purity grade (Merck Grade 9385), pore size 60 Å,
230e240 mesh particle size. The reagents were purchased from
Sigma-Aldrich, Merck (EDC and NMO), Fluka (TBTU and BF3(OEt)2)
and Alfa Aesar (1-bromo-5-fluoropentane). Compounds 4 and 5
were purchased from Chiron AS. Compounds 14e16, 26, 29e316
have been reported in our previous publication in Tetrahedron
Letters, but they were also reported here for convenience of the
readers. 1H NMR and 13C NMR spectra of the synthesized potential
metabolites can be found in the appendix while the results of the
high-resolution mass measurements by RMV using LC-QTOF-MS
can be found in the experimental.

J. Wallgren et al. / Tetrahedron 74 (2018) 2905e2913
2909
4.2. Synthesis
123.1, 122.8, 122.6, 109.2 (aromatic C), 71.7, 66.7 (COH), 52.1, 49.2,
42.0, 36.5, 30.1, 29.6, 26.1 (aliphatic C).
4.2.1. N-(adamantan-1-yl)-1H-indazole-3-carboxamide (2)
Compound 1 (1.0 g, 6.17 mmol) was dissolved in DMF (57 mL). To
this solution EDC (1.74 g, 9.57 mmol), HOBt (1,03 g, 7.6 mmol) and
Et3N (3.7 mL, 26.3 mmol) was added. The solution was left to stir for
10 min in room temperature, thereafter compound 13 (1.32 g,
7.05 mmol) was added to the solution and it was left to stir over-
night at room temperature. The progression of the reaction was
monitored by TLC. The reaction mixture was then diluted with
EtOAc (30 mL) and washed with water (100 mL). The water phase
was extracted with EtOAc (3 ꢁ 20 mL). The collected organic phases
were dried using magnesium sulfate, filtered and concentrated in
vacuo. The residue was thereafter chromatographed on silica using
a mobile phase of EtOAc/n-Hep (3:7) and the fractions containing 2
were concentrated in vacuo to afford compound 2 (880 mg, 48%).
Rf ¼ 0.44 (3:7 EtOAc/n-Hep). HRMS (ESI, [MþH]þ): Calcd. for
4.2.4. N-(adamantan-1-yl)-1-(pent-2-en-1-yl)-1H-indazole-3-
carboxamide (7)
Compound 2 (150 mg, 0.51 mmol) was dissolved in DMF/THF
(1:5, 6 mL). The solution was put to 0 ꢀC using an ice bath and left to
stir for 5 min. To the solution t-BuOK (85 mg, 0.76 mmol) was added
and the solution was stirred for 15 min. As the mixture was brought
to room temperature 1-bromo-2-pentene (79 ml, 0.76 mmol) was
added to the solution and the reaction mixture was left to stir over
the weekend. The development of the reaction was monitored on
TLC. Water (20 mL) was added to the solution and thereafter the
solution was extracted using EtOAc (3 ꢁ 20 mL). The organic phases
were collected and concentrated in vacuo. The remaining residue
was applied to a silica column with a mobile phase composition of
EtOAc/n-Hep (1:2) and chromatographed. The fractions containing
7 were concentrated in vacuo to give compound 7 (182.5 mg, yield
C
18H22N3Oþ2 : 296.1758. Found: 296.1754. 1H NMR (CDCl3, 300 MHz)
: 10.1 (s, 1H), 8.42 (dd, J ¼ 8.1 Hz, 1.2 Hz, 1H), 7.50e7.40 (m, 2H),
7.30 (dd, J ¼ 8.1 Hz, 1.2 Hz, 1H), 6.85 (s, 1H), 2.20e2.15 (m, 6H),
2.15e2.10 (m, 3H),1.80e1.70 (m, 6H). 13C NMR (CD3OD 75.4 MHz)
d
98%). Rf ¼ 0.68 (1:2 EtOAc/n-Hep). 1H NMR (CDCl3, 300 MHz)
d: 8.38
(td, J ¼ 8.1 Hz, 1.8 Hz, 0.9 Hz, 1H), 7.40e7.30 (m, 2H), 7.22 (m, 1H),
6.82 (s, 1H), 5.78e5.56 (m, 2H), 4.93 (dd, J ¼ 5.1 Hz, 1.2 Hz, 2H),
2.21e2.16 (m, 6H), 2.16e2.08 (m, 3H), 2.08e1.98 (m, 2H), 1.80e1.65
d:
164.4 (CONH), 143.0, 140.3, 127.9, 123.4, 122.9, 111.4 (aromatic C),
53.0, 42.7, 37.5, 31.0 (aliphatic C).
(m, 6H), 0.96 (t, J ¼ 7.2 Hz, 3H). 13C NMR (CDCl3, 75.4 MHz)
d: 162.0
(CONH), 140.8, 138.2, 136.7, 126.6, 123.2, 123.1, 122.9, 122.4, 109.6,
51.9, 51.8, 41.9, 36.5, 29.6, 25.2, 13.2.
4.2.2. N-(adamantan-1-yl)-1-(pent-4-en-1-yl)-1H-indazole-3-
carboxamide (3)
Compound 2 (50 mg, 0.17 mmol) was dissolved in DMF/THF
(1:5, 6 mL). The solution was cooled to 0 ꢀC and stirred for 5 min. To
the solution t-BuOK (30 mg, 0.26 mmol) was added and stirred for
15 min. The mixture was brought to room temperature and 5-
4.2.5. N-(adamantan-1-yl)-1-(2,3-dihydroxypentyl)-1H-indazole-
3-carboxamide (8)
Compound 7 (43 mg, 0.12 mmol) was dissolved in a solvent
system comprised of THF/H2O (3:1, 8 mL). OsO4 (4 wt% in H2O, 73 ml,
bromopent-1-ene (40
m
l, 0.34 mmol) was added to the solution
0.012 mmol) and NMO (24 mg, 0.177 mmol) was added to the so-
lution and left to stir overnight at room temperature. The devel-
opment of the reaction was observed on TLC. When the reaction
was finished, saturated NaHCO3(aq) (1.5 mL) was added to the re-
action mixture prior to extraction using EtOAc (3 ꢁ 10 mL). The
organic layers were gathered and concentrated using a rotary
evaporator. The remaining residue was applied to a silica column
and chromatographed using a mobile system of 2:1 (EtOAc/n-Hep).
The fraction vials containing 8 were gathered and concentrated in
vacuo to afford compound 8 (37.7 mg, yield: 80%). Rf ¼ 0.34 (2:1
EtOAc/n-Hep). HRMS (ESI, [MþH]þ): Calcd. for C23H32N3O3þ:
which was left to stir over the weekend. Progression of the reaction
was monitored on TLC. The solvents were evaporated, water
(20 mL) was added and the solution was extracted using EtOAc
(3 ꢁ 20 mL). The combined organic phases were then dried using
magnesium sulfate, filtered and concentrated in vacuo. The residue
was thereafter chromatographed on silica using a mobile phase of
EtOAc/n-Hep (1:4) and the fractions containing 3 were concen-
trated in vacuo to yield compound 3 (54 mg, yield 87%). 1H NMR
(CDCl3, 300 MHz)
d
: 8.42 (dd, J ¼ 8.1 Hz, 1.2 Hz, 1H), 7.50e7.40 (m,
2H), 7.30 (dd, J ¼ 8.1 Hz, 1.2 Hz, 1H), 6.85 (s, 1H), 5.81 (m, 1H), 5.05
(m, 2H), 4.36 (t, J ¼ 7 Hz, 2H), 2.20e2.15 (m, 6H), 2.15e2.10 (m, 3H),
2.05 (dt, J ¼ 7 Hz, 6 Hz, 2H), 2.00 (q, J ¼ 7 Hz, 2H), 1.80e1.70 (m, 6H).
398.2439. Found: 398.2440. 1H NMR (CDCl3, 300 MHz)
d: 8.34 (dd,
J ¼ 8.4 Hz, 1.2 Hz, 1H), 7.50e7.35 (m, 2H), 7.23 (m, 1H), 6.73 (s, 1H),
4.49 (dd, J ¼ 7.2 Hz, 1.2 Hz, 2H), 4.06 (m, 1H), 3.43 (m, 1H), 2.88 (m,
1H), 2.30 (m, 1H), 2.20e2.16 (m, 6H), 2.16e2.10 (m, 3H), 1.80e1.66
(m, 6H), 1.70e1.58 (m, 2H), 0.99 (t, J ¼ 6.9 Hz, 3H). 13C NMR (CDCl3,
4.2.3. N-(adamantan-1-yl)-1-(4,5-dihydroxypentyl)-1H-indazole-
3-carboxamide (6)
Compound 3 (110.9 mg, 0.30 mmol) was dissolved in a solvent
mixture containing THF/H2O (3:1, 20 mL). To the solution OsO4
75.4 MHz) d: 161.8 (CONH), 141.9, 139.1, 127.2, 123.2, 122.8, 122.7,
109.5 (aromatic C), 73.3, 72.5 (COH), 52.2, 52.1, 42.0, 36.6, 29.7, 26.8,
10.2 (aliphatic C).
(4 wt% in H2O, 186 ml, 0.03 mmol) and NMO (61.9 mg, 0.46 mmol)
was added. The solution was left to stir at room temperature
overnight. The progression of the reaction was monitored on TLC.
When the reaction had run its course, saturated NaHCO3(aq)
(10 mL) was added to the reaction mixture and the reaction mixture
was then extracted using EtOAc (3 ꢁ 20 mL). The organic layers
were collected and concentrated in vacuo. The remaining residue
was purified by column chromatography with EtOAc as the eluent.
The fractions which contained 6 were collected and concentrated in
vacuo to give compound 6 (79.2 mg, yield 66%). Rf ¼ 0.37 (EtOAc).
HRMS (ESI, [MþH]þ): Calcd. for C23H32N3Oþ3 : 398.2439. Found:
4.2.6. N-(adamantan-1-yl)-1-(2-hydroxypentyl)-1H-indazole-3-
carboxamide (9) and N-(adamantan-1-yl)-1-(3-hydroxypentyl)-
1H-indazole-3-carboxamide (10)
Compound 7 (139 mg, 0.382 mmol) was put into a round bottom
flask which was purged with nitrogen. To the flask BH3$THF (1 M,
3 mL) was added and the reaction mixture was left for 2 h at 0 ꢀC.
The progression of the reaction was monitored on liquid chroma-
tography. As the reaction mixture was brought to room tempera-
ture, it was quenched using water (2 mL). Following the quenching
NaOH (31 mg, 0.764 mmol) together with H2O2 (1 mL) was added
and the reaction mixture was left to react overnight. When the
reaction was done, checked using liquid chromatography, the re-
action mixture was extracted using EtOAc (3 ꢁ 20 mL). The organic
layers were collected and filtered before being concentrated in
vacuo. The crude mixture was then dissolved in DCM and applied to
398.2440. 1H NMR (CDCl3, 300 MHz)
d: 8.33 (td, J ¼ 8.4 Hz, 2.4 Hz,
1.2 Hz, 1H), 7.40e7.30 (m, 2H), 7.21 (m, 1H), 6.81 (s, 1H), 4.36 (t,
J ¼ 7.2 Hz, 2H), 3.68 (m, 1H), 3.55 (dd, J ¼ 11.1 Hz, 3 Hz, 1H), 3.38 (dd,
J ¼ 11.1 Hz, 6.9 Hz, 1H), 2.68 (s, 2H), 2.20e2.15 (m, 6H), 2.14e2.08
(m, 3H), 2.10e1.90 (m, 2H), 1.80e1.66 (m, 6H), 1.46e1.38 (m, 2H).
13C NMR (CDCl3, 75.4 MHz)
d: 162.2 (CONH), 141.0, 138.2, 126.8,

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J. Wallgren et al. / Tetrahedron 74 (2018) 2905e2913
a silica column and chromatographed using an eluent system of 1:2
(EtOAc/n-Hep). The fractions containing 9 were gathered and dried
in vacuo to give compound 9 (101 mg, yield 69%). Rf ¼ 0.56 (1:2
EtOAc/n-Hep). HRMS (ESI, [MþH]þ): Calcd. for C23H32N3O2þ:
(CO), 162.0 (CONH), 141.2, 138.5, 127.1, 123.1, 122.9, 122.8, 109.5
(aromatic C), 69.4 (COH), 54.4, 49.6, 44.3, 43.7, 41.6, 40.7, 36.6, 35.1,
30.9, 7.8 (aliphatic C).
382.2490. Found: 382.2505. 1H NMR (CDCl3, 300 MHz)
d: 8.36 (ddd,
J ¼ 8.1 Hz, 2.4 Hz, 1.2 Hz, 1H), 7.46e7.34 (m, 2H), 7.23 (m, 1H), 6.73
(s, 1H), 4.40e4.25 (m, 2H), 4.15 (m, 1H), 2.58 (s, 1H), 2.20e2.16 (m,
6H), 2.16e2.10 (m, 3H), 1.80e1.66 (m, 6H), 1.62e1.40 (m, 4H), 0.96
4.2.9. 3-Amino-1-adamantol (14)
To a round bottom flask containing compound 13 (100 mg,
0.53 mmol)
a mixture of concentrated sulfuric acid (2 mL,
(t, J ¼ 6.9 Hz, 3H). 13C NMR (CDCl3, 75.4 MHz)
d: 161.8 (CONH),141.8,
37.1 mmol) and concentrated nitric acid (0.2 mL, 4.46 mmol) was
added at 0 ꢀC. The mixture was left to stir for two hours at 10 ꢀC. Ice-
cold water was then carefully added to the reaction mixture,
following this addition the reaction mixture was made basic using
solid sodium hydroxide until precipitation could be seen. The so-
lution was thereafter filtered, and the wet solid collected and dis-
solved in DCM and left to stir for 30 min. Next, the solid was
removed from the solution by filtration and washed with DCM. The
DCM-solution containing 14 was concentrated in vacuo to afford
138.9, 127.1, 123.2, 122.7, 109.5 (aromatic C), 70.9 (COH), 55.0, 52.1,
42.0, 36.7, 36.6, 29.7, 18.8, 14.1 (aliphatic C).
The fractions containing 10 were combined and purified once
again on a silica column using EtOAc as the mobile phase. The
fraction vials containing 10 were collected and dried in vacuo to
afford (11.4 mg, yield: 8%). Rf ¼ 0.85 (EtOAc). HRMS (ESI, [MþH]þ):
Calcd. for C23H32N3Oþ2 : 382.2490. Found: 382.2507. 1H NMR (CDCl3,
300 MHz)
d
: 8.38 (td, J ¼ 8.1 Hz, 2.1 Hz, 1.2 Hz, 1H), 7.50e7.35 (m,
2H), 7.24 (m, 1H), 6.76 (s, 1H), 4.64e4.46 (m, 2H), 3.45 (m, 1H),
2.22e2.16 (m, 6H), 2.16e2.10 (m, 3H), 2.14 (m, 1H), 1.92 (m, 1H),
1.80e1.65 (m, 6H), 1.56e1.44 (m, 2H), 0.92 (t, J ¼ 7.2 Hz, 3H). 13C
compound 14 (70 mg, yield 81%). 1H NMR (CDCl3 300 MHz)
d:
2.24e2.16 (m, 2H), 1.62e1.58 (m, 4H), 1.54e1.50 (m, 2H),
1.49e1.43(m, 6H). 13C NMR (CDCl3, 75.4 MHz)
d: 69.6 (HOC), 53.9,
NMR (CDCl3, 75.4 MHz)
d: 162.1 (CONH), 141.2, 138.5, 126.9, 123.3,
50.5, 44.9, 44.2, 34.9, 31.1 (aliphatic C).
122.9, 122.6, 109.3 (aromatic C), 70.4 (COH), 52.0, 46.1, 42.1, 36.6,
36.5, 30.7, 29.7, 9.9 (aliphatic C).
4.2.10. N-(3-hydroxyadamantan-1-yl)-1H-indazole-3-carboxamide
(15)
4.2.7. N-(adamantan-1-yl)-1-(3-oxopentyl)-1H-indazole-3-
carboxamide (11)
Compound 14 (41 mg, 0.246 mmol) was dissolved in THF
Compound 2 (52.5 mg, 0.18 mmol) was dissolved in THF (10 mL).
To the solution t-BuOK (30 mg, 0.27 mmol) and 1-chloro-3-
pentanone (31 ml, 0.27 mmol) were added. The solution was heat-
(15 mL). To the solution Et3N (69 ml, 0.5 mmol), compound 1 (27 mg,
0.164 mmol) and TBTU (79 mg, 0.246 mmol) were added. The re-
action mixture was left to stir overnight at room temperature.
Progression of the reaction was monitored on TLC. Following the
evaporation of solvents, the remaining residue was chromato-
graphed on silica using a mobile phase system of EtOAc/n-Hep
(4:1). The fractions which contained 15 were pooled and concen-
trated to yield compound 15 (41.5 mg, yield 81%). Rf ¼ 0.38 (4:1
EtOAc/n-Hep). HRMS (ESI, [MþH]þ): Calcd. for C18H22N3O2þ:
ed to 120 ꢀC using microwave irradiation and stirred for one hour.
Progress of the reaction prior to purification was monitored using
LC as well as TLC. The solvents were evaporated, water (20 mL) was
added and the solution was extracted using EtOAc (3 ꢁ 20 mL). The
combined organic phases were concentrated in vacuo, dissolved in
MeOH and purified with the aid of preparative LC. The fractions
containing 11 were concentrated in vacuo to yield compound 11
(16.4 mg, yield 24%). Rf ¼ 0.23 (1:3 EtOAc/n-Hep). HRMS (ESI,
[MþH]þ): Calcd. for C23H30N3O2þ: 380.2333. Found: 380.2329. 1H
312.1707. Found: 312.1709. 1H NMR (CD3OD 300 MHz)
d: 8.18 (dd,
J ¼ 9.6 Hz, 1.2 Hz, 1H), 7.55 (dd, J ¼ 9.3 Hz, 1.2 Hz, 1H), 7.47 (s, 1H),
7.40 (m, 1H), 7.23 (m, 1H) 2.34e2.26 (m, 2H), 2.18e2.14 (m, 2H),
2,14e2.08 (m, 4H), 1.80e1.56 (m, 6H). 13C NMR (CD3OD, 75.4 MHz)
d: 164.4 (CONH), 143.0, 140.2, 127.9, 123.4, 122.8, 111.4 (aromatic C),
69.7 (HOC), 55.6, 55.5, 49.8, 49.7, 44.9, 41.4, 36.1, 32.2 (aliphatic C).
NMR (CDCl3, 300 MHz)
d
: 8.36 (dd, J ¼ 8.1 Hz, 1,2 Hz, 1H), 7.50e7.35
(m, 2H), 7.26 (m, 1H), 6.73 (s, 1H), 4.64 (t, J ¼ 7.2 Hz, 2H), 3.08 (t,
J ¼ 7.2 Hz, 2H), 2.44 (q, J ¼ 7.8 Hz, 2H), 2.22e2.18 (m, 6H), 2.18e2.10
(m, 3H), 1.82e1.66 (m, 6H), 1.05 (t, J ¼ 7.8 Hz, 3H). 13C NMR (CDCl3,
75.4 MHz) d: 208.7 (CO), 161.9 (CONH), 141.1, 138.7, 127.0, 123.2,
122.9, 122.7, 109.4 (aromatic C), 52.0, 43.6, 42.0, 41.6, 36.6, 29.7, 7.8
(aliphatic C).
4.2.11. N-(3-hydroxyadamantan-1-yl)-1-pentyl-1H-indazole-3-
carboxamide (16)
Compound 15 (32 mg, 0.1 mmol) was dissolved in DMF/THF
(1:5, 7.2 mL) and left to stir at 0 ꢀC. Following 5 min of stirring t-
BuOK (17 mg, 0.15 mmol) was added and the solution was stirred
4.2.8. N-(3-hydroxyadamantan-1-yl)-1-(3-oxopentyl)-1H-
indazole-3-carboxamide (12)
Compound 15 (30 mg, 0.09 mmol) was dissolved in THF (10 mL).
To the solution t-BuOK (18 mg, 0.15 mmol) and 1-chloro-3-
pentanone (12 ml, 0.09 mmol) were added. The solution was heat-
for an additional 15 min. Finally, 1-Bromopentane (13 ml, 0.1 mmol)
was added to the solution, brought to room temperature and stirred
overnight. The progression of the reaction was monitored by TLC.
Water (30 mL) was added to the solution and extracted using EtOAc
(3 ꢁ 20 mL). The combined organic phases were concentrated in
vacuo and the residue was chromatographed on silica using an
eluent composition of 2:1 EtOAc/n-Hep. The fractions which con-
tained 16 were gathered and concentrated to afford compound 16
(35.4 mg, yield 90%). Rf ¼ 0.44 (2:1 EtOAc/n-Hep). HRMS (ESI,
[MþH]þ): Calcd. for C23H32N3O2þ: 382.2490. Found: 382.2483. 1H
ed to 120 ꢀC for one hour using microwave irradiation. The pro-
gression of the reaction was checked using liquid chromatography
prior to purification. The solvents were evaporated, water (20 mL)
was added and the solution was extracted using EtOAc (3 ꢁ 20 mL).
The combined organic phases were concentrated in vacuo, the
remaining residue was dissolved in MeOH and purified using pre-
parative liquid chromatography. The fractions containing 12 were
collected and concentrated to give (10.9 mg, yield 28%). Rf ¼ 0.60
(EtOAc). HRMS (ESI, [MþH]þ): Calcd. for C23H30N3Oþ3 : 396.2282.
NMR (CDCl3 300 MHz)
d
: 8.36 (dd, J ¼ 8.1 Hz, 1.2 Hz, 1H), 7.42e7.37
(m, 2H) 7.25 (m, 1H), 6.88 (s, 1H) 4.35 (t, J ¼ 6.9 Hz, 2H) 2.37e2.30
(m, 2H) 2.21e2.18 (m, 2H) 2.14e2.10 (m, 4H) 1.93 (dt, J ¼ 7 Hz, 2H)
1.80e1.50 (m, 6H), 1.42e1.24 (m, 4H) 0.89 (t, J ¼ 6.3 Hz, 3H). 13C
Found: 396.2282. 1H NMR (CDCl3, 300 MHz)
d
: 8.33 (dd, J ¼ 8.4 Hz,
1,2 Hz, 1H), 7.50e7.38 (m, 2H), 7.25 (m, 1H), 6.80 (s, 1H), 4.64 (t,
J ¼ 6.6 Hz, 2H), 3.08 (t, J ¼ 6.9 Hz, 2H), 2.44 (q, J ¼ 7.8 Hz, 2H),
2.38e2.30 (m, 2H), 2.20e2.16 (m, 2H), 2.13e2.08 (m, 4H), 1.82e1.54
NMR (CDCl3, 75.4 MHz) d: 162.3 (CONH), 141.0, 137.9, 126.7, 123.1,
122.9, 122.5, 109.3 (aromatic C), 69.4 (COH), 54.4, 49.6, 49.5, 44.3,
40.7, 35.1, 30.9, 29.6, 29.0, 22.4, 14.1 (aliphatic C).
(m, 6H), 1.05 (t, J ¼ 7.8 Hz, 3H). 13C NMR (CDCl3, 75.4 MHz)
d: 208.6

J. Wallgren et al. / Tetrahedron 74 (2018) 2905e2913
2911
4.2.12. 1-(pent-4-en-1-yl)-N-(3-hydroxyadamantan-1-yl)-1H-
indazole-3-carboxamide (17)
Compound 15 (130 mg, 0.42 mmol) was dissolved in DMF/THF
(1:5, 6 mL). The solution was put to 0 ꢀC using an ice bath and left to
stir for five minutes. t-BuOK (70 mg, 0.63 mmol) was added to the
solution which was stirred for an additional 15 min. As the mixture
further purification using preparative LC. The fractions containing
the mixture of 19 (91%) and 20 (9%) were collected and dried in
vacuo to afford compound 19 and 20 (34.8 mg, yield 35%). Rf ¼ 0.20
(EtOAc). HRMS (ESI, [MþH]þ): Calcd. for C23H32N3Oþ3 : 398.2439.
Found: 398.2434. 1H NMR (CDCl3, 300 MHz)
d: 8.34 (dd, J ¼ 8.1 Hz,
1.2 Hz, 1H), 7.40e7.36 (m, 2H), 7.24 (m, 1H), 6.89 (s, 1H), 4.34 (t,
J ¼ 7.2 Hz, 2H), 3.62 (t, J ¼ 6.3 Hz, 2H), 2.36e2.30 (m, 2H), 2.22e2.16
(m, 2H), 2.14e2.08 (m, 4H), 1.96 (quin, J ¼ 7.5 Hz, 2H), 1.82e1.50 (m,
was brought to room temperature 5-bromopent-1-ene (50 ml,
0.42 mmol) was added to the solution and the reaction mixture was
left to stir over the weekend. The progression of the reaction was
checked on TLC. Water (20 mL) was added to the solution and
thereafter the solution was extracted using EtOAc (3 ꢁ 20 mL). The
organic phases were collected and concentrated in vacuo. The
remaining residue was applied to a silica column with a mobile
phase composition of EtOAc/n-Hep (3:1) and chromatographed.
The fractions containing 17 were concentrated in vacuo to afford
compound 17 (125.3 mg, yield 79%). Rf ¼ 0.38 (3:1 EtOAc/n-Hep). 1H
8H),1.46e1.34 (m, 2H). 13C NMR (CDCl3, 75.4 MHz)
d: 162.2 (CONH),
141.0, 137.9, 126.8, 123.1, 122.9, 122.6, 109.2 (aromatic C), 69.4, 62.6
(COH), 54.4, 49.5, 49.3, 44.3, 40.6, 35.1, 32.2, 30.8, 29.6, 23.2
(aliphatic C).
4.2.15. 1-(4-hydroxypentyl)-N-(3-hydroxyadamantan-1-yl)-1H-
indazole-3-carboxamide (20)
NMR (CDCl3, 300 MHz)
7.23 (m, 1H), 6.88 (s, 1H), 5.78 (m, 1H), 5.04 (m, 1H), 5.00 (m, 1H),
d
: 8.35 (d, J ¼ 8.1 Hz, 1H), 7.38e7.32 (m, 2H),
To the solution of compound 17 (29 mg, 0.077 mmol) in 3 mL
DCM was added 70% m-CPBA (21 mg, 0.085 mmol) of and stirred
overnight (about 18 h). The reaction was then quenched with
saturated NH4Cl (aq) and extracted with ethyl acetate (3 ꢁ 10 mL).
The solvent was then evaporated, and the product was dissolved in
MeOH (1.25 mL) and purified using preparative LC to give epoxide
4.34 (t, J ¼ 6.9 Hz, 2H), 2.34e2.26 (m, 2H), 2.20e2.16 (m, 2H),
2.15e1.96 (m, 8H), 1.82e1.50 (m, 6H). 13C NMR (CDCl3, 75.4 MHz)
d:
162.1 (CONH), 140.9, 137.9, 137.1, 126.7, 123.1, 122.8, 122.5, 115.9,
109.2, 69.3 (COH), 54.3, 49.5, 48.5, 44.2, 40.6, 35.0, 30.80, 30.76,
28.8.
(21 mg, yield 66%). 1H NMR (CDCl3 300 MHz)
d
: 8.36 (dt, J ¼ 8.1 Hz,
0.9 Hz, 1H), 7.43e7.36 (m, 2H), 7.28e7.21 (m, 1H), 6.87 (br s, 1H),
4.51e4.33 (m, 2H), 2.99e2.87 (m, 1H), 2.75 (dd, J ¼ 5.1, 3.9 Hz, 1H),
2.47 (dd, J ¼ 5.1, 2.7 Hz, 1H), 2.37e2.29 (m, 2H), 2.21e2.08 (m, 8H),
4.2.13. 1-(4,5-dihydroxypentyl)-N-(3-hydroxyadamantan-1-yl)-
1H-indazole-3-carboxamide (18)
Compound 17 (30.3 mg, 0.08 mmol) was dissolved in a solvent
mixture of THF:H2O (3:1, 8 mL). To the solution OsO4 (4 wt% in H2O,
1.82e1.40 (m, 9H). 13C NMR (CDCl3, 75.4 MHz)
d: 162.2, 141.1, 138.2,
126.9, 123.2, 122.9, 122.7, 109.2, 69.5, 54.4, 51.8, 49.5, 48.9, 47.0,
44.3, 40.7, 35.1, 30.9, 29.7, 26.5.
49 ml, 0.008 mmol) and NMO (16.2 mg, 0.12 mmol) were added and
left to stir overnight at room temperature. The development of the
reaction was monitored on TLC. When the reaction was completed
a saturated NaHCO3 solution (1 mL) was added to the reaction
mixture. Following this, the reaction mixture was extracted using
EtOAc (3 ꢁ 10 mL). The organic phases were combined and
concentrated in vacuo. The residue was then chromatographed on
silica using EtOAc as the eluent. The fractions containing 18 were
gathered and dried to afford compound 18 (27 mg, yield 82%).
Rf ¼ 0.08 (EtOAc). HRMS (ESI, [MþH]þ): Calcd. for C23H32N3O4þ:
To the resulted epoxide (21 mg, 0.053 mmol) added 2-propanol
(1.2 mL) and NaBH4 (4 mg, 0.106 mmol). The mixture was stirred at
60 ꢀC for about 3 h. Then quenched with 10 mL saturated NH4Cl (aq)
and extracted with ethyl acetate (3 ꢁ 10 mL), concentrated and
purified on silica gel with EtOAc to give compound 20 (15.7 mg, 75%
yield).
1H NMR (CDCl3 300 MHz)
d
: 8.36 (dt, J ¼ 8.1, 1.0 Hz, 1H),
7.43e7.37 (m, 2H), 7.24 (m, 1H), 6.86 (br s, 1H), 4.39 (t, J ¼ 8.1 Hz,
2H), 3.83 (m, 1H), 2.36e2.30 (m, 2H), 2.22e1.93 (m, 7H), 1.82e1.42
414.2388. Found: 414.2384. 1H NMR (CDCl3, 300 MHz)
d: 8.34 (dd,
(m, 9H), 1.18 (d, J ¼ 6.3 Hz, 3H). 13C NMR (CDCl3, 75.4 MHz)
d: 162.2,
J ¼ 8.4 Hz, 1.2 Hz, 1H), 7.42e7.35 (m, 2H), 7.24 (m, 1H), 6.87 (s, 1H),
4.40 (t, J ¼ 7.2 Hz, 2H), 3.73 (m, 1H), 3.62 (m, 1H), 3.42 (m, 1H),
2.38e2.28 (m, 2H), 2.20e2.16 (m, 2H), 2.16e2.06 (m, 4H), 2.10e2.00
(m, 2H), 1.82e1.50 (m, 6H) 1.45 (q, J ¼ 7.8 Hz, 2H). 13C NMR (CDCl3,
141.0, 138.0, 126.8, 123.2, 123.0, 122.6, 109.3, 69.4, 67.7, 54.4, 49.6,
49.3, 44.3, 40.7, 36.2, 35.1, 30.9, 26.2, 23.9.
75.4 MHz)
d: 162.3 (CONH), 141.0, 138.0, 126.9, 123.1, 122.9, 122.7,
4.2.16. 1-(pent-2-en-1-yl)-N-(3-hydroxyadamantan-1-yl)-1H-
indazole-3-carboxamide (21)
Compound 15 (150 mg, 0.48 mmol) was dissolved in a solvent
mixture containing DMF/THF (1:5, 6 mL). The solution was cooled
to 0 ꢀC and stirred for five minutes before addition of t-BuOK
(81 mg, 0.72 mmol) followed by 15 min of stirring. As 1-bromo-2-
109.3 (aromatic C), 71.8, 69.4, 66.7 (COH), 54.5, 49.5, 49.2, 44.2,
40.6, 35.1, 30.9, 30.1, 26.1 (aliphatic C).
4.2.14. 1-(5-hydroxypentyl)-N-(3-hydroxyadamantan-1-yl)-1H-
indazole-3-carboxamide (19) and 1-(4-hydroxypentyl)-N-(3-
hydroxyadamantan-1-yl)-1H-indazole-3-carboxamide (20) mixture
Compound 17 (95 mg, 0.25 mmol) was put into a round bottom
flask which was flushed with nitrogen. BH3$THF (1 M, 2 mL) was
added to the flask and the reaction mixture was left to stir for three
hours at 0 ꢀC. The progression of the reaction was monitored using
TLC. As the reaction mixture was brought to room temperature, it
was first quenched using water (2 mL). Following the quenching
NaOH (20 mg, 0.5 mmol) together with H2O2 (1 mL) were added to
the reaction mixture which was then left to react overnight. When
the reaction was finished, which was checked with the use of liquid
chromatography, the reaction mixture was extracted using EtOAc
(3 ꢁ 10 mL). The organic layers were gathered before being
concentrated in vacuo. The crude mixture was then dissolved in
DCM and applied to a silica column and chromatographed using
EtOAc as the eluent. The fractions containing the mixture of 19 and
20 were accumulated, concentrated and dissolved in methanol for
pentene (57 ml, 0.48 mmol) was added to the solution the solution
was brought to room temperature and was left to stir over night.
The development of the reaction was monitored on TLC. The reac-
tion mixture was diluted with water (20 mL) and extracted using
EtOAc (3 ꢁ 20 mL). The combined organic phases were concen-
trated in vacuo and chromatographed on a silica column using a
mobile phase system composed of 3:1 EtOAc/n-Hep. The fractions
containing 21 were gathered and dried to give compound 21
(149.2 mg, yield 82%). Rf ¼ 0.35 (3:1 EtOAc/n-Hep). 1H NMR (CDCl3,
300 MHz)
d
: 8.36 (dd, J ¼ 8.1 Hz, 1.2 Hz, 1H), 7.40e7.30 (m, 2H), 7.24
(m, 1H), 6.88 (s, 1H), 5.80e5.55 (m, 2H), 4.94 (dd, J ¼ 6.6 Hz, 1.2 Hz,
2H), 2.35e2.28 (m, 2H), 2.20e2.18 (m, 2H), 2.14e1.98 (m, 4H),
2.10e2.00 (m, 2H), 1.80-1-50 (m, 6H), 0.97 (t, J ¼ 7.2 Hz, 3H). 13C
NMR (CDCl3, 75.4 MHz) d: 162.2 (CONH), 140.9, 138.0, 136.8, 126.7,
123.2, 123.1, 122.9, 122.6, 109.7, 69.4 (COH), 54.4, 52.0, 49.6, 44.3,
40.6, 35.1, 30.9, 25.2, 13.2.

2912
J. Wallgren et al. / Tetrahedron 74 (2018) 2905e2913
4.2.17. 1-(2,3-dihydroxypentyl)-N-(3-hydroxyadamantan-1-yl)-
1H-indazole-3-carboxamide (22)
4.2.19. N-(4-oxoadamantan-1-yl)-1H-indazole-3-carboxamide
(26)
Compound 21 (34.4 mg, 0.091 mmol) was dissolved in a solvent
mixture of THF/H2O (3:1, 8 mL). To the solution osmium tetroxide
Compound 25 (50 mg, 0.3 mmol) was dissolved in MeCN (220
4.2 mmol). To the solution boron trifluoride diethyl etherate (114
m
m
l,
l,
(OsO4, 4 wt
%
in H2O) (55
m
l, 0.0091 mmol) and 4-
0.9 mmol) and TFA (184 ml, 2.4 mmol) were added and the vial was
Methylmorpholine N-oxide monohydrate (NMO) (18.4 mg,
0.136 mmol) were added and left to stir overnight at room tem-
perature. The progress of the reaction was monitored on TLC. When
the reaction was completed saturated NaHCO3 solution (1 mL) was
added to the reaction mixture. Following this, the reaction mixture
was extracted using EtOAc (3 ꢁ 10 mL). The organic phases were
combined and concentrated in vacuo. The residue was then chro-
matographed on silica using EtOAc as the eluent. The fractions
containing 22 were gathered and dried to afford compound 22
(30.7 mg, yield 82%). Rf ¼ 0.26 (EtOAc). HRMS (ESI, [MþH]þ): Calcd.
for C23H32N3Oþ4 : 414.2388. Found: 414.2383. 1H NMR (CDCl3,
sealed with a cap. The solution was heated at 70 ꢀC for three hours
before being let cool to room temperature and left overnight. The
solvents were evaporated prior to the addition of a saturated
NaHCO3(aq) solution (3 mL). Additional water was added, and the
solution was extracted using chloroform (3 ꢁ 20 mL). The organic
phases were concentrated and the crude carboxamide was dis-
solved in concentrated HCl (3 mL). The solution was heated for one
hour at 150 ꢀC using microwave irradiation. The solvent was then
evaporated. The crude 5-aminoadamantan-2-one was dissolved in
THF (10 mL). Compound
1 (32 mg, 0.2 mmol), TBTU (96 mg,
0.3 mmol) and Et3N (84 l, 0.6 mmol) were added to the solution.
m
300 MHz)
d
: 8.31 (dd, J ¼ 8.1 Hz, 1.2 Hz, 1H), 7.50e7.35 (m, 2H), 7.23
The reaction mixture was left to stir overnight at room tempera-
ture. The advancement of the reaction was followed using TLC.
Following the evaporation of solvents the remaining residue was
chromatographed on silica using a mobile phase system of EtOAc/
n-Hep (4:1). The fractions which contained 26 were pooled and
concentrated to give 26 (27.8 mg, yield 30%). Rf ¼ 0.57 (4:1 EtOAc/n-
(m, 1H), 6.82 (s, 1H), 4.52e4.46 (m, 2H), 4.06 (m, 1H), 3.44 (m, 1H),
3.02 (m, 1H), 2.40 (m, 1H), 2.34e2.26 (m, 2H), 2.15e2.10 (m, 2H),
2.10e2.04 (m, 4H), 1.80e1.50 (m, 6H), 1.70e1.50 (m, 2H), 0.99 (t,
J ¼ 6.9 Hz, 3H). 13C NMR (CDCl3, 75.4 MHz)
d: 161.9 (CONH), 141.9,
138.7, 127.3, 123.0, 122.9, 122.8, 109.7 (aromatic C), 73.6, 72.5, 69.4
(COH), 54.5, 52.3, 49.4, 44.2, 40.62, 40.59, 35.1, 30.8, 26.8, 10.3
(aliphatic C).
Hep). 1H NMR (CDCl3, 300 MHz)
d: 10.56 (s, 1H), 8.27 (m, 1H),
7.54e7.40 (m, 2H), 7.29 (m, 1H), 6.98 (s, 1H), 2.72e2.64 (m, 2H),
2.58e2.42 (m, 6H), 2.32 (m, 1H), 2.16e1.94 (m, 4H). 13C NMR
(CD3OD, 75.4 MHz) d: 219.0 (CO), 164.7 (CONH), 143.0, 139.9, 127.9,
122.6,122.5,122.4,111.5 (aromatic C), 52.1, 48.1, 42.9, 41.3, 39.4, 30.2
(aliphatic C).*
4.2.18. 1-(2-hydroxypentyl)-N-(3-hydroxyadamantan-1-yl)-1H-
indazole-3-carboxamide (23) and 1-(3-hydroxypentyl)-N-(3-
*Results from a crude 13C-NMR where the strongest signals thought
to originate from the product were recorded.
hydroxyadamantan-1-yl)-1H-indazole-3-carboxamide (24)
Compound 21 (107 mg, 0.282 mmol) was put into a round bot-
tom flask which was flushed with nitrogen. To the flask BH3$THF
(1 M, 2.5 mL) was added and the reaction mixture was left to stir for
three hours at 0 ꢀC. The progression of the reaction was checked
using liquid chromatography. As the reaction mixture was brought
to room temperature, it was first quenched with water (1.5 mL).
Following the quenching NaOH (23 mg, 0.764 mmol) together with
H2O2 (1 mL) was added and the reaction mixture was left to react
overnight. When the reaction was over, which was checked with
the use of liquid chromatography, the reaction mixture was
extracted using EtOAc (3 ꢁ 10 mL). The organic layers were
collected and filtered before being concentrated in vacuo. The crude
mixture was then dissolved in DCM and applied to a silica column
and chromatographed using EtOAc as the eluent. The fractions
containing 23 were collected and dried in vacuo to yield compound
23 (72 mg, yield 64%). Rf ¼ 0.61 (EtOAc). HRMS (ESI, [MþH]þ):
Calcd. for C23H32N3Oþ3 : 398.2439. Found: 398.2437. 1H NMR (CDCl3,
4.2.20. N-((1s,3R,4s,5S,7s)-4-hydroxyadamantan-1-yl)- 1H-
indazole-3-carboxamide (27) and N-((1s,3R,4r,5S,7s)-4-
hydroxyadamantan-1-yl)- 1H-indazole-3-carboxamide (28)
Compound 26 (37.7 mg, 0.122 mmol) was dissolved in methanol
(10 mL). To the solution NaBH4 (25 mg, 0.66 mmol) was added, the
solution was stirred for one hour at room temperature. The
advancement of the reaction was monitored using liquid chroma-
tography. Once the reaction had finished the reaction mixture was
concentrated before addition of HCl(aq) (1 M, 10 mL). The acidic
solution was extracted using EtOAc (3 ꢁ 10 mL). After concentrating
the combined organic layers, the resulting residue was dissolved in
DMF and purified by preparative liquid chromatography. The frac-
tions containing 27 were combined and concentrated to afford 27
(11.7 mg, yield 31%).Rf ¼ 0.75 (EtOAc). 2D-NMR were recorded for
structural elucidation. HRMS (ESI, [MþH]þ): Calcd. for C18H22N3O2þ:
312.1707. Found: 312.1708. 1H NMR (CD3OD, 300 MHz)
d: 8.18 (dd,
J ¼ 8.4 Hz, 1.2 Hz, 1H), 7.56 (m, 1H), 7.48 (m, 1H), 7.24 (m, 1H), 3.95
300 MHz)
d
: 8.35 (dd, J ¼ 8.1 Hz, 1.2 Hz, 1H), 7.48e7.38 (m, 2H), 7.25
(m, 1H), 2.28e2.00 (m, 11H), 1.58e1.48 (m, 2H). 13C NMR (CD3OD,
(m, 1H), 6.80 (s, 1H), 4.44e4.24 (m, 2H), 4.16 (m, 1H), 2.53 (m, 1H),
2.35e2.25 (m, 2H), 2.18e2.14 (m, 2H), 2.10e2.05 (m, 4H), 1.80e1.40
75.4 MHz) d: 164.5 (CONH), 143.0, 140.1, 127.9, 123.4, 122.8, 111.4
(aromatic C), 74.2 (COH), 52.4, 42.8, 41.2, 36.5, 31.0, 30.3 (aliphatic
C).
(m, 10H), 0.96 (t, J ¼ 6.9 Hz, 3H). 13C NMR (CDCl3, 75.4 MHz)
d: 161.9
(CONH), 141.9, 138.7, 127.2, 123.1, 122.9, 109.6 (aromatic C), 70.9,
69.4 (COH), 55.1, 54.5, 49.5, 44.3, 40.6, 36.7, 35.1, 30.8, 18.8, 14.1
(aliphatic C).
The fractions containing 28 were combined and concentrated to
afford compound 28 (8 mg, yield 21%). Rf ¼ 0.79 (EtOAc). HRMS (ESI,
[MþH]þ): Calcd. for C18H22N3O2þ: 312.1707. Found: 312.1709. 1H
The fractions containing 24 were combined and dried in vacuo to
give compound 24 (8 mg, yield 7%). Rf ¼ 0.45 (EtOAc). HRMS (ESI,
[MþH]þ): Calcd. for C23H32N3O3þ: 398.2439. Found: 398.244. 1H
NMR (CD3OD, 300 MHz)
d
: 8.19 (dd, J ¼ 8.4 Hz, 1.2 Hz, 1H), 7.56 (m,
1H), 7.40 (m, 1H), 7.24 (m, 1H), 3.78 (m, 1H), 2.46e2.00 (m, 9H),
1.92e1.66 (m, 4H). 13C NMR (CD3OD, 75.4 MHz)
d: 164.4 (CONH),
NMR (CDCl3, 300 MHz)
d
: 8.36 (d, J ¼ 7.5 Hz, 1H), 7.50e7.36 (m, 2H),
143.0, 140.2, 127.9, 123.4, 122.8, 111.4 (aromatic C), 73.6 (COH), 52.6,
42.3, 37.2, 36.3, 36.2, 29.8 (aliphatic C).
7.25 (m, 1H), 6.83 (s, 1H), 4.64e4.46 (m, 2H), 3.45 (m, 1H),
2.38e2.30 (m, 2H), 2.22e2.16 (m, 2H), 2.17e2.10 (m, 5H), 1.92 (m,
1H), 1.82e1.44 (m, 8H), 0.92 (t, J ¼ 7.5 Hz, 3H). 13C NMR (CDCl3,
4.2.21. N-(4-oxoadamantan-1-yl)-1-pentyl-1H-indazole-3-
carboxamide (29)
75.4 MHz) d: 162.2 (CONH), 141.2, 138.2, 127.0, 123.1, 122.9, 122.7,
109.4 (aromatic C), 70.4, 69.5 (COH), 54.5, 49.6, 46.1, 44.3, 40.7, 36.6,
35.1, 30.9, 30.7, 9.9 (aliphatic C).
Compound 26 (27.8 mg, 0.09 mmol) was dissolved in DMF/THF
(1:5, 7.2 mL) and left to stir at 0 ꢀC for five minutes. t-BuOK (15 mg,

J. Wallgren et al. / Tetrahedron 74 (2018) 2905e2913
2913
0.135 mmol) was added and the solution was stirred for 15 min. As
the solution was brought to room temperature 1-bromopentane
(11 ml, 0.09 mmol) was added and the solution was left to stir
Following the color change HCl (aq) (1 M) was added until the so-
lution became clear. The clear solution was extracted using DCM
(3 ꢁ 10 mL). The organic phase was collected and then extracted
using NaOH (aq) (1 M, 3 ꢁ 10 mL). The basic water phases were
gathered and acidified to pH ¼ 1 using concentrated HCl (aq) before
being extracted using DCM (3 ꢁ 10 mL). The organic phases were
collected, concentrated in vacuo and the remaining residue was
dissolved in DCM and purified by flash chromatography using a
mobile phase system containing EtOAc with <0.5% formic acid. The
fractions containing 32 were, accumulated and concentrated in
vacuo to afford compound 32 (9.0 mg, yield 58%). Rf ¼ 0.39 (EtOAc,
>0.5% formic acid). HRMS (ESI, [MþH]þ): Calcd. for C23H30N3O4þ:
overnight. The progression of the reaction was monitored by TLC.
Water (20 mL) was added to the solution which was extracted using
EtOAc (3 ꢁ 20 mL). The combined organic phases were concen-
trated in vacuo and the residue was chromatographed on silica
using an eluent composition of 1:1 EtOAc/n-Hep. The fractions
which contained 29 were gathered and concentrated to afford
compound 29 (24.6 mg, yield 72%). Rf ¼ 0.53 (1:1 EtOAc/n-Hep).
HRMS (ESI, [MþH]þ): Calcd. for C23H30N3Oþ2 : 380.2333. Found:
380.2331. 1H NMR (CDCl3, 300 MHz)
d
: 8.33 (dd, J ¼ 8.1 Hz, 1.2 Hz,
1H), 7.42e7.36 (m, 2H), 7.25 (m, 1H), 6.87 (s, 1H), 4.34 (t, J ¼ 7.1 Hz,
2H) 2.70e2.62 (m, 2H) 2.58e2.38 (m, 6H), 2.31 (m, 1H), 2.16e1.95
(m, 4H), 2.00e1.85 (m, 2H), 1.40e1.24 (m, 4H), 0.88 (t, J ¼ 6.9 Hz,
412.2231. Found: 412.2227. 1H NMR (CDCl3, 300 MHz)
d: 8.35 (d,
J ¼ 8.1 Hz, 1H), 7.42e7.36 (m, 2H), 7.25 (m, 1H), 6.90 (s, 1H), 4.38 (t,
J ¼ 6.9 Hz, 2H), 2.40 (t, J ¼ 6.9 Hz, 2H), 2.36e2.30 (m, 2H), 2.21e2.16
(m, 2H), 2.16e2.08 (m, 4H), 2.06e1.94 (m, 2H), 1.83e1.52 (m, 8H).
3H). 13C NMR (CDCl3, 75.4 MHz)
d: 216.7 (CO), 162.5 (CONH), 141.0,
137.5, 126.8, 123.0, 122.8, 122.7, 109.3 (aromatic C), 50.9, 49.5, 46.7,
42.3, 40.7, 38.5, 29.6, 29.0, 28.9, 22.4, 14.1 (aliphatic C).
13C NMR (CDCl3, 75.4 MHz)
d: 177.6 (COOH), 162.2 (CONH), 141.0,
138.0, 126.9, 123.2, 122.9, 122.7, 109.1 (aromatic C), 69.6 (COH), 54.4,
49.5, 48.8, 44.3, 40.6, 35.1, 33.2, 30.9, 29.1, 22.0 (aliphatic C).
4.2.22. N-((1s,3R,4s,5S,7s)-4-hydroxyadamantan-1-yl)-1-pentyl-
1H-indazole-3-carboxamide (30) and N-((1s,3R,4r,5S,7s)-4-
hydroxyadamantan-1-yl)-1-pentyl-1H-indazole-3-carboxamide
(31)
4.3. Confirmation of analytes in authentic urine samples
The method used for liver microsome incubations was pre-
Compound 29 (21.9 mg, 0.058 mmol) was dissolved in methanol
(10 mL). To the solution NaBH4 (15 mg, 0.4 mmol) was added, the
solution was left to stir for one hour at room temperature. The
progression of the reaction was monitored using liquid chroma-
tography. Once the reaction had finished the reaction mixture was
concentrated prior to the addition of HCl(aq) (1 M, 10 mL). The
acidic solution was extracted using EtOAc (3 ꢁ 10 mL). After com-
bination and concentration of the organic phases, the residue was
dissolved in methanol, filtered through a pipette using cotton and
purified by preparative liquid chromatography. The fractions con-
taining 30 were combined and concentrated to give compound 30
(8 mg, yield 36%). Rf ¼ 0.38 (1:1 EtOAc/n-Hep). 2D-NMR were
recorded for structural elucidation. HRMS (ESI, [MþH]þ): Calcd. for
sented in detail earlier.5 In brief, AKB-48 (1
mg/ml) was incubated
with human liver microsomes and the incubation was terminated
by addition of acetonitrile after 60 min. The samples were analyzed
by liquid chromatography Time-of-Flight mass spectrometry (LC-
QTOF-MS).
To optimize the separation of analogs a separation on a Cortecs
UPLC C18 column (100 ꢁ 2.1 mm, Waters) was developed. A 7.5 min
gradient from 26 to 67% ACN in 10 mM ammonium acetate was
used. Retention times of synthesized analogs were compared to
those of a urine sample treated with glucuronidase/arylsulfatase.
Acknowledgements
C
23H32N3Oþ2 : 382.2490. Found: 382.2489. 1H NMR (CDCl3,
300 MHz)
Financial support from Eurostar project Psychomics and the
National Board of Forensic Medicine in Sweden are gratefully
acknowledged.
d
: 8.36 (dd, J ¼ 8.1 Hz, 1.2 Hz, 1H), 7.42e7.38 (m, 2H), 7.24
(m, 1H), 6.81 (s, 1H), 4.35 (t, J ¼ 7.4 Hz, 2H), 4.02 (m, 1H), 2.34e2.04
(m, 11H), 1.98e1.86 (m, 2H), 1.66e1.50 (m, 2H), 1.42e1.24 (m, 4H),
0.89 (t, J ¼ 6.9 Hz, 3H). 13C NMR (CDCl3, 75.4 MHz)
d: 162.4 (CONH),
Appendix A. Supplementary data
141.0, 137.9,126.7, 123.1,122.9,122.5,109.3 (aromatic C), 73.6 (COH),
51.2, 49.5, 42.1, 40.4, 35.5, 30.1, 29.6, 29.1, 28.9, 22.4, 14.1 (aliphatic
C).
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.tet.2018.04.026.
The fractions containing 31 were combined and concentrated to
yield compound 31 (4.5 mg, yield 20%). Rf ¼ 0.40 (1:1 EtOAc/n-
Heptane). HRMS (ESI, [MþH]þ): Calcd. for C23H32N3Oþ2 : 382.2490.
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d
: 8.37 (d, J ¼ 8.1 Hz,
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