Synthesis of deuterium and 15N-labelled 2,5-Bis[5-amidino-2- pyridyl]furan and 2,5-Bis[5-(methoxyamidino)-2-pyridyl]furan

Article abstract of DOI:10.1002/jlcr.1111

The acetate salt of 2,5-bis[5-amidino-2-pyridyl]furan-d₂/ ¹⁵N₂ (4) was synthesized from 2,5-bis[5-cyano-2-pyridyl] furan-d₂ (2), through the bis-O-acetoxyamidoxime followed by hydrogenation. Compound 2 was obtained via a Stille coupling reaction of 6-chloronicotinonitrile with 2,5-bis[tri-n-butyltin]-furan-d₂ (1). 2,5-bis[5-amidino-2-pyridyl)furan-d₆ (10) was synthesized from 2,5-bis[5-cyano-2-pyridyl)furan-d₆ (9) via a direct reaction with lithium bis(trimethylsilyl)amide, followed by deprotection with ethanolic HCl. ¹⁵N and/or deuterium-labelled methoxy-amidines 5a-d₂/ ¹⁵N₂, 5b-d₈, 12, 14-d₆ were prepared in good yield via direct methylation of their respective diamidoximes with either dimethylsulfate-d₀ or dimethylsulfate-d₆ in DMF solution and using LiOH as a base. Copyright

Full text of DOI:10.1002/jlcr.1111

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 985–996.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1111
Research Article
15
Synthesis of deuterium and N-labelled 2,5-Bis[5-
amidino-2-pyridyl]furan and 2,5-Bis[5-
(methoxyamidino)-2-pyridyl]furan
Mohamed A. Ismail1,2 and David W. Boykin1,*
1 Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA
2 Department of Chemistry, Faculty of Science, Mansoura University, Mansoura 35516,
Egypt
Summary
The acetate salt of 2,5-bis[5-amidino-2-pyridyl]furan-d2/15N2 (4) was synthesized from
2,5-bis[5-cyano-2-pyridyl]furan-d2 (2), through the bis-O-acetoxyamidoxime followed
by hydrogenation. Compound 2 was obtained via a Stille coupling reaction of
6-chloronicotinonitrile with 2,5-bis[tri-n-butyltin]-furan-d2 (1). 2,5-bis[5-amidino-
2-pyridyl)furan-d6 (10) was synthesized from 2,5-bis[5-cyano-2-pyridyl)furan-d6 (9)
via a direct reaction with lithium bis(trimethylsilyl)amide, followed by deprotection
with ethanolic HCl. 15N and/or deuterium-labelled methoxy-amidines 5a-d2/15N2,
5b-d8, 12, 14-d6 were prepared in good yield via direct methylation of their
respective diamidoximes with either dimethylsulfate-d0 or dimethylsulfate-d6 in
DMF solution and using LiOH as a base. Copyright # 2006 John Wiley & Sons,
Ltd.
Key Words: deuterium-labelled; 15N-isotopes; prodrug; bromination; Stille coupling
Introduction
2,5-Bis[4-methoxyamidinophenyl]furan (Ia), an orally administered prodrug,
is currently in Phase II clinical trials against malaria, and Phase III trials
against Pneumocystsis pneumonia and human African trypanosomiasis
(HAT).1–7 The prodrug undergoes a multistep bioconversion in vivo to yield
the active drug furamidine (IIa).8–11 The establishment of the bioconversion
*Correspondence to: David W. Boykin, Department of Chemistry, Georgia State University, Atlanta,
GA 30303, USA. E-mail: dboykin@gsu.edu
Contract/grant sponsor: Bill and Melinda Gates Foundation
Copyright # 2006 John Wiley & Sons, Ltd.

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M. A. ISMAIL AND D. W. BOYKIN
pathway of Ia into IIa was significantly aided by the synthesis of deuterium
labelled Ia and IIa.12
As part of an effort to develop antitrypanosomal compounds that are more
effective than Ia/IIa we have found that aza-analogs of these compounds show
excellent activity against Trypanosoma brucei rhodesiense (T. b. r.) both in vitro
and in vivo in a mouse model.13 Specifically, Ib which is a prodrug of IIb, has
shown very promising oral activity giving cures in the virulent STIB900 mouse
model T. b. r. at the low dosage of 5 mg/kg which is superior to the activity of
Ia in the same model. The establishment of the bioconversion pathway of Ib
into IIb was significantly aided by the synthesis of deuterium labelled Ib and
IIb.14,15 In view of the efficacy of III against T. b. r. and its putative simpler
metabolism than Ib, preclinical toxicity and metabolism studies have been
initiated in anticipation of further trials. It is expected that labelling of the
furan group of III and IV with deuterium (d2-analog) plus two nitrogen atoms
with the 15N-isotope will serve as reasonable mass spectroscopy internal
standards for metabolism studies. For comparative metabolism studies we also
require the d6 and d8 analog of III. It is hypothesized that metabolism of III
will likely be similar to that of Ia, because III is a symmetric molecule as in Ia.
Consequently, to aid with these studies we describe the syntheses of the novel
isotopically labelled compounds 3a-d2/15N2, 4-d2/15N2, 5a-d2/15N2, 5b-d8, 10,
11, 12 and 14-d6 for use in preclinical studies (Figure 1).
NOMe
NH2
NH
O
O
MeON
H2N
HN
H2N
X
X
NH2
Ia; X = CH
b; X = N
IIa; X = CH
b; X = N
NOMe
NH2
NH
O
O
N
MeON
H2N
HN
H2N
N
N
N
NH2
IV
III
Figure 1.
Results and discussion
As shown in Scheme 1, the acetate salt of 2,5-bis[5-amidino-2-pyridyl]furan-
d2/15N2 (4) was synthesized from 2,5-bis[5-cyano-2-pyridyl]furan-d2 (2),
through the bis-O-acetoxyamidoxime followed by hydrogenation. Compound
2 was obtained via a Stille coupling reaction of 6-chloronicotinonitrile with
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 985–996
DOI: 10.1002/jlcr

DEUTERIUM AND 15N-LABELLED BIS-AZAFURAMIDINE ANALOGS
987
d4
O
(i)
d2
d2
(ii)
Cl
NC
+
Bu3Sn
SnBu3
O
O
N
NC
CN
N
N
1
2
d2
(iii-a), (iii-b)
HON
H2N
NOH
NH2
O
N
N
3a, 15N2
3b, 14N2
(v)/3a
(vi)/3b
(iv)
d2
d2
NH
HN
O
RON
NOR
NH2
O
N
N
N
N
H2N
NH2
H2N
4
5a, R = CH3
5b, R = CD3
Scheme 1. Reagents and conditions: (i) sec.BuLi, Bu3SnCl; (ii) Pd(PPh3)4 1,4-
dioxane; (iii-a) 15NH2OH.HCl/KO-t-Bu, (iii-b) NH2OH.HCl/KO-t-Bu; (iv-a)
AcOH/Ac2O, (iv-b) H2/Pd-C; (v) (CH3)2SO4/LiOH; (vi) (CD3)2SO4/LiOH
2,5-bis[tri-n-butyltin]-furan-d2 (1), which was prepared from commercially
available furan-d4 via lithiation with sec-butyllithium and a subsequent
treatment with tri-n-butyltin chloride as previously described for furan-d0.16
2,5-Bis[5-(methoxyamidino)-2-pyridyl]furan-d2/15N2 (5a) was prepared in
good yield via methylation of the diamidoxime 3a-d2/15N2, a product of the
treatment of the dinitrile 2 with 15N-labelled hydroxylamine hydrochloride and
potassium tert-butoxide in DMSO at ambient temperature, with dimethylsul-
fate in DMF solution and using LiOH as a base. In a similar way, 2,5-bis
[5-(methoxyamidino)-2-pyridyl]furan-d8 (5b) was prepared via methylation of
the diamidoxime 3b-d2 with dimethylsulfate-d6.
As outlined in Scheme 2, 2,5-bis[5-amidino-2-pyridyl]furan-d6 (10) was
synthesized from 2,5-bis[5-cyano-2-pyridyl]furan-d6 (9). The dinitrile 9 was
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

988
M. A. ISMAIL AND D. W. BOYKIN
d4
d3
d3
(ii)
(i)
ND2
NH2
(iii)
Br
Br
Br
N
N
N
7
6
d3
d3
d3
d3
(iv)
O
O
Br
Br
N
N
CN
NC
N
N
9
8
(v)
(vi)
d3
d3
d3
d3
RON
H2N
NOR
O
NH
N
N
HN
O
N
N
NH2
NH2
H2N
10
11; R = H
12; R = CH3
(vii)
Scheme 2. Reagents and conditions: (i) NBS, DMF; (ii) aq. HBr, Br2, NaNO2,
08C to Rt; (iii) 2,5-bis[tri-n-butyltin]-furan, Pd(PPh3)4; (iv) Cu(I)CN, DMF; (v)
.
a) LiN(TMS)2, THF, (v-b) HCI(g)/EtOH; (vi) NH2OH HCl/KO-t-Bu; (vii)
(CH3)2SO4/LiOH
allowed to react with lithium bis(trimethylsilyl)amide17 in THF, followed by
deprotection of the silylated amidines with ethanolic HCl to furnish a
hydrochloride salt of the diamidine 10. Compound 9 was obtained in four
steps starting with bromination of the commercially available
2-aminopyridine-d6 with N-bromosuccinimide in DMF solution to give
5-bromo-2-aminopyridine-d3 (6). Treatment of 6 with hydrobromic acid,
bromine and sodium nitrite,18 gave 2,5-dibromopyridine-d3 (7) in 76% yield. A
Stille coupling reaction of two equivalents of 7 with one equivalent of
2,5-bis[tri-n-butyltin]-furan-d0 at 70–808C furnished the dibromo-compound
8, the regioselectivity of 7 is consistent with similar reactions reported
previously for 2,5-dibromopyridine-d0.13,19–22 Cyanation of 8 with Cu(I)CN at
140–1508C gave the dinitrile 9. Treatment of 9 with hydroxylamine
hydrochloride and potassium tert-butoxide in DMSO at ambient temperature
gave the diamidoxime 11 in 93% yield. A subsequent methylation of
Copyright # 2006 John Wiley & Sons, Ltd.
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DEUTERIUM AND 15N-LABELLED BIS-AZAFURAMIDINE ANALOGS
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11 with dimethylsulfate-d0 afforded 2,5-bis[5-(methoxyamidino)-2-pyridyl]
furan-d6 (12).
2,5-Bis[5-(methoxyamidino)-2-pyridyl]furan-d6 (14) which bears deuterium-
labelled methoxy groups was prepared by direct methylation of the respective
diamidoxime (13)13 with dimethylsulfate-d6 (Scheme 3). The hydrochloride
salts of all the oximes, 3a-d2/15N2, 5a-d2/15N2, 5b-d8, 11, 12, 14-d6 were made
by passing hydrogen chloride gas into ethanolic solutions of their free bases.
(i)
D3CON
H2N
NOCD3
NH2
HON
H2N
O
O
NOH
NH2
N
N
N
N
14
13
Scheme 3. Reagents and conditions: (i) (CD3)2SO4/LiOH
Conclusion
An efficient five step synthesis of 4-d2/15N2 (IV-isotope) and four step syntheses
of 5a-d2/15N2 and 5b-d8 (III-isotopes) starting from furan-d4 have been
developed. In addition, labelling of III has been achieved in two more different
ways, a six-step approach for the synthesis of 12-d6 starting from
2-aminopyridine-d6 has been described and direct methylation of 13 with
dimethylsulfate-d6 provided 14-d6. No detectable exchange of deuterium was
observed during the syntheses of all of the target compounds. The use of these
deuterium labelled compounds in metabolism and pharmacokinetic studies
will be described in due course.
Experimental section
Melting points were recorded using a Thomas-Hoover (Uni-Melt) capillary
melting point apparatus and are uncorrected. TLC analysis was carried out on
1
silica gel 60 F254 precoated aluminum sheets and detected under UV light. H
3
and C NMR spectra, were recorded employing a Varian Unity Plus 300
spectrometer (Varian, Inc., Palo Alto, California, USA), and chemical shifts
(d) are in ppm relative to TMS as internal standard. Mass spectra were
recorded on a VG analytical 70-SE spectrometer (VG Analytical, Ltd.,
Manchester, UK). Elemental analyses were obtained from Atlantic Microlab
Inc. (Norcross, GA) and are within Æ 0.4 of the theoretical values. The
compounds reported as salts frequently analyzed correctly for fractional moles
of water and/or ethanol of solvation. In each case proton NMR showed the
presence of indicated solvent(s). All non-labelled chemicals and solvents were
purchased from Aldrich Chemical Co. or Fisher Scientific. Isotopically
labelled compounds, furan-d4 (98 atom % D), hydroxylamine.HCl (98 atom
%
15N), 2-aminopyridine-d6 (98 atom % D) and dimethylsulfate-d6 (99 atom
% D) were obtained from Aldrich Chemical Co., Cambridge Isotopes or Icon
Copyright # 2006 John Wiley & Sons, Ltd.
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M. A. ISMAIL AND D. W. BOYKIN
Isotopes. NBS was recrystallized from nitromethane prior to use. All solvents
were reagent grade.
2,5-Bis(tri-n-butylstannyl)furan-d2 (1)
To a solution of furan-d4 (2.2 ml, 30 mmol), tetramethylethylenediamine
(11.3 ml, 75 mmol), and hexane (75 ml) under nitrogen was added sec.butyl-
lithium (53.5 ml, 75 mmol) dropwise at 08C. After 1 h at 08C, the reaction
mixture was warmed to room temperature and kept stirring for 8 h, thereafter
the reaction mixture was cooled to 08C and a solution of n-tributyltin chloride
(21.8 ml, 81 mmol) was added dropwise. The reaction mixture was warmed to
room temperature and kept stirring for 48 h, then 75 ml of saturated
ammonium chloride was added and the product was extracted using hexanes.
The organic layer was washed with aqueous CuSO4 (2.5%), dried (Na2SO4)
and the solvents removed under reduced pressure to give 1 as an oil in 85%
yield. 1H NMR (CDCl3); d 1.58 (m, 12 H), 1.36 (m, 12 H), 1.06 (t, J = 8.1 Hz,
12 H), 0.91 (m, 18 H). EIMS (m/z, relative intensity (rel.int.)); 647 (M++1, 20),
646 (M+, 15), 591 (75), 535 (30), 481 (15), 421 (20), 367 (20), 177 (90), 57
(1 0 0).
2,5-Bis[5-cyano-2-pyridyl)furan-d2 (2)
A mixture of 6-chloronicotinonitrile (4.14 g, 30 mmol), 2,5-bis(tri-n-butylstan-
nyl)furan-d2 (9.93 g, 15 mmol) and tetrakis(triphenyl-phosphine)palladium(0)
(900 mg) in dry 1,4-dioxane (40 ml) was heated under nitrogen at 80–908C for
24 h. The solvent was evaporated under reduced pressure and the residue was
dissolved in methylene chloride and the solution was passed through celite to
remove Pd. The solution was evaporated, the precipitate was triturated with
ethanol, filtered, washed with hexanes to furnish 2 in 75% yield, m.p.
308–3108C (DMF), identical to authentic unlabelled compound by TLC
1
(Rf = 0.48 using hexanes/ethyl acetate, 60:40). H NMR (DMSO-d6); d 8.14
(d, J = 8.4 Hz, 2 H), 8.38 (dd, J = 8.4, 1.8 Hz, 2 H), 9.02 (d, J = 1.8 Hz, 2 H).
13C NMR (DMSO-d6); 153.3, 152.5, 149.9, 140.7, 118.6, 116.7, 107.1. MS
(ESI) m/e (rel. int.): 275 (M++1, 100). High resolution mass calculated for
C16H7D2N4O: 275.0902. Observed 275.0890. Analytically Calculated for
C16H6D2N4O-0.1H2O: C, 69.60; H + D as H, 3.02. Found. C, 69.49;
H + D as H, 2.97.
2,5-Bis[5-(N-hydroxyamidino)-2-pyridyl]furan-d2/15N2 (3a)
A mixture of hydroxylamine hydrochloride, 15N-isotope, (2.11 g, 30 mmol,
10 eq.) in anhydrous DMSO (25 ml) was cooled to 58C under nitrogen and
potassium t-butoxide (3.36 g, 30 mmol, 10 eq.) was added in portions. The
mixture was stirred for 30 min. To this mixture was added the dinitrile-d2 2
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DEUTERIUM AND 15N-LABELLED BIS-AZAFURAMIDINE ANALOGS
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(822 mg, 3 mmol, 1 eq.). The reaction mixture was stirred for 24 h at room
temperature. The reaction mixture was then poured slowly onto ice–water.
The precipitate was filtered and washed with water and then ethanol to afford
1
3a (free base) in 98% yield; m.p. 265–2678C. H NMR (DMSO-d6); d 6.30
(s, 4H), 7.96 (d, J = 8.4 Hz, 2H), 8.15 (dd, J = 8.4, 2.4 Hz, 2H), 8.91
(d, J = 2.4 Hz, 2H), 10.00 (s, 2H). 13C NMR (DMSO-d6); d 153.5, 149.9,
148.4, 147.1, 134.2, 126.6, 118.2. MS (ESI) m/e (rel. int.): 343 (M++1, 100),
225 (10). High resolution mass calculated for C16H13D124N415N2O3: 343.1272.
Observed 343.1259. (3a hydrochloride salt); m.p. 291–293.58C dec. Analytically
Calculated for C16H12D124N415N2O3-4.0 HCl: C, 39.33; H + D as H, 3.73; N,
17.21. Found. C, 39.49; H + D as H, 3.91; N, 17.29.
2,5-Bis[5-(N-hydroxyamidino)-2-pyridyl]furan-d2 (3b)
The same procedure described for 3a was used by employing 14NH2OH.HCl
instead of 15NH2OH.HCl. Yield 95%; m.p. 286–2888C. 1H NMR (DMSO-d6);
d 5.89 (s, 4H), 7.93 (d, J = 8.1 Hz, 2H), 8.12 (dd, J = 8.1, 2.4 Hz, 2H), 8.90 (d,
J = 2.4 Hz, 2H), 9.78 (s, 2H). 13C NMR (DMSO-d6); d 153.5, 148.7, 148.0,
146.8, 133.7, 127.6, 118.1. MS (ESI) m/e (rel. int.): 341 (M++1, 100), 324 (20),
307 (25), 241 (10), 214 (10), 171 (80). High resolution mass calculated for
C16H13D2N6O3: 341.1331. Observed 341.1330.
2,5-Bis[5-amidino-2-pyridyl)furan-d2/15N2 acetate salt (4)
To a solution of 3a (342 mg, 1 mmol) in glacial acetic acid (10 ml) was slowly
added acetic anhydride (0.35 ml). After stirring overnight TLC indicated
complete acylation of the starting material. The reaction mixture was poured
onto ice-water, the precipitate was filtered, washed with water and dried. To
the precipitate in glacial acetic acid (13 ml), and ethanol (20 ml) was added
10% palladium on carbon (150 mg). The mixture was placed in a Parr
hydrogenation apparatus at 50 psi for 8 h at room temperature. The mixture
was filtered through hyflo and the filter pad washed with water. The filtrate
was evaporated under reduced pressure and the precipitate was collected and
washed with ether to give 4 in 73% yield, m.p. 242–2448C dec. 1H NMR (D2O/
DMSO-d6); d 1.78 (s, 3.5 Â CH3 of acetate) 8.06 (d, J = 8.1 Hz, 2H), 8.25 (dd,
J = 8.1, 1.8 Hz, 2H), 8.96 (d, J = 1.8 Hz, 2H). MS (ESI) m/e (rel. int.): 311
(M++1, 40), 294 (10), 156 (1 0 0). High resolution mass calculated for
C16H13D124N145N2O: 311.1374. Observed 311.1371. Analytically Calculated for
C16H12D124N145N2O-3.5CH3CO2H-1.75H2O: C, 49.99; H + D as H, 5.70; N,
15.22. Found. C, 49.80; H + D as H, 5.73; N, 15.46.
Copyright # 2006 John Wiley & Sons, Ltd.
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M. A. ISMAIL AND D. W. BOYKIN
2,5-Bis[5-(methoxyamidino)-2-pyridyl]furan-d2/15N2 (5a)
To a suspension of the diamidoxime 3a (376 mg, 1.1 mmol) in DMF (15 ml)
was added LiOH.H2O (554 mg, 13.2 mmol, in 5 ml H2O) which was followed
by dimethylsulfate (1.38 g, 11 mmol). The reaction mixture was kept stirring
overnight after which it was poured onto ice/water and the precipitate was
filtered, washed with water and dried to give the desired compound in 66%
1
yield; m.p. 233–234.58C. H NMR (DMSO-d6): d 3.78 (s, 6H), 6.31 (s, 4H),
7.97 (d, J = 8.4 Hz, 2H), 8.12 (dd, J = 8.4, 2.1 Hz, 2H), 8.87 (d, J = 2.1 Hz,
2H). 13C NMR (DMSO-d6); d 153.5, 148.9, 148.3, 146.9, 134.1, 126.8, 118.0,
60.7. MS (ESI) m/e (rel. int.): 371 (M++1, 100), 343 (10), 253 (10). High
resolution mass calculated for C18H17D124N415N2O3: 371.1585. Observed
371.1588. (5a hydrochloride salt); m.p. 204–2068C dec. Analytical Calculated
for C18H16D124N415N2O3-4.0HCl-1.8H2O: C, 39.37; H + D as H, 4.66; N,
15.31. Found. C, 39.25; H + D as H, 4.62; N, 15.21.
2,5-Bis[5-(methoxyamidino)-2-pyridyl]furan-d8 (5b)
The target compound was prepared adopting the same procedure described for
5a, starting with diamidoxime 3b and employing dimethylsulfate-d6 instead of
dimethylsulfate-d0. Yield 64%, m.p. 234–2368C. 1H NMR (DMSO-d6): d 6.29
(s, 4H), 7.96 (d, J = 8.4 Hz, 2H), 8.11 (dd, J = 8.4, 2.1 Hz, 2H), 8.87 (d,
J = 2.1 Hz, 2H). 13C NMR (DMSO-d6); d 153.5, 148.9, 148.3, 147.0, 134.2,
126.9, 118.1. MS (ESI) m/e (rel. int.): 375 (M++1, 100), 188 (35). High
resolution mass calculated for C18H11D8N6O3: 375.2021. Observed 375.2015.
(5b hydrochloride salt); m.p. 198–2008C dec. Analytical Calculated for
C18H10D8N6O3-4.0HCl-1.5H2O: C, 39.50; H+D as H, 4.58; N, 15.35. Found.
C, 39.27; H + D as H, 4.72; N, 15.13.
5-Bromo-2-aminopyridine-d3 (6)
To a solution of 2-aminopyridine-d6 (5.00 g, 50 mmol) in DMF (20 ml) was
added portionwise N-bromosuccinimide (8.90 g, 50 mmol) with stirring. The
reaction mixture was stirred overnight at room temperature, then poured onto
cold-water. The precipitate was collected, washed with water and dried then
purified using chromatography (SiO2, hexanes/ethyl acetate, 40: 60) to furnish
6 in 81% yield, m.p. 132–1338C, identical to authentic unlabelled compound
by TLC (Rf = 0.60 using hexanes/ethyl acetate, 40:60). 1H NMR (DMSO-d6):
d 6.12 (s, 2H). MS (ESI) m/e (rel. int.): 176, 178 (M++1, 100, 90). High
resolution mass calculated for C5H3D3BrN2: 175.9903. Observed 175.9902.
2,5-dibromopyridine-d3 (7)
To a solution of 6 (5.46 g, 31 mmol) in 48% aqueous hydrobromic acid (48 ml)
was added 5.0 ml of bromine at 08C and 24.2 ml of an aqueous solution of
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DEUTERIUM AND 15N-LABELLED BIS-AZAFURAMIDINE ANALOGS
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NaNO2 (3.25 M). The reaction mixture was stirred at room temperature for
1 h and then neutralized with NaOH (1N, 435 ml). The unreacted bromine was
quenched with Na2S2O3, the product was extracted with ethyl acetate. The
organic layers were combined, washed with water, dried over anhydrous
Na2SO4 and concentrated to dryness. The residue was chromatographed on
silica gel using hexanes/ethyl acetate (95 : 5) as an eluent to give 7 in 76% yield,
m.p. 94.5–95.5 8C, identical to authentic unlabelled compound by TLC (Rf =
0.81 hexanes/ethyl acetate, 95:5). MS (ESI) m/e (rel. int.): 239, 241 (M++1,
40, 100), 175 (10). High resolution mass calculated for C5HD3Br2N: 238.8899.
Observed 238.8894. Analytical Calculated for C5D3Br2N: C, 25.03; H + D as
H, 1.25. Found. C, 25.17; H + D as H, 1.27.
2,5-Bis[5-bromo-2-pyridyl)furan-d6 (8)
2,5-dibromopyridine-d3 (7) (4.78 g, 20 mmol, 2 eq.) and 2,5-bis(tri-n-butyl-
stannyl)furan-d0 (6.60 g, 10 mmol, 1 eq.) were reacted under the above
mentioned Stille coupling conditions to give the target compound 8 in 61%
yield, m.p. 211–2138C (EtOH). 1H NMR (DMSO-d6): d 7.28 (s, 2H).
13C NMR (DMSO-d6); d 152.8, 146.4, 118.1, 111.6. MS (ESI) m/e (rel. int.):
385, 387 (M++1, 40, 100), 317 (35). High resolution mass calculated for
C14H3D6Br2N2O: 384.9458. Observed 384.9457. Analytical Calculated for
C14H2D6Br2N2O: C, 43.55; H + D as H, 2.10. Found. C, 43.63; H + D as H,
2.09.
2,5-Bis[5-cyano-2-pyridyl)furan-d6 (9)
A mixture of 2,5-bis[5-bromo-2-pyridyl)furan-d6 (8) (2.20 g, 5.75 mmol) and
Cu(1)CN (2.04 g, 23 mmol) in DMF (40 ml) was heated at 140–1508C for 24 h.
The reaction mixture was poured onto water and the solid which formed was
extracted with methylene chloride (300 ml, 3 Â ) from aq. NH4OH. The solvent
was evaporated and the precipitate was purified by chromatography (SiO2,
ethyl acetate/hexanes 90:10). Yield 55%, m.p. 304–3068C, identical to
1
authentic unlabelled compound by TLC. H NMR (DMSO-d6): d 7.50 (s,
2H). 13C NMR (DMSO-d6); d 153.3, 149.9, 116.6, 114.4, 106.9. MS (ESI) m/e
(rel. int.): 279 (M++1, 100), 254 (70). High resolution mass calculated
for C16H3D6N4O: 279.1146. Observed 279.1017. Analytical Calculated
for C16H2D6N4O-0.25H2O: C, 67.93; H + D as H, 3.03. Found. C, 67.66;
H + D as H, 3.20.
2,5-Bis[5-amidino-2-pyridyl)furan-d6 (10)
The dinitrile-d6 9 (278 mg, 1 mmol), suspended in freshly distilled THF (5 ml),
was treated with lithium trimethylsilylamide (1 M solution in THF, 4 ml,
4 mmol) and the reaction was allowed to stir overnight. The reaction mixture
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994
M. A. ISMAIL AND D. W. BOYKIN
was then cooled to 08C to which was added HCl saturated ethanol (70 ml)
whereupon a precipitate started forming. The mixture was left to run
overnight whereafter it was diluted with ether and the resultant solid was
collected by filtration. The diamidine was purified by neutralization with 1N
NaOH followed by filtration of the resultant solid and washing with water
(3X). Finally, the free base was stirred with ethanolic HCl overnight, diluted
with ether, and the solid formed was filtered and dried to give the diamidine
salt 10 in 90% yield; m.p. >3008C. 1H NMR (DMSO-d6): d 7.54 (s, 2H), 9.44
(s, 4H), 9.70 (s, 4H). 13C NMR (DMSO-d6); d 163.4, 153.7, 151.2, 122.6, 114.4.
MS (ESI) m/e (rel. int.): 313 (M++1, 70), 230 (25), 157 (1 0 0). High resolution
mass calculated for C16H9D6N6O: 313.1684. Observed 313.1689. (10 hydro-
chloride salt) Analytical Calculated for C16H8D6N6O-4.0HCl-1.0H2O-
0.6C2H5OH: C, 41.00; H + D as H, 4.68; N, 16.67. Found. C, 41.22;
H + D as H, 4.58; N, 16.32.
2,5-Bis[5-(N-hydroxyamidino)-2-pyridyl]furan-d6 (11)
The same procedure described for 3a was used, starting with dinitrile-d6 9 and
employing 14NH2OH.HCl instead of 15NH2OH.HCl. Yield 93%, m.p.
1
265–2678C, H NMR (DMSO-d6): d 6.07 (s, 4H), 7.31 (s, 2H), 9.92 (s, 2H).
13C NMR (DMSO-d6); d 153.6, 148.7, 147.9, 127.4, 111.7. MS (ESI) m/e (rel.
int.): 345 (M++1, 100), 330 (15), 240 (55), 176 (50). High resolution mass
calculated for C16H9D6N6O3: 345.1582. Observed 345.1575. (11 hydrochloride
salt); m.p. 285–2878C dec. Analytical Calculated for C16H8D6N6O3-4.0HCl-
0.8C2H5OH: C, 40.11; H + D as H, 4.32; N, 15.95. Found. C, 40.41; H + D
as H, 4.26; N, 16.20.
2,5-Bis[5-(methoxyamidino)-2-pyridyl]furan-d6 (12)
The same procedure described for 5a was used, starting with diamidoxime-d6
1
11. Yield 60%, m.p. 228–2298C, H NMR (DMSO-d6): d 3.78 (s, 6H), 6.29
(s, 4H), 7.33 (s, 2H). 13C NMR (DMSO-d6); d 153.6, 148.9, 148.3, 126.7, 112.0,
60.8. MS (ESI) m/e (rel. int.): 373 (M++1, 100), 357 (10), 319 (10), 284 (20).
High resolution mass calculated for C18H13D6N6O3: 373.1895. Observed
373.1897. (12 hydrochloride salt); m.p. 230–231.58C dec. Analytical Calculated
for C18H12D6N6O3-4.0HCl-1.25H2O-1.0EtOH: C, 40.93; H + D as H, 5.19;
N, 14.30. Found. C, 40.97; H + D as H, 4.87; N, 14.00.
2,5-Bis[5-(methoxyamidino)-2-pyridyl]furan-d6 (14)
The same procedure described for 5a was used, starting with diamidoxime
13,13 and employing dimethylsulfate-d6 instead of dimethylsulfate-d0. Yield
65%, m.p. 236–2378C, 1H NMR (DMSO-d6): d 6.29 (s, 4H), 7.32 (s, 2H), 7.96
(d, J = 8.4 Hz, 2H), 8.11 (dd, J = 8.4, 1.8 Hz, 2H), 8.87 (d, J = 1.8 Hz, 2H).
Copyright # 2006 John Wiley & Sons, Ltd.
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DEUTERIUM AND 15N-LABELLED BIS-AZAFURAMIDINE ANALOGS
995
13C NMR (DMSO-d6); d 153.6, 148.9, 148.3, 147.0, 134.2, 126.9, 118.1, 112.0.
MS (ESI) m/e (rel. int.): 373 (M++1, 100), 225 (25). High resolution mass
calculated for C18H13D6N6O3: 373.1895. Observed 373.1892. (14 hydrochloride
salt); m.p. 205–2068C dec. Analytical Calculated for C18H12D6N6O3-4.0HCl-
2.0H2O: C, 39.00; H + D as H, 4.69; N, 15.15. Found. C, 38.98; H + D as H,
4.65; N, 14.99.
Acknowledgements
This work was supported by an award from the Bill and Melinda Gates
Foundation.
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