Synthesis of analogs of forodesine HCl, a human purine nucleoside phosphorylase inhibitor-Part II

Article abstract of DOI:10.1016/j.bmcl.2009.04.018

Forodesine HCl is being investigated as a potential therapeutic target for the control of T-cell proliferation. During the filing process for a New Drug Application (NDA) it became evident that there was a need to synthesize some stereo-isomers of forodes

Full text of DOI:10.1016/j.bmcl.2009.04.018

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2627–2629
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of analogs of forodesine HCl, a human purine nucleoside
phosphorylase inhibitor—Part II
*
Vivekanand P. Kamath , Jie Xue, Jesus J. Juarez-Brambila
BioCryst Pharmaceuticals Inc., 2190 Parkway Lake Drive, Birmingham, AL 35244, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Forodesine HCl is being investigated as a potential therapeutic target for the control of T-cell prolifera-
tion. During the filing process for a New Drug Application (NDA) it became evident that there was a need
to synthesize some stereo-isomers of forodesine HCl. Herein we present the synthesis of these three
novel compounds (2–4).
Received 6 March 2009
Revised 2 April 2009
Accepted 3 April 2009
Available online 9 April 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
PNP inhibitor
T-cell proliferation
Purine nucleoside phosphorylase (PNP) is a purine metabolizing
enzyme that catalyzes the reversible phosphorylysis of purine
nucleosides such as 20-deoxyguanosine (dGuo) to the respective
literature by Schramm and co-workers.9 The structures of the
stereo-isomers are shown (see Fig. 2).
As part of the NDA filing process there is a need to establish a
validated commercial route for large scale manufacturing. FDA
regulations require identification, isolation, characterization and
validation of analytical methods for all possible impurities
observed in the process. Comparison of the HPLC chromatograms
of the parent compound with those of the stereo-isomers and other
impurities supports establishment of the purity of the final
product.
In Part I of our Letter we described the synthesis of some impu-
rities which were identified as by-products of the commercial
process.10 In this Letter we describe the synthesis of the stereo-iso-
mers, compounds 2–4.
Scheme 1, describes the synthesis of compound 2, starting from
the previously reported compound 5.11 Treatment of 5 with
N-chlorosuccinimide for 1 h followed by reaction with DBU gave
the imine, 6 as a syrup in 65% yield.14 The unsaturated compound
was reduced using a macroporous polystyrene-bound equivalent
base and deoxyribose-a
-1-phosphate.1–4 The importance of PNP
to the integrity of the immune system became apparent when
children lacking the PNP enzyme developed severe T-cell
immunodeficiency.5
The unique sensitivity of human T-cells to PNP deficiency is
attributed to a relatively high level of kinase and a low level of
nucleotidase activity compared to other cell types. This unique
enzyme ratio is especially characteristic of immature T-cells. Based
upon these observations, several novel classes of PNP inhibitors
have been designed for the treatment of T-cell mediated diseases
such as cutaneous T-cell lymphoma (CTCL) and acute lymphoblas-
tic leukemia (ALL) and other autoimmune disorders such as psori-
asis, Crohn’s disease and rheumatoid arthritis. One such compound
investigated by BioCryst Pharmaceuticals Inc. is forodesine HCl, a
PNP inhibitor which is currently in advanced stages of clinical trials
(see Fig. 1).6,7
During the filing process for a New Drug Application (NDA) it
became evident that there was a need to synthesize some stereo-
isomers of forodesine HCl. In our earlier communication we
described the synthetic route for making forodesine HCl.8 The
process involved using both basic and acidic conditions. Under
these conditions the risk of epimerization at the stereogenic cen-
tres would be substantial. Hence it was necessary to synthesize
the stereo-isomers at C-20, C-30 and C-40 positions. The synthesis
O
H
N
NH
N
H
HO
N
HCl
of the L-enantiomer of fordesine HCl has been described in the
OH
HO
1
* Corresponding author. Tel.: +1 205 444 4600; fax: +1 205 444 4640.
E-mail address: vkamath@biocryst.com (V.P. Kamath).
Figure 1. Structure of forodesine HCl.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.018

2628
V. P. Kamath et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2627–2629
BOM
O
N
BOM
H
N
O
N
H
N
H
N
HO
H
BOC
N
BOC
N
N
N
OMe
OMe
NH
NH
HO
1N HCl
N
N
RO
HO
H
N
MeOH
95%
OH
N
HO
H
N
N
N
HO
O
O
HO
OH
8. R = TBDMS
9
HN
N
H
HO
OH
3
HO
OH
BOM
O
2
4
BOC
N
N
OMe
Figure 2. Structures of stereo-isomers of forodesine HCl.
TIPDSCl2
55%
1. Swern
2. NaBH4
N
O
N
Si
O
N
OH
O
(92% - 2 steps)
Si
10
BOM
BOM OMe
O
H
N
BOM
N
N
NH
N
BOC
N
OMe
1. NCS
H
OMe
N
N
N
H
HO
N
N
conc. HCl
43%
N
RO
N
O
Si
O
.HCl
N
RO
N
2. DBU
65%
O
O
O
OH
11
Si
HO
O
O
6
OH
3
5. R = TBDMS
BOM
Scheme 2.
N
OMe
H
N
MP-TABH
N
TBDMSO
N
168 h, 98%
BOM
BOM
O
O
BOC
N
N
BOC
N
N
OMe
OMe
7
1. TrCl
2. Bu2SnO
H
N
N
N
HO
RO
H
HO
3. pMeOBnCl
27%- 3 steps
N
N
. HCl
HO
OH
HO
OPMB
9
12. R = Trityl
1. Pd(OH)2/C, H2
OH
N
HO
O
H
N
2. 4M HCl/MeOH
53%
BOM
NH
HN
BOC
N
OMe
N
N
H
H
N
HO
O
2
N
1. Swern
conc. HCl
76%
N
.HCl
RO
N
2. NaBH4
79%
HO
OPMB
13
Scheme 1.
HO
OH
4
Scheme 3.
tetraalkylammonium triacetoxyborohydride (MP-TABH) reagent
[purchased from Biotage, 1.8–2.4 mmol/g] with stirring for one
week at rt, to furnish compound 7 in 98% yield. Hydrogenation of
the intermediate using Pd(OH)2/C in MeOH pre-saturated with
ammonia followed by acid hydrolysis using 4 M HCl/MeOH under
reflux conditions for 2 h, furnished a solid. The solid was recrystal-
lized in water/EtOH mixture to give the desired compound 2, in
53% yield.14 The structure of compound 2 was further supported
by X-ray crystallography data.
stannylation chemistry followed by addition of p-MeOBnCl. The
reaction mixture was purified by column chromatography to
isolate compound 12 (27%—three steps).13 Swern oxidation, fol-
lowed by reduction of the ketone using NaBH4 gave 13 in 79%
yield. Finally, acid hydrolysis was carried out using concd HCl
under reflux conditions. Workup of the reaction mixture gave a
solid residue. The solid was recrystallized in water/EtOH mixture
to give compound 4 in 76% yield as a white solid.16
The final compounds were tested for PNP activity. The IC50 val-
ues were observed to be in the range of 100–300 nM and although
the compounds did show some activity it was not as potent as the
parent compound, forodesine HCl.
On similar lines (Scheme 2) compound 811 was treated with 1 N
HCl in MeOH and heated to 40 °C for 24 h to furnish 9 in 95% yield.
The intermediate was treated with 1,3-dichloro-1,1,3,3-tetra-
isopropyldisiloxane (TIPDSCl2) that would allow the hydroxyl
groups, O-60 and O-40 to be bridged together to furnish 10 in 55%
yield. Swern oxidation, followed by reduction of the ketone using
NaBH4 furnished 11 in 92% yield (two steps).12 Finally, acid hydro-
lysis was carried out using concd HCl under reflux conditions for
1 h followed by workup of the reaction mixture to furnish a solid.
The solid was recrystallized in water/EtOH mixture to give com-
pound 3 as a white solid in 43% yield.15
Acknowledgements
We thank Dr. Ken Belmore at The University of Alabama, Tusca-
loosa for the high-field NMR spectral analysis; Ms. Vallarie Tate for
checking the manuscript; Dr. Krishnan Raman for obtaining the
crystals and Dr. Nattamai Bhuvanesh at Texas A&M University for
obtaining the X-ray crystal structure.
Scheme 3 describes the synthesis of compound 4. Compound 9
was treated with trityl chloride to protect the primary hydroxyl at
O-60 position. The intermediate was subjected to standard

V. P. Kamath et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2627–2629
2629
40), 3.81 (dd, 1H, J = 12.0, 3.0 Hz, H-60), 3.72 (dd, 1H, J = 12.0, 6.0 Hz, H-60), 3.45
(br, 1H, H-50); 13C NMR: (DMSO-d6, 75.5 MHz) d 152.83, 143.15, 129.13, 117.16,
106.60, 71.76, 71.67, 62.48, 58.27, 55.30. HRMS: calcd for C11H15N4O4 (M+H+):
267.1082; found: 267.1080.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.04.018.
15. To a solution of 8 (65.6 g, 100 mmol) in MeOH (260 mL) was added 1 N HCl
(145 mL) and the reaction was heated to 40 °C for a period of 24 h. Upon
completion of the reaction as indicated by TLC the reaction mixture was
worked up to furnish 9 (47.5 g, 95%). A small portion of the sample 9 (10 g,
20 mmol) was stirred in dry DMF (50 mL) in presence of imidazole (5.45 g,
80 mmol) and TIPDSCl2 reagent (7.04 mL, 22 mmol). The reaction mixture was
stirred at rt for 20 h. Workup of the reaction followed by column
References and notes
1. Horenstein, B. A.; Parkin, D. W.; Estupinan, B.; Schramm, V. L. Biochemistry
1991, 30, 10788.
2. Horenstein, B. A.; Schramm, V. L. Biochemistry 1993, 32, 7089.
3. Miles, R. W.; Tyler, P. C.; Evans, G. B.; Furneaux, R. H.; Parkin, D. W.; Schramm,
V. L. Biochemistry 1999, 38, 13147.
4. (a) Evans, G. B.; Furneaux, R. H.; Lewandowicz, A.; Schramm, V. L.; Tyler, P. C. J.
Biol. Chem. 2003, 34, 31465; (b) Miles, R. W.; Tyler, P. C.; Bagdassarian, C. K.;
Furneaux, R. H.; Schramm, V. L. Biochemistry 1998, 37, 8615; (c) Boyer, P. D.
Annu. Rev. Biochem. 1997, 66, 717.
chromatography gave 10 (8.3 g, 55%). To
a solution of DMSO (2.8 mL,
39.2 mmol) in DCM (72 mL) at ꢀ70 °C was added TFAA (2.8 mL, 20.2 mmol)
and stirred for 10 min. To this was added compound 10 (2.6 g, 3.5 mmol) pre-
dissolved in DCM (60 mL) followed by addition of triethylamine (4.2 mL,
56.7 mmol). The reaction was stirred at that temperature for 15 min. and then
allowed to warm to rt. Usual workup of the reaction furnished the crude
ketone. The crude ketone was suspended in MeOH (100 mL) followed by
addition of NaBH4 (1.4 g, 35 mmol) and stirred at rt for 30 min. Evaporation of
the solvent followed by column chromatography furnished 11 (2.4 g, 92%—two
steps). Compound 11 (2.4 g, 3.22 mmol) was taken in concd HCl (5 mL) and
refluxed for 1 h. The solvent was evaporated to give a solid which was then
recrystallized from water/EtOH to give compound 3 (0.42 g, 43%) as a white
solid. 1H NMR (300 MHz, D2O): d 7.82 (s, 1H), 7.60 (s, 1H), 4.98 (s, 1H), 4.22 (s,
1H), 4.04 (s, 1H), 3.83 (dd, J 12.0, 5.0 Hz, 1H), 3.72 (dd, J 12.0, 12.0 Hz, 1H), 3.52
(br, 1H); 13C NMR: (D2O, 75.5 MHz) d 155.1, 143.4, 142.5, 130.2, 117.4, 105.3,
76.3, 75.9, 67.1, 59.6, 57.5. HRMS: calcd for C11H15N4O4 (M+H+): 267.1082;
found: 267.1085.
5. Markert, M. L. Immunodeficiency Rev. 1991, 3, 45.
6. Bantia, S.; Miller, P. J.; Parker, C. D.; Ananth, S.; Horn, L. L.; Kilpatrick, J. M.;
Morris, P. E., Jr.; Hutchison, T. L.; Montgomery, J. A.; Sandhu, S. Int.
Immunopharmacol. 2001, 1, 1199.
7. Kilpatrick, J. M.; Morris, P. E., Jr.; Serota, D. J.; Phillips, D.; Moore, D. R.; Bennett,
J. C.; Babu, Y. S. Int. Immunopharmacol. 2003, 3, 541.
8. (a) Tyler, P. C.; Limberg, G.; Furneaux, R. H.; Schramm, V. L. Tetrahedron 1997,
53, 2915; (b) Tyler, P. C.; Furneaux, R. H.; Schramm, V. L. Bioorg. Med. Chem.
1999, 7, 2599; (c) Evans, G. B.; Tyler, P. C.; Furneaux, R. H.; Hutchison, T. L.;
Kezar, H. S.; Morris, P. E.; Schramm, V. L. J. Org. Chem. 2001, 66, 5723.
9. Clinch, K.; Evans, G. B.; Fleet, G. W. J.; Furneaux, R. H.; Johnson, S.; Lenz, D. H.;
Mee, S. P. H.; Rands, P. R.; Schramm, V. L.; Taylor, E. A.; Tyler, P. C. Org. Biomol.
Chem. 2006, 4, 1131.
10. Kamath, V. P.; Xue, J.; Brambila, J. J.; Morris, C. B.; Ganorkar, R.; Morris, P. E., Jr.
Bio. Med. Chem. Lett. 2009, 19. doi:10.1016/j.bmcl.2009.04.017.
11. (a) Evans, G. B.; Furneaux, R. H.; Gainsford, G. J.; Schramm, V. L.; Tyler, P. C.
Tetrahedron 2000, 56, 3053; (b) Tyler, P. C.; Furneaux, R. H. J. Org. Chem. 1999,
64, 8411.
16. To a solution of compound 9 (5.0 g, 10 mmol) in DCM (30 mL) was added N,N
di-isopropylethylamine (6.89 mL, 39.6 mmol), catalytic amount of DMAP
(0.2 g) and trityl chloride (3.36 g, 12.0 mmol). The reaction mixture was
stirred at rt for 16 h. Upon completion of the reaction (as indicated by TLC) the
reaction mixture was evaporated to furnish the crude intermediate. The crude
was taken in toluene (100 mL) followed by addition of dibutyl tin oxide (2.2 g,
8.81 mmol) and refluxed using a Dean–Stark apparatus for 1 h. This was
followed by addition of tetrabutylammonium bromide (2.82 g, 8.81 mmol) and
p-MeO benzyl chloride (21.7 mL, 159.6 mmol) and the reaction refluxed
further for 4 h. Workup of the reaction followed by column chromatography
gave the desired compound 12 (3.1 g, 27%—three steps). To a solution of DMSO
(2.5 mL, 35 mmol) in DCM (72 mL) at ꢀ70 °C was added TFAA (2.5 mL,
18 mmol) and stirred for 10 min. To this was added compound 12 (3.1 g,
2.68 mmol) pre-dissolved in DCM (60 mL) followed by addition of
triethylamine (3.7 mL, 50 mmol). The reaction was stirred at that
temperature for 15 min. and then allowed to warm to rt. Evaporation of the
solvent furnished the crude ketone. The crude ketone was suspended in MeOH
(100 mL) followed by addition of NaBH4 (1.2 g, 30 mmol) and stirred at rt for
30 min. Evaporation of the solvent followed by column chromatography
furnished 13 (2.45 g, 79%—two steps). Compound 13 (2.45 g, 3.22 mmol) was
taken in concd HCl (5 mL) and refluxed for 1 h. The solvent was evaporated to
give a solid which was then recrystallized from water/EtOH to give compound
4 (0.62 g, 76%) as a white solid. 1H NMR (300 MHz, D2O) d 7.97 (s, 1H), 7.64 (s,
1H), 4.74 (d, J 5.0 Hz, 1H), 4.62 (dd, J 5.0, 3.0 Hz, 1H), 4.34 (dd, J 4.2, 3.0 Hz, 1H),
3.82–3.96 (m, 3H); 13C NMR: (D2O, 75.5 MHz) d 155.0, 143.1, 142.7, 129.4,
117.7, 108.2, 79.4, 74.6, 62.8, 58.9, 57.1. HRMS: calcd for C11H15N4O4 (M+H+):
267.1082; found: 267.1081.
12. Kezar, H. S.; Kilpatrick, D. P.; Kellog, D.; Zhang, P. E.; Morris, P. E. Nucelosides,
Nucleotides Nucleic Acids 2005, 24, 1817.
13. Evans, G. B.; Furneaux, R. H.; Lewandowicz, A.; Schramm, V. L.; Tyler, P. C. J.
Med. Chem. 2003, 46, 3412.
14. To a solution of 5 (45.4 g, 82 mmol) in dry mTBE (3.0 L) at 0 °C was added NCS
(16.4 g, 123 mmol) and stirred for 1 h. The solids were filtered and then the
filtrate was treated with DBU (18.4 mL, 123 mmol). And stirred at rt for 3 h.
Upon completion of the reaction the unsaturated product was purified by
chromatography to furnish 6 (23 g, 65%). MP-triacetoxyborohydride (100 g,
240 mmol) was added to the unsaturated product dissolved in THF (300 mL)
and stirred for 1 week at rt. The resin was filtered and concentrated to 7 (33 g,
98%). The syrup was suspended in MeOH (700 mL), pre-saturated with
ammonia followed by addition of Pd(OH)2/C (2.5 g) and hydrogenated at
150 psig. The catalyst was filtered over Celite pad and washed with MeOH.
Evaporation of the filtrate followed by acid hydrolysis using 4 M HCl (160 mL)
and methanol (250 mL) under reflux conditions for 2 h furnished 2 (20.3 g,
53%) as a white solid. 1H NMR (300 MHz, DMSO-d6) d 12.76 (s, 1H, H-7), 9.59
(br, 1H, H-1), 8.43 (s, 1H, H-2), 7.78 (d, 1H, J = 3.0 Hz, H-8), 6.98 (br, 4H), 4.96 (t,
1H, J = 3.0 Hz, H-20), 4.23 (dd, 1H, J = 9.0, 3.0 Hz, H-30), 4.08 (t, 1H, J = 3.0 Hz, H-