Convenient preparation of pinometostat and related 5′-deoxy-5′-amino adenosine derivatives as well as their activity against DOT1L

Article abstract of DOI:10.1016/j.tetlet.2017.12.064

From adenosine and 2′-C-Me adenosine, a 3-step route towards nucleoside DOT1L inhibitors, including pinometostat, EPZ5677, and FED1, was established. With useful structural-activity relationship information, the newly prepared 2′-C-Me adenosine derivative

Full text of DOI:10.1016/j.tetlet.2017.12.064

Accepted Manuscript
Convenient preparation of pinometostat and related 5’-deoxy-5’-amino adeno-
sine derivatives as well as their activity against DOT1L
Tongchao Liu, Huanming Ren, Cong Li, Guohua Chen, Maosheng Cheng,
Dongmei Zhao, Jingkang Shen, Jia Li, Yubo Zhou, Bing Xiong, Yue-Lei Chen
PII:
S0040-4039(17)31596-4
https://doi.org/10.1016/j.tetlet.2017.12.064
TETL 49566
DOI:
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
7 November 2017
14 December 2017
21 December 2017
Please cite this article as: Liu, T., Ren, H., Li, C., Chen, G., Cheng, M., Zhao, D., Shen, J., Li, J., Zhou, Y., Xiong,
B., Chen, Y-L., Convenient preparation of pinometostat and related 5’-deoxy-5’-amino adenosine derivatives as
well as their activity against DOT1L, Tetrahedron Letters (2017), doi: https://doi.org/10.1016/j.tetlet.2017.12.064
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Convenient preparation of pinometostat and
related 5’-deoxy-5’-amino adenosine
derivatives as well as their activity against
DOT1L
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Tongchao Liu, Huanming Ren, Cong Li, Guohua Chen, Maosheng Cheng, Dongmei Zhao, Jingkang Shen, Jia
Li, Yubo Zhou, Bing Xiong and Yue-Lei Chen

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Convenient preparation of pinometostat and related 5’-deoxy-5’-amino
adenosine derivatives as well as their activity against DOT1L
a,b
b,d
c,e
d
a
a,*
Tongchao Liu , Huanming Ren , Cong Li , Guohua Chen , Maosheng Cheng , Dongmei Zhao ,
b,e
c,e
c,e,*
b,e,*
b,e, ∗
Jingkang Shen , Jia Li , Yubo Zhou , Bing Xiong
and Yue-Lei Chen
a
Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, 103 Wenhua Lu, Shenyang
10016, P. R. China.
1
b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203; P. R.
China.
c
The National Center for Drug Screening, 189 Guoshoujing Road, Shanghai 201203, P. R. China
d
School of Pharmacy, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, P. R. China.
e
University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, P. R. China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
From adenosine and 2’-C-Me adenosine, a 3-step route towards nucleoside DOT1L inhibitors,
including pinometostat, EPZ5677, and FED1, was established. With useful structural-activity
relationship information, the newly prepared 2’-C-Me adenosine derivatives contribute to the
limited repertoire of ribose-modified nucleoside DOT1L inhibitors. In general, this new
synthetic method will facilitate not only the study of nucleoside DOT1L inhibitors, but also the
synthetic and medicinal chemistry research of 5’-deoxy-5’-amino adenosine derivatives.
Keywords:
2
009 Elsevier Ltd. All rights reserved.
DOT1L inhibitor
pinometostat
amination
5
’-deoxy-5’-amino adenosine derivative
structure activity relationship
NH2
Disruptor of Telomeric Silencing 1-Like (DOT1L) is a
validated therapeutic target against mixed lineage leukemia gene
7
6
NH2
N
N 1
N
N
Me
S
9
N
N
N
N
5'
rearranged (MLL-r) subtypes of acute myelogenous leukemia
HOOC
N
O 1'
3' 2'
1
3
O
N
(
AML) and acute lymphoblastic leukemia (ALL). Nucleoside
NH2
DOT1L inhibitors resemble S-adenosyl methionine (SAM, Fig. 1)
t-Bu
NH
pinometostat
HO OH
SAM HO OH
as the cofactor of DOT1L, and their structure-activity
2
relationships have been studied. Taking a typical DOT1L
side-chain
nucleoside moiety
1b
Figure 1. Structure of SAM and pinometostat (EPZ-5676), a DOT1L
inhibitor pinometostat (EPZ5676, Fig. 1) as a representative
example, both side-chains on N-5’ were extensively investigated
and found to be critical for the activity and selectivity. On the
contrary, modifications to the sugar moiety of the adenosine
inhibitor under clinical trials.
3b,4
During the investigation of new DOT1L inhibitors,
discovered a convenient route towards pinometostat and its
analogues. Adapted from early procedure employing consecutive
we
3
scaffold are rare, presumably due to synthetic difficulties.
1d, 2a-e,
3
1d, 2a-e, 3
According to literature reports,
the preparation of
reductive amination steps,
the new route proceeds in only
nucleoside DOT1L inhibitors usually involves lengthy protection
and deprotection operations, leading to lower overall yields and
higher process complexity. Thus, it would be ideal to directly
incorporate 5’-N-side-chains to the nucleoside scaffold, with a
minimum number of protection and deprotection steps.
3 steps from unprotected nucleoside starting materials. Using this
method, new DOT1L inhibitors have been prepared and assayed
for their enzymatic activity; these compounds contribute to the
limited repertoire of ribose-modified nucleoside DOT1L
inhibitors.
Using a literature procedure, adenosine was converted into 5’-
2a
chloride 1 in one-pot via transient sulfite protection. Since the
—
——
∗
Corresponding authors. e-mail: D. Z.: dongmeiz-67@163.com, +86-24-43520219; Y. Z.: ybzhou@simm.ac.cn, +86-13661970096; B. X.:
bxiong@simm.ac.cn, +86-15000819768; Y.-L. C.: chenyuelei@gmail.com, +86-13482754275.

6
-NH
2
, 2’-OH, and 3’-OH positions of compound 1 possess low
Scheme 2. Preparation of DOT1L inihibitors cis-7 and trans-7 bearing the
pinometostat side-chain. Reagents and conditions: a) SOCl , pyridine, MeCN,
0 °C, 4 h, then rt, overnight, then aqueous NH , 88%; b) isopropylamine,
reactivity, we envisioned that direct displacement of the 5’-
chloride by an external amine nucleophile might be possible.
Thus, the reaction of compound 1 and isopropylamine was
examined in different solvents at 100 °C in a sealed tube. It was
found that compound 1 remained unchanged in solvents of lower
2
3
100 °C, 48 h, 85%; c) 3, AcOH, MeCN, 60 °C, 10 min, then STAB, 72 h,
65% to cis-7, 13% to trans-7.
2 2
polarity including CH Cl , ethyl acetate, and THF. Some
Intermediates 2 and 6 reacted faster with the less hindered
side-chain 8, which is present in the DOT1L inhibitors
conversion to the desired product 2 was observed with more
polar solvents including 1,4-dioxane, DMF, ethanol, and water.
Since isopropylamine is inexpensive, it was further found that
stirring compound 1 in neat isopropylamine at 100 °C in a sealed
tube for 2 days gave product 2 with a satisfying 83% yield.
Compound 2 was then subjected to reductive amination with
1a
2c,8
EPZ004777
and FED1.
Again, the high yields towards
FED1 (80%) and 9 (85%) indicated that the unprotected 2’,3’-
dihydroxyl groups played no negative roles in the reductive
amination step (Scheme 3). Thus, from adenosine, FED1 was
prepared in 3 steps in 60% yield, again representing a significant
5
2c,8
side-chain 3. The reaction proceeded slowly, probably due to
improvement over literature methods.
the low solubility of 2 in acetonitrile and the steric hindrance on
the 5’-N. Fortunately, the conversion was clean after 3 days. In
the crude product mixture, the cis-/trans- ratio of the cyclobutane
ring was ~5.6:1 according to HPLC analysis, slightly higher than
5
the ratio previously obtained from 2’, 3’-acetonide protected 2.
After normal phase preparative HPLC purification, pinometostat
2
g
and EPZ5677
were obtained in 63% and 12% yield,
respectively. (Scheme 1) Thus, from adenosine, pinometostat was
prepared in 3 steps in 47% overall yield, representing a
5
significant improvement over the literature methods.
Scheme 3. Preparation of DOT1L inihibitors FED1 and 9. Reagents and
conditions: a) 8, AcOH, MeCN, 50 °C, 10 min, then STAB, overnight, 80%
to FED1, 85% to 9.
Although 5’-N-isopropyl proved to be most effective for the
DOT1L inhibitory activity, other alkylamines were used to
inspect the substrate scope of the synthetic method.
Cyclobutylamine was chosen because the size of cyclobutanyl
group is similar to the isopropyl group, and because several
features of cyclobutylamine challenged the efficiency of the
amination step. Since cyclobutylamine is less reactive and much
more expensive than isopropylamine, ethanol was used as a co-
solvent and the amination reaction was carried out at 120 °C for 2
days in a sealed tube. Under these conditions, compound 10 was
prepared from 1 in 80% yield, while compound 12 was prepared
from 5 in 75% yield; both yields satisfied our needs. Next,
reductive amination of compound 10 with side-chain 3 gave
compound 11 as a mixture of cis/trans isomers, in a ~4.0:1 ratio
Scheme 1. Preparation of pinometostat and EPZ5677. Reagents and
conditions: a) SOCl
aqueous NH , 90%; b) isopropylamine, 100 °C, 48 h, 83%; c) 3, AcOH,
MeCN, 60 °C, 10 min; then sodium triacetoxyborohydride (STAB), 72 h,
2
, pyridine, MeCN, 0 °C, 4 h, then rt, overnight, then
3
1
according to H-NMR. Separation of the cis- and trans-
6
3% to pinometostat, 12% to EPZ5677.
cyclobutane isomers was found prohibitively difficult. Therefore
the diastereomeric mixture of 11 was used for biological
evaluation. Meanwhile, reductive amination of compound 12
with side-chain 8 gave compound 13 in 86% yield (Scheme 4).
To further demonstrate the usefulness of this method, 2’-C-Me
adenosine 4 which was readily prepared according to a literature
6
procedure, was employed for the synthesis of ribose-modified
nucleoside DOT1L inhibitors (Scheme 2). Using the chlorination
conditions and amination protocol as previously described,
compound 6 was obtained in high yield. The final reductive
amination step gave a crude mixture of cis-7 and trans-7 in a
~
4.5:1 ratio according to HPLC analysis. To separate the isomers,
supercritical fluid chromatography (SFC) was used, affording
cis-7 (65%) and trans-7 (13%).
7
Scheme 4. Preparation of DOT1L inihibitors 11 and 13. Reagents and
conditions: a) cyclobutylamine, EtOH, 120 °C, 48 h, 80%; b) 3, AcOH,
MeCN, 60 °C, 10 min, then STAB, 72 h, 79%; c) cyclobutylamine, EtOH,
1
20 °C, 48 h, 75%; d) 8, AcOH, MeCN, 50 °C, 10 min, then STAB,
overnight, 86%.

1
It should be noted that intermediates 2, 6, 10, and 12 are
potentially useful for the syntheses of other 5’-deoxy-5’-amino
adenosine derivatives, especially when utilizing reductive
amination reactions.
Experimental procedures as well as copies of the H-NMR
and C-NMR spectra of new compounds are provided.
13
References and notes
Novel compounds cis-7, trans-7, 9, 11, and 13, along with
1
2
.
.
(a) Daigle SR, Olhava EJ, Therkelsen CA, et al. Cancer Cell.
011;20:53. (b) Daigle SR, Olhava EJ, Therkelsen CA, et al.
Blood. 2013;122:1017. (c) Shukla N, Wetmore C, Brien MM, et al.
Blood. 2016;128;2780. (d) Olhava EJ, Chesworth R, Kuntz KW,
et al. WO2012075381A1; 2012.
pinometostat, EPZ5677, and FED1, were assayed for inhibitory
2
9
activity against DOT1L (Table 1). As expected, pinometostat
and EPZ5677 had very similar enzymatic activities (Table 1,
entries 1 and 2). Although the enzymatic activity of 2’-C-Me
analogues cis-7 and trans-7 were still good, they are 6 to 7 times
less active than pinometostat and EPZ5677, respectively (Table 1,
entries 3 and 4). Compound 9, as a 2’-C-Me analogue of FED1,
showed reduced enzyme activity compared to FED1 (Table 1,
entries 5 and 6). According to the data of 11 and 13, the
(a) Anglin JL, Deng L, Yao Y, et al. J Med Chem. 2012;55:8066.
(
b) Yu W, Chory EJ, Wernimont AK, et al. Nat Commun.
2
2
012;3:1288. (c) Yu W, Smil D, Li F, et al. Bioorg Med Chem.
013;21:1787. (d) Anglin JL, Song Y. J Med Chem. 2013;56:8972.
(e) Klaus C, Raimondi MA, Daigle SR, Pollock RM.
WO2014153001; 2014. (f) Basavapathruni A, Jin L, Daigle SR, et
al. Chem Biol Drug Des. 2012;80:971. (g) Kaniskan HÜ, Martini
ML, Jin J. Chem Rev. 2017. DOI: 10.1021/acs.chemrev.6b00801.
5
5
replacement of N -isopropyl by N -cyclobutyl had a strong
negative impact on the DOT1L inhibitory activity (Table 1,
entries 7 and 8)
(
h) Recently, non-nucleoside DOT1L inhibitors with high activity
was also disclosed, see for example: Möbitz H, Machauer R,
Holzer P, et al. ACS Med Chem Lett. 2017;8:338.
(a) Deng L, Zhang L, Yao Y, et al. MedChemComm. 2013;4:822.
Table 1. 5’-C-Me Nucleosides assayed for DOT1L inhibitory activity, in
comparison with known DOT1L inhibitors.
3
.
(
b) Liu T, Xie W, Li C, et al. Bioorg Med Chem. Lett.
a
Entry
Compound
DOT1L Activity (IC50
)
2017;22:4960.
4
5
6
.
.
.
(a) Ren H, Xie W, Xiong B, et al. Tetrahedron. 2017;73:2290.
Olhava, EJ. WO2014152566; 2014.
McGuigan C, Perrone P, Madela K, et al. Bioorg Med Chem. Lett.
2009; 19:4316.
1
2
3
4
5
6
7
8
pinometostat
EPZ5677
cis-7
7.81±0.91 nM
10.00±0.00 nM
45.76±6.52 nM
67.34±35.15 nM
0.10±0.06 µM
2.35±0.11 µM
3.49±0.09 µM
trans-7
FED1
9
7. The DOT1L binding of cis-7 and pinometostat was also evaluated
by computational approaches. The crystal structure (PDB entry:
4
HRA) was used to prepare the receptor model for docking study
with Schrodinger software. Molecular docking followed the
standard Glide protocol. A receptor grid file was generated based
on the prepared protein structure. The Glide grid file was then
generated from an enclosing box centered at the ligand
pinometostat with a size of 18 Å, and extra precision molecular
docking was carried out with the receptor grid file for cis-7. The
result suggested that cis-7 has slightly better binding energy
11
13
5.31±4.33% @ 20µM
a
The activity of DOT1L was detected by using the AlphaLISA DOT1L
Histone H3 Lysine-N-methyltransferase Assay. The dose response of
inhibition test was carried out in triplicate.
(
GScore -11.12 kcal/mol) than pinometostat (GScore -9.40
kcal/mol).
In summary, from adenosine and 2’-C-Me adenosine 4, a 3-
step route towards selected nucleoside DOT1L inhibitors,
including pinometostat, EPZ5677, and FED1 is reported.
Avoiding multiple protection and deprotection steps, the new
method proved more efficient and straightforward compared to
literature methods, leading to nucleoside DOT1L inhibitors.
Meanwhile, the newly prepared 2’-C-Me adenosine derivatives
contributed to the limited repertoire of ribose-modified
nucleoside DOT1L inhibitors. Among these new compounds, cis-
8
9
.
.
Chesworth R, Kuntz KW, Olhava EJ, et al. WO2012082436A2;
2
012.
See ESI for details.
7
and trans-7 demonstrated good enzymatic activity and may be
worthy of further investigation. In general, we believe that this
new synthetic method should facilitate the synthetic and
medicinal chemistry research of 5’-deoxy-5’-amino adenosine
derivatives, especially the research of nucleoside DOT1L
inhibitors.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (81673309), National Science
Technology Major Project “Key New Drug Creation and
Manufacturing Program”, China (2014ZX09507-002),
Personalized Medicines - Molecular Signature-based Drug
&
"
Discovery and Development", Strategic Priority Research
Program of the Chinese Academy of Sciences, Grant No.
XDA12020310, the program for Innovative Research Team of
the Ministry of Education, and program for Liaoning Innovative
Research Team at Shenyang Pharmaceutical University.
Supplementary data

Highlights:
*
*
*
*
Pinometostat was made in 3 steps from adenosine
in 47% yield.
Six other nucleoside DOT1L inhibitors were made
efficiently, too.
2‘-C-Me Nucleoside DOT1L inhibitors were
evaluated for the enzyme activities.
Rapid access to various 5’-deoxy-5’-amino
adenosine derivatives.