Synthesis, activity and metabolic stability of non-ribose containing inhibitors of histone methyltransferase DOT1L

Article abstract of DOI:10.1039/c3md00021d

Histone methyltransferase DOT1L is a drug target for MLL leukemia. We report an efficient synthesis of a cyclopentane-containing compound that potently and selectively inhibits DOT1L (K_i = 1.1 nM) as well as H3K79 methylation (IC₅₀ ～

Full text of DOI:10.1039/c3md00021d

MedChemComm
View Article Online
View Journal | View Issue
CONCISE ARTICLE
Synthesis, activity and metabolic stability of non-ribose
containing inhibitors of histone methyltransferase
DOT1L†
Cite this: Med. Chem. Commun., 2013,
4, 822
Lisheng Deng,‡a Li Zhang,‡a Yuan Yao,a Cong Wang,a Michele S. Redell,b Shuo Dongc
a
*
and Yongcheng Song
Received 16th January 2013
Accepted 12th March 2013
Histone methyltransferase DOT1L is a drug target for MLL leukemia. We report an efficient synthesis of a
cyclopentane-containing compound that potently and selectively inhibits DOT1L (Ki ¼ 1.1 nM) as well as
H3K79 methylation (IC50 ꢀ 200 nM). Importantly, this compound exhibits a high stability in plasma and
liver microsomes, suggesting it is a better drug candidate.
DOI: 10.1039/c3md00021d
www.rsc.org/medchemcomm
leukemia relevant gene expression, and induce differentiation
of MLL leukemia cells.13 This further pharmacologically vali-
Introduction
In addition to genetic changes such as DNA mutation and
translocation, aberrant post-translational modications of
histone sidechains play important roles in cancer initiation and
progression by dysregulating relevant gene expression.1,2
Compounds targeting histone-modifying enzymes that can
correct these epigenetic errors could therefore represent novel
cancer therapeutics.3,4 Histone methyltransferase (HMT)
DOT1L specically methylates the residue Lys79 of histone H3
(H3K79), using S-adenosyl-L-methionine (SAM) as the enzyme
cofactor (Fig. 1).5,6 Recent biological studies have demonstrated
that DOT1L is a novel drug target for acute leukemia with MLL
(mixed lineage leukemia) gene translocation.7–9 This subtype of
leukemia accounts for ꢀ75% infant and ꢀ10% adult acute
leukemia with a particularly poor prognosis.10 The majority of
the translocated gene products, MLL-oncoproteins, are able to
recruit DOT1L, which causes H3K79 hypermethylation leading
to overexpression of leukemia relevant genes and eventually the
cancer. Inhibitors of DOT1L should block the process and may
potentially reverse the progress of MLL leukemia.6
dated DOT1L to be a target for this type of leukemia. At almost
the same time, we used a structure based design to nd N6-
substituted SAH (S-adenosyl-L-homocysteine), such as
compound 2, are highly selective inhibitors of DOT1L.11
A
mechanism based inhibitor design produced several aziridium
analogs of SAM, such as 3 (Fig. 1) which almost quantitatively
inactivates DOT1L. However, these compounds with a polar and
charged amino acid moiety do not have cell activity, presumably
due to limited cell membrane permeability. In addition, our
independent effort in nding competitive DOT1L inhibitors
resulted in the identication of compounds 4 and 5 with a
Previous medicinal chemistry studies by us11,12 and
others13–15 have led to the discovery of several highly potent
inhibitors of DOT1L with Ki values of <1 nM, as representatively
shown in Fig. 1. EPZ004777 (1) is the rst disclosed DOT1L
inhibitor, which can inhibit H3K79 methylation, reduce
aDepartment of Pharmacology, Baylor College of Medicine, 1 Baylor Plaza, Houston,
Texas 77030, USA. E-mail: ysong@bcm.edu; Fax: +1 713-798-3145; Tel: +1 713-798-
7415
bDepartment of Pediatrics, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas
77030, USA
cDepartment of Medicine, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas
77030, USA
† Electronic supplementary information (ESI) available: Supplementary Fig. S1
and the detailed Experimental section. See DOI: 10.1039/c3md00021d
‡ These authors contributed equally.
Fig. 1 DOT1L catalyzed reaction and inhibitors.
822 | Med. Chem. Commun., 2013, 4, 822–826
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Concise Article
MedChemComm
similar structure and activity to 1.12 Structure activity relation-
ship (SAR) investigation showed that the urea group is critically
important for high potency, with each of the two NH moieties
offering ꢀ25 to >50-fold activity enhancement. A crystallo-
graphic study revealed the binding structure of 1 in DOT1L.14
While the adenosine part of 1 adopts almost the same binding
pose as that of SAM, DOT1L undergoes a large conformational
change to favorably hold the tert-butylphenyl substituted urea
group, with the –NHCONH– forming two hydrogen bonds with a
sidechain of the enzyme.
A problem with these ribose-containing inhibitors, e.g., 1
and 4, is their metabolic instability, resulting in a short half-life
in plasma.13 Compound 1 has to be infused continuously using
a subcutaneously implanted osmotic pump to achieve a stable
plasma drug concentration of ꢀ0.5 mM. This might be also
responsible for a relatively weak in vivo efficacy in prolonging
the lifespan of the experimental animals in a mouse model of
MLL translocated leukemia.13 Here, we report the synthesis,
biological activity and metabolic stability of two non-ribose
containing DOT1L inhibitors.
Results and discussion
Inhibitor design and synthesis
Since the adenosine or deaza-adenosine moiety can be recog-
nized by many enzymes,16,17 leading to a rapid cleavage of
adenine and/or the 50-substituent, a possible solution is to
synthesize compounds 6 and 7 by replacing the metabolically
labile ribose (or more accurately ribofuranose) group in 1 and 4
with a cyclopentane or cyclopentene ring.
Scheme 1 Synthesis of compound 6. Reagents and conditions: (i) cyclohexa-
none, cat. H2SO4; (ii) CH2]CHMgBr, THF, ꢁ78 ꢂC, 70% for 2 steps; (iii) NaIO4,
MeOH/H2O; (iv) Ph3PCH3Br, t-BuOK, THF, 87% for two steps; (v) 2nd generation
Grubbs' catalyst (5 mmol%), CH2Cl2; (vi) DMP, CH2Cl2, 86% for two steps; (vii)
CH2]CHMgBr, TMSCl, hexamethylphosphor-amide, CuBr$Me2S, THF, ꢁ78 ꢂC,
89% (viii) NaBH4, CeCl3$7H2O, MeOH, 0 ꢂC, 96%; (ix) 6,6-di-Boc-adenine, Ph3P,
DIAD, THF, 73%; (x) O3, CH2Cl2, ꢁ78 ꢂC, then NaBH4/MeOH, 92%; (xi) trifluoro-
acetic acid, CH2Cl2, 96%; (xii) phthalimide, PPh3, DIAD, THF, 92%; (xiii) NH2NH2,
EtOH, 80 ꢂC, 99%; (xiv) acetone, NaCNBH3, MeOH, 95%; (xv) methyl acrylate,
MeOH, 65 ꢂC, 99%; (xvi) LiAlH4, THF, ꢁ15 ꢂC, 90%; (xvii) 4-tBuPhNCO, CH2Cl2,
95%; (xviii) HCl, MeOH, 92%.
preferably occur from the less hindered up side of the cyclo-
pentane ring.18 Thus, 1,4-addition of a vinyl group to 10 and the
ensuing NaBH4-mediated reduction gave exclusively the key
cyclopentane intermediate 11. A Mitsunobu reaction using di-
Boc(tert-butyloxycarbonyl) protected adenine with 11 afforded
compound 12, which was treated with O3 at ꢁ78 ꢂC followed by
NaBH4 to give 13 with a 50-OH (according to the corresponding
ribofuranose nomenclature). Using a Mitsunobu reaction with
A 20-step synthesis of compound 6 is shown in Scheme 1, phthalimide followed by treatment with NH2NH2, the 50-OH of
starting from readily available D-ribose, with the key steps being 13 was converted to an –NH2, which was then mono-substituted
to construct the central cyclopentane ring with the correct with an isopropyl group using acetone/NaCNBH3 to produce
stereochemistry of its substituents. The 20,30-dihydroxyls of D- compound 14. Conjugated addition of 14 to methyl acrylate
ribose were selectively protected with cyclohexanone and the followed by LiAlH4 reduction gave compound 15. Its –OH was
product was treated with vinylmagnesium bromide to give vinyl- again converted to a primary –NH2, affording compound 16,
substituted ribose derivative 8 (ESI, Experimental section†). Its which was treated with 4-tert-butylphenyl isocyanate followed
vicinal 40,50-diol was oxidatively cleaved by NaIO4 and the by deprotection of 20,30-hydroxyls to give the nal product 6 with
resulting 40-CH]O was treated with CH2]PPh3 to afford diene an overall yield of 19.3% from D-ribose.
9, which was subjected to a ring-closing metathesis reaction,
Compound 7 as well as epi-6, a diastereomer of 6 that can be
followed by oxidation using Dess-Martin periodinane (DMP) to used to determine the importance of the stereochemistry at the
produce cyclopentenone 10. Thanks to a high stereoselectivity 40-position, were synthesized according to Scheme 2. The 20,30-
rendered by the bicyclic ring of 10, nucleophilic attacks dihydroxyls of D-ribose were protected with an acetonide and
This journal is ª The Royal Society of Chemistry 2013
Med. Chem. Commun., 2013, 4, 822–826 | 823

View Article Online
MedChemComm
Concise Article
Table 1 Ki (mM) against DOT1L and other HMTs
DOT1L
PRMT1
CARM1
SUV39H1
SAH
4
6
7
epi-6
0.16
0.40
>50
>50
>50
>50
0.86
>50
>50
>50
>50
4.9
0.00072
0.0011
0.0013
>50
>50
>50
>50
>50
50-position and the urea group in 7 could allow both its adeni-
nylcyclopentene and N-tert-butylphenyl urea moieties to occupy
the optimal binding sites in DOT1L. There is therefore little
binding affinity difference among compounds 4, 6 and 7.
However, epi-6 was found to be inactive against DOT1L with a Ki
value of >50 mM, showing that the corresponding two groups of
epi-6 cannot be in the favorable positions due to the trans-
oriented 50-sidechain (relative to 10-adeninyl). Next, the HMT
enzyme selectivity of compounds 6 and 7 was evaluated. As with
their adenosine-containing analog 4, these two potent DOT1L
inhibitors 6 and 7 were found to have no activity against three
representative histone methyltransferases CARM1, PRMT1 and
SUV39H1 (Table 1). This is in contrast to another DOT1L
inhibitor SAH, which has broad activity against all these HMTs.
The excellent selectivity of compounds 6 and 7 should also be
due to the hydrophobic urea-containing sidechain. Previous
crystallographic studies show the SAM amino acid binding
pocket of DOT1L undergoes a large conformational change.14,15
The urea functionality of these inhibitors forms two hydrogen
bonds with Asp161 and the 4-tert-butylphenyl group is favorably
located in a newly formed hydrophobic pocket of DOT1L,
showing the structural basis for the high selectivity over other
histone methyltransferases.
Scheme 2 Synthesis of compounds 7 and epi-6. Reagents and conditions: (a)
acetone, cat. H2SO4, 85%; (b) TBDPSCl, Et3N, 4-dimethylaminopyridine, DMF,
98%; (c) Ph3PMeBr, t-BuOK, THF, 92%; (d) SO3$Py, Et3N, CH2Cl2, 97%; (e) CH2
]
CHMgBr, THF, ꢁ78 ꢂC, 93%; (f) 2nd generation Grubbs catalyst (5 mmol%),
˚
CH2Cl2, reflux, 95%; (g) PDC, 4 A molecular sieve, DMF, 87%; (h) NaBH4,
CeCl3$7H2O, MeOH, 0 ꢂC, 98%; (i) 6-chloropurine, Ph3P, DIAD, THF, 95%; (j) 7 M
NH3 in MeOH, 100 ꢂC; (k) tetrabutylammonium fluoride, THF, 93% for two steps;
(l) H2, 10% Pd/C, MeOH, 91%.
the 50-OH subsequently with a tert-butyldiphenylsilyl (TBDPS)
group. A Wittig reaction of the product with CH2]PPh3 gave
compound 17, whose 40-OH was oxidized with SO3–pyridine,
followed by an addition of a vinyl group to give diene 18. A ring
closing metathesis reaction produced cyclopentene compound
19 with a tertiary allylic –OH, which was subjected to a pyr-
idinium dichromate (PDC) mediated oxidation, affording
cyclopentenone 20. It was stereoselectively reduced to
compound 21 with a b-OH at the 10-position. A Mitsunobu
reaction of 21 with 6-chloropurine, followed by aminolysis of
the obtained chloride 22 and 50-deprotection of the silyl group,
produced the key cyclopentene intermediate 23, corresponding
to the cyclopentane analog 13 in Scheme 1. Using steps (xii)–
(xviii) in Scheme 1, compound 23 was transformed to the
product 7 with an overall yield of 29.2% from D-ribose. Hydro-
genation of 7 from the less hindered up side of the cyclopentene
ring gave compound epi-6 in 91% yield.
Fig. 2 shows that compounds 6 and 7 are able to block H3K79
methylation in human leukemia cell line MV4-11 in a dose-
dependent manner. Inhibitor 4 was also used in the study. The
IC50 values of compounds 6 and 7 were estimated to be
ꢀ0.2 mM, showing a similar cell activity to 4. It is remarkable
that the IC50 values of these competitive inhibitors are much
higher than their corresponding enzyme Ki values (ꢀ1 nM),
which is mainly due to a considerably higher concentration of
Biological activity evaluation
Compounds 6, 7 and epi-6 were tested for their inhibitory
activities against recombinant human DOT1L, together with 4
as a control. As shown in Table 1 and ESI Fig. S1†, carbocyclic
analogs 6 and 7 exhibited a very high potency against the
enzyme with Ki values of 1.1 and 1.3 nM, respectively, showing a
comparable activity to inhibitor 4 (Ki ¼ 0.72 nM). In addition to
Fig. 2 Western blot showing inhibition of H3K79 methylation in MV4-11 cells by
6 (with a similar structure to 4), the exible linker between the compounds 4, 6 and 7.
824 | Med. Chem. Commun., 2013, 4, 822–826
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Concise Article
MedChemComm
SAM in cells (ꢀ300 mM)19 as compared to that used in the
enzyme inhibition assay (0.76 mM ¼ the Km value). In addition,
cell membrane permeability as well as other factors including
stability could also affect cell activities of these compounds.
Conclusion
In conclusion, cyclopentane-containing compound 6, an analog
of a potent DOT1L inhibitor 4, was synthesized efficiently with
an overall yield of 19.3%, starting from readily available D-
ribose. 6 potently inhibits human DOT1L with a Ki value of
1.1 nM, but is inactive against other HMTs. In addition, it
possesses potent activity in inhibiting cellular H3K79 methyla-
tion with an IC50 of ꢀ200 nM. Of particular interest is the
metabolic stability of compound 6 without degradation by
human plasma and liver microsomes, showing the promise for
this class of compounds to be further developed targeting MLL
leukemia. In addition, cyclopentene analog 7 was also synthe-
sized, which has almost the same biological activities as those
of 6, but lacks desired metabolic stabilities. epi-6 with a trans-
oriented urea sidechain is completely devoid of DOT1L inhibi-
tory activity.
Metabolic stability evaluation
One of the objectives to make the carbocyclic DOT1L inhibitors 6
and 7 is to address the metabolic instability of ribose-containing
inhibitors. We next tested the in vitro metabolic stability of
potent DOT1L inhibitors 6 and 7 in human plasma and liver
microsomes, the latter of which are mainly responsible for drug
metabolism. These two assays, especially the liver microsome
stability, are standard indicators for predicting in vivo pharma-
cokinetic parameters of a compound.20,21 Compound 4 was
included in the study for a comparison. As shown in Fig. 3,
although the ribose-containing compound 4 is reasonably stable
in human plasma with ꢀ90% remaining aer 1 h, it is quickly
degraded in the presence of human liver microsomes with only
ꢀ50% unchanged aer 1 h. The intrinsic clearance (CLint) of 4 is
24.0 mL minꢁ1 mgꢁ1 protein (microsomes). This is in line with a
study for compound 1, showing a quick degradation and a short
half-life in vivo.13 The cyclopentane-containing analog 6 exhibits,
however, a very high metabolic stability in both plasma and liver
microsomes, with a CLint value of only 0.36 mL minꢁ1 mgꢁ1
protein. Unlike 6, the cyclopentene analog 7 can also be metab-
olized by microsomes with ꢀ50% remaining aer 1 h treatment
(CLint ¼ 22.5 mL minꢁ1 mgꢁ1 protein), although it is stable in
human plasma containing few metabolic enzymes (Fig. 3). This
might be due to the C]C double bond in 7 that may be oxidized
by, e.g., cytochrome P450 in microsomes. These results show
changing the metabolically labile ribose ring to the cyclopentane
group could be an effective strategy to produce better drug
candidates with favorable pharmacokinetic properties.
Acknowledgements
This work was supported by a grant (RP110050) from the Cancer
Prevention and Research Institute of Texas (CPRIT) and, in part,
a grant (R01NS080963) from the National Institute of Neuro-
logical Disorders and Stroke (NINDS/NIH) to Y.S.
Notes and references
1 T. Kouzarides, Cell, 2007, 128, 693.
2 P. A. Jones and S. B. Baylin, Cell, 2007, 128, 683.
3 P. A. Cole, Nat. Chem. Biol., 2008, 4, 590.
4 R. A. Copeland, M. E. Solomon and V. M. Richon, Nat. Rev.
Drug Discovery, 2009, 8, 724.
5 Q. Feng, H. Wang, H. H. Ng, H. Erdjument-Bromage,
P. Tempst, K. Struhl and Y. Zhang, Curr. Biol., 2002, 12, 1052.
6 J. Min, Q. Feng, Z. Li, Y. Zhang and R. M. Xu, Cell, 2003, 112,
711.
7 Y. Okada, Q. Feng, Y. Lin, Q. Jiang, Y. Li, V. M. Coffield, L. Su,
G. Xu and Y. Zhang, Cell, 2005, 121, 167.
8 A. V. Krivtsov and S. A. Armstrong, Nat. Rev. Cancer, 2007, 7,
823.
9 A. V. Krivtsov, Z. Feng, M. E. Lemieux, J. Faber, S. Vempati,
A. U. Sinha, X. Xia, J. Jesneck, A. P. Bracken,
L. B. Silverman, J. L. Kutok, A. L. Kung and
S. A. Armstrong, Cancer Cell, 2008, 14, 355.
10 J. M. Hilden, P. A. Dinndorf, S. O. Meerbaum, H. Sather,
D. Villaluna, N. A. Heerema, R. McGlennen, F. O. Smith,
W. G. Woods, W. L. Salzer, H. S. Johnstone, Z. Dreyer and
G. H. Reaman, Blood, 2006, 108, 441.
11 Y. Yao, P. Chen, J. Diao, G. Cheng, L. Deng, J. L. Anglin,
B. V. V. Prasad and Y. Song, J. Am. Chem. Soc., 2011, 133,
16746.
12 J. L. Anglin, L. Deng, Y. Yao, G. Cai, Z. Liu, H. Jiang,
G. Cheng, P. Chen, S. Dong and Y. Song, J. Med. Chem.,
2012, 55, 8066.
13 S. R. Daigle, E. J. Olhava, C. A. Therkelsen, C. R. Majer,
C. J. Sneeringer, J. Song, L. D. Johnston, M. P. Scott,
J. J. Smith, Y. Xiao, L. Jin, K. W. Kuntz, R. Chesworth,
Fig. 3 Metabolic stability of DOT1L inhibitors in human liver microsomes (up)
and plasma (down).
This journal is ª The Royal Society of Chemistry 2013
Med. Chem. Commun., 2013, 4, 822–826 | 825

View Article Online
MedChemComm
Concise Article
M. P. Moyer, K. M. Bernt, J. C. Tseng, A. L. Kung, 16 J. E. Lee, G. D. Smith, C. Horvatin, D. J. Huang, K. A. Cornell,
S. A. Armstrong, R. A. Copeland, V. M. Richon and
M. K. Riscoe and P. L. Howell, J. Mol. Biol., 2005, 352,
R. M. Pollock, Cancer Cell, 2011, 20, 53.
559.
14 A. Basavapathruni, L. Jin, S. R. Daigle, C. R. Majer, 17 X. Yang, Y. Hu, D. H. Yin, M. A. Turner, M. Wang,
C. A. Therkelsen, T. J. Wigle, K. W. Kuntz, R. Chesworth,
R. M. Pollock, M. P. Scott, M. P. Moyer, V. M. Richon,
R. T. Borchardt, P. L. Howell, K. Kuczera and
R. L. Schowen, Biochemistry, 2003, 42, 1900.
R. A. Copeland and E. J. Olhava, Chem. Biol. Drug Des., 18 M. Yang, W. Ye and S. W. Schneller, J. Org. Chem., 2004, 69,
2012, 80, 971. 3993.
15 W. Yu, E. J. Chory, A. K. Wernimont, W. Tempel, A. Scopton, 19 P. Chiba, C. Wallner and E. Kaiser, Biochem. Biophys. Acta,
A. Federation, J. J. Marineau, J. Qi, D. Barsyte-Lovejoy, J. Yi, 1988, 971, 38.
R. Marcellus, R. E. Iacob, J. R. Engen, C. Griffin, A. Aman, 20 R. S. Obach, J. G. Baxter, T. E. Liston, B. M. Silber, B. C. Jones,
E. Wienholds, F. Li, J. Pineda, G. Estiu, T. Shatseva, T. Hajian,
R. Al-Awar, J. E. Dick, M. Vedadi, P. J. Brown, C. H. Arrowsmith,
J. E. Bradner and M. Schapira, Nat. Commun., 2012, 3, 1288.
F. MacIntyre, D. J. Rance and P. Wastall, J. Pharmacol. Exp.
Ther., 1997, 283, 46.
21 K. Ito and J. B. Houston, Pharm. Res., 2004, 21, 785.
826 | Med. Chem. Commun., 2013, 4, 822–826
This journal is ª The Royal Society of Chemistry 2013