Discovery of new S-adenosylmethionine decarboxylase inhibitors for the treatment of Human African Trypanosomiasis (HAT)

Article abstract of DOI:10.1016/j.bmcl.2009.04.096

Modification of the structure of trypanosomal AdoMetDC inhibitor 1 (MDL73811) resulted in the identification of a new inhibitor 7a, which features a methyl substituent at the 8-position. Compound 7a exhibits improved potencies against both the trypanosomal AdoMetDC enzyme and parasites, and better blood brain barrier penetration than 1.

Full text of DOI:10.1016/j.bmcl.2009.04.096

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2916–2919
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of new S-adenosylmethionine decarboxylase inhibitors
for the treatment of Human African Trypanosomiasis (HAT)
Bradford Hirth a, Robert H. Barker Jr. a, Cassandra A. Celatka a, Jeffrey D. Klinger a, Hanlan Liu a, Bakela Nare b,
Amarjit Nijjar a, Margaret A. Phillips c, Edmund Sybertz a, Erin K. Willert c, Yibin Xiang a,
*
a Drug and Biomaterial R&D, Genzyme Corporation, 153 Second Avenue, Waltham, MA 02451, United States
b SCYNEXIS, Inc., 3501C Tricenter Boulevard, Durham, NC 27713, United States
c University of Texas Southwestern Medical Center, Department of Pharmacology, 6001 Forest Park Road, Dallas, TX 75390-9041, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Modification of the structure of trypanosomal AdoMetDC inhibitor 1 (MDL73811) resulted in the identi-
fication of a new inhibitor 7a, which features a methyl substituent at the 8-position. Compound 7a exhib-
its improved potencies against both the trypanosomal AdoMetDC enzyme and parasites, and better blood
brain barrier penetration than 1.
Received 10 March 2009
Revised 16 April 2009
Accepted 21 April 2009
Available online 24 April 2009
Ó 2009 Published by Elsevier Ltd.
Keywords:
Sleeping sickness
Trypanosomiasis
S-Adenosylmethionine decarboxylase
(AdoMetDC)
Inhibitor
Blood brain barrier
Purine
Sleeping sickness, or Human African Trypanosomiasis (HAT) is
caused by the protozoan pathogen Trypanosoma brucei, which in-
fects between 50,000 and 150,000 people each year across sub-
Saharan Africa, and is fatal if left untreated.1 Current drugs such
as Melarsoprol or Eflornithine have either significant toxicity or
highly impractical dosing regimens for rural Africa due to the
requirement of extended intravenous infusion. New therapies are
urgently needed, yet because of the extreme poverty in countries
with endemic disease, for many years there has been little interest
within the pharmaceutical industry in discovering and developing
new drugs for this indication.
Polyamines, such as spermine and spermidine, are small cat-
ionic molecules that are critical to the survival of eukaryotes,
including trypanosomes.2,3 It has been postulated that the rate lim-
iting step for the biosynthesis of polyamines is catalyzed by S-
adenosylmethionine decarboxylase (AdoMetDC), which converts
S-adenosylmethionine (AdoMet) to decarboxylated S-adenosylme-
thionine (dcAdoMet).4,5 It has been reported that the T. brucei Ado-
MetDC differs substantially from the human host enzyme, and it is
allosterically activated by heterodimer formation with an inactive
paralog, termed prozyme.6,7 This regulatory mechanism is found
only in the trypanosomatids. In 1989, Casara et al. reported the dis-
covery of a mechanism based inhibitor 1 (MDL73811) (Fig. 1),
which potently inhibits AdoMetDC in vitro and notably improves
the survival of T. brucei infected mice.8,9 However, the pharmacoki-
netic and tissue distribution characteristics of compound 1, espe-
cially its apparent low penetration of the blood brain barrier, are
not adequate to meet the improved target product profile for a
new anti-trypanosomal drug.
We undertook a medicinal chemistry effort to modify the struc-
ture of 1 with the goal of improving the potency and pharmacoki-
netic characteristics of the compound, especially the ability to
cross the blood brain barrier. Because it was reported that the
NH2
N
N
N
N
OH
OH
O
H2N
N
Me
* Corresponding author. Tel.: +1 781 434 3653; fax: +1 781 672 5823.
E-mail address: yibin.xiang@genzyme.com (Y. Xiang).
Figure 1. 1 (MDL73811).
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.04.096

B. Hirth et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2916–2919
2917
N-(Z)-4-aminobutenyl)-N-methyl group at the 50-position is criti-
cal to the mechanism of AdoMetDC inhibition by forming a Schiff
base with the pyruvoyl prosthetic group at the active site,6 our ef-
forts were mostly focused on the modification of the ribose and
purine moieties. We reasoned that in order to increase penetration
of the blood brain barrier, it would be critical to improve com-
pound stability and increase exposure levels in the systemic circu-
lation. To achieve this, substitution with a halogen, especially a
fluorine atom, at the 20-position of the ribose would be a reason-
able measure to take, as such a change should have positive impact
on the stability of the glycosidic bond. Other considerations in-
clude modifying the substitution pattern on the purine base of
compound 1. In 2001, Steven Ealick et al. reported an interesting
discovery in their study of purine nucleoside inhibitors to human
AdoMet DC, in which they revealed that such compounds adopt
an unusual syn-conformation to bind to the enzyme.10 Considering
the similarity of human and trypanosomal AdoMetDCs, it is rea-
sonable to assume that the inhibitor 1 might adopt the syn-confor-
mation in the binding site too. Changes of the purine substitution
pattern might not only decrease the susceptibility of the com-
pounds to purine deaminase, but also alter their conformational
preference. Particularly by adding a substituent at the 8-position,
the energy penalty for adopting the syn-conformation could be
reduced.
cantly improved ability to penetrate the blood brain barrier as
demonstrated by its presence at higher concentrations in the mice
brain than compound 1 following parenteral injection.
The synthetic methods used to construct derivatives of 1 with
the desired changes on either the ribose or purine moieties are
highlighted in Schemes 1 and 2. In Scheme 1, the purine nucleoside
2 (a–c) was treated with either mesyl chloride or tosyl chloride in
pyridine at 0 °C. The resulting 50-mesyl or tosyl nucleosides under-
went a displacement by (Z)-tert-butyl 4-(methylamino)but-2-
enylcarbamate11 in DMSO in the presence of triethyl amine to form
compound 3 (a–c), which, in 3a and 3b, was subjected to deprotec-
tion of the N6-benzoyl group using ammonia in methanol. Final
deprotection of the Boc group with HCl in ethyl acetate produced
compounds 4 (a–c). In Scheme 2, the starting nucleoside 5 (a–d)
was reacted with thionyl chloride and pyridine in acetonitrile at
0 °C, followed by treatment with ammonium hydroxide in metha-
nol.12 The resulting 50-chloro-adenosine derivatives were reacted
with methylamine in ethanol at elevated temperature to produce
6 (a–d). N-alkylation of 6 (a–d) with (Z)-tert-butyl 4-chlorobut-2-
enylcarbamate in the presence of potassium carbonate and a cata-
lytic amount of sodium iodide in DMSO, followed by deprotection
of the Boc group with HCl in either methanol or dioxane, gave the
desired compound 7 (a–d).8
The preparation of some commercially unavailable starting
nucleosides 5 (b–d) is illustrated in Schemes 3–5. 8-Ethyl-adeno-
sine (5b) was prepared by a sequence highlighted in Scheme 3:
8-bromo-adenosine (9) was treated with hexamethyldisilazane
(HMDS) in the presence of ammonium sulfate in dioxane, followed
by reaction with triethylaluminum in the presence of palladium-
Here we report the discovery of a new compound 7a, which fea-
tures a methyl group at the 8-position of the purine. Compound 7a
demonstrates improved potency against AdoMetDC and is more
effective in T. brucei rhodeesiense and T. brucei brucei growth inhibi-
tion assays than 1. More interestingly, compound 7a has a signifi-
NHR1
NH2
N
NHR1
N
N
N
N
N
N
N
N
N
R4
N
R4
R4
N
R2
R3
R2
R3
c, d
a, b
R2
R3
O
O
BocHN
H2N
O
N
Me
N
Me
HO
R1 R2
R3
OH
OH
R4
H
H
R2
4a α-F
4b
R3
OH
OH
R4
H
H
R1 R2
3a Bz α-F
R3
OH
OH
R4
H
H
2a 18 Bz α-F
2b19
Bz β-F
β-F
3b
3c
Bz β-F
2c 20
H
R2,
R
3:α-acetonide Cl
4c α-OH OH
Cl
2
3 α
H
R , R : -acetonide Cl
Scheme 1. Reagents and conditions: (a) MsCI (or TsCI), pyridine, 0 °C; (b) (Z)-tert-butyl 4-(methylamino)but-2-enylcarbamate, Et3N, DMSO, 65 °C (31–42%, two steps);
(c, only for 3a–b) NH3, CH3OH (65–95%); (d) HCI, EtOAc (35–53%). (see above-mentioned references for further information.)
NH2
N
NH2
N
NH2
N
N
N
N
N
N
N
R2
OH
R2
OH
N
N
N
R2
OH
R1
R1
R1
d, e
a, b, c
O
O
H2N
O
OH
OH
OH
HN
Me
R1
N
Me
R1
HO
R1
R2
R2
R2
5a 21
5b
5c
H
H
H
Me
Et
n-Pr
H
7a
7b
7c
H
H
H
Me
Et
n-Pr
H
6a
6b
6c
H
H
H
Me
Et
n-Pr
H
5d Me
7d Me
6d Me
Scheme 2. Reagents and conditions: (a) SOCI2, pyridine, ACN, 0 °C; (b) NH4OH, CH3OH/H20 (35–75%, two steps); (c) CH3NH2, EtOH, 120 °C (50–55%); (d) (Z)-tert-butyl
4-chlorobut-2-enylcarbamate, Nal, K2C03, DMSO, 65 °C; (e) HCI, CH3OH or dioxane (38–65%, two steps). (see above-mentioned references for further information.)

2918
B. Hirth et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2916–2919
Table 1
Activity against purified Tryp
NH2
N
NH2
N
N
N
N
kinact/Kiappa against Tryp
T. b. r.b IC50
M)
T. b. bc IC50
M)
d
d
Br
Compound
No.
Et
N
AdoMedDC/prozyme (Mꢀ1 minꢀ1
)
(
l
(l
N
N
a, b, c
O
O
1 (MDL73811)
1.50
0.19
0.19
0.02
7.78
ND
0.011
1.4
0.21
>14
OH
OH
4a
4b
4c
7a
7b
7c
7d
HO
HO
2.6
>14
OH
OH
1.7
0.001
ND
ND
ND
7.27
0.027
0.90
11.05
>14
8
5b
Scheme 3. Reagents and conditions: (a) HMDS, (NH4)2S04; (b) Et3Al, Pd(PPh3)4,
THF, 70 °C; (c) NH4CI, MeOH/H20 (74%, three steps).
ND
ND
AdoMetDC and trypanosome parasites.
a
Expression of kinetic enzyme inhibition. kinact: first order of rate constant of
inactivation of the enzyme; Kiapp: determined by the equation of kobs = kinact[I]/
(Kiapp + [I]).10
NH2
NH2
b
T. brucei rhodeesiense.
T. brucei brucei.
N
N
N
N
c
N
Br
nPr
d
N
Compound concentration required to reduce parasite viability by 50%.22
N
N
a, b, c, d
O
O
OH
OH
HO
HO
OH
in the presence of palladium triphenylphosphine, and (3) deprotec-
tion with ammonium chloride.
OH
8
5c
Table 1 summarizes the inhibitory activities of these new 50-(4-
aminobutenyl-N-methylamino)-adenosine derivatives against the
purified AdoMetDC enzyme and in growth inhibition assays with
two trypanosomal strains, Trypanosoma brucei rhodesiense and Try-
Scheme 4. Reagents and conditions: (a) HMDS, (NH4)2S04; (b) allyltributyl tin,
Pd(PPh3)4, NMP, wave at 190 °C; (c) NH4CI, MeOH/H20 (48%, three steps); (d) Pd/C,
cat. TFA, EtOH/H2O.
l
panosoma brucei brucei. Enzyme inhibition is expressed in kinac
/
Kiapp, a method which measures kinetic inhibition of an enzyme,
as first reported by Kitz and Wilson.15 It is interesting to note that,
although most changes on either the base or sugar moieties of the
nucleoside resulted in decrease of activity, the addition of a methyl
group at the C8-position of the purine (7a) significantly increased
potency in both the AdoMetDC and parasite growth inhibition as-
says relative to 1. A similar observation has been reported by Ste-
ven Ealick et al. in their recent study of human AdoMetDC
inhibition.16 A possible explanation has been postulated in the pa-
per that a small substituent at the 8-position of purine results in
favouring syn-conformation, and therefore increases the binding
affinity for the active site of AdoMetDC.
NH2
NH2
N
N
N
N
N
N
N
I
N
Me
a, b, c
O
O
OH
OH
HO
HO
OH
OH
9
5d
Scheme 5. Reagents and conditions: (a) HMDS, (NH4)2S04; (b) 2.0 equiv AIMe3,
Pd(PPh3)4, THF, 70 °C; (c) NH4CI, MeOH/H20 (57%, three steps).
We have also evaluated several compounds in mice plasma PK
and assessed their ability to penetrate the blood brain barrier fol-
lowing parenteral administration. As indicated in Table 2, 7a has
a prolonged t1/2 in circulating blood in comparison to 1. More inter-
estingly, it also exhibits increased blood brain penetration relative
to compound 1 as evidenced by its higher brain Cmax and AUC. At
the present time, the reason for the improvements is not clear,
though it is possible that the increased hydrophobicity of com-
pound 7a versus 117, as well as its possibly altered conformational
preference could be factors. Compound 7a has subsequently been
evaluated in vivo in T. b. brucei infected mice, and demonstrated
greater efficacy than 1 in the reduction of parasitemia. A separate
manuscript describing studies in the disease model has been sub-
mitted for publication (C. Bacchi, personal communication).
triphenylphosphine in THF at 70 °C. Cleavage of all TMS groups
with ammonium chloride in methanol produced 5b.13 8-Propyl-
adenosine (5c) was synthesized from 8-bromo-adenosine (9) by
the sequence highlighted in Scheme 4: (1) treatment with HMDS
in dioxane in the presence of ammonium sulfate; (2) palladium tri-
phenylphosphine mediated coupling with allyltributyl tin in NMP
(N-methylpyrrolidinone);14 (3) deprotection of TMS ethers with
ammonium chloride and (4) hydrogenation using Pd on carbon
in the presence of TFA in ethanol/water. 2-Methyl-adenosine (5d)
was prepared from 2-iodo-adenosine (9) using the sequence high-
lighted in Scheme 5: (1) treatment with HMDS in the presence of
ammonium sulfate; (2) reaction with excess of trimethylaluminum
Table 2
PK parameters and brain penetration of 1 and 7a following a single dose i.p administration in mice
AUCa in bloodb
(l
g * h/mL)
t1/2 (h)
AUC in brain (lg * h/mL)
AUCbrain/AUCblood (%)
Cmax in blood (
l
g/mL)
Cmax in brain (lg/g)
c
d
Compound
1 (MDL 73811)
7a
20.69
17.92
2.48
7.43
0.36
1.30
1.70
7.28
44.80
36.90
0.08
0.66
a
AUC: area under the curve.
b
c
lg * hr/mL: microgram times hours/mL.
AUCbrain/AUC
(%): area under the curve for brain divided by area under the curve for blood times 100.
blood
d
Cmax: maximum concentration.

B. Hirth et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2916–2919
2919
8. Casara, P.; Marchal, P.; Wagner, J.; Danzin, C. J. Am. Chem. Soc. 1989, 111, 9111.
9. Bitonti, A. J.; Byers, T. L.; Bush, T. L.; Casara, P. J.; Bacchi, C. J.; Clarkson, A.
B., Jr.; McCann, P. P.; Sjoerdsma, A. Antimicrob. Agents Chemother. 1990, 34,
1485.
10. Tolbert, W. D.; Ekstrom, J. L.; Mathews, I. I.; Secrist, J. A.; Kapoor, P.; Pegg, A. E.;
Ealick, S. E. Biochemistry 2001, 40, 9484.
11. (Z)-tert-butyl 4-(methylamino)but-2-enylcarbamate was prepared based on
the literature procedures (see Ref. 8).
12. Hirota, K.; Kitade, Y.; Kanbe, Y.; Maki, Y. J. Org. Chem. 1992, 57, 5268.
13. Robins, M. J.; Hansske, F.; Wnuk, S. F.; Kanai, T. Can. J. Chem. 1991, 69, 1468.
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P. A. J. Med. Chem. 1993, 36, 2938.
In conclusion, we have discovered a new trypanosomal Ado-
MetDC inhibitor 7a, which has a methyl group at the 8-position
of the purine. This substitution makes the compound 7a a more po-
tent inhibitor of AdoMetDC, and also increases its ability to pene-
trate the blood brain barrier penetration in mice. This discovery
may indicate a new direction for further improvement of this class
compounds as antitrypanosomal agents in the future.
Acknowledgements
15. Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245.
16. McCloskey, D. E.; Bale, S.; Secrist, J. A., III; Tiwari, A.; Moss, T. H., III;
Valiyaveettil, J.; Brooks, W. H.; Guida, W. C.; Pegg, A. E.; Ealick, S. E. J. Med.
Chem. 2009, 52, 1388.
17. The LogDs of compound 7a and 1 are ꢀ1.4 and ꢀ1.8, respectively. These values
were determined by measuring the partitioning of the compounds between
octonal and PBS pH 7.4 after shaking and phase separation at the Genzyme
DMPK department.
The authors would like to thank Drs. Patrick Casara, Simon
Croft, Reto Brun and Robert Don for helpful discussions throughout
these studies. This work was supported in part by funding from
DNDi and by resources provided by Genzyme. Genzyme is commit-
ted to assisting in discovery of drugs for neglected diseases in col-
laboration with other partners and seeks no profit from drugs used
for these applications (Bacchi et al., mss submitted).
18. Compound 2a was prepared from 9-b-D-arabinofuranosyladenine based on a
literature procedure: Kawasaki, A. M.; Casper, M. D.; Freier, S. M.; Lesnik, E. A.;
Zounes, M. C.; Cummins, L. L.; Gonzalez, C.; Cook, P. D. J. Med. Chem. 1993, 36,
831.
19. Compound
References and notes
2b
was
prepared
from
9-(2-deoxy-2-fluoro-b-D-
arabinofuranosyl)adenine based on literature procedures: (a) Pankiewicz, K.
W.; Krzeminski, J.; Ciszewski, L. A.; Ren, W.-Y.; Watanabe, K. A. J. Org. Chem.
1992, 57, 553; (b) Krainer, E.; Naider, F.; Becker, J. Tetrahedron Lett. 1993, 34,
1713.
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dimethoxypropane in the presence of p-toluenesulfonic acid in acetone and
DMF.
21. Compound 5a was prepared based on procedures reported in the Ref. 12.
22. Seebacher, W.; Brun, R.; Weis, R. Eur. J. Pharmcol. Sci. 2004, 21, 225.
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