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COMMUNICATION
Journal Name
New Approach to MK-4482:
3
, and 4 was isolated in 78% yield from cytidine. A traditional
DOI: 10.1039/D0CC05944G
O
(5 Equiv.)
N
chemically catalyzed acylation was developed to provide a non-
enzymatic option to reach 4. Though inexpensive this option
O
NH2
2 2 4
NH OH•H SO
(4.5 Equiv.)
O
O
N
11
5
O
N
O
OH
iPrOH,
78 °C, 20 h
96% IY
Novozyme 435
200 wt%)
,4 Dioxane,
HO
might not be preferable to the enzymatic route. More reagents
are added to the reaction system and a greater number of
byproducts are formed which might hamper efforts to purify
the intermediate at scale. Conversion was stopped at 90% to
halt over-acylation of product, resulting in an isolated yield of
76% of 4. Similarly, an alternative route to 3 was developed
(
1
(75% Over
2 Steps)
4
6
0 °C, 43 h, 78%
O
NHOH
NH2
N
Route Features:
O
O
N
N
O
N
HO
•
2-Steps to API
• Increases yield to 75%
Avoids protecting groups
• Replaces uridine with cytidine
O
OH
O
HO
HO
OH
Cytidine, 2
•
MK-4482
(
EIDD-2801, 1)
1
1
from uridine.
O
(3 Equiv.)
N
(37% Over
This completes two concise routes to MK-4482, which differ in
the order of synthetic transformations. When conducting
esterification first, MK-4482 is obtained in 75%, and it is made
in 37% when hydroxyamination is conducted first. The step
count is reduced from five transformations to two, and the
more expensive uridine is replaced with cytidine. The use of
protecting groups and derivatization is eliminated. We plan to
further report on the optimization of this preliminary result to
refine catalyst loadings, solvent selection, and yield while
developing process-amenable isolation sequences.
2 Steps)
NH2OH•HOAc
3 Equiv.)
NHOH
O
(
O
N
5
HO
N
H2O, pH 5.5
0 °C, 48 h, 50%
Novozyme 435 (200 wt%)
1,4 Dioxane,
O
OH
4
HO
40 °C, 2 h, 74%
3
Figure 2: A new route to MK-4482 from cytidine.
We began our exploration by examining direct transamination
7
of cytidine with hydroxylamine.
Older literature studies
suggested that mono-hydroxamination can be achieved under
the right concentration, temperature and pH, while minimizing
We thank the Bill and Melinda Gates Foundation for their
longstanding support of our research. Grace Ahlqvist would
also like to acknowledge support by the National Science
Foundation Graduate Research Fellowship under Grant No.
7
a,b
over-reaction of substrate.
More recently this finding was
7
c
repeated by Purohit with preparative HPLC separation while
Painter claimed difficulties with the procedure leading to 20%
yield.7 In our hands, with slight adjustment of reaction
conditions, N(4)-hydroxycytidine (NHC, 3) was synthesized in
d
1
745302.
7
0% assay yield (AY). Importantly, upon concentration pure
NHC was obtained by simple crystallization directly from the
reaction mixture in 50% isolated yield.
We also explored transamination of cytidine isobutyryl ester 4,
Conflicts of interest
“There are no conflicts to declare”.
and surprisingly, with use of NH
2 2
OH·H SO
4
in iPrOH
dihydroxamination was avoided completely. We were quite
pleased to find that the ester remained intact. MK-4482 was
obtained from 4 in 96% isolated yield (IY), demonstrating
viability of direct hydroxamination from either cytidine reaction
pathway.
Notes and references
1
2
020). b) An Open Letter from Daniel O’Day, Chairman &
Selective acylation remained as the largest technical
uncertainty toward production of a shorter, protecting group
free route to MK-4482. The esterification of NHC 3 would need
to be selective for one of four hydroxyl groups, and the
literature suggests the N-hydroxy group is most reactive toward
2
L. Zhang, S. Neville, E. Carra, W. Lew, B. Ross, Q. Wang, L.
Wolfe, R. Jordan, V. Soloveva, J. Knox, J. Perry, M. Perron,
K.M. Stray, O. Barauskas, J.Y. Feng, Y. Xu, G. Lee, A.L.
Rheingold, A.S. Ray, R. Bannister, R. Strickley, S.
7
d,8
acylation by chemical means.
Enzyme catalyzed
esterification of cytidine has achieved this goal by making use
9
of vinyl esters and anhydride acyl donors. The use of oxime
Swaminathan, W.A. Lee, S. Bavari, T. Cihlar, M.K. Lo, T.K
Warren, R.L Mackman, J. Med. Chem., 2017, 60, 1648. b) C.
De Savi, D.L Hughes, L. Kvaerno, Org. Process Res. Dev., 2020,
ester transfer agents were of particular interest due to the
structural similarity with N-hydroxycytidine, 3, and excellent
selectivity was observed with uridine though cytidine was
2
4, 940. c) T. Vieira, A.C. Stevens, A. Chtchemelinine, D. Gao,
P. Badalov, L. Heumann, Org. Process Res. Dev., 2020, ASAP,
DOI:10.1021/acs.oprd.0c00172. c) F. Xue, X. Zhou, R. Zhou,
X. Zhou, D. Xiao, W. Zhong, E. Gu, W. Guo, J. Xiang, K. Wang,
L. Yang, Y. Qin, Org. Process Res. Dev., 2020, ASAP,
DOI:10.1021/acs.oprd.0c00310.
a) T.P. Sheahan, A.C. Sims, S. Zhou, R.L. Graham, A.J.
Pruijssers, M.L. Agostini, S.R. Leist, A. Schafer, K.H. Dinnon III,
L.J. Stevens, J.D. Chappel, X. Lu, T.M Hughes, A.S. George,
C.S. Hill, S.A. Montgomery, A.J. Brown, G.R. Bluemling, M.G.
Natchus, M. Saindane, A.A. Kolykhalov, G. Painter, J.
Harcourt, A. Tamin, N.J. Thornburg, R. Swanstrom, M.R.
Denison, R.S. Baric, Sci. Transl. Med., 2020, 12, 1-15. b) B.
1
0
unfortunately reported to give the O,N-diacylated product.
We were curious whether this approach would work to form the
desired α-branched esters.
Surprisingly immobilized CALB (Candida Antarctica Lipase B)
provided the desired selectivity not only for N-hydroxycytidine
but also for cytidine. Isobutyric oxime ester 5 was used as the
acyl transfer agent with solid supported enzyme (200 wt%, 1.5
mol%). A sufficient excess of the oxime ester was necessary to
drive the reaction to completion, and early results have been
best with 1,4-dioxane. MK-4482 was isolated in 74% yield from
3
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Vasudevan
