Synthesis of two isotopically labeled versions of NEDD8-activating enzyme (NAE) inhibitor

Article abstract of DOI:10.1016/j.tetlet.2011.02.033

The synthesis of two isotopically labeled versions of an investigational NAE inhibitor (MLN4924) is reported. ¹⁴C-MLN4924 was synthesized from ¹⁴C-thiourea in eight steps. D₈-MLN4924 was prepared from D₉-3-pheny

Full text of DOI:10.1016/j.tetlet.2011.02.033

Tetrahedron Letters 52 (2011) 1807–1810
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Tetrahedron Letters
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Synthesis of two isotopically labeled versions of NEDD8-activating enzyme
(NAE) inhibitor
⇑
Yuexian Li , Mihaela Plesescu, Shimoga R. Prakash
Department of Drug Metabolism and Pharmacokinetics, Isotope Chemistry Group, Millennium Pharmaceuticals, Inc., 35 Landsdowne Street, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of two isotopically labeled versions of an investigational NAE inhibitor (MLN4924) is
reported. 14C-MLN4924 was synthesized from 14C-thiourea in eight steps. D8-MLN4924 was prepared
from D9-3-phenylpropanoic acid in seven steps.
Received 25 January 2011
Revised 4 February 2011
Accepted 7 February 2011
Available online 12 February 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
NAE inhibitor
MLN4924
14C-6-chloro-7-deazapurine
D8-(S)-1-aminoindan
NEDD8 (Neural precursor cell expressed, developmentally
down-regulated 8)-activating enzyme (NAE) is an essential compo-
nent of the NEDD8 conjugation pathway that controls the activity
of the cullin-RING subtype of ubiquitin ligases, thereby regulating
the turnover of a subset of proteins upstream of the proteasome.1
Substrates of cullin-RING ligases have important roles in cellular
processes associated with cancer cell growth and survival path-
ways.1 MLN4924 (1), ((1R,2R,4S)-4-{4-[(1R)-2,3-dihydro-1H-in-
den-1-ylamino]-7H-pyrrolo[2,3-d]pyrimidin-7-yl}-2-hydroxycycl-
opentyl)methyl sulfamate, is a potent and selective inhibitor of
NAE.1–3 By deregulation of S-phase DNA synthesis, MLN4924 dis-
rupts cullin-RING ligase-mediated protein turnover leading to
apoptotic death in human tumor cells.1 MLN4924 is a mecha-
nism-based inhibitor of NAE and creates a covalent NEDD8-
MLN4924 adduct catalyzed by the enzyme.3 A recent publication
in Blood demonstrates that MLN4924 has antitumor activity in pre-
clinical DLBCL (diffuse large B-cell lymphoma) models through the
HN
N
N
O
N
O
S
H2N
O
HO
1
Figure 1. Structure of MLN4924.
stable isotope labeled version was also needed as an internal stan-
dard for mass spectrometry based bioanalytical assays (Fig. 1).5
The structure of MLN4924 (1) provides three potential sites for
labeling: on (S)-1-aminoindan (2), or 6-chloro-7-deazapurine (3),
or triol 4 (Scheme 1).2 Preclinical metabolism results indicated that
radiolabeling any one of the three portions of the molecule would
be adequate for the intended ADME (Absorption, Distribution,
Metabolism, and Excretion) studies in animals. For expedient syn-
thesis, we chose labeling (S)-1-aminoindan (2) for stable isotope
labeled version and 6-chloro-7-deazapurine (3) for the radiola-
beled version (Scheme 1).
To synthesize the key C-14 labeled intermediate 3A, C-14 la-
beled 6-chloro-7-deazapurine, we chose commercially available
14C-thiourea (7A) as the radiolabeled precursor (Scheme 2). Cyclo-
condensation of 14C-thiourea (7A) with 6 provided 8 in 76% yield.6
Desulfurization of 8 with Raney Nickel led to 9 that was converted
inhibition of the NF-
j
B pathway.4 In addition, the publication
B depen-
showed that MLN4924 has antitumor activity in non-NF-
j
dent DLBCLs through other mechanisms including DNA re-replica-
tion.4 Currently, novel investigational inhibitor MLN4924 is in
Phase 1 clinical trials.
The biological activity of MLN4924 led to detailed investiga-
tions on its disposition characteristics. The radiolabeled version
was needed to assist such investigations, especially in metabolite
profiling and mass balance studies in experimental animals. The
⇑
Corresponding author. Tel.: +1 617 444 1275; fax: +1 617 444 1480.
E-mail address: yli@mpi.com (Y. Li).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.02.033

1808
Y. Li et al. / Tetrahedron Letters 52 (2011) 1807–1810
COOH
NH2
2
5
HN
N
Cl
N
O
N
NH2
O
N
O
N
O
CH(OC2H5)2
N
S
S
H2N
H
O
NH2
CN
3
4
HO
7
6
1
OH
HO
HO
Scheme 1. Retro-synthesis of MLN4924.
OH
OH
NH2
14C
i
N
14C
CH(OC2H5)2
NH2
ii
iii
N
C
CH(OC2H5)2
NH2
S
H14
NH2
HS
N
N
7A
8
9
Cl
OH
N
C
v
N
C
iv
vi
HN
H14
H14
N
N
N
N
H
N
H
10
14CH
3A
N
N
H
11
HN
HN
vii
HN
viii
N
14CH
N
14CH
N
14CH
N
N
N
N
O
N
O
N
TBSO
TBSO
S
HO
TBSO
H2N
O
13
14
.HCOOH
HO
1A
Scheme 2. Reagents and conditions: (i) ethyl 2,2-diethoxyethylcyanoacetate (6), NaOEt, HOEt, 85 °C, 18 h, 76%; (ii) Raney Ni, H2O, 110 °C, 1 h; (iii) 1 N HCl, rt, 3 h, 74% in two
steps; (iv) POCl3, 120 °C, 1.5 h, 80%; (v) (S)-1-aminoindanÁHCl (2), 2-butanol, (i-Pr)2NEt, microwave, 150 °C, 5 h, 68%; (vi) (1S, 3S, 4S)-3-{[tert-butyl(dimethyl)silyl]oxy}methyl
cyclopentyl methanesulfonate (12), Cs2CO3, DMF, 90 °C, 6 h, 92%; (vii) HOAc, THF, H2O, 40 °C, 18 h, 51%; (viii) (1) Ph2NC(O)NHSO2Cl, TEA, 0 °C, 1 h; (2) HCl, rt, 1 h; (3) prep
HPLC, 23%.
to 10 via acid-catalyzed hydrolysis and cyclization in 74% (for two
steps) yield.7 Compound 3A was prepared by chlorination of 10
with POCl3 in 80%.7 Coupling of 3A and amine 2 required two days
to complete by traditional refluxing.2 To hasten the reaction,
microwave assisted coupling of 3A and 2 afforded 11 in 5 h with
a yield of 68%. Replacement of the methanesulfonate group of 12
by 11 provided compound 13 in 92% yield. Partial deprotection
of 13 using HOAc followed by sulfamation of 14 and deprotection
of both tert-butyl(dimethyl)silyl and diphenylcarbomoyl groups
gave C-14 labeled MLN4924 (1A) in 12% yield (for two steps).2
To ensure complete separation of labeled and unlabeled molec-
ular ion clusters during mass spectrometric assays of MLN4924, a

Y. Li et al. / Tetrahedron Letters 52 (2011) 1807–1810
1809
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
i
ii
D
COOH
D
D
D
OH
N
O
Ph
D
D
17
5B
15
D
D
D
D
D
D
D
D
D
D
D
D
D
D
iv
D
D
iii
D
v
D
D
D
D
D
D
OH
N
N
Ph
NH2
Ph
H
D
19
18
2B
D
D
D
D
D
D
D
D
D
D
vi
D
D
vii
D
HN
D
HN
D
D
N
N
O
N
O
N
N
N
S
H2N
O
HO
.HCl
HO
HO
21
1B
Scheme 3. Reagents and conditions: (i) polyphosphoric acid, 70 °C, 3.5 h, 93%; (ii)
CH3COOD, 0 °C, 1.5 h, 63%; (iv) Pb(OAc)4, MeOD, 0 °C, 0.5 h; (v) DCl, D2O, EtOD, 95 °C, 18 h, 75% in two steps; (vi) (1S, 2S, 4R)-4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-
(hydroxyl-methyl)cyclopentanol (20), 2-BuOH, (i-Pr)2NEt, microwave, 150 °C, 4.5 h, 49%; (vii) (1) Ph2NC(O)NHSO2Cl, TEA, 0 °C, 1 h; (2) HCl, rt, 1 h, 28%.
D
-(À)-alpha-phenylglycinol (16), 4-MeC6H4SO3H, toluene, reflux, 3 h, 88%; (iii) NaBH4, THF,
labeled version, that is, at least 3 amu higher than the unlabeled
version is required. To increase the molecular weight, labeling of
(S)-1-aminoindan (2) or 6-chloro-7-deazapurine (3) was required.
Labeling the (S)-1-aminoindan (2) with deuterium was chosen to
get the increase in amu.
MLN4924 (1B) was prepared from D9-3-phenylpropanoic acid in
an overall yield of 5.3%.
Acknowledgments
To synthesize the key labeled intermediate product 2B, com-
mercially available D9-3-phenylpropanoic acid (5B) was used as
the labeled precursor (Scheme 3). Cyclization of 5B with polyphos-
Thanks are due to Xiaofeng Yang of the Protein Science Depart-
ment of Millennium Pharmaceuticals, Inc. for the provision of
HRMS data. Thanks are also due to Mark Milton of the Analytical
Chemistry Department of Millennium Pharmaceuticals, Inc. for
providing 500 MHz NMR spectra of C-14 labeled compound.
phoric acid provided 15 in 93% yield.8–11 Reaction of 15 with
D
-(À)-
alpha-phenylglycinol (16) in the presence of catalytic amounts of
p-toluenesulfonic acid gave imine 17 (88%).10 Reduction of 17 by
sodium borohydride yielded amine 18 (63%).10 To prevent back ex-
change of deuterium by hydrogen in the rings, CH3COOD was used
in the reduction. Oxidative cleavage of 18 with Pb(OAc)4 and sub-
sequent hydrolysis afforded 2B in 75% (for two steps).12 To prevent
back exchange of deuterium, deuterium labeled solvents/reagent
(MeOD, DCl, D2O, EtOD) were utilized in the above two steps. To
hasten the reaction, the use of microwave assisted coupling of 2B
with 20 provided diol 21 in 4.5 h in 49% yield. D8-MLN4924 (1A)
was synthesized by sulfamation of 21 and deprotection of the
diphenylcarbomoyl group of the resulting product in 28% yield.2,13
In conclusion, practical methods for the synthesis of carbon-14
and stable isotope labeled NAE inhibitor, MLN4924, were devel-
oped. The key intermediate for the C-14 synthesis, 14C-6-chloro-
7-deazapurine (3A), was synthesized from 14C-thiourea (7) in four
steps. The key intermediate for the stable isotope labeled synthe-
sis, D8-(S)-1-aminoindan (2B), was prepared from D9-3-phenyl-
propanoic acid (5B) in five steps. 14C-MLN4924 (1A) was
synthesized from 14C-thiourea in an overall yield of 3.3%. D8-
References and notes
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Mizutani, H.; Narayanan, U.; Olhava, E. J.; Peluso, S.; Rezaei, M.; Sintchak, M. D.;
Talreja, T.; Thomas, M. P.; Traore, T.; Vyskocil, S.; Weatherhead, G. S.; Yu, J.;
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5. An account of this work was presented at the 11th Tetrahedron Symposium,
Beijing, China, Jun 22–25, 2010.
6. Davoll, J. J. Chem. Soc. 1960, 131.

1810
Y. Li et al. / Tetrahedron Letters 52 (2011) 1807–1810
7. Reigan, P.; Gbaj, A.; Chinje, E.; Stratford, I. J.; Douglas, K. T.; Freeman, S. Bioorg.
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1H), 5.90 (dd, J = 8.5, J = 15.6, 1H), 5.35 (dt, J = 9.8, J = 14.4, 1H), 4.32 (s, 1H),
4.22 (dd, J = 6.3, J = 9.7, 1H), 4.04 (dd, J = 8.0, J = 9.7, 1H), 3.00 (m, 1H), 2.85 (m,
1H), 2.71 (m, 1H), 2.62 (m, 1H), 2.15 (m, 2H), 2.05 (m, 1H), 1.94 (m, 2H);
chemical purity, 99.0% (UV at 280 nm, HPLC); radiochemical purity, 98.7%;
specific activity, 43.5 mCi/mmol; LC–MS/MS: m/z 446.1 (M+H).
Compound 1B: 1H NMR (300 MHz, DMSO-d6): d 9.59 (s, 1H), 8.40 (s, 1H), 7.63
(d, J = 3.0, 1H), 7.46 (s, 2H), 7.02 (d, J = 3.0, 1H), 5.75 (dd, J = 7.6, J = 15.1, 1H),
5.43 (dt, J = 8.4, J = 14.2, 1H), 5.34 (m, 1H), 4.24 (dd, J = 7.0, J = 9.6, 1H), 4.05 (dd,
8.0, 9.6, 1H), 2.73 (m, 1H), 2.19 (m, 2H), 1.96 (m, 2H); 13C NMR (100 MHz,
DMSO-d6): d 149.45, 145.97, 143.96, 142.03, 141.55, 129.05, 127.40, 125.57,
125.23, 124.96, 102.97, 102.50, 71.39, 69.91, 57.84, 53.83, 43.20, 43.09, 34.26,
33.39, 30.49; HRMS m/z (M+H): 448.1950 (3.35%, D4), 449.2010 (12.48%, D5),
450.2071 (26.05%, D6), 451.2130 (33.46%, D7), 452.2189 (24.66%, D8) (D6 peak
was used in the bioanalytical studies).
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13. All new compounds were characterized on the basis of 1H, 13C NMR and HRMS
data. Analytical data for selected compounds are given below:
Compound 1A: 1H NMR (500 MHz, DMSO-d6): d 8.16 (s, 1H), 7.38 (s, 2H), 7.26 (t,
J = 7.5, 1H), 7.24 (d, J = 3.5, 1H), 7.19 (m, 2H), 7.13 (t, J = 7.5, 1H), 6.62 (d, J = 3.5,