Synthesis of deuterated-BCX-1777, a potent inhibitor of purine nucleoside phosphorylase

Article abstract of DOI:10.1002/jlcr.533

BCX-1777, a novel inhibitor of the enzyme purine nucleoside phosphorylase, mimics the charged ribosyl oxocarbenium ion formed during the transition state of the enzyme-catalyzed C-N bond cleavage of nucleosides. BCX-1777 is a slow-onset, tight-binding inh

Full text of DOI:10.1002/jlcr.533

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2002; 45: 71–78.
DOI: 10.1002/jlcr.533
Research Article
Synthesis of deuterated-BCX-1777, a potent
inhibitor of purine nucleoside phosphorylase
1
2
Hollis S. KezarIII , Tracy L. Hutchison1, PeterC. Tyler and
Philip E. Morris Jr.1,*
1 BioCryst Pharmaceuticals, Inc., 2190 Parkway Lake Drive, Brimingham,
Alabama 35244, USA
2 Carbohydrate Chemistry, Industrial Research Limited, P.O. Box 31310,
Lower Hutt, New Zealand
Summary
BCX-1777, a novel inhibitor of the enzyme purine nucleoside phosphorylase,
mimics the charged ribosyl oxocarbenium ion formed during the transition
state of the enzyme-catalyzed C–N bond cleavage of nucleosides. BCX-1777 is
a slow-onset, tight-binding inhibitorwith a Kiꢀ of 23 pM and is one of the most
potent inhibitors known for the enzyme. In support of our BCX-1777 program,
a mass spectrometric assay has been developed utilizing 50-[2H]-BCX-1777 as
an internal standard. The synthesis of 50-[2H]-BCX-1777 is described in this
report. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: BCX-1777; purine nucleoside phosphorylase; T-Cell
Introduction
The enzyme purine nucleoside phosphorylase (PNP, EC 2.4.2.1)
catalyzes the reversible cleavage of purine nucleosides to the corre-
sponding purine base and sugar phosphate in the purine salvage
pathway as shown.1
*Correspondence to: P. E. Morris, Biocryst Pharmaceuticals, Inc., 2190 Prakway Lake Drive,
Birmingham, Alabama, AL 35244 USA. E-mail: pmorris@biocryst.com
Copyright # 2002 John Wiley & Sons, Ltd.
Received 18 June 2001
Revised 4 October 2001
Accepted 12 October 2001

72
H. S. KEZAR ET AL.
In the absence of PNP, nucleoside substrates such as 20-deoxygua-
nosine (dGuo) accumulate. dGuo accumulation has been observed in
children with inherited PNP deficiency and as a consequence, these
children exhibit severe T-cell immunodeficiency but retain normal or
exaggerated B-cell function.2 T-cell cytotoxicity is due to phosphoryla-
tion of dGuo (via 20-deoxycytidine kinase, dCK, (EC 2.7.1.74)) to 20-
deoxyguanosine triphosphate (dGTP). dGTP allosterically inhibits the
enzyme ribonucleotide diphosphate reductase (EC 1.17.4.1), preventing
DNA synthesis and hence T-cell proliferation.3 The relatively unique
sensitivity of T-cells is attributed to their relatively high level of dCK
compared to other cells. This observation has led to the development of
PNP inhibitors for the treatment of T-cell cancers and T-cell
autoimmune indications. The biochemcial basis forthe use of PNP
inhibitors as well as the various classes of inhibitors developed has been
reviewed.4 More recently, clinical trial experience in psoriasis with the
5
PNP inhibitorBCX-34 has been described.
Using transition state analysis, a new class of PNP inhibitors has been
developed.6 One of these inhibitors, BCX-1777 (1) is a clinical trial
candidate. In support of our clinical program, we needed to develop a
rapid and sensitive method of determining drug levels in biological
matrices such as plasma and urine. One method evaluated for this
purpose was an LC-MS-MS method utilizing an isotopically-labeled
analog as an internal standard. In this paper, we present the synthesis of
50-[2H]-BCX-1777 (2), which we have used in these assays.
Copyright # 2002 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2002; 45: 71–78

SYNTHESIS OF DEUTERATED-BCX-1777
73
Results and discussion
We have recently described the convergent synthesis of aza-C-nucleo-
sides via addition of lithiated 9-deazapurines to carbohydrate-derived
cyclic imines.7 One such aza-C-nucleoside reported by us though this
route was 3, which we have utilized as our starting material for the
labeled analog as shown in Scheme 1. Treatment of 3 with 1 M
tetrabutylammonium fluoride in THF afforded the corresponding
alcohol 4 in excellent yield. Dess-Martin oxidation of alcohol 4 gave
2
the corresponding aldehyde 5 in 96% isolated yield. The H-label was
introduced through reduction of aldehyde 5 with NaB[2H]4 in
CH3O[2H] as the solvent giving 6 as the product. While we have
demonstrated that 6 can be converted directly to the target compound 2
by treatment with strong acid under reflux, this route tended to give
product that was highly colored. Most of this color developed during
the extended reflux conditions required to completely remove the N-9
benzyloxymethyl protecting group. We circumvented this problem by
using the same chemistry we had employed previously for the non-
labeled material in which the N-9 benzyloxymethyl protecting group
was hydrogenated in the presence of Pearlman’s catalyst to give 8. After
filtering the catalyst through Celite, thin layer chromatographic analysis
of the filtrate indicated that in addition to 8, a small amount of the N-9
CH2OH product 7 remained. Residual 7 was conveniently converted in
situ to the desired N-9 NH product 8 by treating the filtrate with a small
amount of NH4OH. In this manner, 8 was isolated in 84% yield from 6.
Final deprotection was achieved by refluxing 8 with concentrated
aqueous HCl/MeOH for2 h giving 2, which was isolated as the
hydrochloride. The overall yield of 2 was 60.4% from 3 and the final
product had an isotopic purity of 98.4%. The position of the [2H]-label
was confirmed at the 50-C through comparison of the 50-CH2 resonances
of 1 (determined by a DEPT 135 experiment to be d 58.88 for 1) and 2.
In the 13C NMR of 2, this signal was greatly diminished
in intensity and very broad due to C–[2H] coupling.y In addition, the
50-CH2 resonance for 2 was shifted upfield to d 58.47 relative to 1
consistent with a C–[2H] bond.
y The diminution of this signal is expected and is due to loss of NOE and a longerT time for
1
C–[2H] vs. C-H.
Copyright # 2002 John Wiley & Sons, Ltd.
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H. S. KEZAR ET AL.
Scheme 1. Reagents and conditions: a tetrabutylammonium fluoride, THF, ice-
water bath; b. Dess-Martin periodinane, CH2Cl2, rt; c. NaB[2H]4, CH3O[2H]; d.
H2, 20% Pd(OH)2/C, EtOH; e. trace NH4OH; f. HCl, MeOH, reflux
Experimental
General: Melting points were determined on a Meltemp II melting
point apparatus and are uncorrected. The 1H NMR spectra were
Copyright # 2002 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2002; 45: 71–78

SYNTHESIS OF DEUTERATED-BCX-1777
75
reported on a Bruker AMX-360 at 360 MHz or a Bruker Avance 300
2
at 300.13 MHz spectrometer. The H NMR spectra were recorded on a
Bruker AMX-500 spectrometer at 76.77 MHz. The 13C NMR spectra
were recorded on a Bruker AMX-360 at 90.56 MHz or a Bruker Avance
300 at 75.5 MHz spectrometer. Chemical shifts (ppm) are referenced to
1
internal tertramethylsilane for H and 13C spectra and either CDCl3
(d ¼ 7:27) orDMSO-d
(d ¼ 2:52) for 2H spectra. Spectra were
6
recorded at ambient temperature unless otherwise noted. IR spectra
were obtained on a Bio-Rad FTS-7 FT-IR. Mass spectra were recorded
on a Micromass ZMD in the positive electrospray mode with a scan
range of 0–1000 m/z and cone voltage setting of 20 V. A solution of the
sample (ffi100 mg/ml) in methanol (100%) was introduced into the
source via a Waters 2690 autosampler. Flash chromatographic
(
separations were performed on Whatman silica gel, 60 A orusing a
(
Biotage Flash 40i or75 i with pre-packed silica get (60 A) cartridges.
Thin layer chromatography (TLC) was performed using aluminum
backed silica gel 60 plates from E. Merck. Sodium borodeuteride (98
atom % D) was obtained from Aldrich Chemical Co. (Milwaukee, WI).
(1S)-1-C-(5-N-Benzyloxymethyl-4-methoxypyrrolo[3,2-d]pyrimidin-
7-yl)-N-tert-butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-
D-ribitol (4): A sample of the silyl ether 37 (15.1 g, 23.1 mmol) was
dissolved in anhydrous tetrahydrofuran (450 ml) under an Ar atmo-
sphere and cooled in an ice/water bath. To this was added
tetrabutylammonium fluoride (1 M THF, 25 ml, 25 mmol, ꢁ1.1 eq.).
After30 min an additional 12.5 ml of TBAF was added followed by
another12.5 ml 30 min later( ꢁ50 ml total, ꢁ2.2 eq. total). The mixture
was concentrated and the residue partitioned with CH2Cl2 and
saturated aqueous NH4Cl. The organic layer was washed with water,
dried (Na2SO4), and concentrated to give a crude oil. The crude product
was purified by chromatography (Flash 75, 200 g SiO2, gradient
30 ! 40% EtOAc-hexane) and the relevant fractions combined and
evaporated to give 12.21 g (22.6 mmol, 98%) of 4 as a straw-colored
foam. An analytical sample was obtained from a center fraction to give
4 as a white foam. IR (KBr) 3195, 2979, 2936, 1693, 1612, 1538 and
1365 cmꢂ1; MS (m/z, ES+) 541.4 (100%); 1H-NMR (360 MHz, DMSO-
d6, 508C) d 8.45 (s, 1H), 7.64 (s, 1H), 7.29–7.18 (m, 5H), 5.73 (s, 2H),
5.13 (brs, 1H), 5.09–5.06 (brs, 1H, D 2O exchangeable), 5.05 (d,
J ¼ 5:2 Hz, 1H), 4.82 (d, J ¼ 5:5 Hz, 1H), 4.49 (s, 2H), 4.06 (s, 3H),
4.02–3.99 (m, 2H), 3.61–3.45 (m, 2H), 1.47 (s, 3H), 1.30 (s, 12H);
13C-NMR (90.56 MHz, DMSO-d6, 508C) d 155.66, 153.52, 149.11,
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H. S. KEZAR ET AL.
147.85, 137.24, 132.20, 127.85, 127.22, 127.04, 115.73, 114.92, 110.59,
84.11, 81.67, 78.77, 76.99, 69.46, 65.93, 60.92, 60.72, 53.18, 27.76, 27.13,
25.12. Analysis calculated forC 28H36N4O7: C, 62.21; H, 6.71; N, 10.36.
Found: C, 62.32; H, 6.87; N, 10.09.
5-Aldehydo-(1S)-1-C-(5-N-benzyloxymethyl-4-methoxypyrrolo[3,2-
d]pyrimidin-7-yl)-N-tert-butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-O-
isopropylidene-D-ribitol (5): A sample of alcohol 4 (11.75 g, 21.7 mmol)
was dissolved in CH2Cl2 (300 ml) underan Aratmospheer and Dess-
Martin periodinane (18.4 g, 43.5 mmol, 2.0 eq.) added. After 3 h, the
mixture was concentrated and the residue taken up in ether. The
resulting organic layer was washed with NaHCO3/Na2S2O3 (sat aq
NaHCO3 : 10% aq Na2S2O3 – 1:1) until all solids had dissolved. The
organic layer was then washed with saturated brine, dried (Na2SO4),
and concentrated to give 11.27 g (20.9 mmol, 96%) of 5. An analytical
sample was obtained by chromatography (Flash 40, 40 g SiO2, gradient
20 ! 30% EtOAc-hexane) to give 3 as a light yellow glass. IR (KBr)
2981, 2939, 1734, 1697, 1612, 1514, 1392 and 1367 cmꢂ1; MS (m/z,
ES+) 539.3 (100%); 1H-NMR (300 MHz, DMSO-d6) d 9.42 (d,
J ¼ 11:5 Hz, 1H), 8.36 (s, 1H), 7.88 (d, J ¼ 3:0 Hz, 1H), 7.30–7.19
(m, 5H), 5.75 (d, J ¼ 12:6 Hz, 2H), 5.30–5.22 (m, 2H), 4.86 (d,
J ¼ 5:1 Hz, 1H), 4.51–4.46 (m, 2H), 4.27 (d, J ¼ 8:7 Hz, 1H), 4.13
(s, 3H), 1.48 (s, 3H), 1.32 (s, 9H), 1.29 (s, 3H). 13C-NMR (90.56 MHz,
CDCl3) d 200.77, 156.31, 153.61, 150.10, 149.89, 136.82, 132.42, 131.08,
128.46, 127.98, 127.76, 127.54, 115.04, 112.35, 84.87, 83.27, 80.84, 73.81,
70.23, 59.88, 53.61, 28.95, 28.30, 25.26. Analysis calculated for
C28H34N4O7: C, 62.44, H, 6.36; N, 10.40. Found: C, 62.40; H, 6.41;
N, 9.98.
5-[2H]-(1S)-1-C-(5-N-Benzyloxymethyl-4-methoxypyrrolo[3,2-
d]pyrimidin-7-yl)-N-tert-butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-O-
isopropylidene-d-ribitol (6): A sample of 5 (6.95 g, 12.8 mmol) was
dissolved in CH3O[2H] (ꢁ60 ml) underan Aratmosphere. To this was
added NaB[2H]4 (0.75 g, 17.9 mmol, 1.4 eq.). After1 h, the volatiles were
removed and the residue taken up in MeOH (ꢁ50 ml) followed by
removal via rotovap (3 ꢃ ). The crude product was purified by
chromatography (Flash 40, 90 g SiO2, gradient 25 ! 40% EtOAc-
hexane) and the relevant fractions combined and evaporated to give
6.32 g (11.7 mmol, 91%) of 6 as a straw-colored foam. An analytical
sample was obtained from a center fraction to give 6 as a white foam. IR
(KBr) 3195, 2979, 2936, 2159(w), 1693, 1612, 1538 and 1366 cmꢂ1; MS
(m/z, ES+) 542.4 (100%); 1H-NMR (360 MHz, DMSO-d6, 508C) d 8.45
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF DEUTERATED-BCX-1777
77
(s, 1H), 7.63 (s, 1H), 7.31–7.19 (m, 5H), 5.73 (s, 2H), 5.13 (s, 1H)
5.05–5.04 (m, 1H), 4.81 (d, J ¼ 5:5 Hz, 1H), 4.49 (s, 2H), 4.06 (s, 3H),
4.00 (d, J ¼ 7:1 Hz, 1H), 3.55–3.48 (m, 1H), 1.47 (s, 3H), 1.30 (s, 12H);
13C-NMR (90.56 MHz, DMSO-d6, 508C) d 155.66, 153.53, 149.11,
147.83, 137.24, 132.20, 127.86, 127.22, 127.06, 115.76, 114.92, 110.59,
84.10, 81.65, 78.77, 76.99, 69.46, 65.87, 60.70, 60.33, 53.20, 27.78, 27.14,
2
25.12; H-NMR (76.77 MHz, DMSO) d 3.50. Analysis calculated for
C28H35D1N4O7: Calculated: (H+D as H) C, 62.21; H, 6.71; N, 10.36.
Found: C, 62.32; H, 6.87; N, 10.09.
5-[2H]-(1S)-1-C-(4-Methoxypyrrolo[3,2-d]pyrimidin-7-yl)-N-tert-
butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-d-ribitol (8):
A mixture of 6 (5.71 g, 10.5 mmol) and 20% Pd(OH)2 on C (5.7 g) in
EtOH (ꢁ75 ml) was shaken underan H atmosphere (Parr shaker, 40
2
psig) for 22 h. The mixture was filtered through Celite and the Celite
washed with EtOH. To the combined filtrates was added conc. aq
NH4OH (1 ml) and after 1 h the mixture concentrated. The crude
product was purified by chromatography (Flash 40, 90 g SiO2, 50%
EtOAc-hexane) and the relevant fractions combined and evaporated to
give 3.72 g (8.82 mmol, 84%) of 8 as a white foam. IR (KBr) 3219, 2982,
2938, 2164(w), 1664, 1630, 1541, 1400 and 1383 cmꢂ1; MS (m/z, ES+)
422.2 (100%); 1H-NMR (360 MHz, DMSO-d6, 508C) d 11.78 (brs, 1 H,
D2O exchangeable), 8.42 (s, 1H), 7.44 (s, 1H), 5.12 (brs, 3H), 4.83 (d,
J ¼ 4:9 Hz, 1H), 4.09 (s, 3H), 3.99 (d, J ¼ 6:2 Hz, 1H), 3.49 (d,
J ¼ 4:21 Hz, 1H), 1.47 (s, 3H), 1.30 (s, 12H); 13C-NMR (90.56 MHz,
DMSO-d6, 508C) d 155.31, 153.53, 148.38, 146.58, 128.42, 114.56
(coincident with anotherpeak), 110.50, 84.16, 81.61, 78.59, 65.78, 60.85,
60.51, 52.83, 27.76, 27.16, 25.14; 2H-NMR (90.56 MHz, DMSO) d 3.52.
Analysis calculated forC 20H27D1N4O6: Calculated: (H+D as H) C,
57.00; H, 6.70; N, 13.29. Found: C, 57.12; H, 6.64; N, 13.11.
5-[2H]-(1S)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-di-
deoxy-1,4-imino-D-ribitol hydrochloride (2): A mixture of 8 (3.22 g,
7.64 mmol) in MeOH (25 ml) and concentrated aqueous HCl (25 ml)
was refluxed under Ar for 2 h. The solution was concentrated and the
residue taken up in a small amount of water. EtOH was added and the
resulting mixture concentrated and the treatment with EtOH repeated
three times. The crude product was recrystallized from water/EtOH to
give 1.95 g (6.42 mmol, 84%) of 6 as an off-white solid, m.p.>2508C
(dec). IR (KBr) 3413, 3087, 3042, 2945, 1693, 1661 and 1596 cmꢂ1; MS
1
(m/z, ES+) 268.1 (100%); H-NMR (300 MHz, DMSO-d6) d 12.38
(d, J ¼ 2:7 Hz, 1H), 12.17 (s, 1H), 10.41 (brs, 1H), 8.41 (brs, 1H), 7.90
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H. S. KEZAR ET AL.
(s, 1H), 7.64 (d, J ¼ 3:1 Hz, 1H), 5.60 (d, J ¼ 4:2 Hz, 1H), 5.50
(d, J ¼ 3:1 Hz, 1H), 5.42 (d, J ¼ 4:6 Hz, 1H), 4.63 (d, J ¼ 7:2 Hz, 1H),
4.44 (d, J ¼ 3:0 Hz, 1H), 4.18 (d, J ¼ 2:6 Hz, 1H), 3.71 (s, 1H), 3.47 (m,
1
1H, coincident with HDO), H-NMR (300 MHz, DMSO-d6/D2O) d
7.90 (s, 1H), 7.58 (s, 1H), 4.65 (d, J ¼ 8:1 Hz, 1H), 4.48 (dd, J ¼ 8:1,
4.8 Hz, 1H), 3.72–3.69 (m, 1H), 3.54 (dd, J ¼ 4:4, 4.4 Hz, 1H); 13C-
NMR (75.5 MHz, DMSO-d6) d 153.52, 143.02, 142.22, 127.47, 118.09,
109.27, 74.04, 70.38, 65.12, 58.20, 56.65; 2H-NMR (90.56 MHz, DMSO)
d 3.64. Analytical Calculations forC 11H13D1N4O4HCl: Calculated:
(H+D as H) C, 43.50; H, 4.65; N, 18.45. Found: C, 43.84; H, 4.94; N,
18.21.
Acknowledgements
We thank Dr. Mike Kilpatrick of BioCryst Pharmaceuticals for the
isotopic purity determination.
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Copyright # 2002 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2002; 45: 71–78