Novel carboxaldehyde mediated synthetic pathway for 5′-amino adenosine analogues

Article abstract of DOI:10.1080/15257770.2014.1001074

Modifications of the 5′-position of adenosine have been prepared via a novel 5′-carboxaldehyde synthon. The described methodology should prove useful for making related compounds in which amine-derived moieties off the 5′-position of adenosine (or related nucleoside congeners) can be easily incorporated via reductive amination, especially for the incorporation of aromatic amines.

Full text of DOI:10.1080/15257770.2014.1001074

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Novel Carboxaldehyde Mediated
Synthetic Pathway for 5′-Amino
Adenosine Analogues
Garrett S. Gibbonsa, Hollis D. Showalterb & Zaneta Nikolovska-
Coleskaa
a Department of Pathology, University of Michigan Medical School,
Ann Arbor, MI, USA
b Vahlteich Medicinal Chemistry Core, College of Pharmacy,
University of Michigan, Ann Arbor, MI, USA
Published online: 15 Apr 2015.
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To cite this article: Garrett S. Gibbons, Hollis D. Showalter & Zaneta Nikolovska-Coleska (2015)
Novel Carboxaldehyde Mediated Synthetic Pathway for 5′-Amino Adenosine Analogues, Nucleosides,
Nucleotides and Nucleic Acids, 34:5, 348-360, DOI: 10.1080/15257770.2014.1001074
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Nucleosides, Nucleotides and Nucleic Acids, 34:348–360, 2015
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2014.1001074
NOVEL CARBOXALDEHYDE MEDIATED SYNTHETIC PATHWAY
FOR 5ꢀ-AMINO ADENOSINE ANALOGUES
Garrett S. Gibbons,1 Hollis D. Showalter,2 and Zaneta Nikolovska-Coleska1
1Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA
2Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann
Arbor, MI, USA
Modifications of the 5ꢁ-position of adenosine have been prepared via a novel 5ꢁ-carboxaldehyde
2
synthon. The described methodology should prove useful for making related compounds in which
amine-derived moieties off the 5ꢁ-position of adenosine (or related nucleoside congeners) can be easily
incorporated via reductive amination, especially for the incorporation of aromatic amines.
Keywords Synthetic methodology; reductive amination; modified nucleosides; enzyme
inhibition
INTRODUCTION
Nucleoside derivatives are highly sought after as therapeutic agents for
the treatment of various disease states, ranging from antibiotics and antivi-
rals to treat infections[1,2] to DNA methyltransferases in cancer.[3] Diverse
biological molecules are amenable to targeting via a nucleoside scaffold such
as protein kinases,[4] histone methyltransferases,[5–7] reverse transcriptase,[2]
poly(ADP-ribose) polymerase (PARP),[8] and many others. In conjunction
with ongoing projects in our laboratory to develop small molecule inhibitors
against cancer targets, we needed to establish a synthetic strategy for intro-
ducing different amino derivatives at the 5ꢁ-position of adenosine. Current
synthetic strategies for 5ꢁ-modified adenosine analogues typically proceed via
replacement of the 5ꢁ-hydroxyl with a chlorine leaving group and subsequent
displacement with a thiol or amine. Activation via Mitsunobu reaction with
phthalimide followed by hydrazine treatment to give the primary amine has
also been utilized.[6,9,10] Within these strategies, subsequent homologation
of installed amines is then carried out by alkylation or reductive amination
Received 6 October 2014; accepted 14 December 2014.
Address correspondence to Zaneta Nikolovska-Coleska, Department of Pathology, University
of Michigan Medical School, Ann Arbor, Michigan 48105, USA. E-mail: showalh@med.umich.edu;
zanetan@med.umich.edu
348

Modifications of the 5ꢁ-Position of Adenosine
349
FIGURE 1 Strategies for synthesizing 5ꢁ-deoxy-5ꢁ-amino derivatives of adenosine via reductive amination.
methodologies. The latter is generically exemplified by manifold a (Figure 1),
with many examples of the installation of R’CH2 groups (R’ = aryl) given
in a recent paper.[11] Due to the mild conditions of reductive amination, we
chose this methodology to access our target amines designed as bioisosteres
of S-adenosylmethionine (SAM; 1), shown in Figure 2. While it would be pos-
sible to access 4 and 5 by manifold a, we were interested in accessing all target
compounds from a single carboxaldehyde intermediate as shown in manifold
b. Examples of nucleoside 5ꢁamine installation by manifold b are sparse, and
indeed an exhaustive literature search uncovered no examples of reductive amination
of a nucleoside aldehyde with an aromatic amine, required for target compounds
2 and 3. Thus, we devised a synthetic strategy utilizing a convergent approach
FIGURE 2 Target model compounds of 5ꢁ-adenosine modifications.

350
Garrett S. Gibbons et al.
SCHEME 1 Reagents and conditions: (a) 1. HMDS, DMAP, TMSOTf; 2. Boc2O, THF; 3. 5:1 MeOH: TEA
(100%, 3 steps); (b) Dess-Martin periodinane, DCM, 0◦C - rt (99%); (c) 15, DCE, NaBH(OAc)3 (26%);
(d) LiOH, MeOH (39%); (e) aq. TFA (35% for 9); (f) hplc purification; (g) 1. Fuming HNO3, conc.
H2SO4; 2. Boc2O, NaHCO3, 1,4-dioxane/H2O [14]; (h) MeI, K2CO3, THF (65%); (i) H2, 10% Pd/C,
MeOH (87%).
in which a protected adenosine 5ꢁ-carboxaldehyde (8, Scheme 1) would be
condensed with a requisite amine side chain via a reductive amination, pro-
viding a relatively novel and efficient method to access 5ꢁ-deoxy-5ꢁ-amino
derivatives of adenosine.
SYNTHESIS
Based on the use of S-adenosylmethionine (SAM; 1) as a methyl donor by
many methyltransferase enzymes, we sought to synthesize several model com-
pounds 2–5 using our reductive amination strategy to provide bioisosteres

Modifications of the 5ꢀ-Position of Adenosine
351
of the homocysteine tail of SAM (Figure 2). This required the synthesis of
the novel adenosine synthon 8 as shown in Scheme 1. Accordingly, hexam-
ethyldisilazane (HMDS) persilylation of 2ꢁ,3ꢁ-(isopropylidene)adenosine (6)
followed by treatment with di-t-butyl dicarbonate was carried out by modi-
fication of a procedure used to introduce N 6-Boc functionality onto nucle-
osides.[12] The literature procedure mixes reactants using milling in a flask
with Pyrex glass beads. However, we found this was not necessary and simply
conducted our reaction with conventional magnetic stirring. This gave an
intermediate N 6,N 6-bis(tert-butoxycarbonyl)-5ꢁ-O-(trimethylsilyl)adenosine,
which was not isolated but was reacted directly with methanol:triethylamine
(5:1) to simultaneously cleave the TMS ether and one of the N 6-Boc groups.
This provided 7 in quantitative yield with the overall sequence representing
a much superior method to make this reported compound.[13] Subsequent
conversion of 7 to the 5ꢁ-carboxaldehyde 8 was then carried out by oxi-
dation with Dess-Martin periodinane. This intermediate was then used for
synthesis of designed compounds. For target compound 2 the side chain
was derived from (S)-(+)-phenylglycine 12 in a series of standard reac-
tions. Nitration followed by α-amine Boc protection was carried out by
a literature procedure to provide compound 13.[14] Methyl ester forma-
tion gave 14, which was then hydrogenated to amine 15. Reductive amina-
tion of aldehyde 8 with 15 then provided compound 9 in modest overall
yield.
HPLC analysis of this product showed a ∼1:1 mixture of diastere-
omers, indicating that racemization had taken place in the earlier ni-
tration step of building up the side chain as previously documented.[15]
Hydrolysis of the methyl ester with LiOH gave 10, which was subjected to
aqueous TFA hydrolyis to simultaneously remove the Boc and isopropy-
lidene protecting groups. Upon HPLC purification, two diastereomers, 2
and 11, were separated (Supplementary Figure 1). Alternatively, 9 was also
subjected to TFA deprotection to provide methyl ester 3 as a mixture of
diastereomers.
For compound 5, the same general synthetic approach was uti-
lized (Scheme 2). Compound 19 was prepared via amidation of 1,3-
propanediamine (17) with N -Boc-glycine methyl ester using the method
described by Morandeau et al.[16] Coupling of 19 with aldehyde 8 was
performed under the same reductive amination conditions shown above
providing the Boc and isopropylidene protected compound 21. Subse-
quent global hydrolysis of the protecting groups provided 5 in good over-
all yield. The same sequence of steps was repeated on ethylenediamine
(16) to make compound 4 with the truncated linker. All compounds
reported in this paper were rigorously purified and their structural assign-
ments are supported by mass spectrometry (ESI and HRMS target com-
1
pounds) and H NMR spectroscopy (Experimental and Supplementary
Figures 2–16).

352
Garrett S. Gibbons et al.
SCHEME 2 Reagents and conditions: (a) N -(Boc)-glycine methyl ester, MeOH, 0◦C – rt (50–52% yield);
(b) 18 or 19, NaBH(OAc)3, DCE (43–48% yield); (c) aq. TFA (54–56% yield).
Evaluation of in vitro DOT1L Inhibition
As several SAM analogues with 5ꢁ-adenosine modifications have been
demonstrated to potently inhibit the histone methyltransferase Disruptor of
Telomeric silencing 1 Like (DOT1L),[5,7,17] we tested the ability of the syn-
thesized compounds to inhibit DOT1L histone methyltransferase activity in
3
vitro. Briefly, the assay utilized H-methyl-S-adenosylmethionine (3H-SAM)
as the source of radioisotopically labelled methyl donor. In the presence
3
of DOT1L the H-methyl group is transferred to the substrate histone H3
lysine 79 (H3K79) in a milieu of extracted core histones. The dose depen-
dent curves together with the IC50 values of synthesized and characterized
compounds are presented in Figure 3. S-adenosylhomocysteine (SAH) was
used as a positive control and showed inhibition of the methyltransferase
activity of DOT1L with IC50 of 1.6 μM, consistent with previously reported
results. Compound 2 showed the most potent inhibition of DOT1L histone
methyltransferase activity in comparison with other synthesized nucleoside
analogues with an IC50 value of 220 μM. However, it was significantly less
active in comparison to SAH. Interestingly compound 11, the diastereomer
of 2, showed five-fold lower potency with IC50 of 1,055 μM. Based on the in
vitro histone methyltransferase activity, compound 2 was assigned the natural
2-(S)-stereochemistry in the side chain since it showed higher potency, while
compound 11 was assigned as the 2-(R)-diastereomer. Compound 3, which
is a diastereomeric mixture of the methyl ester of 2/11, showed less potency
(IC50 > 1,500 μM).
These findings are consistent with the reported DOT1L crystal struc-
ture[18] which demonstrates that the co-factor SAM forms a key hydrogen
bond with the protein through the carboxylic acid moiety. Replacing the

Modifications of the 5ꢁ-Position of Adenosine
353
FIGURE 3 DOT1L inhibition by designed compounds. IC50 values of synthesized inhibitors assessed by
in vitro radiometric DOT1L histone methyltransferase (HMTase) assay.
amino acid tail of SAM with an α-amino acetamide bioisostere resulted in a
detrimental effect on the activity of compounds 4 and 5 with IC50 values >
1,500 μM. In addition, the different length of the linker in these two com-
pounds seemed to have no effect on their potency. We believe that much
of the difference in potency for these compounds compared with SAH can
be attributed to disruption of the hydrogen bond network formed between
SAM and DOT1L. In addition, a structure activity relationship (SAR) study
of SAM analogues published while our work was in progress confirmed that
slight alterations of the structure of SAM can dramatically affect the ability
of small molecules to inhibit DOT1L.[9]
CONCLUSIONS
Herein we present a novel synthetic methodology utilizing a 5ꢁ-
carboxaldehyde adenosine intermediate that can undergo reductive ami-
nation with representative amines to provide 5ꢁ-deoxy-5ꢁ-amino derivatives
of adenosine. This strategy was applied to the synthesis of five model SAM
analogues, which were tested for in vitro inhibition of the histone methyl-
transferase DOT1L. The newly synthesized SAM analogues demonstrate that

354
Garrett S. Gibbons et al.
a rigid linker between adenosine and the amino acid tail portion of SAM is
more favorable than a flexible α-amino acetamide bioisoteric replacement
of the amino acid moiety for inhibition of DOT1L. Our synthetic strategy will
be useful for making related compounds in which moieties off the 5ꢁ-position
of adenosine (or related nucleoside congeners) can be easily constructed
via reductive amination of a precursor 5ꢁ-carboxaldehyde. Thus, the utility
clearly extends beyond SAM analogues, as adenosine is a useful scaffold for
the development of numerous potential classes of inhibitors.
EXPERIMENTAL
General Chemistry Procedures
All starting materials were obtained from commercial suppliers and were
used without further purification. 1H NMR spectra were recorded on a Var-
ian 400 instrument. Chemical shift values are recorded in δ units (ppm).
Mass spectra were recorded on a Micromass TofSpec-2E Matrix-Assisted,
Laser-Desorption, Time-of-Flight Mass Spectrometer in positive ESI mode
unless otherwise noted. High resolution mass spectrometry (HRMS) analy-
sis was performed on an Agilent Q-TOF system. Analytical HPLC was run on
a reverse-phase column (Restek Ultra C18, 5 μm, 150 × 4.6 mm column;
flow rate of 1 mL/min using a gradient of 40–80% acetonitrile in water
over 20 min). Thin-layer chromatography (TLC) was performed on silica
gel GHLF plates (250 microns) purchased from Analtech. Column chro-
matography was carried out in the flash mode utilizing silica gel. Extraction
solutions were dried over MgSO4 prior to concentration.
tert-Butyl (9-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahyd-
rofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-yl)carbamate (7).[13] Using a mod-
ification of a literature procedure,[12] a suspension of 6 (1.54 g, 5 mmol), hex-
amethyldisilazane (HMDS; 2 mL), and 4-dimethylaminopyridine (DMAP;
120 mg) was magnetically stirred together at 25◦C in an oven-dried flask.
To this was added drop-wise trimethylsilyl trifluoromethanesulfonate (TM-
SOTf; 20 μL) and the resultant suspension was heated at 75◦C for 2 h.
The mixture was concentrated to oil that was dissolved in 3 mL of dry THF
with gentle warming. The solution was ice-cooled and treated with 3.27 g
(15 mmol) of di-t-butyl dicarbonate and an additional 7 ml of THF, and
allowed to stir at 25◦C for 4 h. The solution was concentrated and then
treated with 18 mL of 5:1(v/v) methanol: triethylamine. Following vigor-
ous evolution of CO2, the solution was stirred at 55◦C for 16 h and then
concentrated to an oil that was distributed between dichloromethane and
water. The dichloromethane phase was dried and concentrated to an oil
that was purified by flash chromatography, eluting with a 1:1 mixture of
hexane and ethyl acetate. Combined product fractions were concentrated
to an oil that was pumped in vacuo at 60◦C to leave 7 (2.53 g; 100% yield)

Modifications of the 5ꢁ-Position of Adenosine
355
as a white foam: NMR and mass spec data were identical to those previously
reported.[13]
tert-Butyl (9-((3aR,4R,6S,6aS)-6-formyl-2,2-dimethyltetrahydrofuro[3,4-
d][1,3]dioxol-4-yl)-9H-purin-6-yl)carbamate (8). To an ice-cooled stirred sus-
pension of Dess-Martin periodinane (674 mg, 1.6 mmol) in dichloromethane
(7.5 mL) was added 7 (589 mg, 1.45 mmol) and the resultant mixture was al-
lowed to warm to room temperature over 3 h. The mixture was concentrated
to a white powder that was suspended in ethyl acetate, and the insoluble Dess-
Martin reagent was removed by filtration. The filtrate was concentrated and
additional residual amounts of Dess-Martin reagent were removed by the
same process. The filtrate was concentrated to leave fairly pure 8 (583 mg,
99% crude yield) as a solid, which was suitable for use in the next reaction:
1H NMR (400 MHz, CDCl3): δ 9.25 (s, 1H), 8.54 (s, 1H), 7.96 (s,1H), 6.17
(s, 1H), 5.49 (d, J = 6.1 Hz, 1H), 5.28 (d, J = 6.1 Hz, 1H), 4.62 (s, 1H), 1.54
(s, 3H), 1.50 (s, 9H), 1.34 (s, 3H); MS (ESI): m/z 438.1 (M+Na)+.
Methyl (S)-2-((tert-butoxycarbonyl)amino)-2-(3-((((3aR,4R,6R,6aR)-6-(6-
((tert-butoxycarbonyl)amino)-9H-purin-9-yl) - 2,2-dimethyltetrahydrofuro[3,
4-d][1,3]dioxol-4-yl)methyl)amino)phenyl)acetate and R-diastereomer (9).
To an ice-cold solution of 8 (370 mg, 0.91 mmol) in 1,2-dichloroethane
(DCE; 2.5 mL) was added a solution of 15 (280 mg, 1 mmol) in DCE (2.5 mL)
followed by sodium triacetoxyborohydride (STAB; 270 mg, 1.3 mmol). The
reaction mixture was stirred overnight at room temperature and quenched
with saturated aq. NaHCO3. The mixture was diluted with water and ex-
tracted with ethyl acetate (3x). The combined extracts were dried and
concentrated to an oil that was purified by flash chromatography, elut-
ing with hexanes: ethyl acetate (2:1) to give 9 (157 mg, 26% yield) as a
1
∼1:1 ratio of diastereomers by HPLC: H NMR (400 MHz, CDCl3): δ 8.84,
8.67 (s each, 0.57H & 0.43H, H-8 of each diastereomer), 8.03 (br s, 1H),
7.97, 7.94 (s each, 0.49 & 0.51H, H-2 of each diastereomer), 7.10 (q, J =
8.1 Hz, 1H), 6.65 (t, J = 9.0 Hz, 1H), 6.61–6.51 (m, 2H), 6.03, 5.96 (s
each, 0.44H & 0.56H, anomeric H of each diastereomer), 5.54–5.09 (m,
4H), 4.60–4.50 (m,1H), 3.67 (s, 3H), 3.53–3.45 (m, 2H), 1.61 (s, 3H), 1.55
(s, 9H), 1.42 (s, 9H), 1.36 (s, 3H); MS (ESI): m/z 670.2 (M+1)+, 692.2
(M+Na)+.
(S)-2-((tert-Butoxycarbonyl)amino) - 2 -(3-((((3aR,4R,6R,6aR)-6-(6-((tert-
butoxycarbonyl)amino)-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,
3]dioxol-4-yl)methyl)amino)phenyl)acetic acid and (R)-diastereomer (10).
To a stirred solution of 9 (160 mg, 0.24 mmol) in methanol (1 mL) at
0◦C was added LiOH (11 mg, 0.26 mmol). Cooling was removed and
reaction mixture was stirred at room temperature overnight. TLC showed
incomplete conversion with a new spot of Rf ∼0.4 (2:1 ethyl acetate:
methanol). Additional LiOH (11 mg) was added and stirring was continued
for 4 h. The mixture was concentrated and the residue was distributed
between 5% aq. acetic acid added and ethyl acetate. The combined ethyl

356
Garrett S. Gibbons et al.
acetate extracts were dried and concentrated to oil that was purified by
flash chromatography, eluting with ethyl acetate: methanol (9:1). Product
fractions were combined and concentrated to give 10 (61 mg, 39% yield):
1H NMR (400 MHz, CDCl3): δ 8.66 (s, 1H), 7.98 (s, 1H), 7.11–6.97 (m, 1H),
6.71–6.45 (m, 3H), 6.03 (d, J = 4.4 Hz, 1H), 5.91–5.84 (m, 1H), 5.50–4.99
(m, 3H), 4.57 (m, 1H), 3.40–3.20 (m, 2H), 1.56 (s, 3H), 1.52 (s, 9H), 1.39
(s, 3H), 1.37 (s, 9H); MS (ESI): m/z 656.2 (M+1)+.
Methyl (S)-2-amino-2-(3-((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-
dihydroxytetrahydrofuran-2-yl)methyl)amino)phenyl)acetate and R-diaster-
eomer (3). To a solution of 9 (173.5 mg, 0.26 mmol) in ice-cold water
(3 mL) was added trifluoroacetic acid (TFA; 3 mL) at 0◦C. The mixture was
stirred at room temperature overnight, concentrated and then diluted with
aq. NaHCO3. The mixture was extracted with ethyl acetate (3x), and the
combined extracts were dried and concentrated to a residue that precipi-
tated solids upon addition of ethyl acetate. The solids were collected and
washed well with hexanes. The filtrate was concentrated and processed in
a similar fashion to provide a second crop that was combined with the first
1
to give 3 (39 mg, 35% yield): H NMR (400 MHz, CDCl3): δ 8.23 (s, 1H),
7.86 (s, 1H), 7.12 (t, J = 7.7 Hz, 1H), 6.64 (d, J = 7.5 Hz, 1H), 6.56 (d, J =
10.7 Hz, 1H), 6.40 (d, J = 7.0 Hz, 1H), 6.30 (s, 1H), 6.00 (s, 1H), 5.51 (d, J
= 5.9 Hz, 1H), 5.40 (d, J = 7.0 Hz, 1H), 5.23 (d, J = 5.4 Hz, 1H), 4.60 (m,
1H), 3.68 (s, 3H), 3.60 - 3.35 (m, 2H); MS (ESI): m/z 430.9 (M+1)+, 452.1
(M+Na)+.
(S)-2-Amino-2-(3-((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydr-
oxytetrahydrofuran-2-yl)methyl)amino)phenyl)acetic acid (2) and (R)-dia-
stereomer (11). To a solution of 10 (61 mg, 0.09 mmol) in dichloromethane
(0.4 mL) at 0◦C was added water (0.1 mL) and TFA (0.5 mL). The
mixture was stirred at room temperature overnight and concentrated to a
residue that was dissolved in H2O: methanol (95:5). The solution was then
purified by reverse-phase HPLC (Restek Ultra C18, 5 μm, 150 × 21.2 mm
column; flow rate of 10 mL/min using a gradient of 10–30% acetonitrile
in water over 25 min) followed by concentration and lyophilization of
fractions corresponding to each isomer provided purified diastereomers.
1
Diastereomer 11: H NMR (400 MHz, DMSO-d6 + D2O): δ 8.34 (s, 1H),
8.12 (s, 1H), 7.10 (t, J = 7.8 Hz, 1H), 6.70–6.59 (m, 3H), 5.88 (d, J =
7.4 Hz, 1H), 4.99 (d, J = 11.0 Hz, 1H), 4.73 (s, 1H), 4.65–4.57 (m, 1H),
4.23–4.17 (m, 1H), 3.26–3.20 (m, 2H); MS (ESI): m/z 416.1682 (M+1)+;
Diastereomer 2: 1H NMR (400 MHz, DMSO-d6): δ 8.58 (br s, 2H; exchanges
with D2O), 8.38 (s, 1H), 8.24 (s,1H), 7.69 (br s, 2H; exchanges with D2O),
7.12 (t, J = 7.8 Hz, 1H), 6.68–6.61 (m, 3H), 5.87 (d, J = 6.0 Hz,1H), 5.47
(br s, 1H; exchanges with D2O), 5.27(br s, 1H; exchanges with D2O), 4.88
(d, J = 18.0 Hz,1H; collapses to s with D2O wash), 4.77–4.68 (m, 1H), 4.14
(t, J = 4.3 Hz, 1H), 4.07 (d, J = 3.5 Hz, 1H), 3.32 (d, J = 7.2 Hz, 2H; peak
revealed with D2O wash); MS (ESI): m/z 416.1685 (M+1)+.

Modifications of the 5ꢁ-Position of Adenosine
357
Methyl
(S)-2-((tert-butoxycarbonyl)amino)-2-(3-nitrophenyl)acetate
(14).[19] A stirred solution of partially racemized (S)-2-((tert-butoxycarbo-
nyl)amino)-2-(3-nitrophenyl)acetic acid[14] (13; 600 mg, 2.03 mmol),
iodomethane (140 μL, 2.2 mmol), anhydrous potassium carbonate
(415 mg, 3 mmol) and THF (10 mL) was heated at 60◦C for 18 h. The
mixture was filtered and the filtrate was diluted with water and extracted
with dichloromethane (3x). The combined organic extracts were dried and
concentrated to an oil that was purified by flash chromatography, eluting
with hexanes: ethyl acetate (6:1). Product fractions were combined and
concentrated to leave 14 (409 mg, 65% yield): (400 MHz, CDCl3): δ 8.23 (t,
J = 2.0 Hz, 1H), 8.19–8.15 (m, 1H), 7.72 (d, J = 7.7 Hz, 1H), 7.57–7.49 (m,
1H), 5.78 (s, 1H), 5.42 (d, J = 6.7 Hz, 1H), 3.73 (s, 3H), 1.51 (s, 9H).
Methyl
(S)-2-(3-aminophenyl)-2-((tert-butoxycarbonyl)amino)acetate
(15).[19] A mixture of 14 (720 mg, 2.3 mmol), 10% Pd/C (catalytic)
and methanol (35 mL) was hydrogenated at 50 psi at room temperature
overnight. The mixture was filtered over CeliteꢀR and the filtrate concen-
1
trated to leave 15 (566 mg, 87% yield) as a viscous yellow oil: H NMR
(400 MHz, CDCl3): δ 7.10 (t, J = 7.8 Hz, 1H), 6.70 (dt, J = 7.7, 1.2 Hz, 1H),
6.64 (t, J = 2.0 Hz, 1H), 6.62–6.59 (m, 1H), 5.46 (d, J = 7.5 Hz, 1H), 5.18
(d, J = 7.5 Hz, 1H), 3.69 (s, 3H), 1.41 (s, 9H).
tert-Butyl (2-((2-aminoethyl)amino)-2-oxoethyl)carbamate (18). To ethy-
lenediamine (16; 13.5 mL, 200 mmol) at 0◦C was added drop-wise a solution
of N -Boc-glycine methyl ester (948 mg, 5 mmol) in methanol (20 mL). The
reaction mixture was stirred at room temperature for 6 h and concentrated
to an oil that was partitioned between water and dichloromethane. The com-
bined organic extracts were dried and concentrated to an oil that was purified
by flash chromatography eluting with ethyl acetate: methanol: triethylamine
(50:50:1). Product fractions were concentrated to give 18 (544 mg, 50%
1
yield) as an oil: H NMR (400 MHz, CDCl3): δ 6.58 (br s, 1H), 5.23 (br s,
1H), 3.77 (d, J = 5.8 Hz, 2H), 3.31 (m, 2H), 2.83 (br s, 2H), 1.44 (s, 9H),
1.26 (s, 2H); MS (ESI): m/z 218.2 (M+1)+.
tert-Butyl (2-((3-aminopropyl)amino)-2-oxoethyl)carbamate (19). Reac-
tion of 1,3-propanediamine (17; 16.8 ml, 200 mmol), as described for the
synthesis of 18, gave 19 (600 mg, 52% yield): 1H NMR (400 MHz, CDCl3): δ
7.10 (br s, 1H), 5.29 (br s, 1H), 3.74 (d, J = 5.8 Hz, 2H), 3.35 (q, J = 6.2 Hz,
2H), 2.77 (t, J = 6.3 Hz, 2H), 1.75–1.60 (m, 4H), 1.42 (s, 9H); MS (ESI): m/z
232.1 (M+1)+, 254.1 (M+Na)+.
tert-Butyl (9-((3aR,4R,6R,6aR)-6-(11,11-dimethyl-6,9-dioxo-10-oxa-2,5,8-
triazadodecyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-
6-yl)carbamate (20). To a solution of 8 (203 mg, 0.5 mmol) in DCE (2 mL)
at 0◦C was added 18 (120 mg, 0.55 mmol) dissolved in DCE (1 mL),
followed by sodium triacetoxyborohydride (148 mg, 0.7 mmol). The
reaction mixture was stirred overnight at room temperature, quenched

358
Garrett S. Gibbons et al.
with saturated aq. NaHCO3, and distributed between dichloromethane and
water. The combined organic extracts were dried and concentrated to an oil
that was purified by flash chromatography eluting with dichloromethane:
methanol (19:1). Product fractions were combined and concentrated to
give 20 (130 mg, 43% yield): 1H NMR (400 MHz, CDCl3 + D2O): δ 8.72 (s,
1H), 8.13 (s, 1H), 6.07 (s, 1H), 5.48–5.41 (m, 1H), 4.99 (s, 1H), 4.35 (s,
1H), 3.77 (d, J = 5.3 Hz, 2H), 3.34–3.21 (m, 2H), 2.92–2.60 (m, 4H), 1.61
(s, 3H), 1.56 (s, 9H), 1.42 (s, 9H), 1.39 (s, 3H).
tert-Butyl (9-((3aR,4R,6R,6aR)-6-(12,12-dimethyl-7,10-dioxo-11-oxa-2,6,-
9-triazatridecyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H -pur-
in-6-yl)carbamate (21). Reaction of 19 (127 mg, 0.55 mmol) with 8 followed
by purification, as described for the synthesis of 20, gave 21 (153 mg, 48%
1
yield): H NMR (400 MHz, CDCl3): δ 8.70 (s, 1H), 8.10 (s, 1H), 7.15 (d, J
= 6.5 Hz, 1H), 6.09 (d, J = 2.5 Hz, 1H), 5.47–5.36 (m, 2H), 5.07 (dd, J =
6.4, 3.4 Hz, 1H), 4.50–4.47 (m, 1H), 3.68 (d, J = 5.8 Hz, 2H), 3.30 (d, J =
6.2 Hz, 2H), 3.20–3.08 (m, 2H), 2.81–2.71 (m, 2H), 1.80–1.70 (m, 2H), 1.60
(s, 3H), 1.55 (s, 9H), 1.42 (s, 9H), 1.37 (s, 3H).
2-Amino-N-(2-((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl) -3,4-dihydrox-
ytetrahydrofuran-2-yl)methyl)amino)ethyl)acetamide (4). To a solution of 20
(130 mg, 0.21 mmol) in dichloromethane (0.8 mL) at 0◦C was added water
(0.2 mL) and TFA (1.1 mL). The mixture was stirred at room temperature
overnight and concentrated to a residue that was dissolved in methanol:
water (5:95) and purified by preparative reverse-phase HPLC (Restek Ultra
C18, 5 μm, 150 × 21.2 mm column; flow rate of 10 mL/min using an isocratic
elution of 2% acetonitrile in water for 10 min) followed by concentration and
lyophilization of fractions yielding 4 (44 mg, 56% yield): 1H NMR (400 MHz,
DMSO-d6 + D2O): δ 8.47 (s, 1H), 8.31 (s, 1H), 5.97 (d, J = 5.4 Hz, 1H),
4.65 (t, J = 5.0 Hz, 1H), 4.23–4.19 (m, 2H), 3.51 (s, 2H), 3.46–3.30 (m, 4H),
3.08–2.99 (m, 2H); MS (ESI): m/z 367.1841 (M+1)+.
2-Amino-N-(3-((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl) -3,4-dihydrox-
ytetrahydrofuran-2-yl)methyl)amino)propyl)acetamide (5). To a solution of
21 (185 mg, 0.3 mmol) in dichloromethane (1.2 mL) at 0◦C was added water
(0.3 mL) and TFA (1.5 mL). The mixture was stirred at room temperature
overnight and concentrated to a residue that was dissolved in methanol:
water (5:95) and purified by preparative reverse-phase HPLC (Restek Ultra
C18, 5 μm, 150 × 21.2 mm column; flow rate of 10 mL/min using an isocratic
elution of 2% acetonitrile in water for 10 min) followed by concentration and
lyophilization of fractions yielding 5 (62 mg, 54% yield): 1H NMR (400 MHz,
DMSO-d6 + D2O): δ 8.42 (s, 1H), 8.31 (s, 1H), 5.95 (d, J = 5.3 Hz, 1H),
4.63 (t, J = 5.0 Hz, 1H), 4.25–4.15 (m, 2H), 3.50 (s, 2H), 3.36 (dd, J =
13.2, 9.6 Hz, 1H), 3.24 (dd, J = 13.4, 2.8 Hz, 1H), 3.12 (t, J = 6.8 Hz,
2H), 2.90 (t, J = 7.7 Hz, 2H), 1.79–1.64 (m, 2H); MS (ESI): m/z 381.2000
(M+1)+.

Modifications of the 5ꢁ-Position of Adenosine
359
In vitro DOT1L Histone Methyltransferase Assay
Enzymatic activity of DOT1L was assessed by incubating 125 nM of recom-
binantly expressed and nickel affinity purified GST-DOT1L catalytic domain
(1–420), with N-terminal 6x His-tag, in the presence of 125 nM (0.28 μCi)
3H-S-adenosylmethionine (NET155250UC; Perkin Elmer), and 1 μg of calf
thymus extracted core histones (H9250; Sigma-Aldrich) in histone methyl-
transferase (HMTase) buffer (20 mM Tris pH 7.9, 4 mM EDTA, 1 mM DTT,
0.01% Triton X-100) at a final volume of 26.5 μL. For determination of
inhibitor potency, compounds were added to the reaction mixture prior to
3
initiation of the HMTase reaction with H-SAM. The HMTase reaction was
allowed to proceed for 1 hr at room temperature and was stopped by transfer-
ring 5 μL of reaction mixture to P81 filter paper. After drying, filter papers
were washed three times with 50 mM NaHCO3 pH 9.0. Filter papers were
then dried and radioactivity measured in vials with 10 mL liquid scintillation
fluid on a Tri-Carb 2800 TR liquid scintillation counter (Perkin Elmer).
FUNDING
This work was supported by the Leukemia Research Foundation, New
Investigator Award to Z. Nikolovska-Coleska.
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