Antitubercular Bis-Substituted Cyclam Derivatives: Structure-Activity Relationships and in Vivo Studies

Article abstract of DOI:10.1021/acs.jmedchem.7b01569

We recently reported the discovery of nontoxic cyclam-derived compounds that are active against drug-resistant Mycobacterium tuberculosis. In this paper we report exploration of the structure-activity relationship for this class of compounds, identifying

Full text of DOI:10.1021/acs.jmedchem.7b01569

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Anti-Tubercular bis-Substituted Cyclam Derivatives:
Structure-Activity Relationships and In Vivo Studies
Malcolm Spain, Joseph K.-H. Wong, Gayathri Nagalingam, James M. Batten,
Elinor Hortle, Stefan Oehlers, Xiao Fan Jiang, Hasini E. Murage, Jack T. Orford,
Patrick Crisologo, James A Triccas, Peter J. Rutledge, and Matthew H. Todd
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01569 • Publication Date (Web): 20 Mar 2018
Downloaded from http://pubs.acs.org on March 21, 2018
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AntiꢀTubercular bisꢀSubstituted Cyclam Derivatives:
StructureꢀActivity Relationships and In Vivo Studies
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†
†
‡
†
§
Malcolm Spain, Joseph K.ꢀH. Wong, Gayathri Nagalingam, James M. Batten, Elinor Hortle, Stefan
^
†
†
†
†
‡
Oehlers, Xiao Fan Jiang, Hasini E. Murage, Jack T. Orford, Patrick Crisologo, James A. Triccas,
*
,†
*,†
Peter J. Rutledge and Matthew H. Todd.
†
School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia
‡
Microbial Immunity and Pathogenesis Group, Department of Infectious Diseases and Immunology,
Sydney Medical School, The University of Sydney, Sydney, NSW 2006, Australia
§ Tuberculosis Research Program, Centenary Institute, Royal Prince Alfred Hospital, Missenden Road,
Camperdown, Sydney, NSW 2050, Australia.
^
Central Clinical School, Sydney Medical School, The University of Sydney, Sydney, NSW 2006,
Australia
Tuberculosis, mycobacteria, structure activity relationship, cyclam, in vivo studies.
Abstract. We recently reported the discovery of nonꢀtoxic cyclamꢀderived compounds that are
active against drugꢀresistant Mycobacterium tuberculosis. In this paper we report exploration of
the structureꢀactivity relationship for this class of compounds, identifying several simpler
compounds with comparable activity. The most promising compound identified, possessing
significantly improved water solubility, displayed high levels of bacterial clearance in an in vivo
zebrafish embryo model, suggesting this compound series has promise for in vivo treatment of
tuberculosis.
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INTRODUCTION
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Antimicrobial resistance represents a current worldwide threat to human health and an
1ꢀ4
increasing burden upon healthcare. An estimated 1.7 billion people were infected with latent
tuberculosis (TB) in 2014, leading to 10.4 million incident cases and 1.8 million deaths in
5
,6
2015. In the treatment of tuberculosis, multiꢀdrug therapy of firstꢀline drugs are used over long
7
periods of time (typically 6 months) to address the challenge of resistance. However, poor
adherence to the long treatment regimens has been implicated in the rise of drug resistant TB
7
,8
strains. In 2015, 480,000 cases of multiꢀdrugꢀresistant TB (MDRꢀTB) were reported, requiring
6
,7
longer and more expensive treatment programs. Furthermore, mismanagement of MDRꢀTB
7
has led to extensivelyꢀdrugꢀresistant TB (XDRꢀTB) being reported in 117 countries. The rise in
drugꢀresistant forms of TB is a global public health crisis and requires the urgent development of
7
,9,10
new small molecule drugs.
During M. tuberculosis infection in humans, host phagocytes take up bacteria, which can
replicate within these cells; as the infection progresses, aggregation of further immune cells leads
1
1
to granulomas where bacteria can remain for long periods of time. Commonly, TB results from
reꢀactivation of established granulomas in individuals infected with latent TB, with a 10%
1
1
chance of activation over a lifetime. As such, granulomas represent an important and
characteristic feature of TB infections. Recently, zebrafish (Danio rerio) infected with
Mycobacterium marinum, a close relative of M. tuberculosis, have been used to study
mycobacterial granulomas. The translucency of the zebrafish allows the infection, and hence the
impact of treatment, to be observed over time. The zebrafishꢀM. marinum infection platform is
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thus amenable for investigating the efficacy of novel antibiotic compounds within an in vivo
12
infection context.
We have shown the promising activity of cyclamꢀbased ligands and their copper and zinc
1
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complexes against virulent and drugꢀresistant M. tuberculosis strains. These compounds are
nonꢀtoxic and active against intracellular M. tuberculosis. Herein we present our progress in
determining the structureꢀactivity relationship (SAR) of the cyclam compound 1 (Figure 1),
which possesses a cyclic cyclam core linked to two identical naphthalimide pendants via triazole
linkers.
RESULTS AND DISCUSSION
The SAR was determined through the symmetric modification of the two pendant groups of 1,
the linker groups and investigations into the impact of metal complexation upon biological
activity.
Figure 1. Most important hit compound 1 against M. tuberculosis (from our previous study see
ref. 13), a bisꢀsubstituted cyclam core with pendant naphthalimide groups linked through 1,2,3ꢀ
triazoles.
To investigate the pendant groups, compounds 5aꢀh were synthesized through copperꢀ
catalysed azideꢀalkyne cycloaddition (CuAAC) between bisꢀalkyne 2 and the corresponding
azides 3aꢀh, to yield 4aꢀh followed by Boc deprotection (Scheme 1).
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Scheme 1. Synthesis of compounds to investigate the pendant groups.
14
Naphthalimides represent key structural features in a range bioactive compounds including
antiꢀtumor, antiꢀviral and antiꢀbacterial compounds, while their fluorescent properties make them
13,15
widely used probes.
However, inclusion of the naphthalimide group can lead to compounds
of low water solubility. In an attempt to lower molecular weight and increase water solubility
relative to parent compound 1, a range of pendant groups was tested, maintaining the cyclam
core and triazole linkers (Figure 2). Pleasingly replacing the naphthalimide in 1 (MIC 3.13 µM)
with simple naphthaleneꢀbased pendant groups (5aꢀb) maintained some of the superior activity
1
6
15c
of the naphthalimide. However, single aromatic ring pendants 5cꢀh including a diversity of
aromatic electron density, with or without a methylene spacer, exhibited low potency. Thus
while the naphthalimide can be replaced by simpler 1ꢀnaphthyl groups, single aromatic rings
were not found to be suitable replacements.
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Figure 2. Pendant group SAR. Potencies are MIC vs. H37Rv using concentration of compound
as determined by the resazurin viability assay (see the experimental section for details).
Due to the ease of synthesis and promising activity of simple naphthyl groups, the 1ꢀnaphthyl
pendant group was maintained while other linking groups were investigated. Compound 9a was
synthesized through CuAAC between bisꢀazide 6 and the corresponding alkyne 7a, to yield 8a
followed by Boc deprotection (Scheme 2). To investigate the simple alkyl linker groups
compound 13 was synthesized through alkylation of 10 with bromide 11 followed by hydrolysis
and hydrogenation of intermediate 12 to yield 13 (Scheme 2).
Scheme 2. Synthesis of compounds to investigate the linker group.
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Interestingly, the alkane linker in 13 maintained similar bioactivity (25–50 µM) versus the
triazoleꢀbased compound 5a (Figure 3), suggesting that flexible and nonꢀcoordinating linker
groups can maintain bioactivity. Importantly, introducing an extra methylene group and
changing the 1,2,3ꢀtriazole connectivity to be Cꢀlinked to the pendant group in 9a resulted in a
more potent compound (6.25 µM) suggesting that positioning of the pendants, as opposed to the
flexibility of the linker, is a key determinant of potency.
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Figure 3. Linker group SAR. Potencies are MIC vs. H37Rv
Previous medicinal chemistry studies on the treatment of HIV with cyclamꢀderived compounds
1
7
noted that the coordinating metal ion impacted on biological activity. Thus we investigated a
variety of complexes of 1 (Table 1) using metal ions of different radii and ionic charge.
1
3
2+
2+
Previously, we reported 14aꢀb, the Zn and Cu salts, which both had similar antiꢀtubercular
1
3
2+
3+
activities to the free amine 1 and salt. Metal complexes of Fe (14c, ionic radius 0.61 Å), Fe
3
+
2+
2+
(
14d, 0.55 Å), Sm (14e, 0.96 Å), Co (14f, 0.65 Å) and Mn (14g, 0.67 Å) were synthesized
by complexation with 1 and evaluated showing no significant difference in antiꢀtubercular
activity, strongly suggesting that the identity of the metal centre is not important. However it
remains possible that metal ion exchange is occurring to give an active compound under the
18
2+
2+
assay conditions. Additionally, the Zn (15a) and Cu (15b) complexes of the simpler
naphthyl compound 5a were synthesized and also screened for antiꢀtubercular activity displaying
increased activity relative to the corresponding metalꢀfree compound.
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Table 1. Activity of metal complexes 14aꢀg and 15aꢀb.
1
1
1
1
1
1
1
1
1
1
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0
1
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9
0
1
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a
Compound
Complexation
Ionic
radius MIC
(µM)
vs.
H37Rv
b
c
(Å)
d
1
n/a
n/a
3.13 – 6.25
3.13 – 6.25
6.25
d
1
1
1
1
1
4a
4b
4c
4d
4e
1 + Zn(ClO4)2
0.74
0.73
0.61
0.55
0.96
0.65
0.67
n/a
d
1 + Cu(ClO4)2
1 + Fe(ClO
1 + Fe(ClO
)
)
4
3.13
4
2
3.13
4
3
1 + Sm(ClO
)
1.56 – 3.13
3.13 – 6.25
3.13
3
14f
1 + Co(ClO
1 + Mn(ClO
n/a
4 2
)
14g
)
4 2
5
1
1
a
25 – 50
3.13
5a
5b
5a + Zn(ClO
5a + Cu(ClO )
4 2
4
)
2
0.74
0.73
3.13
a
b
All compounds were complexed as detailed in the SI. Ionic radii based on the hexacoordinate
1
9 c
d
metals. See experimental section for details. See ref. 13 for details.
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Having observed increased potency for the 1ꢀnaphthyl substituted compound 9a (Figure 3)
which has the ‘reversed’ triazole connectivity relative to the parent compound 1 (and 5a), we
investigated a range of pendant groups with this alternative triazole linker (Figure 4).
Compounds 9bꢀe were synthesized through CuAAC between bisꢀazide 6 and the corresponding
alkyne 7bꢀe, followed by Boc deprotection (Scheme 2). Interestingly, the obvious naphthalimideꢀ
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15c
substituted compound 9b exhibited reduced activity (25 µM), but the 2ꢀnaphthyl pendant 9c
(6.25 µM) displayed comparable activity to the original 1ꢀnaphthyl compound 9a. Surprisingly,
15c
the compound with simple phenyl pendant groups (9d) exhibited moderate activity, while
2
+
2+
benzyl groups (9e) were inactive. Additionally Zn and Cu complexes of these compounds
(16aꢀ20b) were synthesized and screened for activity (Table 2), with the results clearly
2+
suggesting Cu complexation is beneficial for antiꢀtubercular activity in this series: for example,
2
+
2+
Cu complex 20b regains high activity, from the inactive ligand 9e whilst the Zn complex 20a
does not.
Figure 4. Pendant group SAR with the alternative linker topology.
2
+
2+
Table 2. Activity of Zn and Cu metal complexes (16aꢀ20b) of compounds 9aꢀe.
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a
Compound Complexation
MIC vs.
b
H37Rv (µM)
9
1
1
9
a
n/a
6.25
6.25
3.13
25
6a
6b
9a + Zn(ClO )
4 2
9a + Cu(ClO )
4 2
c
b
n/a
c
c
17a
9b + Zn(ClO4)2
25
17b
9b + Cu(ClO4)2 6.25
9
1
1
9
1
1
c
n/a
6.25
3.13
3.13
50
8a
8b
9c + Zn(ClO )
4 2
9c + Cu(ClO )
4 2
c
d
n/a
c
9a
9d + Zn(ClO4)2
50
c
9b
9d + Cu(ClO4)2
6.25
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4 2
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Free amines were complexed with the relevant metal perchlorate salts detailed in the SI. See
c
experimental section for details. See ref. 15c for synthetic details.
The SAR suggests that napthalimides can be replaced by simple naphthyl substituents, and
various linker groups and metal complexes can be tolerated without significant changes in
activity. SAR around the optimal linker proved more complicated, and in some cases dependent
2
+
on metal ion complexation, with distinct preference for the Cu ꢀcomplex. The initial hit
compound 1 exhibits a good combination of activity, synthetic simplicity, and importantly
possibilities for further functionalization of the Nꢀsubstituent of the naphthalimide. Therefore,
modifications to increase solubility were sought by simple changes at this position, by
incorporating 3ꢀaminopropanol derived naphthalimides (Table 3). Although some of our results
2
+
suggest a potential benefit of Cu ꢀcomplexes, this was not observed in the related series 14aꢀg,
2
+
therefore Zn ꢀcomplexes were synthesized for their superior fluorescence properties.
1
3
Previously, the kinetic solubility of the Zn(ClO ) complex of 1 had been studied, and
4
2
correlates to the <0.33 mM thermodynamic water solubility measured. The 3ꢀaminopropanol
derived variant 5i (synthesized according to Scheme 1) exhibited improved aqueous solubility
for the Zn(ClO ) derived complex 21a (5.7 mM) and the ZnCl derived complex 21b (10.7
4
2
2
mM). Pleasingly this modification led to increased solubility without significant loss in
biological activity and facilitated progression of 21b towards in vivo studies.
Table 3. Effect of Nꢀfunctionalization on water solubility and biological potency.
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0
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Compound
Complexation
Thermodynamic solubility MIC
vs.
b
c
at pH 7.0 (mM)
H37Rv (µM)
d
1
2
2
4a
1a
1b
1 + Zn(ClO4)2
<0.33
5.7
6.25
6.25
6.25
5i + Zn(ClO )
4 2
5i + ZnCl
10.7
2
a
b
Free amines were complexed with the relevant metal salts as detailed in the SI. Solubility was
c
determined through a filtration assay (see experimental section for details). See experimental
d
section for details. From ref. 13.
Zebrafish embryos were exposed to a range of doses of 21b to determine in vivo compound
toxicity by assessment of embryo survival. Based on this titration, a dose of 10 µM, refreshed
every second day of infection, was chosen to test in vivo efficacy against M. marinum infection;
this dose did not result in any gross morphological changes to embryonic development (Figure
5A). Quantification of bacterial burden demonstrated a statistically significant decrease in M.
marinum burden at 5 days post infection (Figure 5B and Supporting Information), demonstrating
clearly the potential of this simple compound to reduce bacterial burden in a biologicallyꢀ
relevant setting.
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Figure 5. In vivo efficacy of 21b in the zebrafishꢀM. marinum infection model. (a)
Representative images of 5 day post infection zebrafish embryos infected with tdTomatoꢀ
fluorescent M. marinum and treated with 21b , as indicated. (b) Quantification of M. marinum
burden by fluorometry in 5 day post infection zebrafish embryos. * indicates p < 0.05 by tꢀtest. n
=
11 control, 8 treated embryos.
CONCLUSIONS
We have shown through SAR that simple naphthyl groups can be used as replacements for
naphthalimides in this compound series, and that the activity is relatively insensitive to the linker
groups tested. Importantly, the metal coordinating ability of the triazole is not essential. This
links to the observation that in the primary series of compounds 14aꢀg, activity is apparently
2
+
insensitive to the coordinated metal ion or its identity. However, Cu coordination does increase
2+
the activity of several compounds of the ‘reversed’ triazole geometry 18aꢀ20b, and thus Cu
may be involved in the bioactivity of these compounds. Encouragingly, progression of the hit
compound 1 to our current lead compound 21b with good water solubility has shown that these
compounds reduce bacterial load in vivo. Combining the in vivo activity of this unusual class of
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antiꢀtubercular drugs with our previous demonstration of their effectiveness against drugꢀ
resistant M. tuberculosis leads us to prioritize further optimization of potency alongside studies
towards understanding the mechanism of action.
1
1
1
1
1
1
1
1
1
1
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0
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0
EXPERIMENTAL SECTION
General Procedures. Acetonitrile, methanol and THF were collected from a PureSolv MD 7
solvent purification system with anhydrous alumina columns. All commercially available
reagents and solvents were purchased from Sigma Aldrich, Alfa Aesar, Matrix Scientific, Merck
or Ajax Finechem and used without purification. Flash column chromatography was performed
on Davisil Grace Davison 40 – 63 ꢁm (230 – 400 mesh) silica gel. Automated flash column
chromatography was performed on a Biotage Isolera Spektra One using Biotage SNAP KPꢀSil
cartridges (filled with Grace 40ꢀ63 ꢁm (230 – 400 mesh) silica gel) at their default flow rates.
Reversedꢀphase automated chromatography was carried out on a Biotage Isolera Spektra One
using Biotage SNAP KPꢀC18ꢀHS (30 g) column using a flow rate of 25 mL/min; mobile phases
of 0.1% TFA in MilliꢀQ water and 0.1% TFA in MeCN, 0 to 100% over 10 CV was used unless
stated otherwise. Preparative reversedꢀphase HPLC was carried out on a Waters 600 controller
with a Waters 600 pump and a 2998 photodiode array detector. A Waters SunFireTM C18
OBDTM preparative column (5 ꢁm, 19 × 150 mm) was used at a flow rate of 7 mL/min; mobile
phases of 0.1% TFA in MilliꢀQ water and 0.1% TFA in MeCN in different ratios were used.
Analytical high performance liquid chromatography (HPLC) analysis was conducted on a
Waters Alliance 2695 instrument using a SunFire C18 column (5 µm, 2.1 × 150 mm) with a
Waters 2996 photodiode array (PDA) detector and a flow rate of 0.2 mL/min; mobile phases of
0
.1% TFA in MilliꢀQ water and 0.1% TFA in MeCN, 0ꢀ100% over 90 mins were used. Melting
points were recorded on a Stanford Research Systems Optimelt automated melting system and
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1
13
are uncorrected. H and C NMR spectra were recorded on a Bruker Avance DPX 200, 300, 400
or 500 MHz spectrometer. Chemical shifts are reported in ppm relative to tetramethylsilane or
residual solvent resonance as internal standard. Coupling constants J are reported in Hz to the
nearest 0.1 Hz. Mass spectra (LRMS) were acquired on a Thermo Classic LCQ mass
spectrometer or a Bruker amaZon SL mass spectrometer. Accurate mass measurements (HRMS)
were performed on a Bruker Apex Qe 7T Fourier Transform Ion Cyclotron Resonance (FTꢀICR)
mass spectrometer. Infrared spectra were recorded on a Bruker ALPHA FT‐ IR
spectrophotometer (ZnSe or diamond ATR). Elemental analyses were carried out by the
Campbell Microanalytical Laboratory (University of Otago, New Zealand) on a Carlo Erba EA
1108 elemental analyser or by the Chemical Analysis Facility (Macquarie University, Australia)
on a PelkinElmer PE2400 CHN elemental analyser. Synthesis and characterization of
compounds 1 and 14aꢀb have been reported previously in ref. 13 and their purities determined
through elemental analysis, additionally compounds 5c, 9b, 9d, 17aꢀb, 19aꢀb were previously
reported in ref. 15c and their purities determined through elemental analysis. Elemental analysis
or analytical HPLC was used to determine the purity of the compounds tested for biological
activity which are >95% pure by HPLC or within 0.4% by elemental analysis.
0
1
2
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0
Diꢀtertꢀbutyl 4,11ꢀdi(propꢀ2ꢀynꢀ1ꢀyl)ꢀ1,4,8,11ꢀtetraazacyclotetradecaneꢀ1,8ꢀdicarboxylate
(
2). To a solution of 10 (4.50 g, 20.1 mmol) in anhydrous MeCN (250 mL, 0.08 M) was added
propargyl bromide (~80% in toluene, 15.0 mL, 101 mmol, 5 equiv.). The reaction mixture was
stirred at rt overnight (resulting in a light beige suspension). The precipitate was collected by
vacuum filtration and suspended in methanol (60 mL), NaOH (2.5 M, 60 mL, 150 mmol, 7.5
equiv.) was added and the reaction mixture was stirred at rt for 1 h, resulting in a pale yellow
solution. A solution of diꢀtertꢀbutyl dicarbonate (20.0 g, 91.6 mmol, 4.5 equiv.) in methanol
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(
30.0 mL) was added and the reaction mixture was stirred at rt for 2 ꢀ 16 h. Methanol was
removed by rotary evaporation giving a white suspension, the product was collected by vacuum
filtration and washed with H O (3 × 50 mL) to yield 2 as a white solid (8.89 g, 18.7 mmol, 93%,
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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4
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5
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5
5
5
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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0
typically yields vary from 63 – 93% and requires no further purification). R (EtOAc:hexane =
F
15a
1
1
:1) 0.76. m.p. 143 – 148 °C (lit. 153 – 155 °C) H NMR (300 MHz, CDCl ): δ 1.45 (s, 18
3
H), 1.66 – 1.77 (m, 4 H), 2.17 (s, 2 H), 2.52 (t, J 6.5, 4 H), 2.65 (t, J 6.0, 4 H), 3.25 – 3.50 (br. m,
+
1
2 H). LRMS (ESI+) m/z 477.31 ([M + H] , 100%). Spectroscopic properties matched those
15a, 20
previously described.
2
1
1
ꢀAzidonaphthalene (3a). Based upon a literature procedure. To a suspension of 1ꢀ
naphthylamine (1.05 g, 7.4 mmol, 1 equiv.), ice cold H O (10 mL), and conc. HCl (32%, 11
2
mL), was added NaNO (806 mg, 9.6 mmol, 1.3 equiv.) in H O (10 mL) over 2 mins. The
2
2
resulting purple suspension was stirred for 15 min, and a solution of NaN (724 mg, 8.9 mmol,
3
1.2 equiv.) in H O (10 mL) was added over 2 mins (CAUTION: vigorous release of N gas), the
2
2
reaction mixture was allowed to warm to rt over 1.5 h. The product was extracted with diethyl
ether (3 × 50 mL), dried over Na SO and carefully concentrated by rotary evaporation to yield
2
4
1
3
a as a brown oil (0.747 g, 4.4 mmol, 60%). H NMR (200 MHz, CDCl ): δ 7.20 – 7.31 (m, 1
3
1
3
H), 7.40 – 7.58 (m, 3 H), 7.59 – 7.70 (m, 1 H), 7.75 – 7.89 (m, 1 H), 8.03 – 8.18 (m, 1 H) C
NMR (50 MHz, CDCl ): δ 113.9, 122.5, 124.7, 125.6, 126.1, 126.4, 126.9, 127.7, 134.3, 136.5.
3
ꢀ1
FTIR (ATR) νmax/cm : 2102, 1389. Spectroscopic properties matched those previously
2
2
described.
ꢀ(Azidomethyl)naphthalene
23
1
(3b).
Following
the
literature
procedure,
1ꢀ
(chloromethyl)naphthalene (1.12 g, 6.37 mmol, 1 equiv., >97% from SigmaꢀAldrich, contains
1
approx. 17% of 2ꢀ(chloromethyl)naphthalene by HꢀNMR)) and NaN (0.745 g, 11.5 mmol, 1.8
3
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equiv.) in MeCN (10 mL, 0.6 M) were refluxed for 17 h. The reaction was carefully
concentrated, diluted with H O (10 mL) and extracted with diethyl ether (3 × 10 mL), the organic
2
layers were dried over MgSO and carefully concentrated to yield 3b as a colourless oil (0.770 g,
4
0
1
2
3
4
5
6
7
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0
1
2
3
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4
.2 mmol, 64%, contains 2ꢀ(azidomethyl)naphthalene) impurities that could not be separated by
1
HPLC). H NMR (300 MHz, CDCl ): δ 4.71 (s, 2 H), 7.41 – 7.57 (m, 4 H), 7.81 – 7.87 (m, 2 H),
3
1
8
.00 (d, J 8.0, 1 H). LRMS (ESI+): m/z Not found. H NMR matched those previously
2
3
described.
1
ꢀAzidoꢀ4ꢀnitrobenzene (3d). Similar to the procedure for 3a, 4ꢀnitroaniline (443 mg, 3.21
mmol, 1.0 equiv.), H O (2 mL), conc. HCl (1 mL), NaNO (289 mg, 4.19 mmol, 1.3 equiv.) in
2
2
H O (1 mL), NaN (251 mg, 3.87 mmol, 1.2 equiv.) in H O (1 mL) for 1 h extracted, dried over
2
3
2
1
Na SO and concentrated to yield 3d as a yellow solid (525 mg, 3.20 mmol, 99%). H NMR
2
4
1
3
(
300 MHz, CDCl ): δ 7.10 – 7.20 (m, 2 H), 8.24 – 8.30 (m, 2 H) C NMR (50 MHz, CDCl ): δ
3
3
ꢀ1
119.4, 125.6, 144.7, 146.9. FTIR (ATR) νmax/cm : 2120, 2100, 1611. Spectroscopic properties
2
1
matched those previously described.
ꢀAzidoꢀ4ꢀmethoxybenzene (3e). Similar to the procedure for 3a, pꢀanisidine (396 mg, 3.21
mmol, 1.0 equiv.), H O (2 mL), conc. HCl (1 mL), NaNO (289 mg, 4.19 mmol, 1.3 equiv.) in
1
2
2
H O (1 mL), NaN (251 mg, 3.87 mmol, 1.2 equiv.) in H O (1 mL) for 1 h extracted, dried over
2
3
2
1
Na SO and concentrated to yield 3e as a dark brown solid (319 mg, 2.15 mmol, 67%). H NMR
2
4
13
(
300 MHz, CDCl ): δ 3.82 (s, 3 H), 6.90 – 6.94 (m, 2 H), 6.96 – 7.00 (m, 2 H) C NMR (50
3
ꢀ1
MHz, CDCl ): δ 55.6, 115.2, 120.0, 132.4, 157.1. FTIR (ATR) ν /cm : 2108, 1602.
3
max
21
Spectroscopic properties matched those previously described.
2
4
Azidomethyl benzene (3f). Based on the literature procedure. To a solution of benzyl
bromide (1 mL, 8.4 mmol, 1 equiv.) in acetone (1 mL) and H O (1 mL) was added , NaN (1.9 g,
2
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0.2 mmol, 3.6 equiv.) the reaction mixture was stirred at rt for 2 h. The reaction was diluted
with H O (10 mL) and extracted with Et O (3 × 10 mL) and dried over Na SO and concentrated
2
2
2
4
1
to yield 3f as a colourless oil (770 mg, 5.8 mmol, 69%). H NMR (300 MHz, CDCl ): δ 4.37 (s,
3
13
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
H), 7.32 – 7.45 (m, 5 H) C NMR (50 MHz, CDCl ): δ 54.8, 128.3, 128.4, 128.9, 135.5.
3
ꢀ1
FTIR (ATR) νmax/cm : 2089, 1605. Spectroscopic properties matched those previously
2
5
described.
ꢀ(Azidomethyl)ꢀ4ꢀnitrobenzene (3g). Similar to the procedure for 3f, 4ꢀnitrobenzyl bromide
416 mg, 2 mmol, 1 equiv.), NaN (484 mg, 7.4 mmol, 3.7 equiv.), acetone:H O (2 mL, 3:1) for
1
(
3
2
2
h. The reaction was diluted with H O (10 mL) extracted with Et O (3 × 20 mL) and dried over
2 2
1
Na SO and concentrated to yield 3g as a colourless oil (332 mg, 1.86 mmol, 93%). H NMR
2
4
13
(300 MHz, CDCl ): δ 7.52 (d, J 9.0, 2 H), 8.21 (d, J 9.0, 2 H) C NMR (50 MHz, CDCl ): δ
3
3
ꢀ1
5
3.6, 123.8, 128.4, 142.6, 147.6. FTIR (ATR) νmax/cm : 2107, 1607. Spectroscopic properties
2
6
matched those previously described.
2
7
1
ꢀ(Azidomethyl)ꢀ4ꢀmethoxybenzene (3h). Following a literature procedure, to a vial
charged with 4ꢀmethoxybenzyl chloride (313 mg, 2.0 mmol, 1 equiv.) and NaN (484 mg, 7.4
3
mmol, 3.7 equiv.) was added THF (2 mL) and stirred at rt for 1 h. The reaction was diluted with
H O (10 mL) extracted with Et O (3 × 20 mL), dried over Na SO and concentrated to yield 3h
2
2
2
4
1
as a colourless oil (280 mg,1.88 mmol, 94%). H NMR (300 MHz, CDCl ): δ 3.78 (s, 3 H), 4.23
3
1
3
(s, 2 H), 6.89 (d, J 8.5, 2 H), 7.22 (d, J 8.5, 2 H) C NMR (50 MHz, CDCl ): δ 54.4, 55.3,
3
ꢀ1
114.2, 127.4, 129.6, 156.6. FTIR (ATR) νmax/cm : 2096. Spectroscopic properties matched
27
those previously described.
6
ꢀAzidoꢀ2ꢀ(3ꢀhydroxypropyl)ꢀ1Hꢀbenzo[de]isoquinolineꢀ1,3(2H)ꢀdione (3i). Following a
1
5a
literature 2ꢀstep procedure, to a suspension of 4ꢀbromoꢀ1,8ꢀnaphthalic anhydride (7.5 g, 27.1
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5
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6
mmol, 1 equiv.) in ethanol (0.54 M, 50 mL) was added 3ꢀaminopropanꢀ1ꢀol (2.1 mL, 27.1 mmol,
1
.0 equiv.) and heated to reflux for overnight. The reaction was cooled to rt and filtered,
collecting to yield the intermediate 6ꢀbromoꢀ2ꢀ(3ꢀhydroxypropyl)ꢀ1Hꢀbenzo[de]isoquinolineꢀ
1
0
1
2
3
4
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1
,3(2H)ꢀdione as a light brown powder (8.6 g, 25.7 mmol, 95%). H NMR (300 MHz, CDCl ):
3
δ
2.00 (app. quin, J 6.0, 2 H), 2.96 (t, J 6.0, 1 H), 3.61 (app. q, J 6.0, 2 H), 4.36 (t, J 6.0, 2 H),
7.88 (dd, J 8.5, 7.0, 1 H), 8.07 (d, J 8.0, 1 H), 8.45 (d, J 8.0, 1 H), 8.61 (dd, J 8.5, 1.0, 1 H), 8.69
13
(
dd, J 7.0, 1.0, 1 H). C NMR (75 MHz, CDCl ):
δ
31.0, 37.0, 58.9, 121.7, 122.6, 128.1, 128.8,
3
ꢀ1
1
30.5, 130.6, 131.1, 131.4, 132.2, 133.4, 164.1, 164.3. FTIR (ATR) νmax/cm : 1701, 1655.
1
5a
Spectroscopic properties matched those previously described. The intermediate was then
converted to the azide product using a reaction carried out at a maximum of 3.0g scale for safety
reasons. To 6ꢀbromoꢀ2ꢀ(3ꢀhydroxypropyl)ꢀ1Hꢀbenzo[de]isoquinolineꢀ1,3(2H)ꢀdione (3.00 g, 9.0
mmol, 1.0 equiv.) in NMP (10 mL, 0.9 M) was added NaN (3.27 g, 50.2 mmol, 5.6 equiv.) was
3
°
heated to 110 C for 45 mins and cooled to rt. The reaction mixture was diluted with water (60
mL) and filtered collecting the precipitate which was dried under high vacuum to yield 3i as a
1
brown solid (2.58 g, 0.87 mmol, 97%). H NMR (300 MHz, CDCl ):
δ 1.85 – 1.97 (m, 2 H),
3
3
.52 (t, J 5.5, 2 H), 4.26 (t, J 6.0, 2 H), 7.39 (d, J 8.0, 1 H), 7.67 (m, 1 H), 8.37 (d, J 8.5, 1 H),
1
3
8
.50 (d, J 8.0, 1 H), 8.56 (d, J 7.0, 1 H). C NMR (75 MHz, CDCl ):
δ 30.9, 36.8, 58.9, 114.7,
3
118.4, 122.2, 124.2, 124.3, 126.9, 129.0, 132.0, 132.5, 143.3, 162.3, 164.5. FTIR (ATR)
ꢀ1
νmax/cm : 2125, 1697, 1654, 1589. Spectroscopic properties matched those previously
1
5a
described.
Diꢀtertꢀbutyl
tetraazacyclotetradecaneꢀ1,8ꢀdicarboxylate (4a). To a flask charged with N was added 3a
4,11ꢀbis((1ꢀ(naphthalenꢀ1ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ1,4,8,11ꢀ
2
(511 mg, 3.02 mmol, 2.4 equiv.) and 2 (591 mg, 1.25 mmol, 1 equiv.), then THF (13 mL) and
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H O (13 mL) (0.16 M of cyclam in the 1:1 mixture of THF:H O) the reaction mixture was
2
2
backfilled with N (× 5, fast), CuSO ·5H O (63 mg, 20 mol%) and sodium ascorbate (110 mg,
2
4
2
50 mol%) were added and backfilled with N (× 3, fast) and the reaction mixture was stirred at rt
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
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3
3
3
4
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4
4
4
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5
5
5
5
5
6
0
1
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0
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8
9
0
for 20 h. The reaction mixture was concentrated to remove THF and diluted with aqueous
saturated NH Cl (20 mL) and extracted with DCM (3 × 50 mL). The combined organic layers
4
were dried over Na SO and concentrated by rotary evaporation to yield the crude product which
2
4
was purified using automated flash chromatography 0–100% EtOAcꢀPetroleumn benzene over
1
1
0 CV, to yield 4a as a brown foamy solid (584 mg, 58%). m.p. 85 – 96 °C. H NMR (200
MHz, CDCl ): δ 1.34 – 1.40 (br. s, 18 H), 1.70 – 2.00 (m, 4 H), 2.60 (t, J 6.0, 4 H), 2.72 (t, J 6.0,
3
1
3
4 H), 3.24 – 3.61 (m, 8 H), 3.91 (s, 4 H), 7.39 – 7.67 (m, 12 H), 7.85 – 8.05 (m, 4 H). C NMR
(
75 MHz, CDCl ): δ 26.5, 28.4, 46.9, 50.1, 51.9, 53.6, 61.9, 79.3, 122.3, 124.7, 124.9, 126.9,
3
1
27.8, 128.2, 128.4, 130.1, 133.7, 134.0, 140.4, 155.7 (1 × quaternary aromatic signal not seen or
+
overlapping) LRMS (ESI+): m/z 815.49 ([M+H] , 100%).
Diꢀtertꢀbutyl
4,11ꢀbis((1ꢀ(4ꢀnitrophenyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ1,4,8,11ꢀ
tetraazacyclotetradecaneꢀ1,8ꢀdicarboxylate (4d). Similar to the procedure for 4a, 3d (0.84
mmol, 137.2 mg, 2.09 equiv.) and 2 (238 mg, 0.4 mmol, 1 equiv.) in THF (1.7 mL) and H O (1.7
2
mL), CuSO ·5H O (20 mg, 20 mol%) and sodium ascorbate (35 mg, 50 mol%) and stirred at rt
4
2
for 1 h. The reaction mixture was concentrated and purified by flash chromatography (0 – 100%
DCMꢀMeOH, over 5 CV to remove azide) then 100% MeOH (5 CV)) to yield 4d as an orange
1
foamy solid (171 mg, 0.21 mmol, 53%). H NMR (500 MHz, CDCl ): δ 1.19 – 1.53 (m, 18 H),
3
1
3
.76 – 1.86 (br. m, 4 H), 2.53 – 2.59 (br. m, 4 H), 2.60 – 2.71 (br. m, 4 H), 3.26 – 3.60 (m, 8 H),
13
.86 (br. s., 4 H), 7.86 – 8.15 (m, 6 H), 8.26 – 8.47 (m, 4 H) C NMR (126 MHz, CDCl ): δ
3
27.0, 28.4, 46.9, 47.5, 50.2, 52.2, 54.5, 79.5, 120.3, 120.7, 125.4, 141.1, 147.0, 147.2, 155.9.
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4
4
4
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4
5
5
5
5
5
5
5
5
5
5
6
+
+
LRMS (ESI+): m/z 805.46 ([M+H] , 100%). HRMS (ESI+): m/z Calcd. for C H N O
38
53 12
8
8
05.4109, found 805.4109.
Diꢀtertꢀbutyl 4,11ꢀbis((1ꢀ(4ꢀmethoxyphenyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ1,4,8,11ꢀ
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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8
9
0
1
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5
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9
0
1
2
3
4
5
6
7
8
9
0
tetraazacyclotetradecaneꢀ1,8ꢀdicarboxylate (4e). Similar to the procedure to 4a, 3e (0.84
mmol, 125 mg, 2.09 equiv.) and 2 (238 mg, 0.4 mmol, 1 equiv.) in THF (1.7 mL) and H O (1.7
2
mL) CuSO ·5H O (20 mg, 20 mol%) and sodium ascorbate (35 mg, 50 mol%) and stirred at rt
4
2
for 1 h. The reaction mixture was concentrated and purified by flash chromatography (0 – 100%
DCMꢀMeOH, over 5 CV to remove excess 3e) and 100% MeOH (5 CV) to yield 4e as a yellowꢀ
1
green solid (140 mg, 0.18 mmol, 45%). H NMR (500 MHz, CDCl ): δ 1.22 – 1.50 (br. s, 18 H),
3
1.82 – 1.86 (br. m, 4 H), 2.51 – 2.65 (br. m, 4 H), 2.63 – 2.71 (br. m, 4 H), 3.26 – 3.58 (m, 8 H),
3
.81 – 3.86 (br. m, 10 H), 6.90 – 6.95 (br. m, 4 H), 7.56 – 7.62 (br. m, 4 H), 7.74 – 7.80 (br. s, 2
1
3
H) C NMR (126 MHz, CDCl ): δ 26.9, 28.4, 46.8, 47.4, 50.0, 52.0, 55.5, 79.6, 114.6, 115.1,
3
+
121.9, 122.1, 130.1, 155.9, 159.6. LRMS (ESI+): m/z 775.51 ([M+H] , 100%). HRMS (ESI+):
+
m/z Calcd. for C H N O 775.4619 found 775.4620.
4
0
59 10
6
Diꢀtertꢀbutyl
4,11ꢀbis((1ꢀbenzylꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ1,4,8,11ꢀ
tetraazacyclotetradecaneꢀ1,8ꢀdicarboxylate (4f). Similar to the procedure for 4a, 3f (0.84
mmol, 160 mg, 2.09 equiv.) and 2 (238 mg, 0.4 mmol, 1 equiv.) in THF (1.7 mL) and H O (1.7
2
mL) were added CuSO ·5H O (20 mg, 20 mol%) and sodium ascorbate (35 mg, 50 mol%) and
4
2
stirred at rt for 1 h. The reaction mixture was concentrated and purified by flash chromatography
(0 – 15% DCMꢀMeOH, over 10 CV) to yield 4f as a white foamy solid (95 mg, 0.13 mmol,
1
3
2%). H NMR (500 MHz, CDCl ): δ 1.39 (br. s., 18 H), 1.62 – 1.76 (m, 4 H), 2.35 – 2.48 (m, 4
3
H), 2.49 – 2.64 (m, 4 H), 3.14 – 3.41 (m, 8 H), 3.62 – 3.81 (m, 4 H), 5.49 (d, J 14.5, 4 H), 7.24
1
3
(br. s., 4 H), 7.31 – 7.38 (br. m, 8 H) C NMR (126 MHz, CDCl ): δ 26.5, 28.4, 46.4, 47.0,
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0.4, 52.0, 53.6, 54.0, 79.2, 122.3, 127.9, 128.6, 129.0, 134.9, 145.0, 155.7. LRMS (ESI+): m/z
+
43.20 ([M+H] , 100%).
Diꢀtertꢀbutyl
4,11ꢀbis((1ꢀ(4ꢀnitrobenzyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ1,4,8,11ꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
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7
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9
0
1
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4
5
6
7
8
9
0
1
2
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4
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8
9
0
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8
9
0
1
2
3
4
5
6
7
8
9
0
tetraazacyclotetradecaneꢀ1,8ꢀdicarboxylate (4g). Similar to the procedure for 4a, 3g (0.84
mmol, 150 mg, 2.09 equiv.) and 2 (238 mg, 0.4 mmol, 1 equiv.) in THF (1.7 mL) and H O (1.7
2
mL) and CuSO ·5H O (20 mg, 20 mol%) and sodium ascorbate (35 mg, 50 mol%) for 1 h at rt.
4
2
The reaction mixture was concentrated and purified by flash chromatography ((0 – 15% DCMꢀ
1
MeOH, over 10 CV)) to yield 4g as a yellow foamy solid (120 mg, 0.14 mmol, 36%). H NMR
(500 MHz, CDCl ): δ 1.40 (br. s., 18 H), 1.60 – 1.84 (br. m, 4 H), 2.36 – 2.50 (m, 4 H), 2.52 –
3
2.63 (m, 4 H), 3.18 – 3.48 (br. m, 8 H), 3.72 (br. s., 4 H), 5.64 (br. s., 4 H), 7.40 (d, J 8.5, 4 H),
13
7
.45 – 7.65 (br. s., 2 H), 8.22 (d, J 8.5, 4 H) C NMR (126 MHz, CDCl ): δ 26.7, 28.4, 46.6,
3
4
9.8, 50.5, 52.1, 53.0, 54.3, 79.4, 122.7, 124.3, 128.5, 142.0, 148.0, 155.7, 158.1. LRMS (ESI+):
+
+
m/z 833.44 ([M+H] , 100%). HRMS (ESI+): m/z Calcd. for C H N O 833.4422, found
40
57 12
8
8
33.4429.
Diꢀtertꢀbutyl
4,11ꢀbis((1ꢀ(4ꢀmethoxybenzyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ1,4,8,11ꢀ
tetraazacyclotetradecaneꢀ1,8ꢀdicarboxylate (4h). Similar to the procedure for 4a, 3h (0.84
mmol, 137 mg, 2.09 equiv.) and 2 (238 mg, 0.4 mmol, 1 equiv.) in THF (1.7 mL) and H O (1.7
2
mL) and CuSO ·5H O (20 mg, 20 mol%) and sodium ascorbate (35 mg, 50 mol%) for 1 h at rt.
4
2
The reaction mixture was concentrated and purified by flash chromatography (0 – 15% DCMꢀ
1
MeOH, over 10 CV) to yield 4h as a yellow foamy solid (155 mg, 0.19 mmol, 48%). H NMR
(500 MHz, CDCl ): δ 1.34 – 1.43 (br. m, 18 H), 1.63 – 1.74 (br. m, 4 H), 2.38 – 2.46 (br. m, 4
3
H), 2.51 – 2.60 (br. m, 8 H), 3.15 – 3.40 (m, 8 H), 3.64 – 3.74 (br. m, 4 H), 3.78 (s, 6 H), 6.87 (d,
1
3
J 8.5, 4 H), 7.14 – 7.25 (br. m, 4 H), 7.31 (br. s., 2 H) C NMR (126 MHz, CDCl ): δ 27.1,
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2
2
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2
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8.4, 46.5, 49.7, 50.4, 51.9, 53.6, 53.6 (HSQC), 55.3, 79.3, 114.4, 114.8, 122.1, 126.9, 129.6,
+
55.8, 159.8. HRMS (ESI+): m/z Calcd. for C H N O 803.4932, found 803.4934.
4
2
63 10
6
Diꢀtertꢀbutyl
4,11ꢀbis((1ꢀ(2ꢀ(3ꢀhydroxypropyl)ꢀ1,3ꢀdioxoꢀ2,3ꢀdihydroꢀ1Hꢀ
0
1
2
3
4
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0
1
2
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0
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9
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9
0
1
2
3
4
5
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7
8
9
0
benzo[de]isoquinolinꢀ6ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ1,4,8,11ꢀtetraazacyclotetradecaneꢀ
1
,8ꢀdicarboxylate (4i). To a flask charged with 3i (1.86 g, 6.29 mmol, 2.09 equiv.) and 2 (1.5 g,
.15 mmol, 1 equiv.) was added THF (14 mL) and H O (14 mL) (0.11 M of cyclam in the 1:1
3
2
mixture of THF:H O) backfilled with N at this point (× 3). CuSO ·5H O (157 mg, 20 mol%
2
2
4
2
0
.63 mmol) and sodium ascorbate (0.281 g, 50 mol%, 1.6 mmol) were added and the reaction
o
was backfilled with N (× 3). The reaction was stirred at rt (reached using a water bath at 25 C)
2
for 1 h. The reaction mixture was concentrated by rotary evaporation and purified by column
chromatography (5% MeOHꢀDCM, to remove the 3i increasing to 40% MeOHꢀDCM). Fractions
were combined which were >98% purity by HPLC (analytical run, 0 – 100% over 60 mins;
MeCNꢀH O (with 0.1% TFA) over 90 mins) to yield 4i as a yellowꢀbrown solid (1.80 g, 1.68
2
1
mmol, 52%). H NMR (300 MHz, CDCl ): δ 1.34 – 1.43 (br. m, 22 H), 1.64 – 2.03 (br. m, 4 H),
3
2
.55 – 2.59 (br. m, 4 H), 2.70 – 2.75 (br. m, 4 H), 3.20 – 3.42 (br. m, 8 H), 3.61 – 3.65 (br. m, 4
H), 3.91 – 4.02 (br. m, 4 H), 4.35 – 4.39 (br. m, 4 H), 7.85 – 7.92 (br. m, 4 H), 8.26 – 8.39 (br. m,
1
3
4
1
H), 8.72 – 8.70 (m, 4 H) C NMR (75 MHz, CDCl ): δ 28.4, 30.9, 37.2, 45.6, 47.5, 59.1, 79.7,
3
22.6, 123.4, 123.6, 125.1, 126.4, 128.7, 129.1, 130.0, 131.1, 132.4, 138.5, 155.7, 155.8, 163.2.
ꢀ1
FTIR (ATR) νmax/cm : 3055, 1662, 1647. Spectroscopic properties matched those previously
1
5a
described.
,8ꢀBis((1ꢀ(naphthalenꢀ1ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ1,4,8,11ꢀ
1
tetraazacyclotetradecane (5a). To a solution of 4a (489 mg, 0.60 mmol) in 1,4ꢀdioxane (3.0
mL, 0.2 M) was added HCl in dioxane (4.0 M, 4.8 mmol, 1.2 mL) and stirred at rt for 1 h. 1,4ꢀ
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dioxane was removed by rotary evaporation to yield the salt of 5a·0.8 C H O ·3.8 H O·3.4 HCl
4
8
2
2
as pale brown powder (447 mg, 98%). m.p. 250 – 262 °C Anal.: Calculated for C H N ·0.8
3
6
42 10
C H O ·3.8 H O·3.4 HCl: C 53.64 H 6.82 N 15.96; Found: C 53.64 H 6.77 N 15.96. Conversion
4
8
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of the salt (100 mg) to the amine was performed by reverse phase automated chromatography (0
100% MeCNꢀH O (with 0.1% TFA) over 10 CV) and concentrated and extracted from 2.5 M
–
2
aqueous NaOH (10 mL) with DCM (3 × 30 mL). The combined organic layers were dried over
Na SO and concentrated to yield 5a as a brown solid (35 mg, 0.057 mmol, 9.5% (43% based on
2
4
1
the % purified), >95% purity by analytical HPLC). H NMR (300 MHz, CDCl ): δ 1.81 – 1.92
3
(br. s., 4 H), 2.55 – 2.62 (m, 4 H), 2.64 – 2.70 (br. m, 4 H), 2.71 – 2.81 (m, 8 H), 3.86 (s, 4 H),
13
7.45 – 7.60 (m, 10 H), 7.71 (s, 2 H), 7.83 – 7.91 (m, 2 H), 7.91 – 7.99 (m, 2 H) C NMR (75
MHz, CDCl ): δ 25.9, 47.3, 47.4, 49.9, 51.4, 54.2, 122.3, 123.5, 124.9, 125.2, 126.9, 127.7,
3
+
1
28.1, 128.5, 130.1, 133.7, 134.0, 143.2. LRMS (ESI+): m/z 615.38 ([M+H] , 100%). HRMS
(ESI+): m/z Cald. for C H N 615.3672 found 615.3666. Anal.: Calculated for C H N ·0.8
36
43 10
36 42 10
C H O ·3.8 H O·3.4 HCl: C 53.64 H 6.82 N 15.96; Found: C 53.64 H 6.77 N 15.96.
4
8
2
2
1
,8ꢀBis((1ꢀ(naphthalenꢀ1ꢀylmethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ1,4,8,11ꢀ
tetraazacyclotetradecane (5b). Similar to the procedure for 4a, 3b (112 mg, 0.61 mmol, 2.4
equiv., contained 2ꢀ(azidomethyl)naphthalene as a minor impurity), 2 (123mg, 0.26 mmol, 1
equiv.), CuSO ·5H O (7 mg, 10 mol%) and sodium ascobate (9 mg, 20 mol%) were reacted for 6
4
2
h at 50 ºC and concentrated by rotary evaporation. Saturated NH Cl (50 mL) was added and
4
extracted with DCM (3 × 50 mL) and dried over MgSO and concentrated to yield 4b as a crude
4
1
mixture (crude yield 148 mg, 0.24 mmol, 92%). H NMR (300 MHz, CDCl ):
δ 1.32 – 1.43 (br.
3
s, 18 H), 1.46 – 1.60 (br. m, 4 H), 1.61 – 1.77 (br. m, 4 H), 2.24 – 2.38 (br. m, 4 H), 2.38 – 2.51
(br. m, 4 H), 2.95 – 3.27 (br. m, 8 H), 3.49 – 3.69 (br. s., 4 H), 5.75 – 6.03 (br. m, 2 H) 7.34 –
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5
5
5
5
5
6
+
7
8
.54 (br. m, 10 H), 7.84 (d, J 8.0, 4 H), 7.94 (br. s., 2 H) LRMS (ESI+): m/z 843.50 [M+H] , m/z
+
65.44 [M+Na] . The crude products of 4b (148 mg) were dissolved in 1,4ꢀdioxane (4 mL) and
HCl in dioxane (16 equiv., 4.0 M) was added and stirred at rt for 16 h. The reaction mixture was
concentrated and 16 mg of this mixture was purified by preparative RPꢀHPLC (gradient 20% to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
0% MeCN in H O (with 0.1% TFA), over 90 min) to yield 5b·3.6TFA·0.5H O as a white solid
2 2
1
(
4 mg, 0.004 mmol, 3%). H NMR (300 MHz, D O): δ 1.92 (br. m, 4 H), 2.66 (br. m, 4 H), 2.94
2
(
br. m, 8 H), 3.23 (br. m, 4 H), 3.52 – 5.64 (br. s, 4 H), 5.90 – 6.02 (br. s, 4 H), 7.35 – 7.52 (br. s,
2
H), 7.53 – 7.65 (br. m, 2 H), 7.65 – 7.81 (br. m, 6 H), 8.05 – 8.19 (br. m, 6 H).
1
3
5
b·3.6TFA·0.5H O (5 mg) was converted to the amine (5b) for C NMR analysis (by
2
1
extracting the compound with DCM (3 × 3 mL) from 2.5 M aqueous NaOH (5 mL). H NMR
(500 MHz, CDCl3):
δ 1.64 – 1.76 (m, 4 H), 2.38 (apparent t, J 5.5, 4 H), 2.48 – 2.53 (m, 4 H),
2
–
4
1
.53 – 2.59 (m, 8 H), 3.61 (s, 4 H), 5.61 (s, 4 H), 7.29 (dd, J 8.5, 2.0, 2 H), 7.36 (br. s., 2 H), 7.44
1
3
7.50 (m, 4 H), 7.69 (s, 2 H), 7.74 – 7.81 (m, 6 H). C NMR (126 MHz, CDCl ):
δ 25.5, 47.1,
3
8.1, 49.3, 51.7, 53.2, 54.1, 122.6, 125.2, 126.6, 126.7, 127.3, 127.7, 127.9, 129.1, 132.2, 133.1,
+
33.2, 144.4. LRMS (ESI+): m/z 643.37 ([M (free amine) +H] , 100%). HRMS (ESI+): m/z
+
calculated for C H N
47 10
643.3956, found 643.3985 [M+H] . Anal.: Calculated for
3
8
C H N ·3.6CF CO H·0.5H O: C 51.10 H 4.80 N 13.18; Found: C 51.29 H 4.88 N 13.10.
38
46 10
3
2
2
1
,8ꢀBis((1ꢀ(4ꢀnitrophenyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ1,4,8,11ꢀtetraazacyclotetradecane
(5d). To 4d (80 mg, 0.1 mmol, 1 equiv.) in 1,4ꢀdioxane (1 mL) was added HCl in dioxane (0.25
mL, 4.0 M, 8 equiv.) and the reaction mixture stirred at rt for 30 mins (typically precipitation
occurs after 5 mins, the reactions can be followed by TLC an aliquot containing solid was added
to a vial containing NaHCO (sat., 0.5 mL) and EtOAc (0.5 mL) the amine products have an R
3
F
of 0.00 in EtOAc, 10% MeOHꢀDCM and 1% NH OH (aq.) in 10% MeOHꢀDCM). The reaction
4
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mixture was concentrated by rotary evaporation and purified by reverseꢀphase automated
chromatography (0 – 100% MeCNꢀH O (with 0.1% TFA) over 10 CV) the fractions were
2
concentrated by rotary evaporation (to remove MeCN) and aqueous NaOH (5 mL, 2.5 M) was
added and extracted with DCM (1 × 5 mL). The extracts were dried over Na SO and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
concentrated to yield 5d as an orange solid (38 mg, 0.63 mmol, 63%, >98% purity by analytical
1
HPLC). H NMR (500 MHz, CDCl ): δ 1.87 – 1.95 (m, 4 H), 2.58 – 2.63 (m, 4 H), 2.78 – 2.84
3
(m, 4 H) 2.84 – 2.96 (m, 8 H), 3.80 (s, 4 H), 8.09 (d, J 9.0, 4 H), 8.36 (d, J 9.0, 4 H), 8.64 (br. s.,
13
2
1
H) C NMR (126 MHz, CDCl ): δ 24.8, 47.1, 48.5, 50.5, 51.8, 53.4, 120.3, 121.6, 125.4,
3
+
41.1, 145.8, 147.0. LRMS (ESI+): m/z 605.29 ([M+H] , 100%). HRMS (ESI+): m/z Calcd. for
+
4
ꢀ1
C H N O 605.3061, found 605.3060. FTIR (ATR) ν /cm : 2963, 2823, 1598, 1523.
28
37 12
max
1
,8ꢀBis((1ꢀ(4ꢀmethoxyphenyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ1,4,8,11ꢀ
tetraazacyclotetradecane (5e). Similar to the procedure for 5d, 4e (78 mg, 0.1 mmol, 1 equiv.)
in 1,4ꢀdioxane (1 mL) and HCl in dioxane (4.0 M, 10 equiv., 0.25 mL) for 30 mins at rt. The
reaction mixture was concentrated and purified by reverse phase by automated reverse phase
column chromatography ((0 – 100%) MeCNꢀH O (with 0.1% TFA) over 10 CV). After
2
concentration of the fractions and freezeꢀdrying the products were made into the freeꢀamine by
addition of 2.5 M NaOH (5 mL) and extracted with DCM (5 mL). The organic layer was dried
over Na SO and concentrated to yield 5e as a yellow solid (30 mg, 0.052 mmol, 52%, >98%
2
4
1
purity by analytical HPLC). H NMR (500 MHz, CDCl ): δ 1.81 – 1.92 (m, 4 H), 2.59 (t, J 5.5, 4
3
H), 2.75 – 2.84 (m, 8 H), 2.85 – 2.95 (m, 4 H), 3.74 (s, 4 H), 3.82 (s, 6 H), 6.91 – 7.04 (m, 4 H),
13
7
.59 – 7.69 (m, 4 H), 8.05 (s, 2 H) C NMR (126 MHz, CDCl ): δ 24.8, 47.0, 48.4, 48.5, 50.9,
3
+
5
1.7, 55.5, 114.7, 121.2, 121.8, 130.4, 144.6, 159.6. LRMS (ESI+): m/z 575.35 ([M+H] ,
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2
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4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
+
1
00%). HRMS (ESI+): m/z Calcd. for C H N O 575.3571, found 575.3568. FTIR (ATR)
3
0
43 10
2
ꢀ1
νmax/cm : 2948, 2823, 1685, 1513.
1,8ꢀBis((1ꢀbenzylꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ1,4,8,11ꢀtetraazacyclotetradecane
(5f).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Similar to the procedure for 5d, 4f (74 mg, 0.1 mmol, 1 equiv.) in 1,4ꢀdioxane (1 mL) was added
HCl in dioxane (4.0 M, 10 equiv., 0.25 mL) for 30 mins at rt. The reaction mixture was
concentrated and purified by automated reverse phase column chromatography ((0 – 100%)
MeCNꢀH O (with 0.1% TFA) over 10 CV). After concentration of the fractions and freezeꢀ
2
drying the products were made into the freeꢀamine by addition of 2.5 M NaOH (5 mL) and
extracted with DCM (5 mL). The organic layer was dried over Na SO and concentrated to yield
2
4
1
5f as a yellow solid (31 mg, 0.041 mmol, 41%, >98% purity by analytical HPLC). H NMR (500
MHz, CDCl ): δ 1.75 – 1.84 (br. m, 4 H), 2.44 – 2.54 (br. m, 4 H), 2.62 – 2.78 (br. m, 8 H), 2.75
3
–
2.84 (br. m, 4 H), 3.63 (s, 4 H), 5.45 (s, 4 H), 7.19 – 7.27 (m, 4 H), 7.27 – 7.34 (m, 6 H), 7.62
1
3
(s, 2 H) C NMR (126 MHz, CDCl ): δ 24.6, 29.7, 46.8, 48.7, 51.4, 51.8, 54.0, 123.0, 128.1,
3
+
1
28.7, 129.0, 134.9, 144.6 LRMS (ESI+): m/z 543.35 ([M+H] , 100%). HRMS (ESI+): m/z
+
ꢀ1
Calcd. for C H N 543.3672, found 543.3669. FTIR (ATR) νmax/cm : 2935, 2815, 1459.
3
0
43 10
1
,8ꢀBis((1ꢀ(4ꢀnitrobenzyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ1,4,8,11ꢀtetraazacyclotetradecane
(
5g). Similar to the procedure for 5d, 4g (83 mg, 0.1 mmol, 1 equiv.) in 1,4ꢀdioxane (1 mL) and
HCl in dioxane (4.0 M, 10 equiv., 0.25 mL) for 30 mins at rt. The reaction mixture was
concentrated and purified by reverse phase by automated reverse phase column chromatography
((0 – 100%) MeCNꢀH O (with 0.1% TFA) over 10 CV). After concentration of the fractions and
2
freezeꢀdrying the products were made into the freeꢀamine by addition of 2.5 M NaOH (5 mL)
and extracted with DCM (5 mL). The organic layer was dried over Na SO and concentrated to
2
4
1
yield 5g as a yellow solid (37 mg, 0.058 mmol, 58%, >98% purity by analytical HPLC). H
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NMR (500 MHz, CDCl ): δ 1.76 – 1.85 (m, 4 H), 2.46 – 2.55 (m, 4 H), 2.65 – 2.76 (m, 8 H),
3
2
.78 – 2.85 (m, 4 H), 3.68 (s, 4 H), 5.65 (s, 4 H), 7.46 (d, J 8.5, 4 H), 7.83 (s, 2 H), 8.16 (d, J 8.5,
1
3
4 H) C NMR (126 MHz, CDCl ): δ 24.7, 47.0, 48.4, 48.8, 51.2, 51.7, 52.9, 123.5, 124.1, 128.8,
3
+
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
41.9, 144.6, 147.9. LRMS (ESI+): m/z 633.31 ([M+H] , 100%). HRMS (ESI+): m/z Calcd. for
+
4
ꢀ1
C H N O 633.3374, found 633.3372. FTIR (ATR) ν /cm : 2959, 2829, 1678, 1519.
30
41 12
max
1
,8ꢀBis((1ꢀ(4ꢀmethoxybenzyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ1,4,8,11ꢀ
tetraazacyclotetradecane (5h). Similar to the procedure for 5d, 4h (80 mg, 0.1 mmol, 1 equiv.)
in 1,4ꢀdioxane (1 mL) and HCl in dioxane (4.0 M, 10 equiv., 0.25 mL) for 30 mins at rt. The
reaction mixture was concentrated and purified by reverse phase by automated reverse phase
column chromatography ((0 – 100%) MeCNꢀH O (with 0.1% TFA) over 10 CV). After
2
concentration of the fractions and freezeꢀdrying the products were made into the freeꢀamine by
addition of 2.5 M NaOH (5 mL) and extracted with DCM (5 mL). The organic layer was dried
over Na SO and concentrated to yield 5h as a yellow solid (37 mg, 0.061 mmol, 61%, >98%
2
4
1
purity by analytical HPLC). H NMR (500 MHz, CDCl ): δ 1.70 – 1.79 (br. m, 4 H), 2.41 – 2.50
3
(m, 4 H), 2.56 – 2.69 (m, 8 H), 2.71 – 2.78 (m, 4 H), 3.62 (s, 4 H), 3.76 (s, 6 H), 5.38 (s, 4 H),
1
3
6
4
6
6
.83 (d, J 8.5, 4 H), 7.18 (d, J 8.5, 4 H), 7.47 (s, 2 H) C NMR (126 MHz, CDCl ): δ 24.8, 46.8,
3
8.4, 48.7, 51.5, 51.9, 53.5, 55.2, 114.3, 122.5, 126.7, 129.5, 144.3, 159.8. LRMS (ESI+): m/z
+
+
03.36 ([M+H] , 100%). HRMS (ESI+): m/z Calcd. for C H N O 603.3884, found
3
2
47 10
2
ꢀ1
03.3881. FTIR (ATR) νmax/cm : 2948, 2839, 1688, 1610.
6,6'ꢀ(((1,4,8,11ꢀTetraazacyclotetradecaneꢀ1,8ꢀdiyl)bis(methylene))bis(1Hꢀ1,2,3ꢀtriazoleꢀ
,1ꢀdiyl))bis(2ꢀ(3ꢀhydroxypropyl)ꢀ1Hꢀbenzo[de]isoquinolineꢀ1,3(2H)ꢀdione) (5i). 4i (500
4
mg, 0.47 mmol, 1 equiv.) was dissolved in 1,4ꢀdioxane (6 mL, 0.08M) and HCl in dioxane (4.0
M, 0.94 mL, 3.74 mmol, 8 equiv.) was added (instantaneous change giving a yellowꢀwhite
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precipitate upon addition of HCl). The reaction mixture was left to stir at rt for 2 h and
concentrated by rotary evaporation. The residue was dissolved in a mixture of EtOH (200 mL)
®
and CHCl (200 mL) and Ambersep 900 resin (hydroxide form, 200 mg, washed with MeOH (3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
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1
2
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4
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8
9
0
×
10 mL)) was added and the reaction stirred for 1 h at rt. The resin was removed by filtration
(without washing the resin) and the filtrate concentrated by rotary evaporation to yield 5i as a
1
light yellow powder (348 mg, 0.40 mmol, 85%). H NMR (300 MHz, D O):
δ 1.78 – 1.97 (m, 4
2
H), 2.08 – 2.22 (m, 4 H), 2.82 – 3.00 (m, 4 H), 3.03 – 3.17 (m, 4 H), 3.40 – 3.51 (m, 4 H), 3.53 –
.63 (very br. m, 4 H), 3.64 – 3.72 (m, 4 H), 3.94 – 4.03 (m, 4 H), 4.06 – 4.15 (m, 4 H), 7.20 –
3
7.33 (m, 2 H), 7.56 – 7.71 (m, 4 H), 8.06 – 8.13 (m, 2 H), 8.21 – 8.27 (m, 2 H), 8.29 – 8.33 (m, 2
1
H) H NMR (300 MHz, CDCl (10% MeODꢀd4)):
δ
1.54 – 1.77 (m, 8 H), 2.31 – 2.63 (m, 16 H),
3
3
.05 (br. s., 2 H), 3.39 (t, J 6.0, 4 H), 3.61 – 3.78 (br. m, 4 H), 4.00 (t, J 7.0, 4 H), 7.49 – 7.65 (m,
13
4 H), 7.93 (d, J 8.5, 2 H), 7.99 (s, 2 H), 8.35 (d, J 7.5, 4 H) C NMR (75 MHz, CDCl (10%
3
MeODꢀd4)):
δ 24.7, 30.3, 37.3, 46.5, 49.8, 52.3, 53.3, 54.6, 59.1, 122.1, 123.1, 123.3, 125.6,
1
25.9, 128.1, 128.5, 129.2, 130.3, 131.7, 137.8, 143.2, 163.0, 163.6. HRMS (ESI+): m/z Calcd.
+
+
for C H N O [M+H] 869.4211, found 869.4188.
4
6
53 12
6
1
ꢀEthynylnaphthalene (7a). To a solution of 1ꢀbromonaphthalene (285 mg, 1.38 mmol) in
diisopropylamine (10 mL) was added Pd(PPh ) Cl (48 mg, 68 µmol, 5mol%) and CuI (13 mg,
3
2
2
68 µmol, 5 mol%) under N . Trimethylsilylacetylene (292 µL, 2.07 mmol) was added dropwise
2
and the reaction mixture was stirred for 16 h at 80 ºC. The reaction mixture was filtered and
concentrated by rotary evaporation. The residue was dissolved in CHCl (25 mL), washed with
3
H O (2 × 25 mL), dried over Na SO and concentrated by rotary evaporation. The residue was
2
2
4
purified by flash column chromatography (petroleum benzine) to yield the TMSꢀprotected
1
intermediate as a yellow oil (259 mg, 84%). H NMR (400 MHz, CDCl ): δ 0.33 (s, 9 H), 7.40
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(
dd, J 8.0, 7.0, 1 H), 7.51 (ddd, J 8.0, 7.0, 1.5, 1 H), 7.57 (ddd, J 8.5, 7.0, 1.5, 1 H), 7.70 (dd, J
+
7
.0, 1.0, 1 H), 7.79 – 7.86 (m, 2 H), 8.31 – 8.36 (m, 1 H). LRMS (EI): m/z 209.2 ([MꢀCH ] ,
3
+
28
100%), 224.1 (M , 58%). Spectroscopic properties matched those previously described. To a
solution of the TMSꢀprotected intermediate (255 mg, 1.14 mmol) in MeOH (10 mL) was added a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
solution of KOH (77 mg, 1.4 mmol) in H O (2 mL). The reaction mixture was stirred for 16 h at
2
rt. The volatiles were removed and H O (25 mL) was added. The product was extracted with
2
Et O (2 × 25 mL), and the extracts were combined, washed with H O (25 mL), dried over
2
2
Na SO and the solvent was removed by rotary evaporation. The residue was purified by flash
2
4
1
column chromatography (petroleum benzine) to yield 7a as an orange oil (143 mg, 83%). H
NMR (400 MHz, CDCl ): δ 3.47 (s, 1 H), 7.42 (dd, J 8.0, 7.0, 1 H), 7.49 – 7.55 (m, 1 H), 7.55 –
3
7
1
.61 (m, 1 H), 7.74 (d, J 7.0, 1 H), 7.85 (d, J 8.0, 2 H), 7.36 (d, J 8.5, 1 H). LRMS (EI): m/z
+
29
52.2 (M , 100%). Spectroscopic properties matched those previously described.
2ꢀEthynylnaphthalene (7c). To a solution of 2ꢀbromonaphthalene (500 mg, 2.42 mmol) in
diisopropylamine (10 mL) was added Pd(PPh ) Cl (102 mg, 6 mol%) and CuI (28 mg, 6 mol%)
3
2
2
under N . Trimethylsilylacetylene (512 µL, 3.62 mmol) was added dropwise and the reaction
2
mixture was stirred for 16 h at 80 ºC. The reaction mixture was filtered and the solvent was
removed by rotary evaporation. The residue was dissolved in DCM (50 mL), washed with H O
2
(2 × 25 mL), dried over Na SO and concentrated by rotary evaporation. The residue was
2 4
purified by automated flash column chromatography (100% petroleum benzine for 12 CV) to
1
yield the TMSꢀprotected intermediate as a brown oil (460 mg, 85%). H NMR (400 MHz,
CDCl ): δ 0.28 (s, 9 H), 7.43 – 7.53 (m, 3 H), 7.72 – 7.83 (m, 3 H), 8.00 (s, 1 H). LRMS (EI):
3
+
m/z 209.1 ([MꢀCH ] , 100%), 224.0 (M+, 38%). Spectroscopic properties matched those
3
3
0
previously described. To a solution of the TMSꢀprotected intermediate (443 mg, 1.97 mmol) in
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