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

Article abstract of DOI:10.1016/j.bmcl.2009.04.017

Forodesine HCl is being investigated as a potential therapeutic target for the control of T-cell proliferation. During our ongoing process development work on forodesine HCl several novel compounds were identified as possible impurities in the process. He

Full text of DOI:10.1016/j.bmcl.2009.04.017

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2624–2626
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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 I
*
Vivekanand P. Kamath , Jie Xue, Jesus J. Juarez-Brambila, Christopher B. Morris,
Rakesh Ganorkar, Philip E. Morris Jr.
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 our ongoing process development work on forodesine HCl several novel compounds were
identified as possible impurities in the process. Herein we present the synthesis of three novel com-
pounds (2–4).
Received 6 March 2009
Revised 1 April 2009
Accepted 3 April 2009
Available online 9 April 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
PNP inhibitor
Forodesine HCl
Purine nucleoside phosphorylase (PNP) enzyme has been
widely investigated due to its importance in the purine salvage
pathway. PNP catalyzes the reversible phosphorolysis of ribonu-
cleosides and 20-deoxyribonucleosides of guanine and hypoxan-
thine as well as many other related nucleosides analogs.1
Subsequent deprotection steps of the coupled intermediate gave
forodesine HCl (see Fig. 1).
The process described in the literature generated small quanti-
ties of material for toxicology studies.6–9 Hence there was a need
to optimize the process conditions and implement a feasible route
for large scale manufacturing of fordesine HCl. During the course
of this process development some unique compounds were iso-
lated. The structures of these compounds were determined by
NMR, prep HPLC and LC–MS. These compounds have been identi-
fied as by-products of the process and have to be documented in
the new drug application (NDA). To understand the nature of
these compounds they were synthesized, analyzed and tested
for any activity against PNP enzyme. This paper describes the syn-
thesis of these isolated compounds 2–4 (structures as shown in
Fig. 2).
b-Nucleoside þ PO4 $ Base þ
a-ribose 1-phosphate
The relationship between PNP deficiency and certain types of immu-
nological diseases such as T-cell cancer, rheumatoid arthritis, psoria-
sis and other autoimmune diseases has made this an attractive
therapeutic target. Biochemical evidence suggests that the mamma-
lian PNP enzyme is a trimer made up of three identical units of
32 kDa. The protein sequence analysis has shown that the human
PNP is about 25% identical to members of the mammalian purine
nucleoside phosphorylase (PNP) family of trimeric enzymes.2,3
In the past decade, various groups have identified several potent
inhibitors of PNP. Schramm and co-workers have investigated the
kinetic isotope effects on the PNP enzyme to predict the transi-
tion-state structure.4,5 This has led to the development of several
novel classes of inhibitors that are extremely potent against PNP.
These compounds are the aza-C-nucleosides, and one of them,
forodesine HCl is currently in clinical trials.
As outlined in Scheme 1, compound 69 (obtained by quenching
the lithiated deaza-hypoxantine (15) with water) was treated with
n-BuLi (1.6 M solution in hexanes) in anhydrous THF at ꢀ35 °C,
O
H
N
NH
The convergent synthesis of forodesine HCl was first reported
by Tyler and co-workers.6–9 In this process the lithiated deaza-
hypoxanthine (15) was coupled to the imine (8)7a followed by
purification of the intermediate using column chromatography.
N
H
HO
N
HCl
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.2009.04.017

V. P. Kamath et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2624–2626
2625
O-
N+
O
N
O
HN
H
N
H
N
H
N
RO
RO
O
N
RO
N
NH
NH
Urea.H2O2
1. NCS
2. DBU
98%
HO2C
MeReO3
59%
HN
N
O
O
O
O
O
O
H
N
H
N
H
HO
HO
N
13
12
11. R = TBDMS
HO
OH
OMe
HO
OH
BnO
HO
OH
HO
OMe
BnO
2
N
3
4
n-BuLi
N
N
13
N
º
C
º
C
-50
-30
Figure 2. Structure of analogs of forodesine HCl.
N
84%
N
Br
Li
14
15
OMe
N
N
N
BnO
RO
+
N
MeO
BnO
MeO
BnO
OMe
N
BnO
Li
N
N
N
OH
N
n-BuLi
N
H
N
N
N
Zn/HOAc
86%
º
C
-50
O
O
N
N
6
7
8. R = TBDMS
TBDMSO
TBDMSO
O
O
O
O
MeO
N
N
BnO
16
17
N
H
O
TBDMSO
Pd(OH)2, H2
N
THF
H
N
NH3/MeOH
86%
NH
N
º
º
-35 C - 0 C
22%
O
O
6M HCl/MeOH
58%
H
N
9
HCl
N
N
HN
N
MeO
O
HO
HO
OH
HN
HN
3
H
HCl/MeOH
49%
H
N
TBDMSO
HO
N
Scheme 2.
.HCl
OH
O
O
HO
OMe
OMe
N
BnO
N
BnO
2
N
O
10
N
HO2C
Scheme 1.
N
N
H
N
H
RO
RO
1. n-BuLi
N
º
2. CO2, -30 C
58%
followed by addition of the imine, 87a (pre-dissolved in dry THF).
The reaction was stirred for 10 min and then warmed up to 0°°C
and stirred at this temperature for 1 h to furnish 9 in 22% yield.
Hydrogenation of the intermediate using Pd(OH)2 in methanol
pre-saturated with ammonia gave 10 in 86% yield. Finally, acid
hydrolysis of 10 in concd HCl/MeOH under reflux conditions for
48 h, furnished a white solid. The solid was recrystallized in
water/ethanol mixture to give compound 2, in 49% yield.13
O
O
O
18
9.R = TBDMS
O
H
N
NH
HO2C
HO
N
1. Pd(OH)2, H2
H
N
On similar lines (Scheme 2) compound 119 was treated with
N-chlorosuccinimide (NCS) to give the chlorinated compound,
which upon treatment with DBU at rt for 1 h gave the ketimine
12 in quantitative yields.10 Treatment of the ketimine, 12 with urea
hydrogen peroxide and methyltrioxorhenium in MeOH at rt for 1 h
followed by purification by column chromatography gave the
desired nitrone 13 in 59% yield.11 As reported earlier in the litera-
ture, 9-bromo deazahypoxanthine was lithiated using n-BuLi
(1.6 M solution in hexanes) in TBME at ꢀ30 °C followed by addition
of the nitrone 13. The reaction was warmed to 0 °C and stirred for
1.5 h. The reaction mixture was worked up and purified by column
chromatography to give 16 in 84% yield. Reduction of 16 using zinc
dust in acetic acid furnished compound 17 in 86% yield.12 Acid
hydrolysis using 6 M HCl/MeOH under reflux conditions for 20 h
furnished compound 3 as a white solid. The solid was recrystal-
lized in water/ethanol mixture to give compound 3, in 58% yield.14
2. 4M HCl/MeOH
52%
.HCl
OH
HO
4
Scheme 3.
Scheme 3 describes the synthesis of compound 4. Compound 9
(obtained from coupling of the imine (8) with the lithiated deaza-
hypoxanthine, 15) was treated with n-BuLi (1.6 M solution in hex-
anes) in dry THF at ꢀ30 °C and stirred for 20 min. Dry ice was
added slowly so that the internal temperature was maintained
below ꢀ30 °C. The reaction mixture was stirred for 30 min at this
temperature and then warmed to 0 °C and continued stirring for
1 h. Workup of the reaction followed by column chromatography

2626
V. P. Kamath et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2624–2626
catalyst was filtered over Celite pad and washed with copious amount of MeOH
(100 mL). Evaporation of the solvent gave 10 (22.2 g, 86%). The solid was
dissolved in 1:1 (v/v) ratio of MeOH/concd HCl (208 mL) and refluxed for 48 h.
Evaporation of the solvent gave a solid residue. The solid was redissolved in
water and decolorized with activated carbon. The mixture was filtered, washed
with water and then concentrated to a solid. The solid was recrystallized with
water/ethanol mixture to furnish 2 (6.1 g, 49%) as a white solid. 1H NMR
(300 MHz, D2O) d 7.92 (s, 1H, H-2), 6.68 (s, 1H, H-9), 4.78 (d, J = 9 Hz, H-20),
4.65 (m, 1H, H-30), 4.34 (t, 1H, H-40), 3.86 (dd, 1H, J = 6.0, 8.6 Hz, H-60), 3.80 (m,
1H, H-50); 13C NMR (75.5 MHz, D2O) d 155.8, 145.0, 143.4, 134.3, 119.4, 104.1,
74.2, 71.3, 66.4, 59.1, 58.5. HRMS: Calcd for C11H15N4O4 (M+H+): 267.1082;
found: 267.1080.
gave the desired product, 18 in 58% yield. Finally, hydrogenation
and acid hydrolysis of the intermediate followed by crystallization
gave 4 as a white solid. The solid was recrystallized in water/etha-
nol mixture to give compound 4, in 52% yield.15
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.
Acknowledgements
14. To
a solution of 11 (100 g, 348 mmol) in toluene (500 mL) was added
N-chlorosuccinimide (60.4 g, 452 mmol). After stirring for 1 h the solids were
filtered off and DBU (57 mL, 381 mmol) was added to the filtrate. The reaction
mixture was stirred for 1 h and then purified by column chromatography to
furnish 12 (100 g,) as a syrup. Urea hydrogen peroxide (98.2 g, 1044 mmol) and
methyltrioxorhenium (1.73 g, 6.96 mmol) was added to a solution of 12 in MeOH
(660 mL). The reaction mixture was stirred for 1 h, filtered and concentrated. The
residue was purified by column chromatography to furnish the nitrone, 13
(62.4 g, 59%). 9-Bromo deazahypoxanthine, 14 (87.1 g, 250 mmol) was lithiated
using n-BuLi (164 mL) in dry TBME (1.6 L) at ꢀ30 °C. The nitrone, 13 dissolved in
TBME (300 mL) was added slowly to the reaction at ꢀ30 °C over 20 min. and then
warmed to 0 °C and stirred for 1.5 h. Chromatography of the crude product gave
16 (95.8 g, 84%) as a syrup. Reduction of 16 using zinc dust (110.6 g, 1691 mmol)
in acetic acid (400 mL) and stirring for 1 h at rt gave 17 (90 g, 86%) as a oil. Finally,
acid hydrolysis using 6 M HCl/MeOH (300 mL) under reflux conditions for 20 h
followed by workup (as described for compound 2) and crystallization furnished
3 (25.2 g, 58%) as a white solid. 1H NMR: (DMSO-d6) d 12.38 (d, 1H, J = 3.0 Hz, H-
7), 12.18 (d, 1H, J = 3.0 Hz, H-1), 9.56 (br, 1H, H-10), 9.16 (br, 1H, H-10), 7.92 (d, 1H,
J = 3.0 Hz, H-2), 7.64 (d, 1H, J = 3.0 Hz, H-8), 5.81 (br, 1H, OH), 5.47 (br, 1H, OH),
5.28 (br, 1H, OH), 4.66 (d, 1H, J = 6.0 Hz, H-40), 4.18 (m, 1H, H-30), 4.13 (d, 1H,
J = 12.0 Hz, H-60), 4.01 (d, 1H, J = 12.0 Hz, H-60), 3.60 (m, 1H, H-20), 3.09 (m, 1H, H-
20); 13C NMR (75.5 MHz, D2O) d 155.8, 143.6, 142.8, 127.7, 118.4, 110.6, 76.2,
72.4, 70.2, 63.4, 48.6. HRMS: Calcd for C11H15N4O4 (M+H+): 267.1082; found:
267.1085.
We thank Dr. Ken Belmore at The University of Alabama,
Tuscaloosa for the high-field NMR spectral analysis; Ms. Vallarie
Tate for checking the manuscript and Dr. Krishnan Raman for
obtaining the crystals.
References and notes
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Bagdassarian, C. K.; Furneaux, R. H.; Schramm, V. L. Biochemistry 1998, 37, 8615.
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6. Tyler, P. C.; Furneaux, R. H. J. Org. Chem. 1999, 64, 8411.
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15. A cold solution of n-BuLi (2.5 M, 150.8 mL) was added slowly to a solution of 9
(95 g, 171.5 mmol) in dry THF (1.8 L) at ꢀ30 °C. Upon completion of the
reaction as indicated by TLC, dry ice (37.7 g, 858 mmol) was added slowly to
the reaction mixture. at ꢀ30 °C. The reaction was warmed to 0 °C and stirred
for 1 h. Evaporation of the solvent followed by column chromatography gave
18 (60.0 g, 58%). The syrup was suspended in MeOH (700 mL), pre-saturated
with ammonia followed by addition of Pd(OH)2 and hydrogenated at 150 psig.
The catalyst was filtered over a Celite pad followed by evaporation of the
8. Evans, G. B.; Furneaux, R. H.; Lewandowicz, A.; Schramm, V. L.; Tyler, P. C. J.
Med. Chem. 2003, 46, 3412.
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7213; (b) Fleet, G. W. J.; Son, J. C. Tetrahedron 1988, 44, 2637.
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12. Evans, G. B.; Furneaux, R. H.; Hausler, H.; Larsen, J. S.; Tyler, P. C. J. Org. Chem.
2004, 69, 2217.
13. A cold solution of n-BuLi (1.6 M, 208 mL) was added slowly to a solution of 6
(81.6 g, 303 mmol) in dry THF at ꢀ35 °C. Upon completion of the reaction as
indicated by TLC a solution of 8 (66.9 g, 233.1 mmol) in dry THF was added at
ꢀ35 °C. After the last addition the reaction was warmed to 0 °C and stirred for
1 h. Evaporation of the solvent followed by column chromatography gave 9
(28.4 g, 22%). The syrup was suspended in MeOH (300 mL), pre-saturated with
ammonia followed by addition of Pd(OH)2 and hydrogenated at 150 psig. The
filtrate to furnish
a solid residue. Acid hydrolysis using 4 M HCl/MeOH
(300 mL) under reflux conditions for 2 h furnished 4 (16.8 g, 52%) as a white
solid. 1H NMR (300 MHz, DMSO-d6 + 3 drops DCl) d 8.27 (s, 1H, H-2), 5.22 (d,
1H, J = 7.77 Hz, H-20), 4.62 (dd, 1H, J = 5.04, 7.74 Hz, H-30), 4.28 (t, 1H, H-40),
3.80 (m, 2H, H-60), 3.59 (m, 1H, H-50); 13C NMR (75.5 MHz, D2O) d 161.8, 153.2,
143.3, 140.8, 128.1, 119.5, 112.8, 73.1, 69.8, 64.9, 58.1, 56.2. HRMS: Calcd for
C12H14N4O6 (M+H+): 310.2710; found: 310.2714.