Dihalogen and Solvent-Free Preparation of syn- Bimane

Article abstract of DOI:10.1055/s-0036-1591964

Fluorescent bimanes are low molecular weight and low toxicity molecules with applications ranging from biology to LASER dyes. The widespread use of these molecular probes has presumably been stalled by the hazards involved in their current synthetic preparation which involve handling of dangerous halogens like chlorine (gas) and bromine (liq.). The accessibility achieved by the simple and safe dihalogen and solvent-free methodologies described here open the floodgates to additional future practical applications of bimanes.

Full text of DOI:10.1055/s-0036-1591964

SYNLETT
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6
© Georg Thieme Verlag Stuttgart · New York
2018, 29, A–D
letter
A
en
I. Neogi et al.
Letter
Synlett
Dihalogen and Solvent-Free Preparation of syn-Bimane
Cl
N
Ishita Neogi
O
O
Partha J. Das
N
N
O
Cl
Cl
Flavio Grynszpan*
0
0
0
0
0-
0
0
3
2-
4
4
5
5-
4
1
6
K2CO3
Cl2 (gas)
O
O
O
O
Cl
H
Cl
O
solvent-free
solvent-free
N
N
Department of Chemical Sciences, Ariel University, Ariel 40700,
NH
NH
NH
Israel
N
N
N
neogiis@ariel.ac.il
parthajy.das@msmail.ariel.ac.il
flaviog@ariel.ac.il
TANDEM
SAFER CHEMISTRY
Received: 20.12.2017
Accepted: 26.02.2018
Published online: 21.03.2017
DOI: 10.1055/s-0036-1591964; Art ID: st-2017-b0911-l
um carbonate to afford a mixture of the bicyclic anti- and
syn-bimanes. The thermodynamically more stable anti iso-
mer 4 is considered nonfluorescent. In contrast, the desired
syn-bimane 3 is a highly fluorescent material that can be
isolated in good yield using a combination of column chro-
matography and crystallization steps. Unfortunately, the
preparation of the key chloropyrazolinone intermediate 2 is
somehow hazardous and technically nontrivial. Following
Carpino’s procedure a solution of a pyrazolinone 1, ob-
tained from the reaction of a β-keto ester and hydrazine, in
DCM or 1,2-dichloroethane is chlorinated by chlorine (gas)
which can be generated by slowly pouring HCl (C) onto KMnO4
or used from a chlorine tank kept in the hood (Scheme 1).10
Abstract Fluorescent bimanes are low molecular weight and low toxic-
ity molecules with applications ranging from biology to LASER dyes.
The widespread use of these molecular probes has presumably been
stalled by the hazards involved in their current synthetic preparation
which involve handling of dangerous halogens like chlorine (gas) and
bromine (liq.). The accessibility achieved by the simple and safe dihalo-
gen and solvent-free methodologies described here open the flood-
gates to additional future practical applications of bimanes.
Key words bimane, fluorescent, monobromo-bimane, molecular
economy, easy and safe synthesis
O
The first high-yielding synthetic preparation of the fluo-
rescent syn-9,10-dioxabimanes was achieved by Prof. E.
Kosower and co-workers about four decades ago.1,2 This
procedure has been the only one in use until today. Since
their inception, bimane derivatives have found numerous
applications ranging from biological tags to laser dyes.3–7
Bimane’s high quantum yield combined with its low molec-
ular weight as well as its relatively low toxicity represent
some of its advantages as biolabels over other currently
more popular fluorescent probes. Recently, we showed that
a syn-bimane adduct of α-aminobutyric acid can cross a
rodent’s blood-brain barrier in vivo.8 In a methanol solu-
tion of syn-(Me,Me)bimane (3), for instance, absorbs light
at λex = 356 nm and emits at λem = 458 nm (in MeOH). Upon
chalcogen-donor complexation with Pd(II) the fluorescence
of this fluorophore can be repressed.9 Unfortunately, the
huge potential for the development of practical applica-
tions of syn-bimane derivatives has been hindered by the
hazards involved in the single currently available synthetic
procedure.2 The crucial step in the construction of the bi-
mane scaffold is reportedly very simple. A given chloropyr-
azolinone reacts in the presence of solid hydrated potassi-
NH2NH2
EtOH
H
O
O
NH
O
N
1
O
O
O
Cl2 (g)
N
N
O
3
Cl
K2CO3
NH
N
+
N
N
2
O
4
Scheme 1 Kosower’s synthesis of bimane
According to the Canadian Center for Occupational
Health and Safety, chlorine gas is a green-yellow gas with
pungent odor. It may explode in the presence of oxidizable
materials. It may cause or intensify fire. Chlorine is highly
reactive, therefore incompatible with many common chem-
icals. Chlorine is very toxic, fatal if inhaled. This halogen is
© Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, A–D

B
I. Neogi et al.
Letter
Synlett
corrosive to the respiratory tract. A severe, short-term ex-
posure may cause long-term respiratory effects. Chlorine
causes severe skin burns and eye damage.11 Definitely not
suitable for the faint-of-heart. Chlorine gas should be han-
dled with extreme care making this chlorination step cum-
bersome specially for nonexpert organic chemists and jus-
tifies the relatively steep price of syn-(Me,Me)bimane (3)
and its derivatives when purchased from commercial
sources.
Presumably, the chlorination step occurs at the carbon α
to the carbonyl of 1 after tautomerization followed by
nucleophilic attack at the polarizing diatomic chlorine mol-
ecule (Scheme 2).
and trichloroisocyanuric acid (TCCA)17 were also explored
in an attempt to find an effective and easy-to-handle solid
reagent for the chlorination of 1. In all cases the solid chlo-
rination agent was added to a DCM (or CCl4) solution of 1 at
0 °C and stirred for about 8–12 h at room temperature. Af-
ter removal of the solvent, the CCl4 insoluble succinimide,
hydantoin, or cyanuric acid, respectively, were filtered out.
All the reactions afforded the desired product 2 in about
80% yield without the need for further purification. All
these procedures are safe and extremely easy to perform.
The use of trichlorocyanuric acid as chlorinating agent is fa-
vored by us due to the high yield and intrinsic atom econo-
my18 of the reagent (Scheme 3) which allows the use of just
1/3 equivalents. At this stage, instead of isolating compound
2 from the reaction mixture, it is possible to add K2CO3·1.5
H2O to it and isolate the desired fluorescent compound 3 in
about 75% yield (and about 20% 4) in a convenient one-pot
process. The scalability of this process was demonstrated
by starting the reaction from 0.5 g and 2 g of 1 with similar
overall yield. The yield of our tandem procedure is signifi-
cantly higher than the reported two consecutive steps2
(50.3% overall yield) and it has become our preferred route
to prepare bimanes.
OH
O
tautomerization
H
NH
NH
N
N
1
Cl2 (g)
δ–
Cl
δ+
Cl
H
O
O
Cl
NH
NH
N
N
2
+ HCl
O
O
O
O
Scheme 2 Hazardous key chlorination of pyrazolinone
H
Cl
NaOCl
N
N
NH
NH
N
N
We reasoned that other chlorenium (Cl+) sources, be-
sides the hazardous Cl2 molecule, may also react in the
same fashion in addition or instead to the expected N-chlo-
rination, affording the desired chloropyrazolinone 2 inter-
mediate.12 The first, and perhaps most trivial, source of Cl+
that comes to mind is NaOCl which is commonly found as a
circa 5% water basic solution (pH 12.6) in the form of bleach
for household use. Naively, the first thing we attempted was
the chlorination reaction of 1 using lemon-scented ‘Solo
Bleach’ brand name which was found in the cleaning room
closest to the lab. To our delight, a yellowish product was
readily observed and could be isolated after stirring the re-
action solution at room temperature for 72 h. The isolated
product was not the initially expected intermediate 2 but
the fluorescent bicyclic 3, which was generated under the
existing basic reaction conditions in relatively low yield. Re-
portedly, the highest yields of fluorescent 3 can be obtained
using the heterogeneous K2CO3·1.5 H2O in an organic sol-
vent.2 Encouraged by this positive result we decided to ex-
plore the reaction of 1 with a different hypochlorite deriva-
tive. tert-Butyl hypochlorite is soluble in organic solvents
and can be easily prepared by reacting tert-butyl alcohol
with NaOCl.13 Thus, tert-butyl hypochlorite was added
dropwise to a solution of 1 in CCl4 at 0 °C under an N2 atmo-
sphere and then stirred at room temperature for 8 h to af-
ford the desired intermediate 2 in high yield.14 Three addi-
tional chlorenium sources, namely N-chlorosuccinimide
(NCS),15 1,3-dichloro-5,5-dimethylhydantoin (DCDMH),16
1
2
3
+ 4
t-BuOCl
K2CO3
Cl
N
O
O
O
H
O
Cl
NH
NH
N
N
O
Cl
N
1
2
solvent-
free
N
Cl
O
Cl
N
O
O
N
N
O
H
O
Cl
Cl
Cl
O
NH
NH
N
N
solvent-free
1
2
Scheme 3 Safe and easy access to the key chloropyrazolinone interme-
diate 2 and bimanes using either sodium hypochlorite, tert-butyl hypo-
chlorite, NCS, DCDMH, and TCCA
Organic solvents represent most of the waste produced
by regular synthetic procedures. The use of benign solvents
like water or no solvent at all, even at the expense of a
slightly lower yield, is a central premise while trying to
achieve environmentally responsible chemical processes. In
this case, grinding 1 (previously heated over its reported
melting point of 56 °C) and solid trichlorocyanuric acid at
© Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, A–D

C
I. Neogi et al.
Letter
Synlett
room temperature, in the absence of any additional solvent,
for 15 min affords the desired product in 69% yield (the
same yield was obtained after grinding for 30 min).19 The
fluorescent bicyclic product 3 can then be prepared as re-
ported, via the reaction of 2 in the presence of K2CO3·1.5 H2O
in an heterogeneous system.2
es. We hope this significant improvement will contribute to
uplift the bimane family of fluorophores to a more prepon-
derant place within the arsenal of available fluorescent
probes, where it belongs.
Acknowledgment
Since the chloropyrazolinone intermediate is a liquid at
room temperature we decided to attempt this step in the
absence of any additional solvent. Indeed, the solvent-free
formation of the bicyclic bimane scaffold proceeds smooth-
ly affording syn-bimane in 73% yield.20 This yield is compa-
rable to that reported in the literature. The solvent-free
procedure is very convenient, this reaction ends in less than
5 min and, in contrast to the previously reported procedure
in solution, the resulting mixture of bimanes can be easily
separated from K2CO3 simply by solubilization of the bi-
cyclic compounds in DCM. These two solvent-free steps can
be carried out in tandem by just adding solid potassium
carbonate to the same mortar where liquid 2 is obtained,
without isolation of the intermediate. Following this ‘green’
approach, 3 can be isolated in only 38% overall yield for the
combined two steps. It must be noted that all the described
solvent-free reactions are exothermic and should be per-
formed in a ventilated hood with care.
We thank Dr. K.N. Parida for useful discussions. F.G. is incumbent of
the Pamela and Bard Cosman endowment for organic chemistry re-
search. F.G. thanks the R&D authority of Ariel University for partial
financial support.
References and Notes
(1) Kosower, E. M.; Pazhenchevsky, B.; Hershkowitz, E. J. Am. Chem.
Soc. 1978, 100, 6516.
(2) Kosower, E. M.; Pazhenchevsky, B. J. Am. Chem. Soc. 1980, 102,
4983.
(3) Radkowsky, A. E.; Kosower, E. D. J. Am. Chem. Soc. 1986, 108,
4527.
(4) Lau, C. M.; Thangaraj, K.; Kumar, G.; Ramakrishnan, V. T.;
Stevens, E. D.; Boyer, J. H.; Politzer, I. R.; Pavlopoulos, T. G. Het-
eroat. Chem. 1990, 1, 195.
(5) (a) Mansoor, S. E.; Farrens, D. L. Biochemistry 2004, 43, 9426.
(b) Skjold-Jørgensen, J.; Vind, J.; Svendsen, A.; Bjerrum, M. J. Eur.
J. Lipid Sci. Technol. 2016, 118, 1644.
To take advantage of bimane 3 as a fluorescent probe it
is necessary to convert into an alkylating agent such as
monobromo bimane 5. Conveniently, the fluorescence of 5
is very weak but most of it is recovered when it reacts with
thiol nucleophiles.21 Reportedly, the bromination of a β-alkyl
H of 3 can be achieved via the reaction of 3 with Br2 (liq.).2
Handling liquid bromine may be considered less dangerous
than working with chlorine gas but in order to make the
whole reaction sequence safer and effective, we decided to
replace the use of liquid bromine by solid N-bromosuccin-
imide (NBS, Scheme 4).22 The reaction proceeds smoothly at
room temperature over a period of 12 h affording the de-
sired 5 in yields comparable to the obtained by the original
bromination procedure.
(6) (a) Sosa-Peinado, A.; González-Andrade, M. Biochemistry 2005,
44, 15083. (b) Nicholas, G. M.; Kováč, P.; Bewley, C. A. J. Am.
Chem. Soc. 2002, 124, 3492.
(7) (a) Smirnova, I.; Kasho, V.; Sugihara, J.; Kaback, H. R. Proc. Natl.
Acad. Sci. U.S.A. 2013, 110, 8876. (b) Chaudhuri, A.; Venkatesh,
Y.; Behara, K. K.; Singh, N. D. P. Org. Lett. 2017, 19, 1598.
(8) Lapidot, I.; Baranes, D.; Pinhasov, A.; Gellerman, G.; Albeck, A.;
Grynszpan, F.; Shatzmiller, S. E. Med. Chem. 2016, 12, 48.
(9) Das, P. J.; Diskin-Posner, Y.; Firer, M.; Montag, M.; Grynszpan, F.
Dalton Trans. 2016, 45, 17123.
(10) Carpino, L. A. J. Am. Chem. Soc. 1958, 80, 599.
(11) Canadian Centre for Occupational Health
&
Safety,
https://www.ccohs.ca/oshanswers/chemicals/chem_pro-
files/chlorine.html.
(12) De Luca, L.; Giacomelli, G.; Nieddu, G. Synlett 2005, 223.
(13) Mintz, J.; Walling, C. Org. Synth., Coll. Vol. V 1973, 148.
(14) Synthesis of 3,4-Dimethyl-4-chloro-2-pyrazolin-5-one (2) by
Chlorination of 3,4-Dimethyl-2-pyrazolin-5-one (1) Using
tert-Butyl Hypochlorite
O
O
O
O
NBS
N
N
N
N
3,4-Dimethyl-2-pyrazoline-5-one (1, 0.49 g, 4.37 mmol) was
dissolved in 8 mL of CCl4 under a nitrogen atmosphere. The
solution was cooled to 0 °C. tert-Butyl hypochlorite (0.49 mL,
4.37 mmol, CAS 507-40-4) was slowly added to the reaction
mixture dropwise. The reaction mixture was stirred at rt. After
stirring for 8 h, the solvent was removed using under reduced
pressure at 40 °C, yielding 0.60 g the product 2 (93% yield). 1H
rt, 12 h
Br
3
5
Scheme 4 Use of N-bromosuccinimide in the modified preparation of
monobromo bimane
NMR (CDCl3, 400 MHz): δ = 1.68 (s, 3 H), 2.12 (s, 3 H) ppm. 13
C
In summary, we have shown that fluorescent syn-
(Me,Me)bimane and its reactive bromo derivative can be
prepared using our very simple-and-safe procedures.23 The
methodology presented herein circumvents the need of di-
rect handling of hazardous chlorine (gas) and bromine
(liq.), it draws closer to the concepts of ‘green chemistry’
and can be performed successfully by any scientist with ba-
sic knowledge and experience in organic chemistry practic-
NMR (CDCl3, 100 MHz): δ = 12.6, 21.7, 60.0, 159.9, 173.8 ppm.
ESI-MS: m/z calcd for C5H8ClN2O: 147.0325 [MH+]; found:
147.0352.
(15) Synthesis of 3,4-Dimethyl-4-chloro-2-pyrazolin-5-one (2) by
Chlorination of 3,4-Dimethyl-2-pyrazolin-5-one (1) Using N-
Chlorosuccinimide
3,4-Dimethyl-2-pyrazoline-5-one (1, 0.5 g, 4.45 mmol) was dis-
solved in 10 mL of DCM. The solution was cooled to 0 °C. NCS
© Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, A–D

D
I. Neogi et al.
Letter
Synlett
(0.59 g, 4.45 mmol, CAS 128-09-6) was added to the reaction
mixture over a period of 30 min. The reaction mixture was
stirred at rt. After stirring for 12 h. The solvent was removed
using under reduced pressure at 40 °C. The resulting residue
was dissolved in CCl4, and filtered to remove solid succinimide
and yielding a filtrate. The solvent was removed from the fil-
(0.14 g, 0.59 mmol, CAS 87-90-1) was added to the fine powder
and grinding was continued. The progress of the reaction was
monitored by TLC. The reaction reached its completion after 15
min. Then, DCM was added to the reaction mixture. The insolu-
ble cyanuric acid was removed by filtration. The solvent was
removed under reduced pressure at 40 °C, yielding 0.18 g 3,4-
dimethyl-4-chloro-2-pyrazolin-5-one (2, 69% yield). 1H NMR
(CDCl3, 400 MHz): δ = 1.68 (s, 3 H), 2,12 (s, 3 H) ppm. 13C NMR
(CDCl3, 100 MHz): δ = 12.6, 21.7, 60.0, 159.8, 173.9 ppm. ESI-
MS: m/z calcd for C5H8ClN2O: 147.0325 [MH+]; found: 147.0352.
(20) Solvent-Free Synthesis of 9,10-Dioxa-syn-(Me,Me)bimane (3)
3,4-Dimethyl-4-chloro-2-pyrazolin-5-one (0.2 g, 1.36 mmol)
1
trate yielding 0.52 g the product 2 (80% yield). H NMR (CDCl3,
400 MHz): δ = 1.68 (s, 3 H), 2.12 (s, 3 H) ppm. 13C NMR (CDCl3,
100 MHz): δ = 12.6, 21.7, 60.0, 159.9, 173.8 ppm.
(16) Synthesis of 3,4-Dimethyl-4-chloro-2-pyrazolin-5-one (2) by
Chlorination of 3,4-Dimethyl-2-pyrazolin-5-one (1) Using
1,3-Dichloro-5,5-dimethylhydantoin
3,4-Dimethyl-2-pyrazoline-5-one (1, 0.5 g, 4.45 mmol) was dis-
solved in 10 mL of CCl4. The solution was cooled to 0 °C. 1,3-
Dichloro-5,5-dimethylhydantoin (0.44 g, 2.22 mmol, CAS 118-
52-5) was slowly added to the reaction mixture over a period of
30 min. The reaction mixture was stirred at rt. After stirring for
12 h, a solid precipitate was observed. The precipitate was
removed by filtration. The solvent was removed under reduced
pressure at 40 °C, yielding 0.50 g the product 2 (76% yield). 1H
was melted and added to a scintillation vial containing
K2CO3·1.5 H2O (0.945 mg, 5.71 mmol). The two components
were rapidly mixed with a spatula at rt. The progress of the
reaction was monitored by TLC. The reaction reached comple-
tion after 15 min. DCM was added to the reaction mixture in
five portions and the combined organic extract was evaporated
under reduced pressure. The reaction mixture was purified by
column chromatography eluting with hexane/ethyl acetate
(1:3). The desired 9,10-dioxa-syn-(Me,Me)bimane (3) was iso-
lated (0.096 g, 73% yield). 1H NMR (CDCl3, 400 MHz): δ = 1.80 (s,
3 H), 2.29 (s, 3 H) ppm. ESI-MS: m/z calcd for C10H13N2O2:
193.0977 [MH+]; found: 193.0992.
NMR (CDCl3, 400 MHz): δ = 1.68 (s, 3 H), 2.12 (s, 3 H) ppm. 13
NMR (CDCl3, 100 MHz): δ = 12.6, 21.7, 60.0, 159.8, 173.9 ppm.
C
(17) Synthesis of 3,4-Dimethyl-4-chloro-2-pyrazolin-5-one (2) by
Chlorination of 3,4-Dimethyl-2-pyrazolin-5-one (1) Using
Trichloroisocyanuric Acid
(21) Kosower, N. S.; Kosower, E. M. Methods Enzymol. 1987, 143, 76.
(22) Synthesis of Monobromobimane (5) by Bromination of syn-
(Me,Me)bimane (3) Using NBS
3,4-Dimethyl-2-pyrazoline-5-one (1, 0.5 g, 4.45 mmol) was dis-
solved in 10 mL of DCM. The solution was cooled to 0 °C. Tri-
chloroisocyanuric acid (0.34 g, 1.48 mmol, CAS 87-90-1) was
slowly added to the reaction mixture over a period of 30 min.
The reaction mixture was stirred at rt. After stirring for 12 h, a
solid precipitate of cyanuric acid was observed. The cyanuric
acid precipitate was removed by filtration. The solvent was
removed under reduced pressure at 40 °C, yielding 0.54 g the
syn-(Me,Me)bimane (3, 0.5 g, 2.6 mmol) was dissolved in 50 mL
acetonitrile. The reaction mixture was cooled to 0 °C. Recrystal-
lized NBS (0.46 g, 2.6 mmol, CAS. 128-08-5) was slowly
(pinchwise) added to the cooled reaction mixture over a period
of 30 min. After the addition of N-bromosuccinimide was com-
plete, the reaction mixture was stirred at rt for 12 h. The solvent
was then removed under reduced pressure. The residue was
purified using flash chromatography eluted with hexane/ethyl
acetate (1:1). The product 5 (0.35 g, 50% yield) was isolated. 1H
NMR (CDCl3, 400 MHz): δ = 1.84 (s, 3 H), 1.89 (s, 3 H), 2.45 (s, 3
H), 4.32 (s, 2 H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 6.9, 11.5,
17.8, 113.2, 115.4, 144.2, 146.0, 159.8, 160.7 ppm. ESI-MS: m/z
calcd for C10H12BrN2O2: 271.0082 [MH+]; found: 271.0067.
(23) Neogi, I.; Das, P. J.; Grynszpan, F. US provisional patent applica-
tion 62/597,448, filed December 12, 2, 2017.
1
product 2 (82% yield). H NMR (CDCl3, 400 MHz): δ = 1.68 (s, 3
H), 2.12 (s, 3 H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 12.6, 21.7,
60.0, 159.9, 173.8 ppm.
(18) Trost, B. M. Science 1991, 254, 1471.
(19) Solvent-Free Synthesis of 3,4-Dimethyl-4-chloro-2-pyra-
zolin-5-one (2) by Chlorination of 3,4-Dimethyl-2-pyrazolin-
5-one (1) Using Trichloroisocyanuric Acid
3,4-Dimethyl-2-pyrazoline-5-one (1, 0.2 g, 1.78 mmol) was
finely ground by mortar and pestle. Trichloroisocyanuric acid
© Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, A–D