Multicomponent synthesis of novel coelenterazine derivatives substituted at the C-3 position

Article abstract of DOI:10.1016/j.tet.2015.09.048

Three novel coelenterazine derivatives substituted at the C-3 position were synthesized through a multicomponent strategy based on Groebke-Blackburn-Bienaymé reaction without using protecting groups. An efficient one-pot tert-butyl group cleavage from an

Full text of DOI:10.1016/j.tet.2015.09.048

Accepted Manuscript
Multicomponent Synthesis of Novel Coelenterazine Derivatives Substituted at the C-3
Position
Vito Vece, Giuseppina Vuocolo
PII:
S0040-4020(15)30076-4
10.1016/j.tet.2015.09.048
TET 27145
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 16 July 2015
Revised Date: 9 September 2015
Accepted Date: 21 September 2015
Please cite this article as: Vece V, Vuocolo G, Multicomponent Synthesis of Novel Coelenterazine
Derivatives Substituted at the C-3 Position, Tetrahedron (2015), doi: 10.1016/j.tet.2015.09.048.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Multicomponent Synthesis of Novel Coelenterazine Derivatives Substituted at the
C-3 Position
Vito Vece,* Giuseppina Vuocolo
Imperia-Chemistry, Synthetic Organic Chemistry Laboratory, via Brighei 1, 18100, Imperia, Italy
*
E-mail: vece.imperiachemistry@outlook.com
KEYWORDS: coelenterazine derivatives; multicomponent reaction; tert-butyl group cleavage;
azoylidene carbene; hydrogen abstraction.
---------------------------------------------------------------------------------------------------------
ABSTRACT: Three novel coelenterazine derivatives substituted at the C-3 position were synthesized
through a multicomponent strategy based on Groebke–Blackburn–Bienaymé reaction without using
protecting groups. An efficient one-pot tert-butyl group cleavage from an aminoimidazopirazine and the
first example of direct diazoimidazole derivative hydrogen abstraction in acidified water (traditional
Sandmeyer hydroxylation conditions) were described.
---------------------------------------------------------------------------------------------------------
1
Coelenterazione (1), a natural occurring bioluminescent imidazopyrazine, was found in marine
2
3
organisms such as the jellyfish Aequoria victoria, the sea cactus Cavernularia obesa, the sea pansy
4
5
6
Renilla reniformis, the deep sea shrimp Oplophorus gracilirostris, obelin Obelia longissima and the
7
oceanic squid Symplectoteuthis oualaniensis. The luminescent proprieties have shown utility as
8
signaling reagents in biological and clinical research, indeed, coelenterazine is used in new optical
9
technologies for studying the behavior of several cancers.
1
0
The bioluminescent and antioxidant proprieties of coelenterazine have encouraged the synthesis of
1
1
analogues with a view to improving its physical and biological qualities. Coelenterazine is a quite
1
2
unstable compound, with a half-life of 15 minutes at 37 °C, but its stability can be improved by
1
3
introducing groups at the C-3 position. K. Kikuchi reported the synthesis of coelenterazine-β-galactose

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1
3
conjugates to monitor dual gene expression, enzyme activity and high-throughput screening. Despite
the high interest in producing new derivatives of coelenterazine, all the syntheses described in literature
have in common a final step based on the condensation between coelenteramine 2 (or an analogue) with
1
1,14
a free or protected α-keto aldehyde which require several synthetic steps to be achieved.
Surprisingly, until now, there is no literature precedence for bioluminescence from any 3-deoxy
derivatives of coelenterazine, and also no known synthetic methodology to prepare this class of
compounds.
Hereby we present a new approach to obtain three novel coelenterazine derivatives (4, 5 and 6)
substituted at the C-3 position by means of Groebke–Blackburn–Bienaymé multicomponent reaction
(Scheme 1).
O
OH
O
3
2
O
General
approach
NH2
HO
N
N
N
3a
N
5
9
or
+
6
N
H
8
OEt
OEt
HO
HO
1
2
O
HO
3b
OH
R
3
2
This work:
Groebke-Blackburn-Bienaymé
multicomponet reaction
N
9
N
N
5
N
N
NH
2
O
6
8
NC
+
+
HO
HO
HO
2
7
8
t
4
5
6
: R = NH Bu
: R = NH2
: R = H
Scheme 1. General and multicomponent approaches in the synthesis of coelenterazine derivatives.
Multicomponent reactions (MCRs) are a powerful and efficient synthetic strategy in modern organic
chemistry due to their elegant features to maximize structural complexity and convergence, while saving
1
5
synthetic procedures. As regards the imidazole chemistry, recently we reported a two-step procedure
(Ugi multicomponet reaction followed by Bischler–Napieralski/heterocyclization) to synthesize

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1
6
dihydroimidazo[1’,5’:1,2]pyrido[3,4-b]indol-2-ium salts and indoles in good yields. Encouraged by
these results we aimed our studies at extending the application of MCRs in the synthesis of derivatives
of coelenterazine without any protecting group insertion. To the best of our knowledgeable, Groebke–
1
7
Blackburn–Bienaymé multicomponent reaction
was never investigated in the synthesis of
coelenterazine derivatives.
Starting from commercially available aminopyrazine (9), bromination with 3.0 equivalents of N-
bromosuccinimide in chloroform provided 3,5-dibromopyrazin-2-amine (10) in 69% yield.
Benzylmagnesium bromide, prepared in situ by mixing benzylbromide and magnesium turnings in dry
THF, was first treated with anhydrous zinc chloride to generate the benzylzinc halide, which was then
reacted with 10 at room temperature in the presence of bis(triphenylphosphine)palladium (II) dichloride
1
1d
catalyst to afford 2-amino-3-benzyl-5-bromo-pyrazine (11) in 70% yield. Suzuki coupling of 11 with
1
1b
4
-hydroxyphenylboronic acid 12 gave compound 2 in 94% yield.
Groebke–Blackburn–Bienaymé three-component reaction was accomplished by mixing in anhydrous
1
8
methanol 2 with 2-(4-hydroxyphenyl)acetaldehyde (8) and tert-butylisocyanide (7), the reaction was
catalyzed by glacial acetic acid and conducted at room temperature. After three days it was observed
that a part of the starting material 2 was recovered (60%) and the final yield of the desired product 4 was
only 37%. To improve the yield of this reaction, addition portions of aldehyde 8 (1.1 equivalents) and
isocyanide 7 (1.1 equivalents) were added after 4 hours and 8 hours. Under these modified conditions,
the yield increased to 64% after two days of reaction.
To reach the free amine compound 5, the cleavage of tert-butyl group was investigated. Although
several methods to remove a tert-butyl group from an aminoimidazole derivative to achieve a primary
1
9
20
amine have been described, they required a two-step procedure or a microwave irradiation, and we
were interested to find a direct one-pot procedure applicable also to large-scale synthesis without the use
of microwave reactors. It was found that a solution of 4 in 1:1 37% aqueous hydrochloric acid solution

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and methanol heated to 90 °C for 18 hours afforded the desired product 5, after neutralization with
ammonia solution, in near-quantitative yield (98%).
To prepare compound 6, the reduction of amine 5 to introduce a hydrogen atom in position 3 was
investigated. For structure like aminoimidazoles a hydrogen abstraction was observed as main synthetic
21
process during the diazotization in alcoholic solution. As reported, these reactions involve the
2
2
formation of azoylidene carbenes generated after loss of nitrogen from the diazoimidazole compounds.
We found that in classical Sandmeyer-type hydoxylation condition (sodium nitrite, water and sulforic or
hydrochloric acid) at 0 °C, amine 5 was subjected to a direct hydrogen abstraction to afford compound 6
as unique isolable product in 40% yield (Scheme 2). Any formation of 1 (tautomer of the hydrated
compound) was not detectable by mass spectrometry analysis.
HO
OH
B
1
2
N
N
NH2
OH
CO
N
N
NH2
N
N
NH
2
1) PhCH
2
Br, Mg
NBS (3 equiv.)
2) ZnCl
2
, Pd(PPh
3
)
2
Cl
2
Pd(PPh
3
)
4
, K
2
3
Br
Br
Br
CHCl3
40 °C, 6 h
THF
r.t., 3 d
70%
dioxane / H
80 °C, 18 h
94%
O
2
1
1
9
10
0 °C
69%
O
H
N
OH
NC
N
N
NH2
HO
8
N
N
7
3
7% HCl(aq) / MeOH (1:1)
AcOH
N
9
0 °C, 18 h
8%
MeOH, m.s.
r.t., 2 d
9
HO
HO
6
4%
2
4
OH
H N
OH
2
N
N
N
NaNO (4 equiv.)
N
N
2
H SO / H O
N
2
4
2
0
°C, 3 h
0%
4
HO
HO
5
6
Scheme 2. Synthetic procedure.

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Interesting, to the best of our knowledge, hydrogen abstraction of diazoimidazoles in presence of only
acidified water and nitrite salt (traditional Sandmeyer hydroxylation conditions) was never described
and this reaction is the first of this class. A plausible mechanism is illustrated in Scheme 3.
Diazo-compound 14 spontaneously was converted in carbene 15 after loss of nitrogen. 15 reacted with
nitrous acid, generated in situ from nitrite salt and acid, to afford intermediate 16 which was able to add
a molecule of water to generate intermediate 17. The final step was the elimination of a molecule of
nitric acid and the regeneration of the imidazole aromaticity. In this mechanism nitrous acid plays a role
of reducing agent and it is oxidized to nitric acid in the end of the hydrogen abstraction process.
Scheme 3. Proposed mechanism for the hydrogen abstraction.
In conclusion, three novel coelenterazine derivatives substituted at the C-3 position were synthesized by
means of Groebke–Blackburn–Bienaymé multicomponent reaction. A very efficient one-pot procedure
to remove a tert-butyl group from an aminoimidazole derivative and the first example of direct
hydrogen abstraction of a diazoimidazole derivative in acidified water (traditional Sandmeyer
hydroxylation conditions) are described. The unprecedented strategy described here offers a new and
easy way to prepare a wide range of coelenterazine analogues and explore their chemical and
luminescent proprieties.

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Experimental Section
General Method and Instrument Details. Reactions were carried out in dried glassware and under argon
atmosphere. All reactions were magnetically stirred and monitored by thin-layer chromatography (TLC)
on silica gel 60 F254 (Merck) plates using UV light and/or phosphomolybdic acid, p-anisaldehyde or
1
0% H SO in ethanol solution to visualize the compounds. Solvents were distilled and dried from
2 4
sodium benzophenone ketyl for tetrahydrofuran and from calcium hydride for methylene chloride. Dry
toluene, N,N-dimethylformamide and diethyl ether were purchased from commercial suppliers and used
without further purification. Flash chromatography was performed on silica gel (40-60mm). Yields refer
to chromatographically and spectroscopically pure compounds unless otherwise noted. Melting points
1
13
were measured on a B-540 Büchi apparatus. H and C NMR spectra were recorded at 23°C using
Bruker Avance DRX300, DRX400. Chemical shifts are reported relative to the residue peaks of the
solvent. The following abbreviations are used to denote the multiplicities: s = singlet, d = doublet, dd =
doublet of doublets, t = triplet, m = multiplet, and br = broad. NMR solvents were purchased from
Sigma-Aldrich. Infrared (IR) spectra were recorded with neat liquid or solid samples using a Shimadzu
IR Affinity-1 FT-IR spectrometer. HRMS were recorded using a Thermo Finnigan LCQ spectrometer.
3
,5-Dibromopyrazin-2-amine (10): In an argon atmosphere, aminopyrazine (600.0 mg, 6.31 mmol, 1.0
equiv.) was dissolved in chloroform (25 mL) and then NBS (3.37 g, 18.93 mmol, 3.0 equiv.) was added
over 1h at room temperature. When the addition was completed, the mixture was stirred a further 6 h at
4
1
0 °C. The mixture was poured into saturated Na S O solution (100 mL), extracted with EtOAc (3 ×
2 2 3
00 mL) and the combined organic layers where washed with saturated brine (100 mL). The organic
solution was dried with Na SO and concentrated by rotary evaporation. Finally the residue was purified
2
4
by chromatography on silica gel (gradient elution: PE – EtOAc, 9:1 ꢀ 7:3) to give 10 (1.0856g, 4.29
1
mmol, 69%) as a pale yellow compound. R = 0.65 (silica gel, PE – EtOAc, 7:3); mp 114–116 °C; H
f
1
3
NMR (400 MHz, CDCl
3 3
): δ = 8.02 (s, 1H), 5.17 (br, s, 2H); C NMR (100 MHz, CDCl ): δ = 152.0,
1
43.2, 124.1, 123.6; IR (neat): νmax = 3447, 3281, 3187, 3154, 2933, 1811, 1714, 1621, 1549, 1506,

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-1
1
450, 1359, 1333, 1202, 1151, 1133, 1096, 1079, 1040, 908, 877, 801, 755, 696 cm ; HRMS (ESI):
79
+
3
79
4
81
+
3
calculated for C H Br N 251.8766, found 251.8779; calculated for C H Br BrN 253,8746,
4
4
2
4
81
+
3
found 253.8757; calculated for C H Br N 255.8726, found 255.8735. The spectroscopic data are in
4
4
2
1
4a
good agreement with those reported in the literature.
3
-Benzyl-5-bromopyrazin-2-amine (11): Magnesium turnings (293.9 mg, 12.1 mmol, 3.0 equiv.) and
I2 (153.6 mg, 605 µmol, 0.15 equiv,) were added to a dry 100-mL 2-necked round-bottom flask under
an argon atmosphere. Freshly distilled THF (25 mL) was added using a syringe. The mixture was stirred
at 0 °C until the brown color of the I
equiv.) was slowly added using a syringe. After 10 min ZnCl
0 min. To this mixture, bis(triphenylphosphine)palladium (II) dichloride (141.8 mg, 0.20 mmol, 0.05
2
disappeared. Then, benzylbromide (1.44 mL, 12.1 mmol, 3.0
2
was added and the mixture was stirred for
4
equiv.) and a solution of 2-amino-3,5-dibromopyrazine (1.02 g, 4.03 mmol, 1.0 equiv) in dry THF (25
mL) were added sequentially at room temperature. The resulting orange-colored reaction mixture was
stirred for 3 days at room temperature and then quenched with water (50 mL) at 0 °C. The mixture was
diluted with ethyl acetate (300 mL) and water (100 mL). The organic layer was separated and the
aqueous layer was extracted with ethyl acetate (4 × 200 mL). The combined organic layers were dried
2 4 2 2
over anhydrous Na SO and concentrated on a rotary evaporator. The residue was dissolved in CH Cl
and purified by silica gel column chromatography (gradient elution: PE – EtOAc, 85:15 ꢀ 65:35) to
afford compound 11 (738.2 mg, 2.79 mmol, 70 %) as viscous yellow oil. R = 0.46 (silica gel, PE –
f
1
EtOAc, 7:3); H NMR (400 MHz, CDCl ): δ = 7.93 (s, 1H), 7.26–7.21 (m, 2H), 7.20–7.15 (m, 1H),
3
1
3
7
.15–7.10 (m, 2H), 4.40 (br, s, 2H), 3.99 (s, 2H); C NMR (100 MHz, CDCl ): δ = 152.3, 142.6, 141.9,
3
1
35.8, 129.2, 128.6, 127.4, 126.4, 40.9; IR (neat): νmax = 3472, 3314, 3200, 3060, 3027, 2919, 2850,
1
605, 1555, 1529, 1494, 1424, 1389, 1340, 1261, 1220, 1158, 1117, 1073, 1048, 1028, 1002, 923, 905,
-1
79
+
3
7
91, 759, 726, 696, 634 cm ; HRMS (ESI): calculated for C11
H
11 BrN
264.0131, found 264.0133;
81
+
3
calculated for C11H11 BrN 266.1414, found 266.0113. The spectroscopic data are in good agreement
1
1d
with those reported in the literature.

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4
-(5-Amino-6-benzylpyrazin-2-yl)phenol (2): A mixture of 3-benzyl-5-bromopyrazin-2-amine (637.5
mg, 2.41 mmol, 1.0 equiv.), 4-hydroxylphenylboronic acid (398.6 mg, 2.89 mmol, 1.2 equiv.),
Pd(PPh ) (277.3 mg, 0.24 mmol, 0.1 equiv.) and K CO (1.665 g, 12.0 mmol, 5.0 equiv.) in dioxane
3
4
2
3
(
50 mL) and water (10 mL) was heated to 80 °C and stirred for 18 h under Ar. The mixture was cooled
to room temperature, diluted with water (30 mL) and extracted with EtOAc (3 × 50 mL). The combined
organic layers were washed with brine (25 mL), dried with Na SO , filtered, evaporated and the residue
subjected to flash silica gel column chromatography (gradient elution: PE – EtOAc, 7:3 ꢀ 4:6) to afford
2
4
f
compound 2 (625.5 mg, 2.26 mmol, 94%) as a pale brown solid. R = 0.65 (silica gel PE – EtOAc,
1
2
5:75); mp 202–205 °C; H NMR (400 MHz, DMSO-d ): δ = 9.52 (s, 1H), 8.31 (s, 1H), 7.74 (d, J = 8.6
6
Hz, 2H), 7.34 (d, J = 7.2 Hz, 2H), 7,28 (t, J = 7.2 Hz, 2H), 7.18 (t, J = 7.2 Hz, 1H), 6.81 (d, J = 8.6 Hz,
1
3
2
H), 6.22 (s, 2H), 4.07 (s, 2H); C NMR (100 MHz, DMSO-d ): δ = 157.2, 152.0, 139.7, 139.5, 138.3,
6
1
35.9, 128.9, 128.2, 126.2 (× 2), 126.1, 115.5, 38.7; IR (neat): νmax = 3478, 3346, 3216, 3060, 3026,
2
912, 2806, 2676, 2597, 2476, 2361, 2339, 1633, 1606, 1546, 1513, 1492, 1443, 1389, 1350, 1264,
-1
+
1
223, 1174, 1156, 1131, 1081,1009, 933, 898, 790, 770 cm ; HRMS (ESI): calculated for C17
16 3
H N O
2
78.1288, found 278.1301. The spectroscopic data are in good agreement with those reported in the
1
4b
literature.
2
-(4-Hydroxyphenyl)acetaldehyde (8)
Under anhydrous condition, to a solution of 2-(4-hydroxyphenyl)ethanol (2.00 g, 14.5 mmol, 1.0 equiv.)
in DMSO (20 mL) was added diisopropylethylamine (5.29 mL, 30.4 mmol, 2.1 equiv.) and then a
solution of SO • pyridine (4.54 g, 28.5 mmol, 2.0 equiv.) in DMSO (20 mL) over 30 min. The mixture
3
was stirred for 1 h at room temperature and quenched by the addition of ice-cold water (200 mL) The
aqueous layer was extracted with CH
concentrated under reduced pressure. The crude material was purified using silica flash chromatography
gradient elution: PE – EtOAc, 8:2 ꢀ 6:4). Fractions containing the desired product were combined and
2 2
Cl (3 × 200 mL) and the organic layers were combined and
(

ACCEPTED MANUSCRIPT
f
concentrated under reduced pressure to yield 8 as a colourless oil (649.2 mg, 4.77 mmol, 33%). R =
1
0
8
.24 (silica gel PE – EtOAc, 8:2); H NMR (300 MHz, CDCl ): δ = 9.68 (t, J = 2.4 Hz, 1H), 7.03 (d, J =
3
13
.6 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 7.75–6.10 (br, s, 1H), 3.61 (d, J = 2.4 Hz, 1H); C NMR (75
= 3351 (br), 3022, 2919,
MHz, CDCl ): δ = 201.4, 155.4, 130.8, 123.1, 116.0, 49.6; IR (neat): ν
3
max
2
1
1
834, 2730, 2361, 2338, 1886, 1709, 1612, 1596, 1513, 1442, 1359, 1305, 1261, 1218, 1172, 1158,
-1
• +
105, 1080, 1041, 1014, 957, 935, 827 cm ; HRMS (ESI): calculated for [C H O ] 136.0519, found
8
8
2
1
8
36.0522. The spectroscopic data are in good agreement with those reported in the literature.
4
-(8-Benzyl-3-(tert-butylamino)-2-(4-hydroxybenzyl)imidazo[1,2-a]pyrazin-6-yl)phenol (4): 4-(5-
(237.6 mg, 857 ꢀmol, 1.0 equiv.) and 2-(4-
amino-6-benzylpyrazin-2-yl)phenol
hydroxyphenyl)acetaldehyde (128.3 mg, 943 ꢀmol, 1.1 equiv.) were dissolved in dry MeOH (4 mL). To
this solution molecular sieves 3 Å (240 mg) were added and the mixture was stirred at room
temperature. After 20 min tert-butylisocyanide (250 ꢀL, 2.22 mmol, 2.6 equiv.) and AcOH (350 ꢀL,
6
.11 mmol, 7.1 equiv.) were added. New portions of aldehyde (128.3 mg, 943 ꢀmol, 1.1 equiv.) and
isocyanide (107 ꢀL, 943 ꢀmol, 1.1 equiv.) were added after 4 h and after 8 h. The mixture was stirred at
room temperature for 48 h, then diluted with water (30 mL) and extracted with EtOAc (3 × 50 mL). The
combined organic layers were washed with brine (25 mL), dried with Na SO , filtered, evaporated and
2
4
the residue subjected to flash silica gel column chromatography (gradient elution: PE – EtOAc, 7:3 ꢀ
:6). Fractions containing the desired product were combined and concentrated under reduced pressure
4
to yield 4 as a white solid (260.2 mg, 544 ꢀmol, 64%). R = 0.57 (silica gel PE – EtOAc, 6:4); mp 241–
f
1
2
8
45 °C; H NMR (400 MHz, DMSO-d ): δ = 9.64 (br, s, 1H), 9.14 (br, s, 1H), 8.46 (s, 1H), 7.82 (d, J =
6
.8 Hz, 2H), 7.46 (d, J = 7.0 Hz, 2H), 7.25 (t, J = 7.3 Hz, 2H), 7.17 (t, J = 7.3 Hz, 1H), 7.13 (d, J = 8.6
Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 6.67 (d, J = 8.6 Hz, 2H), 4.51 (s, 1H), 4.41 (s, 2H), 4.02 (s, 2H), 1.14
1
3
(
s, 9H); C NMR (100 MHz, DMSO-d
6
): δ = 157.7, 155.4, 150.9, 142.4, 138.4, 136.7, 134.7, 130.2,
1
29.7, 129.3, 128.2, 127.7, 127.1, 127.1, 126.3, 115.6, 114.8, 110.0, 55.5, 39.0, 32.0, 30.0; IR (neat):

ACCEPTED MANUSCRIPT
ν
max = 3061 (br), 3027, 2966, 2804, 2677, 2595, 2361, 2339, 2163, 1610, 1546, 1513, 1477, 1452, 1402,
1
388, 1363, 1264, 1223, 1203, 1166, 1103, 1084, 1047, 1023, 1003, 931, 911, 821, 785, 751, 726, 667
-1
+
cm ; HRMS (ESI): calculated for C H N O 479.2442, found 479.2421.
3
0
31
4
2
4
-(3-Amino-8-benzyl-2-(4-hydroxybenzyl)imidazo[1,2-a]pyrazin-6-yl)phenol (5): Compound
4
(344.1 mg, 720 ꢀmol, 1.0 equiv.) was dissolved in dry MeOH (15 mL), and then 37% aqueous HCl (15
mL) was slowly added. The reaction mixture was warmed to 90 °C for 18 h. The solvent was removed
under reduced pressure and the crude was directly purified using silica flash chromatography (dry
2 2
deposition, gradient elution: CH Cl – MeOH, 95:5 ꢀ 90:10). Fractions containing the desired product
were combined and concentrated under reduced pressure to yield 5 as a pale yellow solid (296.9 mg,
1
7
03 ꢀmol, 98%). R = 0.59 (silica gel CH Cl – MeOH, 9:1); mp 149–153 °C; H NMR (400 MHz,
f
2
2
DMSO-d ): δ = 9.59 (s, 1H), 9.14 (s, 1H), 8.36 (s, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.45 (d, J = 7.0 Hz,
6
2
6
1
1
2
8
4
4
7
H), 7.25 (t, J = 7.4 Hz, 2H), 7.16 (t, J = 7.4 Hz, 1H), 7.12 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H),
13
.67 (d, J = 8.6 Hz, 2H), 5.44 (s, 2H), 4.36 (s, 2H), 3.98 (s, 2H); C NMR (100 MHz, DMSO-d ): δ =
6
57.5, 155.4, 150.1, 138.8, 136.3, 131.9, 130.6, 130.4, 129.7, 129.4, 129.1, 128.2, 128.1, 126.9, 126.1,
15.5, 114.9, 108.1, 38.9, 31.5; IR (neat): νmax = 3060 (br), 3025, 2922, 2802, 2675, 2591, 2361, 2339,
050, 1608, 1554, 1512, 1492, 1451, 1402, 1353, 1234, 1168, 1103, 1084, 1043, 1021, 992, 913, 837,
-1
+
2
17, 777, 726, 699, 667, 657, 634 cm ; HRMS (ESI): calculated for C H N O 423.1816, found
2
6
23
4
23.1805.
-(8-Benzyl-2-(4-hydroxybenzyl)imidazo[1,2-a]pyrazin-6-yl)phenol (6): A solution of 5 (300 mg,
10 ꢀmol, 1.0 equiv.) in a mixture of 98% H SO (2.0 mL, 36.0 mmol, 50.7 equiv.) and water (10 mL)
2
4
was kept at 0 °C whilst a solution of sodium nitrite (195.0 mg, 2.82 mmol, 4.0 equiv.) in water (5 mL)
was added dropwise during 20 min. The solution was stirred at 0 °C for 3 h, then warmed to room
temperature and neutralized to pH 7 with 25% ammonium hydroxide solution (6 mL). The solvent was
removed under reduced pressure and the crude was directly purified using silica flash chromatography
(
dry deposition, gradient elution: CH Cl – MeOH, 98:2 ꢀ 96:4). Fractions containing the desired
2 2

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product were combined and concentrated under reduced pressure to yield 6 as an orange solid (118.7
1
mg, 281 ꢀmol, 40%). R = 0.60 (silica gel CH Cl – MeOH, 9:1); mp 238–243 °C; H NMR (400 MHz,
f
2
2
DMSO-d ): δ = 9.75 (br, s, 1H), 9.31 (br, s, 1H), 8.84 (s, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.72 (s, 1H),
6
7
.47 (d, J = 7.0 Hz, 2H), 7.28 (t, J = 7.4 Hz, 2H), 7.19 (t, J = 7.4 Hz, 1H), 7.15 (d, J = 8.5 Hz, 2H), 6.87
1
3
(
d, J = 8.8 Hz, 2H), 6.73 (d, J = 8.5 Hz, 2H), 4.48 (s, 2H), 4.05 (s, 2H); C NMR (100 MHz, DMSO-
d6): δ = 158.1, 155.9, 150.5, 147.5 (br), 138.0 (br), 138.0, 137.2 (br), 129.9, 129.2, 129.1, 128.3, 127.2,
1
2
1
27.0, 126.4, 115.7, 115.3, 113.4, 113.0, 39.2, 33.4; IR (neat): νmax = 3129 (br), 3061, 3027, 2920, 2800,
679, 2588, 2361, 2339, 1892, 1735, 1663, 1609, 1513, 1481, 1452, 1414, 1351, 1233, 1168, 1104,
-1
052, 1029, 1004, 920, 816, 783, 733, 700, 667, 660, 649, 635 cm ; HRMS (ESI): calculated for
+
C H N O 408.1707, found 408.1687.
26
22
3
2
ASSOCIATED CONTENT
Supporting Information
Spectra and characterization data for all new compounds. The Supporting Information is available at
DOI:
AUTHOR INFORMATION
Corresponding Author
*
E-mail: vece.imperiachemistry@outlook.com
ACKNOWLEDGMENT
This work was supported by Imperia-Chemistry Foundation Programme. We thank Prof. Leonardo
Vatteone for his valued contribution and useful discussion.
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(

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(
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8
129–8142.
(
(
(
12) Hawkins, E. M. Bioluminescenze and Chemiluminescence: Progress and Current Applications
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(
1

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Supporting Information
Multicomponent Synthesis of Novel Coelenterazine Derivatives
Substituted at the C-3 Position
*
Vito Vece, Giuseppina Vuocolo
Imperia-Chemistry, Synthetic Organic Chemistry Laboratory, via Brighei 1, 18100, Imperia, Italy.
E-mail: vece.imperiachemistry@outlook.com
Contents of Supplementary Information
1
13
1
. H (400 MHz) and C (100 MHz) Spectal Data of 4, 5 and 6 in DMSO-d . Signal Attribution
6
Table...............................................................................................................................................S2
1
13
2
. H, C NMR, FT-IR Spectra and HRMS Data .........................................................................S3
S1

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. H (400 MHz) and C (100 MHz) Spectal Data of 4, 5 and 6 in DMSO-d . Signal
1
13
1
6
Attribution Table
1
6
15
16 15
16 15
1
8
20
1
7
17
17
H
10
2
18
10
2
10
2
21
19
5
OH
14OH
OH
N
H N
14
2
11
14
1
1
3
11
3
3
12
13
12 13
22
12 13
4
4
4
N 1
9
N 1
9
N 1
9
N
N
N
5
5
24
20
23
19
22
23
19
18
2
5
6
30
21
22
26
20
21
25
6
8
6 N
7
8
6
8
N
N
2
7
31
27
7
26
2
8
36
35
24
28
32
31
23
31
30
3
2
HO
27
HO
HO
2
7
2
9
25
24
2
9
3
3
28
3
0
34
5
29
4
6
1
3
a
1
13
a
1
13
a
1
Position
δ C
δ H (J in Hz)
Position
δ C
δ H (J in Hz)
Position
δ C
δ H (J in Hz)
2
3
5
6
8
9
127.1
142.4
110.0
136.7
150.9
134.7
32.0
130.2
114.8
129.7
155.4
2
3
5
6
8
129.7
130.6
108.1
136.3
150.1
131.9
31.5
130.4
114.9
129.4
155.4
2
3
5
6
8
147.5, br
113.0
113.4
137.2, br
150.5
138.0, br
33.4
129.1
115.3
129.9
155.9
7.72, s
8.84, s
8.46, s
8.36, s
9
9
1
1
0
1
4.02, s
10
11
12, 16
13, 15
14
17
18
3.98, s
10
11
12, 16
13, 15
14
17
4.05, s
1
1
2, 16
3, 15
6.67, d (8.6)
7.13, d (8.6)
6.67, d (8.6)
7.12, d (8.6)
6.73, d (8.5)
7.15, d (8.5)
1
1
1
1
4
7
8
9
9.14, br s
4.51, s
9.14, br s
5.44, s
9.31, br s
55.5
30.0
2
0, 21, 22
1.14, s
2
3
127.7
115.6
127.1
157.7
19
20, 24
21, 23
22
25
26
128.2
115.5
126.9
157.5
18
19, 23
20, 22
21
24
25
127.0
115.7
127.2
158.1
2
2
4, 28
5, 27
2
2
3
3
2, 36
3, 35
3
6.87, d (8.8)
7.82, d (8.8)
6.86, d (8.8)
7.81, d (8.8)
6.87, d (8.8)
7.79, d (8.8)
6
9
0
1
9.64, br s
4.41, s
9.59, br s
4.36, s
9.75, br s
4.48, s
39.0
38.9
39.2
138.4
129.3
128.2
126.3
27
138.8
129.1
128.1
126.1
26
138.0
129.2
128.3
126.4
3
3
7.46, d (7.0)
7.25, t (7.3)
7.17, t (7.3)
28, 32
29, 31
30
7.45, d (7.0)
7.25, t (7.4)
7.16, t (7.4)
27, 31
28,30
29
7.47, d (7.0)
7.28, t (7.4)
7.19, t (7.4)
4
1
3
a
C NMR chemical shift values were assigned on the basis of 2D NMR correlations.
S2

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1
13
2
. H, C NMR, FT-IR Spectra and HRMS Data
S3

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N
N
NH2
Br
Br
1
0

ACCEPTED MANUSCRIPT
N
N
NH2
Br
Br
1
0

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N
N
NH2
Br
11

ACCEPTED MANUSCRIPT
N
N
NH2
Br
11

ACCEPTED MANUSCRIPT
N
N
NH2
HO
2

ACCEPTED MANUSCRIPT
N
N
NH2
HO
2

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O
HO
8

ACCEPTED MANUSCRIPT
O
HO
8

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H
OH
N
N
N
N
HO
4

ACCEPTED MANUSCRIPT
H
OH
N
N
N
N
HO
4

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H
OH
N
N
N
N
HO
4

ACCEPTED MANUSCRIPT
H
OH
N
N
N
N
HO
4