Synthesis of 1,2,3-triazole ‘click’ analogues of thalidomide

Article abstract of DOI:10.1016/j.tet.2016.07.019

Click analogues of thalidomide were prepared from 3-azidoglutarimide and a diverse array of arylacetylenes and N-ethynyl/N-propargyl phthalimide derivatives. The sequence necessitated a new and scalable synthesis of the key click intermediate 3-azidoglutarimide. The dipolar cycloaddition reactions between the azidoglutarimide and the alkynyl coupling partners utilized a copper sulfate/sodium ascorbate reagent system in aqueous tetrahydrofuran and were first explored using substituted arylalkynes. Along with the click analogues of thalidomide, the click counterparts of the teratogenic and antiangiogenic thalidomide analogue EM-12 were prepared.

Full text of DOI:10.1016/j.tet.2016.07.019

Accepted Manuscript
Synthesis of 1,2,3-triazole ‘click’ analogues of thalidomide
Jarrid M. Ronnebaum, Frederick A. Luzzio
PII:
S0040-4020(16)30642-1
10.1016/j.tet.2016.07.019
TET 27915
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 25 May 2016
Revised Date: 23 June 2016
Accepted Date: 5 July 2016
Please cite this article as: Ronnebaum JM, Luzzio FA, Synthesis of 1,2,3-triazole ‘click’ analogues of
thalidomide, Tetrahedron (2016), doi: 10.1016/j.tet.2016.07.019.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis of 1,2,3-triazole ‘click’ analogues of thalidomide†
Jarrid M. Ronnebaum and Frederick A. Luzzio*
Department of Chemistry, University of Louisville, 2320 South Brook Street, Louisville, KY
40292 USA
Keywords: angiogenesis, cancer, EM-12, multiple myeloma, teratogenesis, thalidomide, TNF-α
Abstract
Click analogues of thalidomide were prepared from 3-azidoglutarimide and a diverse array of
arylacetylenes and N-ethynyl/N-propargyl phthalimide derivatives. The sequence necessitated a
new and scalable synthesis of the key click intermediate 3-azidoglutarimide. The dipolar
cycloaddition reactions between the azidoglutarimide and the alkynyl coupling partners utilized a
copper sulfate/sodium ascorbate reagent system in aqueous tetrahydrofuran and were first
explored using substituted arylalkynes. Along with the click analogues of thalidomide, the click
counterparts of the teratogenic and antiangiogenic thalidomide analogue EM-12 were prepared.
1. Introduction
In contrast to its notoriety as a potent embryotoxin, the diverse biological effects of thalidomide
1 (Figure 1) have proven beneficial.1-2 The compound is an approved antilepromatic, and
together with some of its analogues are active as immunosuppresants, immunomodulators, anti-
inflammatories and anticancer agents.3 As an experimental therapeutic, the antiangiogenic
properties of thalidomide and closely-related analogues have gained much attention in the
anticancer community.4-8 The implication that 1 undergoes metabolic activation as an
antiangiogenic agent was a consequence of in vivo metabolic studies in the rabbit and ex vivo
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studies with human microsomal preparations.9-13 At present, the structural elucidation and
identity of the active antiangiogenic (or even teratogenic) species, if those indeed exist, has not
been ascertained as well as its interaction with any purported molecular target.14-15 Even though
the suspected metabolites of thalidomide have been bioassay targets, most analogues have been
prepared and evaluated for diverse activities without any considerations of metabolism in
mind.16-17 From the outset, however, a number of basic structural requirements became evident
and were considered during the design of the most potent thalidomide analogues18: (1) the
phthalimide portion of the molecule had to possess an intact five-member phthalimide or
phthalimidine (lactam) ring; (2) the six-member glutarimide portion of the molecule had to
possess an intact glutarimide ring with both carbonyls; (3) the imide (N-H) nitrogen had to
possess the free hydrogen-not replaced with ‘R’ groups; (4) single or multiple substitutions on
the aryl ring of the phthalimide while observing requirements 1-3 (above). While item 4 (above)
has led to analogues of greater activity, this was specifically the case with the therapeutically-
approved thalidomide analogues pomalidomide 2 and lenalidomide 3 (Figure 1), compounds
which are used to treat multiple myeloma.19-21 The structural basis for lenalidomide was derived
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from the well-known thalidomide analogue EM-12 4 (Figure 1), one of the early analogues
investigated as a teratogen22-23 and later found to have both anti-TNF-α and antiangiogenic
activity.24-25 Remarkably, very little change in the structure of 1 was responsible for delivering
analogues of high therapeutic potency in 2 and 3. While analogues involving substitution at just
about every atom of the thalidomide core have been prepared, no thalidomide analogues
designed with a “spacer” in between the phthalimide and glutarimide sections of the molecule
have been examined. The concept is reminiscent of the ‘fleximer’ analogues in nucleoside
chemistry whereby there is a heteroatom or carbon linker between portions of a nucleobase or a
nucleobase and a sugar.26 In the thalidomide series, such spacers could be in the form of an alkyl
group, multivalent heteroatom or a carbocyclic or heterocyclic moiety (Figure 2, 5a-c). In terms
of simplicity in synthesis, a carbon-based ‘homoalkyl’ analogue of thalidomide (5a) would be
the most difficult to synthesize and would require carbon-carbon bond construction within the
glutarimide framework. Heteroatoms such as nitrogen, oxygen, sulfur or phosphorus placed
between the glutarimide and phthalimide rings (5b) may compromise stability while carbocycles
between both rings (5c) will also require a significant synthetic commitment. The 1,2,3-triazole
linker in 6, formed by the click reaction of an N-substituted phthalimido azide, acetylene or
propargyl group with a glutarimide-substituted acetylene or azide would be the most viable
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route.27-29 Moreover, the triazole-linked compounds may yield interesting bioassay results since
these analogues exhibit both intact phthalimide and glutarimide rings and one or both moieties
may associate with the molecular target.30 Due to the ease of preparing many azides and
acetylenes with simple substitution, the click route could be put to practice with relative ease and
would only necessitate the exploration and adjustment of the conditions for the cycloaddition
reaction. We describe herein the preparation of the first triazole spacer click analogues of
thalidomide and its closely-related bioactive analogue EM-12 as well as their click homologues
having a triazole-methylene spacer unit.
2. Results and Discussion
Our route to the 1,2,3-triazole series of thalidomide analogues 6 utilizes the 3-azido-substituted
glutarimide 9 (Scheme 1). Commercially-available glutarimide 7 is reacted with bromine in
chloroform at 100 °C which afforded the sensitive 3-bromoglutarimide 8 as a low-melting
solid.31 The α-bromolactam 8 was directly treated with excess sodium azide in acetone to provide
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the 3-azidoglutarimide 9 as an off-white solid in 53% yield (overall from glutarimide). The
azidoglutarimide 9 was first evaluated for its reactivity with phenylacetylene 10 in a click
reaction mediated by cupric sulfate/sodium ascorbate (Table 1). Using the reaction conditions
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established with 9 and taking into consideration the success realized with earlier-investigated
fluorinated analogues,17 several commercially-available fluoro- and trifluoromethyl-substituted
arylalkynes 11-14 were reacted with the glutarimide azide 9 and afforded the corresponding click
products 15-19 (Table 1). The yield of the click products ranged from 76-98% and all the
compounds were tractable solids and easily purified by column chromatography on silica gel.
The synthesis of the phthalimide/isoindolinone and N-ethynyl/N-propargyl thalidomide reacting
partners 24-27 are detailed in Scheme 2. The N-ethynylphthalimide click partner 24 was
prepared from phthalimide 20 and TMS-acetylene by the method of Davies (Scheme 2).32
Therefore, treatment of 20 with copper acetate, pyridine and ethynyltrimethylsilane in an
atmosphere of oxygen provided the intermediate N-(trimethylsilylethynyl) phthalimide 22 as a
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crystalline solid. Desilylation of the intermediate TMS-ethynylphthalimide 22 was accomplished
with TBAF/acetic acid and provided the unstable ethynyl phthalimide 24 (Scheme 2). Not
surprisingly, isoindolinone 21, with its less-acidic or otherwise less-reactive proton, acted less
favorably in the Davies ethynylation reaction and gave the trimethylsilyl isoindolinone 23 (22%)
under similar conditions as 22. In contrast to 22, desilylation of 23 with TBAF only (THF/rt)
gave the N-ethynyl isoindolinone 25 (92%) as a crystalline solid. The N-propargylation of
phthalimide 20 and isoindolinone 21, which gave the corresponding click partners 26 and 27,
was more straightforward than the N-ethynylation of 20 or 21. Phthalimide and isoindolinone
intermediates 26 and 27 were both prepared by the reaction of 20 or 21 with propargyl bromide
in the presence of cesium carbonate in acetonitrile (Scheme 2).15 Both propargylated
intermediates (26, 69%, 27, 65%) were obtained as crystalline compounds after column
chromatography. The reaction of ethynyl phthalimide 24 and azidoglutarimide 9 gave click
thalidomide derivative 6a as a crystalline solid (83%) after purification by column
chromatography (Table 2). Using the same reaction conditions as 6a, the ethynyl isoindolinone
25 and azidoglutarimide 9 afforded the click EM-12 analogue 6b (78%) as a crystalline solid
after column chromatography. Similarly, the N-propargyl phthalimide 26 and the N-
propargylisoindolinone 26 were clicked with azidoglutarimide 9 giving the homomethylene
thalidomide analogue 6c and the homomethylene EM-12 analogue 6d in 67% and 61% yield
respectively. The regiochemistry of the dipolar cycloaddition click reaction involves products in
which the substitution could be 1,4- or 1,5- on the triazole ring depending on the reaction
conditions or reagents. All of the click products described herein (Tables 1 and 2) were formed
as a result of copper (I) catalysis which gives exclusively the 1,4- or “anti”-substituted 1,2,3-
triazole.28
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3. Conclusions
Synthetic routes to click analogues of thalidomide and its descarbonyl analogue EM-12 are
detailed which utilize the key intermediate 3-azidoglutarimide along with N-ethynyl and N-
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propargyl analogues of phthalimide as reacting components. The dipolar cycloaddition chemistry
of the azidoglutarimide was explored using arylalkynes containing electron-withdrawing groups
in place of the phthalimide group and yielded cycloaddition products in high yield using the
CuSO4/sodium ascorbate reaction conditions. The click thalidomide analogues will be evaluated
in a full range of bioassay types and these results will be reported in due course.
4. Experimental
Solvents and reagents are ACS grade and were used as commercially supplied. Analytical thin-
layer chromatography (TLC) utilized 0.25 mm pre-cut glass-backed plates (Merck, Silica Gel 60
F254). Thin-layer chromatograms were visualized during chromatographic and extraction runs
by rapidly dipping the plates in anisaldehyde/ethanol/sulfuric acid stain or phosphomolybdic
acid/ethanol stain and heating (hot plate). Gravity-column chromatography was carried out using
silica gel 60 (E. Merck 7734, 70-230 mesh). Flash-column chromatography utilized silica gel 60
(E. Merck 9385, 230-400 mesh) with nitrogen gas pressurization. Melting points were taken on a
Thomas Hoover apparatus. Extracts and chromatographic fractions were concentrated with a
l3
Büchi rotavapor under water aspirator vacuum. Nuclear magnetic resonance (1H and C NMR)
spectra were recorded with Varian VNMRS 400 or 700 MHz instruments using CDCl3 as a
solvent and TMS as internal standard. Infrared spectra (FTIR) were recorded with a Perkin-
Elmer Spectrum 100 instrument and spectral values are reported as cm-1. The measurement of
high-resolution mass spectra (HRMS) were performed at the Texas A&M University Laboratory
for Biological Mass Spectrometry. Elemental analysis was performed by Galbraith Laboratories,
Knoxville, TN.
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3-Azidopiperidine-2,6-dione 9: To a 48 mL glass pressure reaction vessel fitted with a Teflon
screw cap was added piperidine-2,6-dione (2.00 g, 17.7 mmol) and Br2 (2.82 g, 0.90 mL, 17.7
mmol) in chloroform (15 mL). The reaction mixture was then heated in a Kugelröhr oven at 110
°C (45 min). The solvent was removed and the crude bromoglutarimide 7 was then dissolved in
acetone (5 mL) followed by the addition of sodium azide (3.44 g, 53.1 mmol) whereupon the
reaction mixture turned blue-purple. The reaction mixture was stirred at room temperature (24 h)
and then directly applied to a silica gel column. Elution with hexane/ethyl acetate (1:1) gave a
mixture of unreacted 3-bromopiperidine-2,6-dione 7 and 3-azidopiperidine-2,6-dione 8 (1:1).
The mixture of azide 8 and unreacted bromide 7 was again dissolved in acetone (5 mL) and
sodium azide (1.15 g, 17.7 mmol) was added and stirring of the blue-purple reaction mixture was
continued at room temperature (24 h). Column chromatography (hexane/ethyl acetate, 1:1) of the
reaction mixture gave pure 3-azidopiperidine-2,6-dione 9 as an off-white amorphous solid (1.44
1
g, 53% from glutarimide): mp 144-145 °C; Rf 0.24 (TLC stains blue with heat); H NMR (400
MHz, CDCl3) δ 4.21 (dd, J=9.6 Hz, 8.4 Hz, 1H), 2.78 (dt, J=18.4 Hz, 5.6 Hz, 1H) 2.63-2.54 (m,
1H), 2.23-2.16 (m, 1H), 2.04-1.95 (m, 1H). 13C NMR (400 MHz, CDCl3) δ 170.7, 169.2, 58.2,
29.1, 24.0. FT-IR (neat) 3090, 2112, 1710, 1676 cm-1; HRMS (ESI-TOF) m/z [M+H]+ calcd for
C5H6N4O2: 155.0491, Found: 155.0569.
General Procedure for the preparation of click compounds 15-19: To a stirred solution of 3-
azidopiperidine-2,6-dione 9 (20.0 mg, 0.13 mmol) and arylalkyne 10-14 (0.143 mmol) in
tetrahydrofuran (750 ꢀL) was added aqueous copper sulfate (52mM, 0.013 mmol, 250 µL).
Sodium ascorbate (12.8 mg, 0.065 mmol) was then added ten minutes after the addition of the
CuSO4 solution. The reaction mixture was stirred (16 h) at room temperature followed by direct
application to a gravity silica gel column and elution with chloroform/methanol (95:5).
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Combination and concentration of the chromatographic fractions gave the pure triazoles 15-19 as
white or off-white amorphous solids:
3-(4-Phenyl-1H-1,2,3-triazol-1-yl)piperidine-2,6-dione 15: 3-(4-Phenyl-1H-1,2,3-triazol-1-
yl)piperidine-2,6-dione 15 was obtained as a white amorphous solid (26 mg, 78%): mp 208-210
°C; Rf 0.28 (chloroform/methanol, 9:1); 1H NMR (400 MHz, DMSO-d6) δ 11.25 (s, 1H), 8.66 (s,
1H), 7.82-7.84 (d, J=7.2 Hz, 2H), 7.42-7.46 (m, 2H), 7.30-7.34 (m, 1H), 5.84 (dd, J=13.2, 5.2
Hz, 1H), 2.83-2.91 (m, 1H) 2.65-2.76 (m, 2H) 2.34-2.36 (m, 1H); 13C NMR (100 MHz, DMSO-
d6 ): δ 172.8, 169.9, 146.7, 131.0, 129.4, 128.4, 125.5, 122.0, 59.6, 31.1, 24.8: IR (neat) 3090,
2930, 1732, 1699, 1676 cm-1; HRMS (ESI-TOF) m/z [M+H]+; calcd for C13H13N4O2: 257.1039,
Found: 257.1140.
3-(4-(4-Fluorophenyl)-1H-1,2,3-triazol-1-yl)piperidine-2,6-dione 16: 3-(4-(4-Fluorophenyl)-
1H-1,2,3-triazol-1-yl)piperidine-2,6-dione 16 was obtained as a white amorphous solid (64 mg,
1
94%): mp 221-222 °C; Rf 0.32 (chloroform/methanol, 9:1); H NMR (400 MHz, DMSO-d6) δ
11.24 (s, 1H), 8.64 (s, 1H) 7.84-7.88 (m, 2H) 7.25-7.30 (m, 2H), 5.83 (dd, J=12.4, 4.8 Hz, 1H),
2.82-2.90 (m, 1H), 2.62-2.70 (m, 2H), 2.32-2.35 (m, 1H) 13C NMR (100 MHz, DMSO-d6): δ
172.8, 169.8, 163.5, 161.0, 145.8, 127.6 (d, J=7.6 Hz), 122.0, 116.4 (d, J=22.0 Hz), 59.6, 31.1,
24.8. IR (neat) 3194, 3105, 2916, 1731, 1708 cm-1. HRMS (ESI-TOF) m/z [M+H]+; calcd for
C13H12FN4O2: 275.0944, Found: 275.1014.
3-(4-(3-Fluorophenyl)-1H-1,2,3-triazol-1-yl)piperidine-2,6-dione 17: 3-(4-(3-Fluorophenyl)-
1H-1,2,3-triazol-1-yl)piperidine-2,6-dione 17 was obtained as a white solid (35 mg, 98%): mp
1
199-201 °C; Rf 0.375 (chloroform/methanol, 9:1); H NMR (400 MHz, DMSO-d6) δ 11.25 (s,
1H), 8.74 (s, 1H), 7.65-7.69 (m, 1H), 7.46-7.51 (m, 2H), 7.13-7.18 (m, 1H), 5.85 (dd, J=12.4, 4.4
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Hz, 1H), 2.83-2.89 (m, 1H), 2.62-2.71 (m, 2H), 2.34-2.37 (m, 1H) 13C NMR (100 MHz, DMSO-
d6): δ 172.9, 169.8, 164.2, 161.8, 145.7, 132.4 (d, J=154.9 Hz), 122.9, 121.6, 115.2 (d, J=21.3
Hz), 112.1 (d, J=15.9 Hz), 59.7, 31.1, 24.8. IR (neat) 3098, 2917, 2853, 1732, 1705 cm-1.
HRMS (ESI-TOF) m/z [M+H]+; calcd for C13H12FN4O2: 275.0944, Found: 275.0837.
3-(4-(2-(Trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)piperidine-2,6-dione 18: 3-(4-(2-
(Trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)piperidine-2,6-dione was obtained as a white
1
solid (36 mg, 84%): mp 155-157 °C; Rf 0.37 (chloroform/methanol, 9:1); H NMR (400 MHz,
DMSO-d6) δ 11.23 (s, 1H), 8.41 (s, 1H), 7.74-7.85 (m, 3H), 7.60-7.63 (m, 1H), 5.89 (dd, J=12.8,
5.2 Hz, 1H), 2.83-2.90 (m, 1H), 2.65-2.78 (m, 2H), 2.31-2.36 (m, 1H) 13C NMR (100 MHz,
DMSO-d6): δ 172.9, 169.8, 143.7, 133.1, 132.2, 129.8 (q, J=2.2 Hz), 129.3, 126.8 (q, J=30.4
Hz), 126.7 (q, J=6.1 Hz), 124.8 (q, J=3.1 Hz), 124.4 (q, J=271.8 Hz), 59.7, 31.2, 24.8. IR (neat)
3220, 3094, 2957, 1732, 1701 cm-1. HRMS (ESI-TOF) m/z [M-H]-; calcd for C14H10F3N4O2
323.0756, Found: 323.0714.
3-(4-(3,5-Bis(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)piperidine-2,6-dione 19: 3-(4-
(3,5-bis(Trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)piperidine-2,6-dione 19 was obtained as
a white solid (38 mg, 76%): mp 115-117 °C; Rf 0.29 (chloroform/methanol, 9:1); 1H NMR (400
MHz, DMSO-d6) δ 11.29 (s, 1H), 9.07 (s, 1H), 8.50 (s, 2H), 8.07 (s, 1H), 5.91 (dd, J=12.8, 5.2
Hz, 1H), 2.84-2.89 (m, 1H), 2.66-2.73 (m, 2H), 2.38-2.41 (m, 1H) 13C NMR (100 MHz, DMSO-
d6): δ 172.8, 169.7, 144.1, 133.5, 131.5 (q, J=32.6 Hz), 125.7, 124.2, 123.2 (q, J=271.9 Hz),
121.7, 59.8, 31.1, 24.7. IR (neat) 3219, 3094, 2872, 1733, 1704 cm-1. HRMS (ESI-TOF) m/z
[M-H]-; calcd for C15H9F6N4O2 391.0630, Found: 391.0707
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Isoindolin-1-one 21: In a 100 mL round bottom flask phthalimide 20 (5.00 g, 34.0 mmol) was
dissolved in acetic acid (25 mL). A slurry of tin metal (9.68 g, 81.6 mmol) and concentrated
hydrochloric acid (12.5 mL) was added to the phthalimide solution. The reaction mixture was
heated under reflux at 120 °C (6 h) then diluted with dichloromethane (75 mL) and washed with
brine (3 x 25 mL). Concentration of the extract followed by gravity- column chromatography
(chloroform/methanol, 95:5) gave pure isoindolin-1-one as an off-white solid (1.75 g, 39%
yield): mp 154-156 ºC (Lit.35 150-152 ºC). The spectroscopic data was consistent with the
1
literature34: H NMR (400 MHz, CDCl3) δ 7.87 (d, J=8Hz, 1H), 7.75-7.59 (m, 1H), 7.47-7.49
(m, 2H), 4.47 (s, 2H).
2-(2-(Trimethylsilyl)ethynyl)isoindoline-1,3-dione 22: Prepared by the method of Davies
(59%):32 mp 92-94 ºC (Lit.33 94-96 ºC). The spectroscopic data was consistent with the
literature.32: 1H NMR (400 MHz, CDCl3) δ 7.90-7.93 (m, 2H), 7.78-7.81 (m, 2H), 0.273 (s, 9H).
2-(2-(Trimethylsilyl)ethynyl)isoindolin-1-one 23: To a 250 mL round bottom flask Cu(OAc)2
(76.4 mg, 0.42 mmol), isoindolin-1-one (1.00 g, 7.51 mmol), Na2CO3 (216 mg, 4.2 mmol), and 4
Å molecular sieves (2.0 g) were combined. A solution of pyridine (324 mL, 4.2 mmol) in toluene
(30 mL) was added to the reaction flask followed by sweeping with three volumes of O2. The
reaction flask was placed in an oil bath and stirred for 1 h (70ºC) until the reaction mixture was a
bright green color. A solution of ethynyltrimethylsilane (298 µL, 2.1 mmol) in dry toluene (2
mL) was added to the mixture over 30 min. The reaction mixture was allowed to stir (4 h) during
which time the reaction mixture changed to a black suspension. The reaction mixture was filtered
through Celite®, concentrated under reduced pressure and purified by flash column
chromatography (hexane/ethyl acetate, 5:1) to yield 23 as a fluffy white amorphous solid (106
1
mg, 22%): mp 134-136 °C; Rf 0.33 (hexane/ethyl acetate, 4:1); H NMR (400 MHz, CDCl3)
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δ 7.89 (d J=7.6 Hz, 1H), 7.62 (dd J=8.0, 7.6 Hz, 1H), 7.50 (dd J=8.0, 7.6 Hz, 1H), 7.44 (d J=8.0
13
Hz, 1H), 4.70 (s, 2H), 0.25 (s, 9H). C NMR (175 MHz, CDCl3) δ 168.6, 140.8, 133.0, 129.6,
128.6, 124.6, 122.8, 92.8, 75.9, 52.5, 0.18. IR (neat) 2954, 2902, 2167, 1712 cm-1; HRMS (ESI-
TOF) m/z [M+Cl]-; calculated for C13H1535ClNOSi 264.0617, Found: 264.0688.
2-Ethynylisoindoline-1,3-dione 24: Prepared by the Method of Davies (94%):32 mp 165-169 ºC
1
(Lit.33 167-172 ºC). The spectroscopic data was consistent with the literature32: H NMR (400
mHz, CDCl3) δ 7.92-7.94 (m, 2H), 7.80-7.83 (m, 2H), 3.33 (s, 1H).
2-Ethynylisoindoline-1-one 25: 2-(2-(Trimethylsilyl)ethynyl)isoindolin-1-one 23 (72.0 mg,
0.313 mmol) was dissolved in THF (3.0 mL) while stirring followed by the addition of tetra-N-
butylammonium fluoride (TBAF, 450 µL, 1 M solution). Stirring was continued (10 min) until
the reaction was complete as indicated by TLC. The crude reaction mixture was directly
submitted to gravity-column chromatography (hexanes/ethyl acetate) and afforded 25 as a white
1
solid (45 mg, 92%): mp 92-94 °C; Rf 0.21 (hexane/ethyl acetate, 4:1); H NMR (400 MHz,
CDCl3) δ 7.91 (d J=7.2 Hz, 1H), 7.64 (dd J=7.2, 7.2 Hz, 1H), 7.51 (dd J=7.2, 7.2 Hz, 1H), 7.46
(d J=7.2 Hz, 1H), 4.71 (s, 2H), 3.08 (s, 1H). 13C NMR (175 MHz, CDCl3) δ 169.1, 140.9, 133.2,
129.4, 128.7, 124.7, 122.9, 73.9, 61.7, 52.1. IR (neat) 3268, 2931, 2139, 1718 cm-1. Anal. Calcd
for C10H7NO (157.17): C, 76.42; H, 4.49; N, 8.91; Found: C, 76.32; H, 4.68; N, 8.69.
2-(Prop-2-yn-1-yl)isoindoline-1,3-dione 26: Prepared by the method of Clayton (69%):34 mp
1
146-148 ºC (Lit.36 149-150 ºC). The spectroscopic data was consistent with the literature36: H
NMR (400 MHz, CDCl3) δ 7.80-7.91 (m, 2H), 7.74-7.76 (m, 2H), 4.47 (s, 2H), 2.24 (s, 1H).
2-(Prop-2-yn-1-yl)isoindolin-1-one 27: Isoindolin-1-one 21 (250 mg, 1.85 mmol) was dissolved
in acetonitrile (9 mL) and cesium carbonate (2.40 g, 7.39 mmol) was added followed by
14

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propargyl bromide (308 µL, 2.77 mmol). The reaction mixture was stirred at reflux (2 h). The
reaction mixture was then dissolved in CH2Cl2 (10 mL) and washed with water (3 x 15 mL). The
organic layer was dried (Na2SO4), filtered and concentrated under reduced pressure. Gravity-
column chromatography of the residual oil (hexane/ethyl/acetate, 2:1) gave 27 as a yellow solid
1
(205 mg, 65%): mp 79-81 °C; Rf 0.66 (ethyl acetate); H NMR (400 MHz, CDCl3) δ 7.82 (d
J=8.0 Hz, 1H), 7.52 (dd J=8.0, 7.2 Hz, 1H) 7.41-7.45 (m, 2H), 4.47 (s, 2H), 4.24 (s, 2H), 2.28 (s,
1H). 13C NMR (100 MHz, CDCl3) δ 167.9, 141.2, 132.1, 131.6, 128.1, 123.8, 122.8, 78.1, 72.5,
49.1, 31.7. IR (neat) 3290, 3243, 2118, 1701 cm-1; HRMS (ESI-TOF) m/z [M+H]+; calculated
for C11H10NO 172.0762, Found: 172.0857.
General Procedure for the preparation of click products 6a-6d: 3-Azidopiperidine-2,6-dione
9 (0.25-0.5 mmol, 38-74 mg) and phthalimide or isoindolinone alkynes 24, 26, 25 or 27 (0.275-
0.55 mmol, 1.1 equiv.) were dissolved in 25% aqueous THF (2.0 mL). The mixture was stirred
(10 min) before addition of solid CuSO4 (0.5 eq.) and sodium ascorbate (0.5 eq.) whereupon the
reaction mixture became a teal-colored suspension and stirring was continued (16 h). The
reaction mixture was concentrated under rotary evaporation while absorbing onto silica gel (70-
230 mesh) and the resulting dry residue was applied to a silica gel chromatography column.
2-(1-(2,6-Dioxopiperidin-3-yl)-1H-1,2,3-triazol-4-yl)isoindoline-1,3-dione 6a: The crude
reaction mixture was submitted to gravity-column chromatography (hexane/ethyl acetate, 1:9) to
give triazole 6a as a white amorphous powder (126 mg, 83%): mp 289-291 °C; Rf 0.38
(THF/hexane, 2:1); 1H NMR δ (400 MHz, DMSO-d6) δ 11.25 (s, 1H), 8.45 (s, 1H), 7.97-8.00 (m,
2H), 7.90-7.94 (m, 2H), 5.90 (dd J=12.8, 5.6 Hz, 1H), 2.82-2.86 (m, 1H), 2.66-2.75 (m, 2H),
13
2.34-2.37 (m, 1H). C NMR (100 MHz, DMSO-d6) δ 172.7, 169.5, 166.5, 136.1, 135.5, 131.8,
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124.2, 122.9, 60.2, 31.2, 24.6. IR (neat) 3146, 3067, 2933, 1721, 1696 cm-1. HRMS (ESI-TOF)
m/z [M+H]+; calculated for C15H12N5O4 326.0889, Found: 326.0961.
3-(4-(1-Oxoisoindolin-2-yl)-1H-1,2,3-triazol-1-yl)piperidine-2,6-dione 6b: The crude reaction
mixture was submitted to gravity-column chromatography (chloroform/methanol, 95:5) and then
recrystallized with methanol to give triazole 6b as a white amorphous powder (70 mg, 78%): mp
1
decomp>300 °C; Rf 0.41 (chloroform/methanol, 9:1); H NMR (700 MHz, DMSO-d6) δ 11.19
(s, 1H), 8.56 (s, 1H), 7.79 (d J = 7.7 Hz, 1H), 7.67-7.71 (m, 2H), 7.53 (dd J=7.7, 7.0 Hz, 1H),
5.85 (dd J=13.3, 5.6 Hz, 1H), 2.76-2.84 (m, 2H), 2.64-2.66 (m, 1H), 2.27-2.28 (m, 1H). 13C
NMR (100 MHz, DMSO-d6) δ 172.8, 169.7, 165.6, 143.6, 142.4, 132.8, 131.6, 128.7, 124.3,
123.6, 113.8, 60.1, 49.4, 31.2, 24.5. IR (neat) 3180, 3087, 2923, 1735, 1697 cm-1. HRMS (ESI-
TOF) m/z [M+H]+; calculated for C15H14N5O3 312.1097, Found: 312.1176.
2-((1-(2,6-Dioxopiperidin-3-yl)-1H-1,2,3-triazol-4-yl)methyl)isoindoline-1,3-dione 6c: The
crude reaction mixture was submitted to gravity-column chromatography (chloroform/methanol,
97:3) to give triazole 6c as a white amorphous powder (56 mg, 67%): mp 230-232 °C; Rf 0.44
(chloroform/methanol, 9:1); 1H NMR (400 MHz, DMSO-d6) δ 11.16 (s, 1H), 8.16 (s, 1H), 7.83-
7.89 (m, 4H), 5.74 (dd J=12.8, 8.0 Hz, 1H), 4.83 (s, 2H), 2.75-2.80 (m, 1H), 2.56-2.63 (m, 2H),
13
2.20-2.23 (m, 1H). C NMR (100 MHz, DMSO-d6) δ 172.8, 169.7, 167.7, 142.7, 135.0, 132.0,
123.7, 123.6, 59.5, 33.3, 31.1, 24.7. IR (neat) 3220, 2957, 2863, 1732, 1701. cm-1; HRMS (ESI-
TOF) m/z [M+H]+; calculated for C16H14N5O4 340.1046, Found: 340.0888.
3-(4-((1-Oxoisoindolin-2-yl)methyl)-1H-1,2,3-triazol-1-yl)piperidine-2,6-dione 6d: The crude
reaction mixture was submitted to gravity-column chromatography (chloroform/methanol, 97:3)
to give triazole 6d as a white amorphous powder (53 mg, 61%): mp 212-215 °C; Rf 0.39
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(chloroform /methanol, 9:1); 1H NMR (400 MHz, DMSO-d6) δ 11.17 (s, 1H), 8.16 (s, 1H), 7.68
(d J=7.2 Hz, 1H), 7.56-7.57 (m, 2H), 7.44-7.49 (m, 1H), 5.76 (dd J=12.8 Hz, 5.2 Hz, 1H), 4.80
(s, 2H), 4.46 (s, 2H) 2.77-2.82 (m, 1H), 2.59-2.64 (m, 2H), 2.22-2.47 (m, 1H). 13C NMR (100
MHz, DMSO-d6) δ 172.8, 169.8, 167.5, 143.5, 142.2, 132.4, 131.9, 128.3, 123.9, 123.8, 123.3,
59.5, 49.9, 37.4, 31.1, 24.7. IR (neat) 3063, 2916, 2836, 1704, 1666 cm-1. HRMS (ESI-TOF) m/z
[M+H]+; calculated for C16H16N5O3 326.1253, Found: 326.1287.
Acknowledgement:
The measurement of high resolution mass spectra by Dr. Bo Wang of the Texas A&M University
Laboratory for Biological Mass Spectrometry is acknowledged.
References and Notes
†Dedicated with best wishes to Professor Gary Posner, Johns Hopkins University.
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Tet 27915
Luzzio Ronnebaum Figurer