Strategies for large-scale synthesis of coelenterazine for in vivo applications

Article abstract of DOI:10.1055/s-0033-1340556

A new application of two Negishi-type coupling reactions for the synthesis of coelenterazine is reported. The synthesis of coelenterazine in high purity on a gram scale will enable numerous approaches to bioluminescence imaging and possibly photodynamic therapy of deep-seated tumors. Coelenterazine is the substrate for several luciferases, among them Gaussia luciferase (gLuc). This synthesis starts with pyrazin-2-amine and uses inexpensive starting materials and catalysts. Georg Thieme Verlag Stuttgart New York.

Full text of DOI:10.1055/s-0033-1340556

646▌
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paper
Strategies for Large-Scale Synthesis of Coelenterazine for in Vivo Applications
Strategies for Large-Scale Synthesis of Coelenterazine
Tej B. Shrestha,*a,b Deryl L. Troyer,b Stefan H. Bossmann*a
a
Kansas State University, Department of Chemistry, 213 CBC Building, Manhattan, KS 66506, USA
Fax +1(785)5326666; E-mail: sbossman@ksu.edu
b
Kansas State University, Anatomy & Physiology, 130 Coles Hall, Manhattan, KS, 66506, USA
E-mail: tbs3@vet.k-state.edu
Received: 25.11.2013; Accepted after revision: 10.12.2013
tumor detection.7 Bioluminescent proteins can also be
Abstract: A new application of two Negishi-type coupling reac-
used for in situ photodynamic therapy for the treatment of
tions for the synthesis of coelenterazine is reported. The synthesis
deep-seated tumors.1b
of coelenterazine in high purity on a gram scale will enable numer-
ous approaches to bioluminescence imaging and possibly photody-
namic therapy of deep-seated tumors. Coelenterazine is the
substrate for several luciferases, among them Gaussia luciferase
is a bioluminescent compound found in very different ma-
(gLuc). This synthesis starts with pyrazin-2-amine and uses inex-
pensive starting materials and catalysts.
Coelenterazine {8-benzyl-6-(4-hydroxyphenyl)-2-[(4-hy-
droxyphenyl)methyl]imidazo[1,2-a]pyrazin-3(7H)-one, 1}
rine organisms, such as Aequorea victoria,8,9 Renilla
reniformis,10,11 Watasenia scintillans,11,12 and Gaussia
Key words: palladium, zinc, organometallic reagents, Grignard re-
action, regioselectivity, Negishi coupling
princeps.12 Like other chemoluminescent compounds (lu-
ciferins), coelenterazine emits blue light (λmax = 480 nm)
in the presence of an enzyme (e.g., Gaussia luciferase)
outside the biological system without ATP.6 The molecu-
Recent advances in life-science and bioengineering per-
lar basis of the chemoluminescence of coelenterazine is
mit the transfection, cellular expression, and real-time im-
shown in Scheme 1.6 Since no excitation light is required
aging of light-emitting proteins, such as Gaussia1 or
for bioluminescent processes, there is no background
Renilla luciferase (Ruc),2 bacterial luciferase (Lux),3 fire-
fluorescence and, consequently, a much improved signal
fly luciferase (Luc),4 green fluorescent protein (GFP),5 or
to noise ratio in imaging experiments.13 However, it
Ruc-GFP fusion protein.3 All of these marker proteins,
should be noted that blue light from Gaussia lumines-
which generate their fluorescence or bioluminescence by
cence has a very low penetration depth in human tissue
chemical processes,6 have been successfully employed for
O
HO
O
N
O
O
–
luciferase, O2
N
HO
N
N
N
N
H
OH
OH
2
1
CO2
*
O
O
+ hν
–
N
HN
HO
HO
N
N
N
N
OH
OH
3
4
Scheme 1 Chemoluminescence of coelenterazine (1): In the presence of Gaussia or Renilla luciferase, triplet O2 is added and then CO2 is
released to form the excited coelenteramide anion 3, which deactivates under emission of blue light (λmax = 480nm). The last step consists of
the protonation of 3 to coelenteramide (4).6,17
SYNTHESIS 2014, 46, 0646–0652
Advanced online publication: 09.01.2014
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1
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DOI: 10.1055/s-0033-1340556; Art ID: SS-2013-M0761-OP
© Georg Thieme Verlag Stuttgart · New York

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Strategies for Large-Scale Synthesis of Coelenterazine
647
OH
TBDMSO
B
H2N
N
N
OH
BnMgCl, ZnCl2
PdCl2(Ph3P)2, 54 h, r.t.
7
H2N
Br
N
Br
PdBn2Cl2
dppb
EtOH, aq Na2CO3
toluene, reflux, 7 h
N
Br
5
6
TBDMSO
H2N
N
HO
O
O
EtO
N
N
N
OEt
9
OTBDMS
N
H
aq HCl–1,4-dioxane, reflux, 5 h
8
OH
1
Scheme 2 Coelenterazine (1) synthesis by Adamczyk et al.19d
(1–2 mm).14 Therefore, to assure a larger penetration tion proceeds at the metal interface and is, therefore, mass
depth, bioluminescent enzymes using coelenterazine as transfer limited and not too exergonic.
substrate should be coupled via Foerster energy resonance
We have studied the effects of scaling-up Negishi cou-
transfer with a second fluorophore or quantum dot causing
pling reactions of aromatic and aliphatic organozinc com-
a bathochromic shift of the emitted light.15 Blue light from
pounds on the yields and purity of the respective
bioluminescent processes is ideally suited to excite potent
intermediates in coelenterazine synthesis. Our results in-
photodynamic agents located in deep-seated tumors, for
dicate that this approach is viable and that highly pure
which a classic photodynamic treatment is very difficult.16
coelenterazine can be prepared in excellent yields.
The total synthesis of coelenterazine (1) was first reported
The approach closest to our synthetic scheme has been
followed by Adamczyk et al., who had synthesized coel-
by Inoue et al. in 1975.18 Since then many methods for the
synthesis of coelenterazine have evolved.19 Although
coelenterazine is commercially available, it is widely re-
garded as insufficiently stable for potential clinical appli-
cations, mainly because unreacted aromatic amines are
enterazine (1) by a sequence of reactions summarized in
Schemes 2 and 3.19d The overall yield of this approach was
48% from 3,5-dibromopyrazin-2-amine (5). The disad-
vantages of this approach are expensive starting materials
and relatively expensive catalysts. Since coelenterazine is
only metastable in aqueous buffers, it is of great impor-
capable of adding to its imidazo[1,2-a]pyrazin-3(7H)-one
ring system.17 We recognized the need to modify the ex-
isting methods for the synthesis of coelenterazine in order
tance that the substance is clean (purity >99.95% by
to provide it in high purity and in large quantities (gram
weight). Otherwise, impurities are able to catalyze the
scale) from inexpensive starting materials. Furthermore, it
thermal decomposition of coelenterazine, thus reducing
the lifetime of its β-cyclodextrin adduct in phosphate-
buffered saline from 72 hours to less than 1 hour. We
was our starting hypothesis that the long-term stability of
coelenterazine can be greatly improved (from a few weeks
to more than two years) by achieving a higher degree of
started the heterocyclic branch of the convergent coelen-
purity than observed to date.
terazine synthesis with pyrazin-2-amine (9). Bromination
The key reaction in achieving these goals is the Negishi of pyrazin-2-amine (9) with 2.5 equivalents of N-bromo-
coupling, which was the first reaction permitting the prep- succinimide is an easy reaction with an excellent yield
aration of unsymmetrical biaryls in good yields and high (Scheme 3).25
flexibility.20,21 In our work, it is the key to the successful
In addition, the side product obtained from the bromina-
scaling-up of coelenterazine production and to avoiding
tion reaction is 4-bromopyrazin-2-amine, which can sub-
reaction byproducts. Compared to the Mizoroki–Heck22
sequently converted into 3,5-dibromopyrazin-2-amine
and Suzuki–Miyaura23 reactions that have been previous-
(5).
ly used in coelenterazine synthesis, the Negishi coupling
The main change is the introduction of the zinc atom. In-
has several distinct advantages: (1) the required starting
stead of the zincation of the previously formed Grignard
materials are significantly easier to prepare; (2) the cata-
reagent, benzylmagnesium chloride, with zinc chloride
lysts used in Negishi coupling reactions are less expensive
for the Negishi coupling reaction (Scheme 2), the direct
and have generally significantly higher turnover num-
zinc insertion approach was used, inspired by Huo’s
method (Scheme 3).26
bers;24 and (3) the use of granular zinc permits easy scale-
up with aromatic/heterocyclic bromides, because the reac-
© Georg Thieme Verlag Stuttgart · New York
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648
T. B. Shrestha et al.
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H2N
N
N
Br
NBS
NBS
Br
TBDMSO
Br
H2N
N
N
H2N
N
N
H2N
Br
N
N
Zn, I2 (5 mol%)
Pd(Cl)2(PPh3)2
11
NBS
Br
1. n-BuLi, ZnCl2
Br
2. Pd(Cl)2(PPh3)2
10
5
6
HO
HO
O
O
H2N
N
N
OEt
OEt
N
N
12
N
H
OTBDMS
aq HCl, EtOH, reflux
OH
8
1
Scheme 3 Improved synthesis of coelenterazine
Zinc was inserted directly into the C–Br bond of benzyl ry zinc into aryl bromide using zinc powder together with
bromide to form the alkylzinc reagent, followed by a zinc pellets was unsuccessful, we followed the approach
Negishi cross-coupling reaction using bis(triphenylphos- to zincation proposed by Buchwald and Milne (Scheme
phine)palladium(II) chloride as catalyst. This coupling re- 3).27 First, TBDMS-protected bromophenol 11 was treat-
action yielded 3-benzyl-5-bromopyrazin-2-amine (6) in ed with n-butyllithium to create the corresponding aryl-
more than 98% isolated yield following column chroma- lithium organometallic compound in situ. Second, the
tography, in addition to a minor fraction of bis-coupling aryllithium compound was treated with anhydrous zinc
product (3,5-dibenzylpyrazin-2-amine).
chloride to create the arylzinc chloride. The resulting ar-
ylzinc chloride was cross-coupled with the aryl bromide 8
using bis(triphenylphosphine)palladium(II) chloride as
the catalyst.
There was no evidence of homocoupling, indicating that
the zinc insertion was quantitative even when commercial
zinc powder, together with zinc pellets, was used during
the scale-up experiments. Iodine (5 mol%) was used to ac- The other component, which is required for the final con-
tivate the zinc surface. Iodine creates a reactive zinc sur- densation step to form coelenterazine, is the acetal-pro-
face by oxidizing zinc to zinc(II) and becoming reduced to tected carbonyl compound 9.19d TBDMS Protection of 4-
iodide.26 Iodide can further facilitate the oxidative zinc in- hydroxybenzaldehyde 13 was not difficult, but the haloge-
sertion by converting benzyl bromide into the more reac- nation of the protected benzyl alcohol 14 proved to be
tive benzyl iodide.
very problematic: The bromo compound proved to be
very unstable, and the chloro compound 15 is volatile, hy-
groscopic, and thermally unstable.28 Both chlorination by
using methanesulfonyl chloride and bromination via the
carbon tetrabromide/triphenylphosphine route19g were un-
successful. Chlorination did not yield any measurable
quantity of product. Chlorination using concentrated hy-
drochloric acid29 and thionyl chloride29 were also unsuc-
cessful, because a water-free chloro-organic reaction
product 15 could not be obtained, thus impeding the sub-
sequent Grignard reaction. Cyanuric chloride–
dimethylformamide30 complex was found to be very help-
ful for this chlorination, but the yield of this reaction was
low (15%) and the chloro compound remained (intrinsi-
The next step was the second Negishi-type cross-coupling
reaction to obtain 3-benzyl-5-[4-(tert-butyldimethylsil-
oxy)phenyl]pyrazin-2-amine (8). Adamczyk et al. ex-
plored the Suzuki–Miyaura coupling reaction for the
synthesis of coelenterazine, using bis(triphenylphos-
phine)palladium(II) chloride as the catalyst (Scheme
2).19d
Synthesizing compound 8 from compound 6 using the
same palladium catalyst, but following a Negishi-type
cross-coupling approach will avoid the necessity of using
the boronic acid derivative 7 and will thus reduce the
number of required steps. Since the insertion of elementa-
Synthesis 2014, 46, 646–652
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Strategies for Large-Scale Synthesis of Coelenterazine
649
cally) volatile (Scheme 4). Changing the protection group
of 4-hydroxybenzyl alcohol 14 from TBDMS to the ben-
zyl group proved to be successful (Scheme 5). Benzyl pro-
tection has distinct advantages: both benzyl protection
and deprotection reactions were virtually quantitative and
easy to carry out; the benzyl-protected compound is a sol-
id and easy to dry; and the benzyl ether 18 is thermally sta-
ble during chlorination reaction, even when performed on
a gram scale. In addition, the benzyl chloride 19 is easy to
dry, which is required for the subsequent Grignard reac-
tion, because compound 19 is a solid and has a much low-
er vapor pressure. The benzyl group was then removed by
catalytic hydrogenation31 before the final condensation
step to yield the unprotected 1,1-diethoxy-3-(4-hydroxy-
phenyl)propan-2-one (12), because the phenolic group is
not a problem under acidic condensation reactions.
Cl
N
Cl
O
N
N
OEt
OH
OH
Cl
EtO
OEt
Mg, THF
Cl
BnBr, K2CO3
DMF, CH2Cl2
OH
OBn
18
OBn
19
17
BnO
HO
Pd/C (10%), H2
r.t., 12 h
O
O
OEt
OEt
OEt
OEt
20
11
Scheme 5 Improved synthesis of 1,1-diethoxy-3-(4-hydroxyphe-
nyl)propan-2-one) (11)
OH
Cl
O
In Scheme 6, the isolated yields of all synthesized com-
pounds and the final product coelenterazine (1) are sum-
marized. For the branch from 3,5-dibromopyrazin-2-
amine (5) to 3-benzyl-5-[4-(tert-butyldimethylsil-
oxy)phenyl]pyrazin-2-amine (8), the molar conversion ef-
ficiency is 70%; for the second branch from 4-
hydroxybenzyl alcohol (17) to 1,1-diethoxy-3-(4-hy-
droxyphenyl)propan-2-one 12, we have achieved 51%.
The final step to give 1 has a good, 65%, yield.
1) 4-DMAP, imidazole
TBDMSCl, CH2Cl2
2) NaBH4
MsCl, Et3N
CH2Cl2
OH
OTBDMS
OTBDMS
13
14
15
O
OEt
EtO
OEt
16
Mg, THF
TBDMSO
Since the synthesis of coelenterazine in amounts that are
required for in vivo imaging purposes is often prohibitive-
ly expensive, the approach described here may make this
compound more accessible to the community of medici-
nal researchers. Furthermore, the coelenterazine synthe-
sized in two-gram batches is >99.6% pure (HPLC
analysis, Figure 1).
O
EtO
OEt
9
Scheme 4 Approach to 1,1-diethoxy-3-[4-(tert-butyldimethylsil-
oxy)phenyl]propan-2-one (9) followed by Adamczyk et al.19d
A new method of synthesis of coelenterazine has been de-
veloped. This synthetic route is able to deliver extremely
pure coelenterazine on a gram scale. It uses inexpensive
starting materials and catalysts. Aminopyrazine is used as
a building block for introducing the pyrazine moiety of
coelenterazine. Negishi-type coupling reactions are used
to couple two different aromatic rings to the pyrazine moi-
The final condensation reaction between compound 12
and compound 8 in hydrochloric acid and ethanol led to
coelenterazine (1) in good yield. This reaction proved to
be unproblematic during scale-up. At the same time as the
final condensation, the protection group was removed
quantitatively in the acidic medium.
Figure 1 Reverse-phase HPLC chromatogram of coelenterazine (1), λabs = 256 nm (100% MeOH)
© Georg Thieme Verlag Stuttgart · New York
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650
T. B. Shrestha et al.
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13C NMR (CDCl3): δ = 123.9, 124.1, 143.4, 152.1.
MS: m/z [M] calcd for C4H3Br2N3: 252.87; found: 251.1 [M – 1]+.
H2N
N
N
HO
O
OEt
OEt
3-Benzyl-5-bromopyrazin-2-amine (6)
+
Zn dust and 30-mesh granular zinc (1:1, 1.605/1.605 g, 49.12 mmol,
3.5 equiv) and I2 (623 mg, 5% mol of Zn) were added to a dry 100-
mL 2-necked round-bottom flask under an argon atmosphere. N,N-
Dimethylacetamide (20 mL, freshly distilled over CaH2) was added
using a syringe. The mixture was stirred at r.t. until the brown color
of the I2 disappeared. Then, freshly distilled BnBr (6.104 g, 35.69
mmol, 2.5 equiv) was added using a syringe, and the mixture was
stirred at 80 °C for 3 h. After insertion of Zn, the mixture was cooled
to r.t. and the suspension of 5 (3.600 g, 14.03 mmol, 1 equiv) and
PdCl2(PPh3)2 (504 mg, 0.712 mmol, 5% of pyrazine) in N,N-di-
methylacetamide (22 mL) was added. The mixture was stirred con-
tinuously for 5 d. Then the mixture was poured into H2O (100 mL)
and extracted with EtOAc (3 × 100 mL). The combined organic lay-
ers were washed with sat. brine (100 mL), dried (anhyd MgSO4),
and concentrated on a rotary evaporator. Purification by column
chromatography (silica gel, n-hexane–EtOAc, 2:1) gave 6 (3.705 g,
14.03 mmol, 98%) as a brown viscous oil; Rf = 0.35 (silica gel, n-
hexane–EtOAc, 2:1).
OTBDMS
12
8
HCl, EtOH
reflux
65%
HO
O
N
N
N
H
OH
1
Scheme 6 Isolated yields for the synthesis of coelenterazine (1) in
two-gram batches
IR (neat): 633, 923, 1072, 1118, 1220, 1388, 1423, 1604, 2921,
3023, 3060, 3203, 3317, 3438 cm–1.
1H NMR (CDCl3): δ = 4.07 (s, 2 H), 4.45 (s, 2 H), 7.20–7.32 (m, 5
H), 8.01 (s, 1 H).
13C NMR (CDCl3): δ = 41.1, 126.4, 127.5, 128.6, 129.3, 135.8,
141.9, 142.6, 152.2.
ety. The final condensation reaction to form coelentera-
zine has a very high yield and produced very pure
materials, which is required for in vivo applications in the
in situ photodynamic therapy of cancer using Gaussia lu-
ciferase as light-generating enzyme.
MS: m/z [M] calcd for C11H10BrN3: 264.12; found: 264 [M]+, 265
[M + 1]+.
3-Benzyl-5-[4-(tert-butyldimethylsiloxy)phenyl]pyrazin-2-
amine (8)
All other chemicals and solvents were ACS grade and used without
distillation. 400 MHz and 200 MHz Agilent Technologies NMR
spectrometers, and a Thermo Scientific Nicolet 380 FT-IR were
used in this study. Mass spectra were recorded using an Applied
Biosystems API-4000 triple quadrupole mass spectrometer with
electrospray and APCI sources. Coelenterazine-containing aqueous
solutions were analyzed using a Gilson/Hewlett Packard HPLC
workstation [Gilson 322 pump (Gilson, Middleton, WI); Hewlett-
Packard series 1100 detector; Agilent; RP-18 column, 4.6 mm di-
ameter (Agilent); MeOH–H2O, 90:10); flow rate: 0.5 mL/min; in-
jection volume: 30 μL]. CHN Analysis was performed using a Carlo
Erba elemental analyzer. The reaction products 1, 6, 8, 11, 12, 18,
and 19 were identical that reported by Adamczyk et al.19d However,
it must be noted that the synthetic strategy reported here is different
from the previously reported approach. Compound 20 is newly syn-
thesized.
A 250-mL round-bottom flask was dried overnight at 150 °C, al-
lowed to cool to r.t. under an argon atmosphere, and then filled with
(4-bromophenoxy)tert-butyldimethylsilane (11, 4.198 g, 14.62
mmol, 2.2 equiv) and THF (30 mL, distilled over Na in the presence
of benzophenone). A septum was used to block the influx of air. The
mixture was cooled to –78 °C, then 1.6 M n-BuLi in hexane (11.6
mL, 18.6 mmol, 2.8 equiv) was added dropwise by syringe. The re-
sulting solution was stirred at –78 °C for 1 h. ZnCl2 (2.715 g, 19.93
mmol, 3 equiv) in THF (25 mL) was added by syringe through the
septum. Again, the mixture was stirred for 0.5 h at –78 °C. Then the
mixture was allowed to warm up to r.t. and the solution was further
stirred for a further 1 h at r.t. A suspension of PdCl2(PPh3)2 (233 mg,
0.332 mmol, 5% mol) and 6 (1.754 g, 6.643 mmol, 1 equiv) in THF
(30 mL) was added and the mixture was stirred at r.t. [TLC moni-
toring (silica gel, n-hexane–EtOAc, 2:1): Rf = 0.55 (6), 0.40 (8), and
0.29 (byproduct)]. There was some starting material discernible af-
ter 5 h, but after 15 h there no starting material remained. There was
a minor fraction of byproduct, which was identified as the product
from the ‘unwanted coupling reaction’ [Rf = 0.29 (silica gel, n-hex-
ane–EtOAc, 2:1)], but the main fraction consisted of the product
[Rf = 0.40 (silica gel, n-hexane–EtOAc 2:1)]. The mixture was
poured into H2O (100 mL) and extracted with EtOAc (3 × 100 mL).
The combined organic extracts were washed with sat. brine (100
mL) and dried (anhyd Na2SO4). After drying the organic fraction,
EtOAc was removed using a rotary evaporator. Column chromatog-
raphy (hexane–EtOAc, 2:1) gave 8 (1.877 g, 4.793 mmol, 72%) as
a yellow solid.
3,5-Dibromopyrazin-2-amine (5)
Pyrazin-2-amine (9, 4.00 g, 42.1 mmol, 1.0 equiv) was completely
dissolved in DMSO (80 mL) in an argon-flushed, 500-mL round-
bottom flask, and then NBS (18.7 g, 26.3 mmol, 2.5 equiv) was add-
ed over 3 h. The mixture was stirred a further 1 h at r.t. The reaction
was complete within 4 h (TLC monitoring). The mixture was
poured into H2O (200 mL), extracted with EtOAc (3 × 200 mL), and
the combined organic layers were washed with H2O (3 × 200 mL),
sat. brine (100 mL), and H2O (2 × 200 mL). The organic solution
was dried (anhyd MgSO4) and concentrated by rotary evaporation.
Finally the product was purified by crystallization (EtOH) to give 5
(8.736 g, 34.94 mmol, 83%) as a pale yellow, needle-shaped, crys-
talline compound; Rf = 0.46 (silica gel, hexane–EtOAc, 2:1); mp
115–116 °C. Spectral results are in agreement with literature val-
ues.31
IR (neat): 728, 834, 918, 1170, 1237, 1251, 1454, 1519, 1609, 2851,
2933, 3145, 3284, 3485 cm–1.
1H NMR (400 MHz, CDCl3): δ = 0.22 (s, 6 H, 2 CH3), 1.005 [s, 9
H, C(CH3)3], 4.17 (s, 2 H, CH2), 4.37 (br s 2 H, NH2), 6.92 (d, J =
8.6 Hz, 2 H), 7.25–7.32 (m, 5 H), 7.81, (d, J = 8.6 Hz, 2 H), 8.32 (s,
1 H, HAr).
IR (neat): 635, 877, 907, 1040, 1096, 1133, 1314, 1450, 1506, 1548,
1620, 3150, 3168, 3280, 3448 cm–1.
1H NMR (CDCl3): δ = 5.11 (br s, 2 H), 8.04 (s, 1 H).
Synthesis 2014, 46, 646–652
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Strategies for Large-Scale Synthesis of Coelenterazine
651
13C NMR (400 MHz, CDCl3): δ = –4.1, 18.5, 25.9, 41.4, 120.7,
127.1, 127.2, 128.7, 129.1, 130.8, 137.0, 137.1, 140.6, 142.9, 151.6,
156.2.
13C NMR (400 MHz, CDCl3): δ = 46.5, 70.3, 115.3, 127.7, 128.3,
128.8, 130.3, 137.0, 159.1.
3-[4-(Benzyloxy)phenyl]-1,1-diethoxypropan-2-one (20)
Mg turnings (750 mg, 30.9 mmol, 4.5 equiv) were suspended in
freshly distilled THF (10 mL) in an argon-flushed, 50-mL two-
necked round-bottom flask. Dibromoethane (0.20 mL) was added to
activate the Mg. After 20 min of activation, a solution of 19 (1.603
g, 6.888 mmol) in THF (10 mL) was added and the mixture was
stirred at r.t. for 30 min. Then mixture was further refluxed for 1 h
to complete the reaction. The pale yellow Grignard reagent was al-
lowed to cool to r.t. and was then kept in an ice bath. Ethyl diethoxy-
acetate (16, 1.289 g, 7.329 mmol, 1.06 equiv) was dissolved in THF
(10 mL) in a separate 100-mL round-bottom flask under an argon
atmosphere and cooled to –78 °C. The Grignard reagent was trans-
ferred dropwise into the cooled flask over 10 min. The mixture was
then stirred for 1.5 h at –78 °C. The reaction was quenched with
H2O (100 mL) and further diluted with EtOAc (200 mL). The EtO-
Ac layer was washed with H2O (3 × 100 mL), followed by sat. brine
(2 × 100 mL). The organic layer was dried (anhyd Na2SO4) and the
solvent was evaporated by rotary evaporation. The viscous
Grignard product was further purified by column chromatography
(silica gel, 10% EtOAc–n-hexane) to give 20 (1.583 g, 4.823 mmol,
70%) as an oily colorless compound; Rf =0.4 (silica gel, n-hexane–
EtOAc, 10:1).
MS: m/z [M] calcd for C23H29N3OSi: 391.58; found: 391.8 [M]+,
392.8 [M + 1]+.
(4-Bromophenoxy)tert-butyldimethylsilane (11)
4-Bromophenol (8.00 g, 46.2 mmol, 1 equiv) and DMAP (0.564 g,
4.62 mmol) were dissolved in CH2Cl2 (120 mL) in a 250-mL round-
bottom flask at 0 °C. After 15 min, imidazole (4.720 g, 69.36 mmol,
1.5 equiv) and TBDMSCl (10.45 g, 69.36 mmol, 1.5 equiv) were
added and the mixture was stirred at 0 °C for a further 1 h. CH2Cl2
was removed using a rotary evaporator, and the resulting solid was
dissolved in Et2O (100 mL). The Et2O solution was first washed
with concd NH3 solution (2 × 100 mL), followed by H2O (3 × 100
mL), and finally brine (2 × 100 mL). Et2O was removed by rotary
evaporation to give 11 (13.2 g, 46.0 mmol, 99%) as a colorless liq-
uid.
1H NMR (400 MHz, CDCl3): δ = 0.16 (s, 6 H), 0.95 (s, 9 H), 6.69
(d, J = 8.8 Hz, 2 H), 7.29 (d, J = 8.8 Hz, 2 H).
13C NMR (400 MHz, CDCl3): δ = –4.3, 18.4, 25.8, 113.8, 122.1,
132.5, 155.1.
4-(Benzyloxy)benzyl Alcohol (18)
4-Hydroxybenzyl alcohol (17, 4.0 g, 32 mmol) and anhyd K2CO3
(22.3 g, 161 mmol, 5 equiv) were dissolved in anhyd DMF (80 mL)
in a 250-mL round-bottom flask. The suspension was purged with
argon and stirred for 30 min at r.t. Then BnBr (13.8 g, 80.6 mmol,
2.5 equiv) was added by syringe to the suspension and the mixture
was stirred for 20 h at r.t. Then mixture was filtered through celite
and the celite cake was washed with Et2O (100 mL). The filtrate was
washed with H2O (3 × 60 mL), followed by sat. brine (2 × 50 mL).
The Et2O solution was dried (anhyd MgSO4), and the solvent was
evaporated using a rotary evaporator and then under high vacuum
to give pure 18 (5.982 g, 27.95 mmol, 87%) as a white solid; Rf =
0.75 (silica gel, EtOAc–n-hexane, 1:1); mp 73–75 °C.
IR (neat): 696, 736, 837, 854, 863, 1026, 1062, 1097, 1162, 1176,
1242, 1381, 1454, 1511, 1607, 1731, 2874, 2975, 3030, 3048 cm–1.
1H NMR (400 MHz, CDCl3): δ = 1.25 (t, J = 7.3 Hz, 6 H), 3.57 (m,
2 H), 3.69 (m, 2 H), 3.84 (s, 2 H), 4.64 (s, 1 H), 5.05 (s, 2 H), 6.94
(d, J = 8.4 Hz, 2 H), 7.14 (d, J = 8.4 Hz, 2 H), 7.43–7.35 (m, 5 H).
13C NMR (400 MHz, CDCl3): δ = 15.4, 43.0, 63.5, 70.2, 115.5,
126.2, 127.7, 128.1, 128.8, 131.0, 132.0, 137.2, 157.94, 203.7.
MS: m/z [M] calcd for C20H24O2: 328.17; found: 329.2 [M + H]+,
351.4 [M + Na]+.
Anal. Calcd for C20H24O2: C, 73.15; H, 7.37; N, 0.0. Found: C,
73.08; H, 7.39; N, 0.02.
IR (neat): 612, 694, 739, 810, 994, 1237, 1380, 1509, 1585, 1605,
1723, 1171, 2864, 2913, 3051, 3060, 3321 cm–1.
1,1-Diethoxy-3-(4-hydroxyphenyl)-2-propan-2-one) (12)
Synthetic procedure: The deprotection of the benzyl group of com-
pound 20 was carried out by reduction using H2 on a Pd/C catalyst.
Compound 20 (1.533 g, 4.671 mmol) was dissolved in MeOH (50
mL) in a 100-mL round-bottom flask. 10% Pd/C (150 mg) was add-
ed. At first the resulting suspension was put under vacuum, and then
H2 gas was passed into the round-bottom flask from a balloon. The
mixture was stirred for 24 h under a H2 atmosphere [TLC monitor-
ing (silica gel, n-hexane–EtOAc, 1:10) Rf = 0.4 (20), 0.16 (12)]. The
black suspension was filtered and the solvent was removed using a
rotary evaporator. The deprotected product was further purified by
column chromatography (silica gel, 50% EtOAc–hexane) to give 12
(1.077 g, 4.519 mmol, 96%) as a colorless oil.
1H NMR (400 MHz, CDCl3): δ = 4.63 (d, J = 5.9 Hz, 2 H), 5.08 (s,
2 H), 6.98 (d, J = 8.7 Hz, 2 H), 7.30 (d, J = 8.7 Hz, 2 H), 7.43–7.37
(m, 5 H).
13C NMR (400 MHz, CDCl3): δ = 65.3, 70.3, 115.20, 127.7, 128.2,
128.9, 129.4, 133.6, 137.1, 158.6.
4-Benzyloxybenzyl Chloride (19)
The chlorination of benzyl alcohol 18 was efficiently carried out by
employing the cyanuric chloride and DMF complexation method.30
Cyanuric chloride (4.940 g, 24.70 mmol) was dissolved in anhyd
DMF (10.0 mL) in a two-necked 250-mL round-bottom flask. The
solution was stirred at r.t. under an argon atmosphere. After stirring
for 30 min at r.t., the white solid of the cyanuric chloride–DMF
complex was formed. Then 4-benzyloxybenzyl alcohol (18, 4.653
g, 24.74 mmol) in CH2Cl2 (70 mL) was added at in one portion us-
ing a syringe to the white solid. The mixture was then stirred at r.t.
[TLC monitoring (silica gel, n-hexane–EtOAc, 10:1) Rf = 0.09 (18),
0.63 (19)]. Chlorination of benzyl alcohol was complete after 4 h.
After the completion of the reaction, the mixture was diluted to 200
mL with CH2Cl2. The white turbid suspension was filtered through
Celite, followed by washing the Celite cake with CH2Cl2 (50 mL).
Solvent was removed from the filtrate by rotary evaporation. This
procedure yielded a white solid. The white solid was subjected to
column chromatography (silica gel, 10% EtOAc–hexane) to give 19
(4.790 g, 20.58 mmol, 89%) a fluffy white solid; mp 74–76 °C.
IR (neat): 517, 825, 1050, 1166, 1221, 1446, 1520, 1539, 1659,
1728, 2884, 2993, 2974, 3403 cm–1.
1H NMR (400 MHz, CDCl3): δ = 1.25 (t, J = 7.0 Hz, 6 H), 3.55 (m,
2 H), 3.71 (m, 2 H), 3.82 (s, 2 H), 4.64 (s, 1 H), 5.11 (br s, 1 H), 6.77
(d, J = 8.6 Hz, 2 H), 7.07 (d, J = 8.6 Hz, 2 H).
13C NMR (400 MHz, CDCl3): δ = 15.4, 43.1, 63.6, 102.4, 115.7,
125.9, 131.1, 154.8, 204.1.
MS: m/z [M] calcd for C13H18O4: 238.28; found: 261.3 [M + Na]+.
Coelenterazine [8-Benzyl-6-(4-hydroxyphenyl)-2-[(4-hydroxy-
phenyl)methyl]imidazo[1,2-a]pyrazin-3(7H)-one, 1]
The final condensation step was adapted from a published meth-
od.19d Compound 12 (0.200 g, 0.839 mmol) was dissolved in de-
gassed EtOH (4.0 mL) (N2, argon, freeze–pump–thaw) in a 50-mL
two-necked flask. Then concd (38%) HCl (0.40 mL) was dissolved
in H2O (1.0 mL) and added to the flask. Finally, a solution of 8
IR (neat): 609, 660, 741, 834, 1004, 1171, 1241, 1380, 1512, 1580,
1609, 2868, 2696, 2933, 3027 cm–1.
1H NMR (400 MHz, CDCl3): δ = 4.57 (s, 2 H), 5.08 (s, 2 H), 6.96
(d, J = 8.7 Hz, 2 H), 7.32 (d, J = 8.7 Hz, 2 H), 7.43–7.37 (m, 5 H).
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, 646–652

652
T. B. Shrestha et al.
PAPER
(0.328 g, 0.891 mmol) in EtOH (4.0 mL) was added into the mixture,
which was then refluxed for 8 h under an argon atmosphere. A dark
brown solution was obtained that was allowed to cool to r.t., and the
solvent was then removed by rotary evaporation. The resulting dark
brown solid was subjected to column chromatography (silica gel,
10% MeOH–CH2Cl2) yielding coelenterazine (1) (0.230 g, 0.543
mmol, 65%); Rf = 0.42 (silica gel, CH2Cl2–MeOH, 10:1).
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IR (neat): 631, 695, 834, 1031, 1099, 1170, 1233, 1339, 1368, 1451,
1508, 1552, 1610, 2851, 2917, 3056, 3170 cm–1.
1H NMR (CD3OD): δ = 4.06 (s, 2 H), 4.39 (s, 2 H), 6.68 (d, J = 8.6
Hz, 2 H), 6.86 (d, J = 8.6 Hz, 2 H), 7.14 (d, J = 8.4 Hz, 2 H), 7.18–
7.22 (m, 2 H), 7.29–7.25 (m, 2 H), 7.38 (d, J = 7.4 Hz, 2 H), 7.48
(d, J = 8.8 Hz, 2 H), 7.62 (s, 1 H).
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MS: m/z [M] calcd for C26H21N3O3: 423.15; found: 424.3 [M + H]+,
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446.3 [M + Na]+.
Anal. Calcd for C26H21N3O3: C, 73.74; H, 5.00; N, 9.92. Found: C,
73.61; H, 5.03; N, 9.85.
Acknowledgment
This research is based upon work supported by the National Science
Foundation under Award No. CBET 933701.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.SnuIomprifgtooatrinSugpIoi
n
nfomartit
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Synthesis 2014, 46, 646–652
© Georg Thieme Verlag Stuttgart · New York