Dienophilicity of imidazole in inverse electron demand Diels-Alder reactions: Cycloadditions with 1,2,4,5-tetrazines and the structure of zarzissine

Article abstract of DOI:10.1016/S0040-4020(01)00476-8

The inverse electron demand cycloadditions of 2-substituted imidazoles with dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate produced imidazo[4,5-d]pyridazines in good yields. This chemistry was applied to the synthesis of 2-amino-1H-imidazo[4,5-d]pyridazine (1), the structure reported for zarzissine, a cytotoxic marine alkaloid. Differences in the ¹H- and ¹³C NMR spectra of 1 with those reported for zarzissine necessitated a structural revision, and zarzissine was then considered to be the corresponding 2-amino-1H-imidazo[4,5-b]pyrazine (2), which was subsequently synthesized from the parent heterocycle.

Full text of DOI:10.1016/S0040-4020(01)00476-8

TETRAHEDRON
Pergamon
Tetrahedron 57 12001) 5497±5507
Dienophilicity of imidazole in inverse electron demand
Diels±Alder reactions: cycloadditions with 1,2,4,5-tetrazines and
the structure of zarzissine
Zhao-Kui Wan, Grace H. C. Woo and John K. Snyderp
Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, MA 02215, USA
Received 3 April 2001;accepted 30 April 2001
AbstractÐThe inverse electron demand cycloadditions of 2-substituted imidazoles with dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate
produced imidazo[4,5-d]pyridazines in good yields. This chemistry was applied to the synthesis of 2-amino-1H-imidazo[4,5-d]pyridazine
11), the structure reported for zarzissine, a cytotoxic marine alkaloid. Differences in the 1H- and 13C NMR spectra of 1 with those reported for
zarzissine necessitated a structural revision, and zarzissine was then considered to be the corresponding 2-amino-1H-imidazo[4,5-b]pyrazine
12), which was subsequently synthesized from the parent heterocycle. q 2001 Elsevier Science Ltd. All rights reserved.
1.Introduction
Imidazole is a fundamental structural unit of numerous
biologically active natural products,1 and its chemistry2
has been investigated extensively. Research into imidazole
chemistry has been quite comprehensive;single site func-
tionalizations and substitutions, as well as various annula-
tion strategies3 have been well documented. We have been
interested in the dienophilicity of heteroaromatic
compounds with latent enamine functionalities in inverse
electron demand Diels±Alder reactions, and the develop-
ment of this chemistry for synthesis, primarily concentrating
on indole,4 and to a lesser extent pyrrole5 and imidazole.
Scheme 1.
The dienophilic potential of imidazole has been largely
neglected except for two reports by Seitz6 using imidazole
between 4-aminoimidazoles and triethyl 1,3,5-triazine-
2,4,6-tricarboxylate leading the corresponding purines
testi®es to the interest in this area in the search for new
drug candidates.10 The cytotoxic marine natural product
zarzissine, deduced to be 2-amino-1H-imidazo[4,5-d]pyrid-
azine 11),11 became our initial target. Zarzissine was
reported to have a broad range of activities, being active
against S. aureus, the fungi Candida albicans and
C. tropicais, as well as against several tumor cell lines.
Given its simple structure, a straightforward synthetic
approach such as a cycloaddition strategy readily amenable
to analogue preparation became our objective.
derivatives as dienophiles in reactions with 1,2,4,5-tetra-
zines, and demonstrations by Horne of the cycloaddition
pathway of 2-aminoimidazole with its in situ generated
Schiff bases in condensation reactions with aldehydes.7 In
addition, our preliminary investigations into the reactions of
imidazoles with 1,2,4-triazines in both inter-8 and intra-
molecular9 cycloadditions leading to the preparation of
3-deazapurines, 8-deazapteridines, and 1,5-naphthyridines
have been communicated.
We have undertaken further investigations into the reactions
between imidazoles and 1,2,4,5-tetrazines as a method for
preparing imidazo[4,5-d]pyridazines as purine analogues
1Scheme 1). The recent report by Dang on the cycloaddition
Other imidazo[4,5-d]pyridazines have been the focus of
chemical and biological investigations,12 but previous
routes to this heterocyclic system can be rather tedious.
For example, the construction of the imidazo[4,5-d]pyrida-
zine system usually requires a 4,5-dicarbonylated imida-
zole13 or 4,5-diaminopyridazine14 as starting material
Keywords: imidazole;inverse electron demand Diels±Alder cyclo-
additions;zarzissine;imidazo[4,5- d]pyridazines;imidazo[4,5- d]pyrazine.
Corresponding author. Tel.: 1617-353-2621;fax: 1617-353-6466;
p
e-mail: jsnyder@chem.bu.edu
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020101)00476-8

5498
Z.-K. Wan et al. / Tetrahedron 57 +2001) 5497±5507
which require several steps to prepare. Seitz had shown that
imidazoles will undergo cycloadditions with 1,2,4,5-tetra-
zines to produce these condensed pyridazine systems, but
imidazole ring-opened products often dominated the
product mixture.6b We now report results examining the
scope of the dienophilicity of imidazoles with 1,2,4,5-tetra-
zines, and the synthesis of 1, whose NMR data differed
signi®cantly from that reported for zarzissine. Zarzissine
was then considered to be the corresponding 2-amino-1H-
imidazo[4,5-b]pyrazine 12), which was also synthesized.
187%), along with 1,4-dihydrotetrazine 74a 181%, Scheme
2). Presumably 7 arises from the dehydrogenation of the
initially formed dihydroadduct 5 by the extra equivalent
of 4a, as also found in the cycloadditions of indole with
4a.4a,20 Hydrolysis of 6a 1KOH, 3 equiv. in 1/1 aq MeOH)
gave unstable dicarboxylic acid 6b 186% crude yield),
which was immediately decarboxylated 1re¯uxing DMSO/
H2O, 1/1, v/v) to 2-phenyl-1H-imidazo[4,5-d]pyridazine 6c
175% from 6a).21 Alternatively, direct demethoxycarbonyl-
ation of 6a in re¯uxing HCl/HOAc 11/1, v/v)22 gave 6c in
quantitative yield.23
1
While the H NMR data for 2 in the protonated state were
quite similar to those reported for zarzissine, there were
some differences in the 13C NMR chemical shifts as well
as in the UV spectra.
With the feasibility of the inverse electron demand cyclo-
addition of 3c established as a route to 2-substituted
imidazo[4,5-d]pyridazines, the purported zarzissine
skeleton, protection of 3b was examined as a means to
coax productive dienophilicity from its chemistry with 4a.
Benzoylation of 3b following neutralization24 of its
commercially available sulfate salt gave a mixture of
2.Results and discussion
2.1. Synthesis of imidazo[4,5-d]pyridazines
mono and dibenzoylated products including the N1 and
0
N2 monoprotected 3d and 3e, respectively, whose ratio
varied considerably from batch-to-batch 1Scheme 3). Puri-
®cation of 3d and 3e by silica gel chromatography proved
impossible due to instability, but reaction of a crude mixture
of 3d and 3e with tetrazine 4a 12 equiv., THF, rt) gave the
desired zarzissine precursor 6d, the yield of which varied
with the amount of 3e in the benzoylation product mixture
130±56% from 3b).
Initial experiments examining the cycloadditions of imida-
zole 13a) and 2-aminoimidazole 13b) with dimethyl 1,2,4,5-
tetrazine-3,6-dicarboxylate 14a)15 were fruitless, yielding
only intractable mixtures of unstable compounds.
Attempted reactions of 3a and 3b with mono- and bis-
methylthiotetrazines 4b16 and 4c,17 respectively, were also
unproductive, giving an intractable mixture of products. The
parent, unsubstituted 1,2,4,5-tetrazine, the cycloaddition of
which with 3b would provide a direct route to 1, is a highly
volatile compound which was prepared18 in low overall
yield due to dif®culty in handling, so this was not
further pursued.19 Since Seitz had been successful in react-
ing 2-methylimidazole with 3,6-bis1tri¯uoromethyl)-
1,2,4,5-tetrazine,6b we turned to other 2-substituted imida-
zoles which might be better behaved in reactions with 4a.
2-Phenylimidazole 13c) reacted cleanly with tetrazine 4a
12 equiv.) in THF to give aromatized cycloadduct 6a
The conclusion was therefore reached that N1-benzoylated
3d did not undergo a cycloaddition with 4a, and that
cycloadduct 6d resulted solely from the reaction of 3e.
0
Acyl migration from N1 to N2 , which has been observed
previously in 1-acylated 2-aminobenzimidazoles,25 was
ruled out by control experiments. N1-Benzoyl-2-amino-
imidazole 13d) was ultimately obtained without any 3e by
variation of the benzoylation conditions, and indeed, 3d
failed to yield a cycloadduct with 4a. Furthermore,
resubjecting 3d to the benzoylation conditions without the
benzoyl chloride did not produce any 3e. It was therefore
concluded that N1-acylated imidazoles were signi®cantly
poorer dienophiles than the N1-unsubstituted compounds.
Seitz has reported the successful cycloaddition of N-methyl-
imidazole with both 4a6a and 3,6-bis1tri¯uoromethyl)-
1,2,4,5-tetrazine,6b so N-1 substitution alone is not respon-
sible for the lowered dienophilicity, which presumably is
also a consequence of the lowered LUMO of 3d. This low
reactivity of 3d contrasts with the excellent dienophilicity of
the corresponding N-benzoylindole in cycloadditions with
4a.4a
Interestingly, the 1H NMR spectrum of 6d 1CDCl3, rt)
showed two distinct, broad singlets for the methoxyl reso-
nances 1d 4.19 and 3.90, both s, 3H) indicative of relatively
slow equilibration of the imidazole proton between the N1
and N3 positions, presumably due to intramolecular hydro-
gen bonding with the amide carbonyl. Coalescence of these
resonances occurred at 508C 1d 4.05). In contrast, the
methoxyl resonances of 6a appeared as one sharp singlet
1d 4.10, s, 6H) indicative of more rapid equilibration.
Cycloadduct 6d was demethoxycarbonylated and
debenzoylated in one pot by re¯uxing in HCl/HOAc 11/1,
v/v, 3 h), followed by removal of the solvent in vacuo and
Scheme 2.

Z.-K. Wan et al. / Tetrahedron 57 +2001) 5497±5507
5499
Scheme 3.
basic work-up to give the desired 1 in a maximum 55%
overall yield from 3b.
only synthetic procedure found in the literature employs a
Hg1II) promoted condensation of phenylmethylamine with
1H-imidazo[4,5-b]pyrazin-2-ol,29 a procedure we wished to
avoid due to the use of mercury. Other efforts to prepare 2
were adapted from the numerous synthetic procedures avail-
able to prepare 2-aminobenzimidazoles3b,30 and related
systems which followed two general strategies: 11) conden-
sations of 2,3-diaminopyrazine,31 or 12) condensations of
2,3-dichloropyrazine.32 These efforts, all of which failed,
are summarized in Chart 1. The low reactivity of 2,3-dia-
minopyrazine in these condensations in comparison to
o-phenylenediamine can be ascribed to the electron
de®ciency of the pyrazine ring, reducing the nucleophilicity
of the amino groups. For example, the requisite intermediate
thioureas were not produced in the procedures described in
Items 1 and 5. Attempts to adapt Buchwald's nickel
catalyzed amination33 to 2,3-dichloropyrazine with N,N0-
di-Boc-N00tri¯ylguanidinium31e 1Item 10) were also
unsuccessful.
The dif®culty in preparing 3e in reproducible yield,
0
however, stimulated a search for another N2 -protecting
group for 2-aminoimidazole. Taylor has successfully
employed N,N-dimethylaminoimine protection of primary
amines in his pteridine and folate studies,26 prepared from
N,N-dimethylformamide dimethyl acetal.27 Adapting this
strategy to 2-aminoimidazole, protection of 3b gave
dimethylaminoimine 3f in 95% yield 1Scheme 4). The
cycloaddition between 3f and 4a proceeded smoothly
1even at 2788C!) to give 6e 186%). Simultaneous
demethoxycarbonylation and deprotection in re¯uxing
HCl/HOAc gave a quantitative yield of 1, in a highly repro-
ducible 75% overall yield from 3b.
The 1H- and 13C NMR spectra of synthetic 1, however, were
not in agreement with the data reported for natural zarzis-
sine, though the UV spectral data were similar.11 Of note,
the resonance of the aromatic proton 1H1/H4) of 1 1DMSO-
d6, d 8.89, s) was signi®cantly down®eld from that reported
for zarzissine 1DMSO-d6, d 8.09, s), though this low®eld
shift of synthetic 1 is typical of other imidazo[4,5-d]pyrida-
zines reported in the literature.28 The 13C chemical shifts of
synthetic 1 as well as its HCl salt were also signi®cantly
different from those reported for zarzissine.
While these attempts were not fruitful, the parent hetero-
cycle imidazo[4,5-b]pyrazine 18) was prepared from
commercially available 2,3-dichloropyrazine following
literature procedures32 1Scheme 5). Protection of 8 with
either MOMCl or BOMCl followed by imidazole deproto-
nation34 and subsequent addition of tosylazide following
Evans' examples for enolate azidation35 gave 10a and 10b
in good yields.36 In both cases, the desired azides were
accompanied by considerable amounts of dimer 11a and
11b,37 respectively, with the BOM protection being the
most effective in our hands. The amount of dimer could
be minimized by performing the reaction in toluene rather
than THF, and using a lithium base for the deprotonation.
Thus, in THF with KHMDS, dimer 11a was the sole product
from the reaction of 9a, isolated in 71% unoptimized yield.
When a freshly generated solution of LDA in THF was
added to a solution of 9a or 9b in toluene, only 16% and
11% yields of 11a and 11b, respectively, were obtained with
the dominant products being the desired azidoimidazo-
pyrazines 10a 157%) and 10b 169%), respectively. Trisyl
azide 12,4,6-triisopropylbenzenesulfonyl azide) failed as
the azide transfer reagent, and the use of the PNB or tri-
phenylmethyl 1trityl) protecting groups 19c and 9d,
respectively) also failed to give the desired azide, resulting
only in recovered imidazopyrazine after work-up.
Given the proton chemical shifts reported for zarzissine, a
more likely structure for zarzissine was thought to be
2-amino-1H-imidazo[4,5-b]pyrazine
12),
which
is
intuitively more satisfying from a biosynthetic viewpoint,
conceivably arising from two glycine molecules and a
guanidinium ion. A comparison of chemical shifts of related
imidazo[4,5-b]pyrazines
with
the
corresponding
imidazo[4,5-d]pyridazines indicated that the resonances of
the aromatic protons of the latter class of compounds appear
approximately 0.8 ppm down®eld from the corresponding
resonances of the former.28 The chemical shift of the sole
aromatic proton reported for zarzissine therefore falls in the
range of the corresponding imidazo[4,5-b]pyrazines,
0.8 ppm up®eld from that found in 1. We therefore set out
to prepare 2 to resolve the structure of zarzissine.
2-Amino-1H-imidazo[4,5-b]pyrazines are very rare. The
Scheme 4.

5500
Z.-K. Wan et al. / Tetrahedron 57 +2001) 5497±5507
Chart 1. Failed condensation reactions attempted to prepare 2. a References give citations from which procedures were adapted.
Reduction 1H2/Pd-C) of 10a and 10b to the amines 12a and
12b, respectively, and deprotection followed by basic work-
up gave the target 2 as the free base.
tivity of the 1H chemical shifts of 1 and 2 and their HCl salts
to concentration showed little variation 1Dd#0.1 ppm) from
0.0005±0.1 M. Varying amounts of water 1H2O) in the
DMSO-d6 also did not affect the chemical shifts.
Comparison of the NMR and UV data of 2 and its HCl salt
with that reported for zarzissine revealed that the chemical
shift of the sole nonexchangeable proton of the HCl salt
matched that reported for zarzissine, but the 13C chemical
shifts of the same sample, which had to be recorded at 508C
due to severe line broadening, and UV absorptions showed
distinct differences 1Table 1). An investigation of the sensi-
Horne has reported large 13C chemical shift differences
between neutral and protonated glycocyamidine derivatives
12-aminoimidazolines) with the guanidine carbons under-
going 12±17 ppm up®eld shifts upon protonation.38 Further-
more, the exact chemical shifts of the protonated
compounds proved to be very sensitive to the degree of
Scheme 5.

Table 1. Comparison of spectroscopic data for 1 and 2 with that reported for zarzissine
1
1´HCl
2
2´HCl
Zarzissinea
UV 1MeOH) l max 1log e)
1H NMRb
206 14.22), 230 1sh),
262 13.71), 308 1sh)
11.5 1bs, NH), 8.94 1bs,
2H), 6.97 1bs, NH2)
159.9, 138.8, 136.1
206 14.26), 225 1sh), 262
13.76), 306 12.84)
9.26 1bs, 2H), 8.20 1bs, NH3)
217 1sh), 244 13.30), 318 14.03)
217 1sh), 244 13.62), 318 14.39)
8.79 1bs, NH3), 8.09 1bs, 2H)
155.0, 141.6, 131.9c
206 14.61), 259 13.91)
11.74 1bs, NH), 7.78 1bs, 2H), 7.12 1bs,
NH2)
12.80 1bs, NH), 8.09 1bs,
2H), 7.11 1bs, NH2)
155.2, 152.4, 139.4
13C NMRb
161.9, 140.7, 132.8
158.7, 146.9, 133.4c
a
As reported in Ref. 11.
Recorded in DMSO-d6.
Recorded at 508C;two up®eld resonances were not observed at rt due to line broadening.
b
c

5502
Z.-K. Wan et al. / Tetrahedron 57 +2001) 5497±5507
protonation, and the identity of the counter anion 1chloride
vs acetate, for example, resulted in the differences of
.20 ppm as a consequence of the ability to form the
dihydrogen chloride salts with HCl, which was not possible
with acetic acid). Given the uncertainty over: 11) the exact
state of protonation of the isolated zarzissine, 12) the
presence of a possible counterion should the natural product
have been isolated in a protonated or partially protonated
state, and 13) the precise conditions under which the NMR
spectra of the natural product were recorded, reproducing
the spectral data becomes a near-impossible task without a
sample of zarzissine in hand. Unfortunately, neither an
authentic sample, nor authentic spectra of zarzissine were
made available for direct comparison. We believe the most
reasonable structure for zarzissine is 2, but the true identity
of zarzissine remains uncon®rmed.
exchanging solvents were identi®ed by the addition of
D2O. Infrared spectra were recorded on NaCl plates or as
KBr pellets. Solid samples were deposited on the NaCl plate
as a solution in an appropriate, volatile solvent 1typically
CH2Cl2) followed by evaporation of the solvent. Only diag-
nostic bands, such as NH, imine, and carbonyl stretching
bands, are reported.
Imidazole 13a, Aldrich), 2-aminoimidazole sulfate salt 13b,
Aldrich), 2-phenylimdazole 13c, Aldrich), and 1,2-dichloro-
pyrazine 1Pfaltz and Bauer) were were commercially avail-
able and used without further puri®cation. Tetrazines 4a,15
4b,16 and 4c17 were prepared according to literature pro-
cedures. All organic solvents were dried and distilled
according to standard procedures42 immediately before
use. Flash chromatography was performed using ¯ash silica
gel 143±60 mm).43
4.1.1. Dimethyl 2-phenyl-1H-imidazo[4,5-d]pyridazine-
4,7-dicarboxylate 16a). A solution of 2-phenylimidazole
13c, 74.3 mg, 0.5 mmol) and dimethyl 1,2,4,5-tetrazine-
3,6-dicarboxylate 14a, 198 mg, 1.0 mmol) in anhydrous
THF 18 mL) under argon was stirred at rt for 48 h, then
re¯uxed 4 h. The resultant orange solution was evaporated
in vacuo and the residue puri®ed by ¯ash chromatography
1EtOAc/hexanes, 1:1 to elute 7, then 2:1 to elute 6a) to give
6a 1140 mg, 87%) as yellow solid. Mp 142±1458C;IR
3.Conclusion
In summary, 2-substituted imidazoles have proven to be
good dienophiles in inverse electron demand Diels±Alder
reactions with 1,2,4,5-tetrazine 4a producing imidazo[4,5-
d]pyridazines in excellent yields. Applying this chemistry,
the structure reported for the marine cytotoxic agent
zarzissine, 2-amino-1H-imidazo[4,5-d]pyridazine 11), was
synthesized, which proved to have spectroscopic data
quite different from that reported for zarzissine. It was
then considered that the biosynthetically more appealing
2-amino-1H-imidazo[4,5-b]pyrazine 12) might be the
correct structure for zarzissine, and after considerable effort,
this compound was also synthesized. While the proton data
reported for zarzissine approximated that of protonated 2,
differences existed in the 13C chemical shifts and UV
1NaCl) 3400±3040, 1734 cm21 1H NMR 1acetone-d6,
;
400 MHz) d 12.5 1bs, NH), 8.45 1dd, Jˆ8.1, 1.7 Hz, 2H),
7.68±7.60 1m, 3H), 4.10 1s, 6H);1CDCl 3, 400 MHz) d 10.95
1bs, NH), 8.24 1dd, Jˆ8.0, 1.2 Hz, 2H), 7.63±7.56 1m, 3H),
4.18 1s, 6H); 13C NMR 1acetone-d6, 67.5 MHz) d 164.8,
159.7, 143.2, 139.6, 132.9, 129.7, 129.6, 128.9, 53.4;1in
CDCl3, 67.5 MHz, not all peaks were resolved);CIHRMS
1NH3, 140 eV) m/z 313.0936 1[M11]1, calcd for
C15H12N4O4, 313.0936);Dimethyl 1,3-dihydro-1,2,4,5-
tetrazine-3,6-dicarboxylate 74a was also isolated 181.3 mg,
81.3%).
spectra. Nevertheless, imidazo[4,5-d]pyridazines are of
general interest as purine analogues
12a,12b,12c,13,14,39
due to
a variety of biological activities including angiotensin II
antagonism40 and cardiotonic activity.28,41 This inverse elec-
tron demand cycloaddition chemistry of imidazole with
1,2,4,5-tetrazine 4a represents a quick and easy route to
this heterocyclic system.
4.1.2. 2-Phenyl-1H-imidazo[4,5-d]pyridazine-4,7-dicarb-
oxylic acid 16b). Diester 6a 145 mg, 0.144 mmol) was
added to a solution of KOH 135.9 mg, 0.6 mmol) in aqueous
MeOH 14 mL, 1:1, MeOH/H2O) and the solution was stirred
at rt for 2 h. The solution was then neutralized with 10%
HCl as monitored by pH paper, inducing precipitation. The
precipitate was collected by vacuum ®ltration and washed
with water 13£2 mL), CH2Cl2 12£2 mL), and dried in vacuo
to give 6b as a light gray solid 135.2 mg, 86%). Mp 143±
4.Experimental
4.1. General methods
Melting points were determined in capillaries and are
1
1458C 1dec.);IR 1KBr): 3400±3250, 1734 cm 21; H NMR
uncorrected; H- and 13C NMR spectra were recorded at
1
93.94 kG 11H 400 MHz, 13C 100 MHz) 70.5 kG 11H
300 MHz, 13C 75 MHz) and 63.41 kG 11H 270 MHz, 13C
67.5 MHz) in CDCl3, DMSO-d6, CD3OD, and acetone-d6
10.5 mL) as indicated. The d 7.24 resonance of residual
CHCl3, d 2.49 protio resonance of residual DMSO-d5, d
3.31 protio resonance of residual CHD2OD, and the d
2.05 protio resonance of residual acetone-d5 were used as
1DMSO-d6, 400 MHz), d 8.43 1dd, Jˆ7.9, 1.8 Hz, 2H),
7.64±7.56 1m, 3H);CIHRMS 1NH 3,140 eV) m/z 285.0610
1[M11]1, calcd for C13H9N4O4 285.0624).
4.1.3.
2-Phenyl-1H-imidazo[4,5-d]pyridazine
16c).
Method A: Diacid 6b 160 mg, 0.211 mmol) was dissolved
in aqueous DMSO 14 mL, 1:1, DMSO/H2O), and re¯uxed
for 8.5 h. The solvent was removed in vacuo and the product
puri®ed by ¯ash chromatography 1EtOAc/MeOH, 25:2) to
provide 6c 136 mg, 87%) as a white solid. Mp 250±2538C
1
internal references for the H spectra recorded in these
solvents, respectively. The center line of the 13CDCl3 triplet
1d 77.0), the center line of the CD3S1O)13CD3 septet 1d
39.5), the center line of the 13CD3OD septet 1d 49.2), and
the center line of the CD3C1O)13CD3 septet 1d 29.9) were
used as internal references for the 13C spectra, respectively.
All exchangeable proton resonances 1NH) in non-
1
1dec.);IR 1NaCl): 3400±3000 cm 21; H NMR 1DMSO-d6,
400 MHz) d 9.09 1s, 2H), 8.29 1d, Jˆ7.3 Hz, 2H), 7.37 1dd,
Jˆ7.3, 7.3 Hz, 2H), 7.28 1t, Jˆ7.3 Hz, 1H); 13C NMR
1DMSO-d6, 75 MHz): d 160.9, 140.5, 140.0, 130.7, 130.6,

Z.-K. Wan et al. / Tetrahedron 57 +2001) 5497±5507
5503
128.8, 127.4;CIHRMS 1butane, 140 eV) m/z 196.0749
1[M]1, calcd for C11H8N4 196.0749).
product was puri®ed by ¯ash chromatography 1CH2Cl2/
Et3N, 20:1) to give 3f as a white solid 1524.5 mg, 95%).
1
Mp 140±1428C;IR 1NaCl): 3101, 1627 cm 21; H NMR
Method B: Diester 5a 170 mg, 0.357 mmol) was dissolved in
HOAc and conc. HCl 16 mL, 1:1 HOAc/HCl), and re¯uxed
for 3 h. The solvent was removed in vacuo, then the residue
was redissolved in H2O 15 mL) and the solution neutralized
with 1 M aq KOH as monitored by pH paper. After evapora-
tion of solvent, the residue was loaded onto a short silica gel
column and eluted with EtOAc/MeOH 125:2) to give 6c as a
white solid 143.8 mg, 991%).
1CDCl3, 400 MHz) d 10.6 1bs, NH) 8.48 1s, 1H), 6.72 1s,
2H), 3.04 1s, 3H), 2.99 1s, 3H); 13C NMR 1CDCl3, 75 MHz)
d 155.9, 152.8, 119.0 1br), 40.4, 34.5;EIHRMS 170 eV), m/z
138.0916 1[M]1, calcd for C6H10N4 138.0905).
4.1.7. Dimethyl 2-[1dimethylamino)methylene]amino-
1H-imidazo[4,5-d]pyridazine-4,7-dicarboxylate
16e).
Protected 2-aminoimidazole 3f 164 mg, 0.46 mmol) and
tetrazine 4a 1115.4 mg, 0.583 mmol) under argon were
mixed in anhydrous THF at 2788C. The temperature was
allowed to slowly rise to rt over a 2 h period, then the
mixture was stirred overnight. The solvent was removed
in vacuo, then the product puri®ed by ¯ash chromato-
graphy 1EtOAc/MeOH, 10:1) to give 6e as a light
yellow solid 1121 mg, 86%). Mp 200±2028C;IR 1NaCl)
4.1.4. Dimethyl 2-benzoylamino-1H-imidazo[4,5-d]pyri-
dazine-4,7-dicarboxylate 16d). A solution of 2-amino-
imidazole sulfate 1681.1 mg, 2.5 mmol) was stirred in a
solution of sodium carbonate 1265 mg, 2.5 mmol) in water
13 mL) at rt for 15 min, then the water was evaporated in
vacuo. The residue was slurried with anhydrous ethanol and
®ltered through Celite, and the solvent was removed in
vacuo to give 2-aminoimidazole as an orange oil 1391 mg,
94%).24 To a solution of 2-aminoimidazole 133.2 mg,
0.4 mmol) in anhydrous THF 14 mL) was added Et3N
10.17 mL, 1.2 mmol), DMAP 14.9 mg, 0.04 mmol) and
benzoyl chloride 146.9 mL, 0.4 mmol), and the mixture
was stirred at rt for 4 h. The precipitate which formed was
removed by vacuum ®ltration, and the solvent removed in
vacuo. The residual yellow solid was dissolved in anhydrous
THF 15 mL), and tetrazine 4a 179.2 mg, 0.4 mmol) was
added. The reaction mixture was stirred at rt for 6 h under
argon. After evaporation of the solvent, the residue was
puri®ed by ¯ash chromatography 1CH2Cl2/MeOH, 25:2) to
give 6d 179.5 mg, 56%) as a yellow solid. Mp 217±2198C;
IR 1NaCl): 3362, 1726, 1702, 1680 cm21; 1H NMR 1CDCl3,
400 MHz): d 12.5 1bs, NH), 11.4 1bs, NH), 7.94 1dd, Jˆ7.9,
1.2 Hz, 2H), 7.61 1tt, Jˆ7.6, 1.2 Hz, 1H), 7.43 1dd, Jˆ7.9,
1
3300±3000, 1734, 1632 cm21; H NMR 1CD3OD, 400 Hz)
d 8.69 1s, 1H), 4.06 1s, 6H), 3.26 1s, 3H), 3.14 1s, 3H);
13C NMR 1CD3OD, 75 MHz, 508C) d 167.1, 165.6,
162.2, 140.9, 139.1, 53.5, 41.8, 35.6;CIHRMS 1butane,
140 eV) m/z 307.1179 1[M11]1, calcd for C12H14N6O4
307.1155).
4.1.8. 2-Amino-1H-imidazo[4,5-d]pyridazine 11). Method
A: Diester 6d 178 mg, 0.22 mmol) was dissolved in HOAc
and conc HCl 14 mL, 1:1), and re¯uxed for 3 h. The solvent
was removed in vacuo, and the residue redissolved in H2O
15 mL), then the solution was neutralized with 1 M aqueous
KOH as monitored with pH paper. After evaporation of the
solvent, the residue was loaded onto a short silica gel
column and eluted with EtOAc/MeOH 15:1) to give 1 as a
white solid 129 mg, 98%). Mp 220±2308C 1dec.);IR 1KBr):
3400 cm21;UV 1MeOH) lmax 1log e) 206 14.22), 230 1sh),
1
7.6 Hz, 2H);4.19 1bs, 3H);3.90 1bs, 3H);
1pyridine-d5, solubility in CDCl3 was too low for 13C
NMR, 67.5 MHz): 170.4, 165.2, 154.8, 141.1,
13C NMR
262 13.71), 308 1sh);1 1NaOH) 216 14.29), 272 13.66); H
NMR 1D2O, 400 MHz) d 8.80 1bs, 2H);1DMSO- d6,
400 MHz) d 11.5 1bs, NH), 8.94 1bs, 2H), 6.97 1bs, NH2);
13C NMR 1D2O, CD3OD as internal reference, 75 MHz) d
169.6, 145.9, 139.1 1DMSO-d6, 75 MHz) d 159.9, 138.8,
136.1;EIHRMS 170 eV) m/z 135.0535 1[M]1, calcd for
C5H5N5 135.0545). The HCl salt of 1 was made as follows.
To a solution of the free base was added 10% aq. HCl to
pH,1. The solvent was removed to give a white solid.
Mp.3008C 1dec);UV 1MeOH) lmax 1log e) 206 14.26),
225 1sh), 262 13.76), 306 12.84);1 1HCl) 210 14.05), 268
d
137.7, 134.2, 134.1, 129.7, 129.6, 53.4;CIHRMS 1NH ,
3
140 eV), m/z 356.0972 1[M11]1, calcd for C16H14N5O5
356.0995).
4.1.5. 2-Amino-N1-benzoylimidazole 13e). To a solution of
2-aminoimidazole 141.4 mg, 0.5 mmol) prepared as
described above for 6d24 in anhydrous THF 18 mL) was
added Et3N 10.21 mL, 1.5 mmol), then benzoyl chloride
152.8 mL, 0.45 mmol). The reaction mixture was stirred
overnight, then diluted with CH2Cl2 120 mL) and washed
with saturated aqueous NaHCO3 120 mL), then the organic
extracts dried over Na2SO4. No further puri®cation was
possible due to instability, and crude 3e, judged to be 95%
1
13.62), 292 13.71); H NMR 1DMSO-d6, 400 MHz) d 9.26
1bs, 2H), 8.20 1bs, NH3); 13C NMR 1DMSO-d6, 67.5 MHz,
508C) d 161.9, 140.7, 132.8.
Method B: Diester 6e 160 mg, 0.196 mmol) was dissolved in
HOAc and conc HCl 14 mL, 1:1), and re¯uxed for 3 h. The
solvent was removed in vacuo, and the residue redissolved
in H2O 14 mL), then the solution was neutralized with 1 M
aqueous KOH as monitored with pH paper. After evapora-
tion of the solvent, the residue was loaded onto a short silica
gel column and eluted with EtOAc/MeOH 15:1) to give 1 as
a white solid 126 mg, 98%).
1
1
pure by H NMR, was used in further steps. H NMR
1CDCl3, 400 MHz) d 7.74±7.72 1m, 2H), 7.64±7.59 1m,
1H), 7.52±7.49 1m, 2H), 6.68 1d, Jˆ2.4 Hz, 1H), 6.54 1d,
Jˆ2.4 Hz, 1H), 6.00 1bs, 2NH).
4.1.6.
2-[1Dimethylamino)methylene]aminoimidazole
13f). 2-Aminoimidazole sulfate 1539.3 mg, 2.0 mmol) and
sodium carbonate 1212 mg, 2 mmol) were dissolved in
water 14 mL) and stirred at rt for 15 min. Water was
removed in vacuo, the residue dissolved in N,N-dimethyl-
formamide dimethyl acetal 110 mL), and the suspension was
stirred at rt overnight. After evaporation of solvent, the
4.1.9. 1H-Imidazo[4,5-d]pyrazine 18). Beginning with 2,3-
dichloropyrazine, copper-promoted amination produced
2,3-diaminopyrazine, which was condensed with triethyl
orthoformate to give 8.32a

5504
Z.-K. Wan et al. / Tetrahedron 57 +2001) 5497±5507
4.1.10. 1-Methoxymethylimidazo[4,5-b]pyrazine 19a). To
a solution of imidazo[4,5-b]pyrazine 18, 180 mg, 1.5 mmol)
in anhydrous THF 110 ml) under argon was added Et3N
10.63 ml, 4.5 mmol) at 08C, then MOMCl 10.165 ml,
1.65 mmol) was added dropwise. The ice bath was removed
and the mixture stirred at rt for 3 h. The precipitate was
removed by ®ltration, and washed with methylene
chloride 13£20 mL). The solvent was removed from
the ®ltrate in vacuo and the residue puri®ed by ¯ash
chromatography 1Et3N/hexanes/EtOAc, 1:50:100) to
give 9a as a light yellow solid 1227 mg, 92%). Mp
4.2. General procedure for azidation of protected
imidazo[4,5-b]pyrazines 9a and 9b
To a solution of the title compounds in anhydrous toluene at
2788C, was added freshly prepared LDA 11.2 equiv.) in
anhydrous THF with stirring. Stirring was continued at
2788C for 20 min, then TsN3 11.5 equiv.) in toluene was
added rapidly. The temperature was allowed to rise to rt
over a one to two hour period, then the reaction was
quenched with saturated aq. NaHCO3, 110 mL), extracted
with CH2Cl2 13£20 mL), and the organic layer dried with
Na2SO4. After decanting from the Na2SO4, the solvent was
removed in vacuo and the residue puri®ed by ¯ash chroma-
tography.
54±578C; 1H NMR 1CDCl3, 400 Hz)
d
8.56 1d,
Jˆ2.9 Hz, 1H), 8.46 1s, 1H), 8.37 1d, Jˆ2.9 Hz), 5.63
1s, 2H);3.37 1s, 3H); 13C NMR 1CDCl3, 75 MHz) d
148.9, 147.0, 140.6, 140.0, 139.1, 74.5, 57.3;EIHRMS
170 eV) m/z 164.0704 1[M]1, calcd for C7H8N4O
164.0698).
4.2.1. 2-Azido-1-methoxymethylimidazo[4,5-b]pyrazine
110a). This compound was synthesized according to the
general procedure from 1-methoxymethylimidazo[4,5-
b]pyrazine 19a, 145 mg, 0.88 mmol in 25 mL toluene)
with LDA 11.06 mmol in 4 mL THF) and TsN3 1261.2 mg,
1.32 mmol in 2 mL toluene). Puri®cation by ¯ash chromato-
graphy 1Et3N/hexanes/EtOAc, 1:50:50) gave 10a as a light
4.1.11. 1-Benzyloxymethylimidazo[4,5-b]pyrazine 19b).
The compound was prepared according to the procedure
above for 9a from imidazo[4,5-b]pyrazine 18, 120 mg,
1.0 mmol), with Et3N 10.42 mL, 3.0 mmol) and BOMCl
10.35 mL, 1.5 mmol). Puri®cation by ¯ash chromatography
1Et3N/hexanes/EtOAc, 1:50:100) gave 9b as a light yellow
solid 1230 mg, 96%). Mp 79±818C; 1H NMR 1CDCl3,
400 MHz) d 8.58 1d, Jˆ2.5 Hz, 1H), 8.46 1s, 1H), 8.39 1d,
Jˆ2.5 Hz, 1H), 7.33±7.25 1m, 5H), 5.74 1s, 2H), 4.60 1s,
2H); 13C NMR 1CDCl3, 75 MHz) d 148.9, 147.0, 140.5,
139.9, 138.9, 136.1, 128.4 12C), 128.2 12C), 127.7, 72.3,
71.8;EIHRMS 170 eV) m/z 240.0998 1[M]1, calcd for
C13H12N4O 240.1011).
yellow oil which solidi®ed upon standing 1104 mg, 57%).
Mp 91±938C;IR 1NaCl) 2173, 2138 cm
21
;
1H NMR
1CDCl3, 400 MHz) d 8.39 1d, Jˆ2.9 Hz, 1H), 8.16 1d,
Jˆ2.9 Hz, 1H), 5.44 1s, 2H), 3.38 1s, 3H); 13C NMR
1CDCl3, 67.5 MHz) d 153.2, 148.2, 141.2, 140.1, 137.3,
72.3, 57.6;EIHRMS 170 eV) m/z 205.0722 1[M]1, calcd
for C7H7ON7 205.0712). The dimer 11a was also isolated
1
as a yellow solid 145 mg, 16%). Mp 241±2428C; H NMR
1CDCl3, 400 MHz) d 8.68 1d, Jˆ2.4 Hz, 1H), 8.53 1d,
Jˆ2.4 Hz, 1H), 6.54 1s, 2H), 3.41 1s, 3H); 13C NMR
1CDCl3, 75 MHz) d 147.7, 145.2, 142.2, 141.5, 141.1,
74.7, 57.5;EIHRMS 170 eV) m/z 326.1255 130%, [M]1,
calcd for C14H14O2N4 326.1239).
4.1.12. 1-14-Nitrobenzyl)imidazo[4,5-b]pyrazine 19c). A
suspension of imidazo[4,5-b]pyrazine 18, 36 mg,
0.3 mmol), Ag2O 169 mg, 0.3 mmol) and 4-nitrobenzyl-
bromide 178.6 mg, 0.36 mmol) in benzene 16 mL) was
re¯uxed for 5 h. Anhydrous CH2Cl2 13£20 mL) was added
and the precipitate was removed by ®ltration. The solvent
was removed in vacuo and the residue puri®ed by ¯ash
chromatography 1EtOAc) to give 9c as a yellow solid
138 mg, 50%, unoptimized). Mp 190±1928C; 1H NMR
1CDCl3, 400 Hz) d 8.59 1d, Jˆ2.6 Hz, 1H), 8.38 1s, 1H),
8.36 1d, Jˆ2.6 Hz, 1H), 8.20 1d, Jˆ8.6 Hz, 2H), 7.46 1d,
Jˆ8.6 Hz, 2H), 5.58 1s, 2H); 13C NMR 1CDCl3, 75 MHz) d
148.8, 148.2, 146.4, 141.9, 140.8, 139.7, 139.2, 128.6 12C),
124.4 12C), 47.0;EIHRMS 170 eV) m/z 255.0761 1[M]1,
calcd for C12H9N5O2 255.0756).
4.2.2. 2-Azido-1-benzyloxymethylimidazo[4,5-b]pyra-
zine 110b). This compound was synthesized according to
the general procedure from 1-benzyloxymethylimidazo[4,5-
b]pyrazine 19b, 162 mg, 0.675 mmol in 20 mL toluene),
with LDA 10.81 mmol in 3 mL THF) and TsN3 1200 mg,
1.01 mmol in 2 mL toluene). Puri®cation by ¯ash chromato-
graphy 1Et3N/hexanes/EtOAc, 1:50:50) gave 10b as a
yellow oil 1131 mg, 69%). IR 1NaCl) 2166, 2130 cm21
;
1H NMR 1CDCl3, 400 MHz) d 8.41 1d, Jˆ2.9 Hz, 1H),
8.19 1d, Jˆ2.9 Hz, 1H), 7.31±7.23 1m, 5H), 5.56 1s, 2H),
4.62 1s, 2H); 13C NMR 1CDCl3, 67.5 MHz) d 153.1, 148.2,
141.0, 140.0, 137.2, 136.5, 128.4, 128.1, 127.6, 72.1, 70.5;
EIHRMS 170 eV) m/z 281.1002 1[M]1, calcd for C13H11ON7
281.1025). The dimer 11b was also isolated as a yellow
4.1.13. 1-Triphenylmethylimidazo[4,5-b]pyrazine 19d).
suspension of imidazo[4,5-b]pyrazine 18, 60 mg,
A
1
solid 136 mg, 11%). Mp 241±2428C; H NMR 1CDCl3,
0.5 mmol), K2CO3 1207 mg, 1.5 mmol) and triphenyl-
methylchloride 1209.1 mg, 0.75 mmol) in anhydrous DMF
was stirred at rt for 6 h. After removal of the precipitate by
®ltration, solvent was removed from the ®ltrate in vacuo and
the residue puri®ed by ¯ash chromatography 1hexanes/
EtOAc, 1:2) to give 9c as a white solid 199 mg, 55%,
unoptimized yield). Mp 196±1988C; 1H NMR 1CDCl3,
400 MHz) d 8.40 1d, Jˆ2.7 Hz, 1H), 8.33 1s, 1H), 8.05 1d,
Jˆ2.7 Hz, 1H), 7.29±7.25 1m, 9H), 7.17±7.14 1m, 6H); 13C
NMR 1CDCl3, 75 MHz) d 149.7, 147.9, 141.1, 140.7 13C),
139.9, 138.4, 129.6 16C), 128.1 16C), 128.0 13C), 76.3;
EIHRMS 170 eV) m/z 362.1555 1[M]1, calcd for C24H19N4
362.1551).
400 MHz) d 8.67 1d, Jˆ2.4 Hz, 1H), 8.53 1d, Jˆ2.4 Hz,
1H), 7.13 1bs, 5H), 6.60 1s, 2H), 4.68 1s, 2H); 13C NMR
1CDCl3, 75 MHz) d 147.7, 145.1, 142.1, 141.3, 141.0,
136.9, 128.2 12C), 127.8, 127.5 12C), 73.0, 72.0;
CIHRMS 1NH3, 140 eV) m/z 479.1948 1[M11]1, calcd
for C26H23O2N8 479.1943).
4.3. General procedure for the reduction of azides 10a
and 10b
The azide was mixed with Pd±C 130±50 wt%) in MeOH in
a round-bottom ¯ask sealed with a septum. A balloon ®lled

Z.-K. Wan et al. / Tetrahedron 57 +2001) 5497±5507
5505
with H2 was ®tted through the septum, the ¯ask was brie¯y
purged with H2, and the reaction mixture stirred at rt for 2 h.
The mixture was ®ltered through a pad of Celite, to remove
the catalyst, and the solvent removed from the ®ltrate in
vacuo to give the product amine, which could be directly
deprotected without further puri®cation. Puri®cation could
be accomplished by ®ltering through a silica gel plug eluting
with MeOH/EtOAc 11:20).
in toluene, 1.2 ml, 1.2 mmol) dropwise. The reaction
mixture was stirred at rt for 1.5 h, then anhydrous MeOH
110 mL) was added and stirring continued for 20 min. The
solvent was removed in vacuo, the residue was washed with
anhydrous acetone, then redissolved in MeOH, and
neutralized with 1% KOH. Puri®cation as above gave 2 as
a white solid 124 mg, 89%).
4.3.1. 2-Amino-1-methoxymethylimidazo[4,5-b]pyrazine
112a). This compound was synthesized according to the
general procedure from 2-azido-1-methoxymethyl-
imidazo[4,5-b]pyrazine 110a, 50 mg, 0.24 mmol) and
Pd±C 125 mg) in MeOH 110 mL) to give 12a as an off-
white solid 143 mg, 991%). Mp 203±2058C; 1H NMR
1CD3OD, 400 MHz) d 7.99 1d, Jˆ2.7 Hz, 1H), 7.85 1d,
Jˆ2.7 Hz, 1H), 5.47 1s, 2H), 3.46 1s, 3H); 13C NMR
1CD3OD, 67.5 MHz) d 160.4, 151.8, 143.5, 137.8, 134.0,
73.0, 57.2;EIHRMS 170 eV) m/z 179.0792 1[M]1, calcd for
C7H9ON5 179.0807).
Acknowledgements
We are grateful to the National Science Foundation for
®nancial support 1CHE-9501069). We also thank Professor
Steve Buchwald for helpful discussions, and Jim Perkins for
a preprint of his paper 1Ref. 31f) prior to publication.
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the general procedure from 2-azido-1-benzyloxymethyl-
imidazo[4,5-b]pyrazine 110b, 65 mg, 0.23 mmol) and
Pd±C 120 mg) in MeOH 18 mL) to give 12b as an off-
white solid 143 mg, 991%). Mp 136±1388C; 1H NMR
1acetone-d6, 400 MHz) d 8.03 1d, Jˆ2.9 Hz, 1H), 7.83 1d,
Jˆ2.9 Hz, 1H), 7.36±7.24 1m, 5H), 7.06 1bs, NH2), 5.67 1s,
2H), 4.64 1s, 2H); 13C NMR 1acetone-d6, 75 MHz) d 159.4,
152.2, 142.8, 138.6, 138.3, 133.4, 129.2 12C), 128.8 12C),
128.7, 71.7, 70.9;EIHRMS 170 eV) m/z 255.1096 1[M]1,
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2. 1a) Hoffman, K. Imidazole and Its Derivatives, The Chemistry
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4.3.3. 2-Amino-1H-imidazo[4,5-b]pyrazine 12). Method A:
2-Amino-1-methoxymethylimidazo[4,5-b]pyrazine
112a,
40 mg, 0.23 mmol) was dissolved in MeOH 110 mL) and
10% aq. HCl 110 mL) and heated to re¯ux for 10 h. The
solvent was removed in vacuo, the precipitate was washed
with anhydrous acetone. The residue was redissolved in
MeOH and neutralized with 1% KOH as monitored by pH
paper. After the removal of solvent, the crude product was
®ltered through a silica gel plug eluting with Et3N/MeOH/
CHCl3 11:2:6) to give 2 as a white solid 133.3 mg, 991%).
The product can be recrystallized from either H2O or MeOH.
Mp.3008C 1dec.);IR 1KBr): 3400 cm 21;UV 1MeOH) lmax
1log e) 217 1sh), 244 13.30), 318 14.03);1 1NaOH) 206
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1
14.31), 230 1sh), 282 1sh), 326 13.59); H NMR 1DMSO-d6,
400 MHz) d 11.74 1bs, NH), 7.78 1bs, 2H), 7.12 1bs, NH2);
13C NMR 1DMSO-d6, 67.5 MHz, tempˆ508C) d 158.7,
146.9, 133.4;EIHRMS 170 eV) m/z 135.054 1[M]1, calcd
for C5H5N5 135.0545). The HCl salt of 2 was made as
follows. To a solution of the free base was added 10% aq.
HCl to pH,1. The solvent was removed to give a white solid;
mp.3008C 1dec.);UV 1MeOH) lmax 1log e) 217 1sh), 244
13.62), 318 14.39);1 1HCl) 204 13.89), 218 1sh), 274 1sh), 306
14.07), 340 1sh); 1H NMR 1DMSO-d6, 400 MHz) d 8.79 1bs,
NH3), 8.09 1bs, 2H); 13C NMR 1DMSO-d6, 75 MHz,
tempˆ508C) d 155.0, 141.6, 131.9.
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imidazo[4,5-b]pyrazine 112b, 50 mg, 0.2 mmol.) in
anhydrous toluene 110 mL) at 08C was added BBr3 11 M

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