Structure-activity relationship of leucyladenylate sulfamate analogues as leucyl-tRNA synthetase (LRS)-targeting inhibitors of Mammalian target of rapamycin complex 1 (mTORC1)

Article abstract of DOI:10.1016/j.bmc.2019.01.037

Leucyl-tRNA synthetase (LRS) plays an important role in amino acid-dependent mTORC1 signaling, which is known to be associated with cellular metabolism and proliferation. Therefore, LRS-targeting small molecules that can suppress mTORC1 activation may provide an alternative strategy to current anticancer therapy. In this work, we developed a library of leucyladenylate sulfate analogues by extensively modifying three different pharmacophoric regions comprising adenine, ribose and leucine. Several effective compounds were identified by cell-based mTORC1 activation assays and further tested for anticancer activity. The selected compounds mostly exhibited selective cytotoxicity toward five different cancer cell lines, supporting the hypothesis that the LRS-mediated mTORC1 pathway is a promising alternative target to current therapeutic approaches.

Full text of DOI:10.1016/j.bmc.2019.01.037

Accepted Manuscript
Structure-Activity Relationship of Leucyladenylate Sulfamate Analogues as
Leucyl-tRNA Synthetase (LRS)-Targeting Inhibitors of Mammalian Target of
Rapamycin Complex 1 (mTORC1)
Suyoung Yoon, Sung-Eun Kim, Jong Hyun Kim, Ina Yoon, Phuong-Thao Tran,
Jihyae Ann, Changhoon Kim, Woong Sub Byun, Sangkook Lee, Sunghoon Kim,
Jiyoun Lee, Jeewoo Lee
PII:
S0968-0896(18)32164-3
https://doi.org/10.1016/j.bmc.2019.01.037
BMC 14729
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
27 December 2018
16 January 2019
29 January 2019
Please cite this article as: Yoon, S., Kim, S-E., Kim, J.H., Yoon, I., Tran, P-T., Ann, J., Kim, C., Byun, W.S., Lee,
S., Kim, S., Lee, J., Lee, J., Structure-Activity Relationship of Leucyladenylate Sulfamate Analogues as Leucyl-
tRNA Synthetase (LRS)-Targeting Inhibitors of Mammalian Target of Rapamycin Complex 1 (mTORC1),
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.01.037
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Structure-Activity Relationship of Leucyladenylate Sulfamate
Analogues as Leucyl-tRNA Synthetase (LRS)-Targeting Inhibitors of
Mammalian Target of Rapamycin Complex 1 (mTORC1)
a,#
a,#
b
b
d
Suyoung Yoon , Sung-Eun Kim , Jong Hyun Kim , Ina Yoon , Phuong-Thao Tran , Jihyae
a
a
a
a
b,c
e,*
Ann , Changhoon Kim , Woong Sub Byun , Sangkook Lee , Sunghoon Kim , Jiyoun Lee
,
a,*
Jeewoo Lee
a
Laboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of
Pharmacy, Seoul National University, Seoul 08826, Korea
b
Medicinal Bioconvergence Research Center, College of Pharmacy, Seoul National University,
Seoul 08826, Korea
c
Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of
Convergence Science and Technology, Seoul National University, Seoul 08826, Korea
d
Department of Medicinal Chemistry, Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hoan
Kiem, Hanoi, Vietnam
e
Department of Global Medical Science, Sungshin University, Seoul 01133, Korea
#
These two authors contributed equally to this work.
*
Corresponding Authors
Jeewoo Lee, Ph.D.
Phone: 82-2-880-7846
Fax: 82-2-888-0649
E-mail: jeewoo@snu.ac.kr
Jiyoun Lee, Ph.D.
E-mail: jlee@sungshin.ac.kr
1

Key Words: Leucyl-tRNA Synthetase, LRS, mTORC1 Inhibitor, Leucyladenylate, anticancer
agents
Abstract
Leucyl-tRNA synthetase (LRS) plays an important role in amino acid-dependent mTORC1
signaling, which is known to be associated with cellular metabolism and proliferation. Therefore,
LRS-targeting small molecules that can suppress mTORC1 activation may provide an alternative
strategy to current anticancer therapy. In this work, we developed a library of leucyladenylate
sulfate analogues by extensively modifying three different pharmacophoric regions comprising
adenine, ribose and leucine. Several effective compounds were identified by cell-based mTORC1
activation assays and further tested for anticancer activity. The selected compounds mostly
exhibited selective cytotoxicity toward five different cancer cell lines, supporting the hypothesis
that the LRS-mediated mTORC1 pathway is a promising alternative target to current therapeutic
approaches.
2

1
. Introduction
Mammalian/mechanistic target of rapamycin (mTOR) is a multiprotein complex that
functions as a serine-threonine protein kinase. mTOR is known to be involved in various signaling
pathways that govern cell growth and immune responses. mTOR consists of two distinct protein
domains, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). It has been reported that
S6 kinase 1 (S6K1) and the translation regulators eukaryotic translation initiation factor 4E (elF4E)-
binding protein 1 (4E-BP1) are the two major substrates for mTORC1 and participate in the signal
1
transduction pathway regulating protein synthesis. Given its crucial role in cell growth and
metabolism, it is not surprising that overactivity of mTORC1 has been linked to a wide range of
2
-4
human diseases, such as diabetes, neurodegenerative diseases, and cancers. Extracellular signals
such as growth hormones and amino acids activate the mTOR pathway. In particular, the
phosphorylation of S6K1 and 4E-BP1 by activated mTORC1 triggers downstream pathways
5
involving biosynthesis and proliferation. Because specific inhibition of mTORC1 can suppress cell
growth and proliferation, rapamycin and their analogues, also known as rapalogs, have been widely
6
,7
tested for anticancer activity. While these compounds are highly specific allosteric inhibitors of
8
,9
mTORC1, rapamycin treatment alone did not appear to generate sufficient anticancer activity.
Several studies have also suggested that a partial blockade of the mTORC1-related pathway could
cause rapamycin-resistance in cancer cells. These results imply that the mTOR pathway constitutes
a promising new drug target. It may also be possible to develop alternative, more effective
therapeutic strategies in addition to rapalogs.
It has been reported that leucine and arginine are the two major amino acids that regulate
1
0,11
the amino acid-dependent activation of the mTORC1 signaling pathway.
Although it is still
unclear how these amino acids activate the mTORC1 pathway, recent studies have proposed that
leucyl-tRNA synthetase (LRS) may play a critical role in leucine sensing by activating RagD
3

1
2,13
GTPase, which subsequently promotes the amino acid-dependent mTORC1 activation.
LRS, a
class І aminoacyl-tRNA synthetase (ARS), catalyzes the ATP-dependent ligation of leucine to its
tRNA to form leucyl tRNA. Since LRS acts as a GTPase-activating protein (GAP) for Rag GTPase,
molecules that bind to LRS and interfere with the Rag GTPase activity are also likely to block the
1
2
mTORC1 activation. As a proof of concept, it has been demonstrated that leucinol, a leucine
analogue, was able to block the amino acid-mediated activation of mTORC1 without affecting the
14,15
catalytic function of LRS.
Considering that this LRS-mediated mTORC1 activation is
independent of the mTOR-rapamycin interaction, LRS inhibitors such as leucinol may provide an
alternative therapeutic option to suppress the mTORC1 activity and overcome rapamycin resistance.
Based on this idea, we have previously developed the leucyladenylate sulfamate analogues
1
6
as mTORC1 inhibitors that specifically bind to LRS. Among the three representative compounds
(1-3, Figure 1) identified in our previous study, leucyladenylate sulfamate 1 blocked the catalytic
activity of LRS but did not affect leucine-induced mTORC1 activation, whereas compound 3
inhibited mTORC1 activation without markedly compromising the catalytic activity of LRS
compared to compounds 1 and 2. More importantly, our mechanistic study indicated that mTORC1
inhibition by compounds 2 and 3 was not due to direct interaction with mTOR but from blocking
the leucine-sensing ability of LRS. While both compounds 2 and 3 demonstrated anticancer activity,
only compound 3 demonstrated specific activity against colon cancer cells expressing overactive
mTORC1.
4

Figure 1. Representative leucyladenylate sulfamate analogues.
In this report, we developed a series of novel leucyladenylate sulfate analogues to optimize
their anticancer activities. We divided the leucyladenylate sulfamate scaffold into three
pharmacophoric regions, the base (adenine), ring (ribose) and side chain (leucyl sulfamate), as
described in Figure 2. We modified each region extensively by adding and deleting functional
groups and synthesized a library of compounds that were further tested for in vitro activity against
leucine-induced mTORC1 activation. Several compounds with high activity were selected to assess
their cytotoxicities against various cancer cell lines.
Figure 2. Structure-activity relationship of leucyladenylate sulfamate.
5

2
2
. Results and discussion
.1. Chemistry
6
For the syntheses of leucyladenylate sulfamate and its N -methyladenylate derivatives
Scheme 1), the key intermediate 5’-O-sulfamoyl-2’,3’-O-diacetyl adenosine was prepared from
(
1
6,17
adenosine in 4 steps.
The sulfamate was coupled with the corresponding acids, and then
deacetylation with sodium methoxide provided the leucyladenylate sulfamate derivatives (5). The
6
6
N -methyladenylate derivatives (6) were synthesized from N -methyladenosine, which was prepared
from adenosine in 3 steps by following the same method described for the synthesis of 4.
6
Scheme 1. Synthesis of leucyladenylate sulfamate and its N -methyl derivatives.
Reagents and conditions: (a) acid, DCC, DMAP, anhyd. CH Cl , r.t., 12 h, 45~80%; (b) 0.02 M NaOMe in
2
2
MeOH, r.t., 2 h, 68~85%; (c) Ac O, pyridine, 60 °C, 8 h; (d) CH Br, DBU, MeCN, r.t.; (e) 7 M NH in
2
3
3
MeOH; (f) MMTCl, anhyd. pyridine, anhyd. DMF, 0 °C to r.t., 24 h; (g) Ac O, TEA, DMAP, CH CN, 0 °C
2
3
to r.t., 4 h; (h) 80% aq. AcOH, r.t., 12 h; (i) i) NaH, anhyd. THF, 0 °C, 1 h and ii) NH SO Cl, anhyd. THF,
2
2
0
°C to r.t., 5 h.
For the syntheses of 2-chloroadenylate, 2-iodoadenylate and inosine derivatives (Scheme
), commercially available 2-chloroadenosine was converted to 2-chloroadenylate derivatives (6) by
2
following the method of Scheme 1. 2-Iodoadenylate derivatives (7) were synthesized from 5’-O-
1
6
sulfamoyl 2-iodoadenosine, which was prepared from guanosine in 4 steps. The synthesis of
inosine derivatives (8) was accomplished from inosine by following the methods described for the
6

synthesis of 4.
Scheme 2. Synthesis of 2-chloroadenylate, 2-iodoadenylate and inosine derivatives.
Reagents and conditions: Route A: (a) MMTCl, anhyd. pyridine, anhyd. DMF, 0 °C to r.t., 24 h; (b) acetic
anhydride, TEA, DMAP, CH CN, 0 °C to r.t., 4 h; (c) 80% aq. AcOH, r.t., 12 h; (d) i) NaH, anhyd. THF, 0 °C,
3
1
h and ii) NH SO Cl, anhyd. THF, 0 °C to r.t., 5 h; (e) acid, DCC, DMAP, anhyd. CH Cl , r.t., 12 h; (f) 0.02
2
2
2
2
M NaOMe in MeOH, r.t., 2 h. Route B: (a) acid, DCC, DMAP, anhyd. CH Cl , r.t., 12 h, 45~80%; (b) 0.02
2
2
M NaOMe in MeOH, r.t., 2 h, 68~85%. Route C: (a) MMTCl, anhyd. pyridine, anhyd. DMF, 0 °C to r.t., 24
h; (b) acetic anhydride, TEA, DMAP, CH CN, 0 °C to r.t., 4 h; (c) 80% aq. AcOH, r.t., 12 h; (d) i) NaH,
3
anhyd. THF, 0 °C, 1 h and ii) NH SO Cl, anhyd. THF, 0 °C to r.t., 5 h; (e) acid, DCC, DMAP, anhyd. CH Cl ,
2
2
2
2
r.t., 12 h; (f) 0.02 M NaOMe in MeOH, r.t., 2 h.
For the syntheses of 2’-deoxyadenylate and 3’-deoxyadenylate arabinose derivatives
Scheme 3), commercially available 2’-deoxyadenosine and 3’-deoxyadenosine, which was
(
prepared from adenosine in 5 steps, were converted to 2’-deoxyadenylate (9) and 3’-deoxyadenylate
10) derivatives, respectively, by following the method in Scheme 1. The synthesis of arabinose
(
derivatives (11) was carried out from commercially available adenine 9-β-D-arabinofuranoside by
following the same route for the corresponding ribose derivatives (4).
7

Scheme 3. Synthesis of 2’-deoxyadenylate derivatives.
Reagents and conditions: Route A: (a) MMTCl, anhyd. pyridine, anhyd. DMF, 0 °C to r.t., 24 h, 53%; (b)
Ac O, TEA, DMAP, CH CN, 0 °C to r.t., 4 h, 89%; (c) 80% aq. AcOH, r.t., 12 h, 48%; (d) i) NaH, anhyd.
2
3
THF, 0 °C, 1 h and ii) NH SO Cl, anhyd. THF, 0 °C to r.t., 5 h, 77%; (e) acid, DCC, DMAP, anhyd. CH Cl ,
2
2
2
2
r.t., 12 h, 25~45%; (f) 0.02 M NaOMe in MeOH, r.t., 2 h, 30~50%. Route B: (a) TBDPSCl, TEA, DMF,
DMAP, r.t., overnight, 73%; (b) Ac O, pyridine, 60 °C, 8 h, 93%; (c) TBAF, THF, 0 °C, 1 h, 89%; (d)
2
NH SO Cl, NaH, anhyd. THF, 0 °C to r.t., 5 h, 75%; (e) acid, DCC, DMAP, anhyd. CH Cl , r.t., 12 h, 65-
2
2
2
2
6
0
9%; (f) 0.02 M NaOMe in MeOH, r.t., 2 h, 30-65%. Route C: (a) MMTCl, anhyd. pyridine, anhyd. DMF,
°C to r.t., 24 h; (b) Ac O, TEA, DMAP, CH CN, 0 °C to r.t., 4 h; (c) 80% aq. AcOH, r.t., 12 h; (d) i) NaH,
2
3
anhyd. THF, 0 °C, 1 h and ii) NH SO Cl, anhyd. THF, 0 °C to r.t., 5 h; (e) acid, DCC, DMAP, anhyd. CH Cl ,
2
2
2
2
r.t., 12 h; (f) 0.02 M NaOMe in MeOH, r.t., 2 h.
For the syntheses of 3’-deoxy-3’-aminoadenylate derivatives (Scheme 4), the N-benzyl-
’,3’-oxazolone derivative of the key intermediate 3’-amino-3’-deoxyadenosine was prepared from
2
1
8
adenosine in 5 steps according to a previous report. The benzyl group of the intermediate was
1
9
readily cleaved by the mild process of oxidative debenzylation using alkali metal bromide. The
removal of the 5’-TBDPS group followed by sulfamoylation provided the 5’-O-sulfamoyl 2’,3’-
oxazolone intermediate, which was converted to 3’-deoxy-3’-aminoadenylate (12) and its
oxazolone (13) derivatives according to the above method.
8

Scheme 4. Synthesis of 3’-deoxy-3’-aminoadenylate derivatives.
a) KBr, oxone, ACN, 70 °C, 24 h, 63%; (b) TBAF, THF, 0 °C to r.t., overnight, 99%; (c) NH SO Cl, NaH,
(
2
2
anhyd. THF, 0 °C to r.t., 5 h, 82%; (d) N-Boc-Leu, DCC, DMAP, anhyd. THF, r.t., 12 h, 64%; (e) 1 N NaOH,
THF, r.t., 48 h; (f) 1 N HCl in MeOH, r.t., overnight; (g) acid, DCC, DMAP, anhyd. THF:DMF, r.t., 12 h, 44-
5
5%; (h) 0.02 M NaOMe, r.t., 4 h, 87-90%.
2
.2. Biological activity
To assess the biological activity of the synthesized derivatives, we first determined the
inhibition of leucine-induced phosphorylation of S6 Kinase (S6K) in HEK293 cells by performing
1
6
Western blot analysis. In this assay, we pretreated HEK293 cells with each compound at a fixed
concentration (200 µM) together with compound 2 (200 µM), rapamycin (100 nM) and leucinol
(800 µM), followed by the addition of leucine for 10 min to activate mTORC1. We first examined a
group of six compounds (14-19) that contained a modified functional group in each pharmacophoric
region including the base, ring, and side chain of the previously reported compound 2. The newly
introduced or modified moieties are marked in red in Figure 3. Based on the band intensity of the
phosphorylated S6K (pS6K), we found that compounds 16 and 17 showed more potent inhibition of
S6K phosphorylation than did compound 2, while compound 18 demonstrated comparable activity.
Compounds 14, 15, and 19 did not inhibit S6K phosphorylation at the same concentration. When
we further analyzed S6K phosphorylation at four different concentrations (20, 50, 100, and 200 M)
of compounds 16 and 17, both compounds demonstrated dose-dependent inhibition, while the effect
of compound 16 appeared to be more potent than 17. None of the tested compounds affected the
9

amount of the total Akt or phosphorylated Akt (pAkt), indicating that these effects are only specific
toward mTORC1. Overall, it appears that the 2’-deoxyribose group is favorable for inhibition
(compound 16), and the 3’-deoxy side chain and (S)-2-hydroxy-4-methylpentanoyl group do not
adversely affect inhibition (compound 17). The stereochemistry of the leucyl side chain and the 3’-
deoxyribose does not have any significant effect on inhibition, while the 2-iodoadenine (compound
1
8) is likely to be beneficial for its effect.
Figure 3. Inhibition of leucine-induced mTORC1 activation in HEK293 cells treated with
compounds 14-19.
Next, we tested compounds 20-27 for additional SAR analysis, and the results are described
in Figure 4. In this group, compounds 22, 26, and 27 exhibited improved activity compared to
compound 2, while compounds 20 and 25 maintained comparable inhibition. On the other hand,
1
0

compounds 21, 23, and 24 did not show any activity. These results suggest that the modification of
the adenine to a hypoxanthine group improved inhibition (compounds 26-27) while the
modification of ribose to arabinose did not affect activity (compounds 21-22). Incorporation of the
2
-chloro group in the adenine also did not appear to enhance inhibition (23-25).
Figure 4. Inhibition of leucine-induced mTORC1 activation in HEK293 cells treated with
compounds 20-27.
We next focused on diversifying the leucyl side chain with various heterocycles and inosine
analogues. As shown in Figure 5, compounds 29-30 showed potent activity compared to compound
2
or leucinol. Compounds 28 and 32-34 appeared to demonstrate similar inhibitory effect compared
to compound 2, whereas compounds 31 and 35-37 did not inhibit mTORC1. Overall, SAR analysis
of this group of compounds indicated that the addition of a rigid cyclopropyl group at the end of the
leucyl side chain (28, 29, 34, and 37) demonstrated comparable activity, except for compound 37. In
11

addition to the cyclopropyl group in the side chain region, the modification of the 2’,3’-dihydroxyl
group of ribose into oxazolone (34) favorably affected its activity. The replacement of the leucyl
group with a bioisosteric 1-methyl pyrazole surrogate also appeared to help maintain their activity
(32, 33). The replacement of the (S)--hydroxyl group with a chlorine improved mTORC1
inhibition (30); however, when the adenine was replaced with hypoxanthine, the inversion of the
same stereocenter with (R)--hydroxyl (35) or the replacement of the (S)--hydroxyl with a
sulfhydryl group (36) adversely affected inhibition.
Figure 5. Inhibition of leucine-induced mTORC1 activation in HEK293 cells treated with
compounds 28-37.
We also tested another group of compounds (38-46) that contained similarly modified
functional scaffolds as described in Figure 6. In this series, only compounds 39, 41 and 42
1
2

maintained mTORC1 inhibition, while the remaining compounds showed much less activity.
Interestingly, we found that compounds 38-40 only differ by one substituent in the 3-position of
pyrazole; however, only 39 showed inhibitory effect. It should also be noted that compounds 41 and
4
3
2 appeared to show better activity compared to their hypoxanthine-containing counterparts (35 and
6), suggesting that the adenine group is important for activity. The replacement of the (S)-2-
hydroxyl group in compound 42 with an N-methyl amino group (43) appeared to adversely affect its
activity. Neither 3’-deoxy-3’-aminoadenylate (44) nor 2’-deoxyadenylate (45) inhibited S6K
phosphorylation, and 2’-deoxyadenylate with a (2R,3S)-2,3-dihydroxy side chain (46) also showed
no activity.
Figure 6. Inhibition of leucine-induced mTORC1 activation in HEK293 cells treated with
compounds 38-46.
In our final group of compounds, we mostly focused on derivatizing the leucyl side chain
1
3

with a slight modification of the ribose or adenine. As shown in Figure 7, compounds in this series
did not inhibit the phosphorylation of S6K in general. However, some of the compounds tested,
particularly compounds 47-49, appeared to inhibit the phosphorylation of AKT, which is catalyzed
by activated mTORC2 but not mTORC1. Contrary to our observations with compound 16, when the
leucyl side chain of the 2’-deoxyadenylate was modified with a (S)-2-hydroxyl side chain (49) or a
(2S,3R)-2-methoxy-3-hydroxyl side chain (51), compounds 49 and 51 lost their activities, probably
due to their significant structural differences with (R)-3-hydroxyl or the leucine group. In addition,
unlike compound 30, which contained 2-chloroadenylate with a (S)-2-chloro side chain, compound
6
5
2 (adenine without chlorine substituent) and compound 53 (N -methyladenylate) also showed no
activity, indicating that the substituents in the adenine group play a significant role in activity.
Compound 56 also lost activity when its ribose was modified to oxazolone. Although we observed
favorable activity with replacement of adenine with hypoxanthine in compound 26 and favorable
interaction with a cyclopropyl side chain at the end of the leucyl side chain, compound 57
con
tain
ing
bot
h
of
the
se
gro
ups
did
1
4

not show any activity.
Figure 7. Inhibition of leucine-induced mTORC1 activation in HEK293 cells treated with
compounds 47-57.
For overall comparison, we repeated the Western blot experiments with our previously
published compounds 1-3 and newly synthesized compounds 17-18 in one panel as described in
Figure 8. In this blot, it is clearly shown that the substitution of the (S)-2-amine group with a (S)-2-
hydroxyl group improved the activity (compound 17) compared to compound 1, and the additional
modification of the 2-iodoadenine further enhanced activity (compound 3); however, the addition of
a (R)-3-hydroxyl group to the same compounds (compounds 2 and 18) did not appear to improve
activity.
Figure 8. Inhibition of leucine-induced mTORC1 activation in HEK293 cells treated with
compounds 1-3, 17 and 18.
1
5

Based on these SAR analyses, we selected a total of 8 compounds to assess anticancer
2
0
activity by performing sulforhodamine B (SRB) colorimetric assays. The selected compounds
were tested in five different cancer cell lines together with etoposide as a positive control, and the
results are summarized in Table 1. Most of the tested compounds generally showed moderate
anticancer activity, having IC values in the low micromolar range, except for compounds 25-28.
5
0
More importantly, these compounds were active against cancer cells while showing marginal to low
cytotoxicity against normal cells (MRC5). We believe that these results again support our
hypothesis that the selective inhibition of the mTORC1 pathway by LRS-targeting molecules is a
viable strategy for the development of anticancer agents.
Table 1. Antitumoral activities of the selected leucyladenylate sulfamates on the growth of various
a
cancer cell lines.
IC (μM)
A549
5.04
1.00
>20
HCT116 MDA-MB-231 SK-HEP-1 SNU638
MRC5
>20
5
0
1
6
7
5
6
7
8
9
0
6.52
3.47
>20
>20
>20
7.27
9.14
7.51
1.22
6.27
2.61
>20
5.74
1.76
>20
>20
>20
5.86
7.26
5.87
0.41
4.72
4.50
>20
>20
>20
4.77
5.70
5.50
0.34
1
2
2
2
2
2
3
16.65
>20
>20
>20
>20
>20
>20
>20
4.84
6.93
6.27
0.47
16.72
17.78
7.75
3.38
>20
>20
>20
Etoposide
12.72
a
A549, lung cancer cells; HCT116, colon cancer cells; MDA-MB-231, breast cancer cells; SK-Hep-1, liver
cancer cells; SNU638, stomach cancer cells; MRC5, normal lung epithelial cells.
3
. Conclusions
In this work, we investigated the structure-activity relationship of a series of
leucyladenylate sulfamate derivatives as LRS-targeted mTORC1 inhibitors. We modified each of
1
6

three pharmacophoric regions in the leucyladenylate sulfamate scaffold consisting of adenine,
ribose and a leucyl side chain, and evaluated their inhibitory effects by cell-based assays. Our SAR
analyses indicated that the structural modifications in any one of these regions, adenine, ribose and
leucine, can affect the biological activity significantly likely due to the small binding pocket in the
LRS. In particular, the modification of the leucyl side chain region appears to be most critical,
although this is not surprising given that these compounds inhibit mTORC1 by affecting leucine-
sensing of LRS. When we further tested several selected compounds in a panel of cancer cell lines,
compounds 16, 17, 28, 29 and 30 were found to be effective at low micromolar IC concentrations
5
0
without significant cytotoxicity toward normal cells. We believe that the leucyladenylate sulfamate
analogues possess highly specific activity toward the LRS-mediated mTORC1 inhibition without
significantly affecting the total expression of mTOR complex or LRS, which is an advantageous
and promising feature for potential anticancer therapy. Currently, we are optimizing the
pharmacophore via ligand-based modeling and cell-based assays to generate LRS-targeting
mTORC1 inhibitors with favorable pharmacokinetic properties for future in vivo applications.
4
4
. Experimental
.1. General Experimental
All chemical reagents were commercially available. Melting points were determined on a Büchi
Melting Point B-540 apparatus and are uncorrected. Silica gel column chromatography was
1
performed on silica gel 60, 230-400 mesh, Merck. H NMR spectra were recorded on a JEOL JNM-
LA 300 at 300MHz, Bruker Avance DE/AVANCE Digital 500 at 500 MHz and a JEOL JNM-ECA-
6
00 at 600MHz. Chemical shifts are reported in ppm units with Me Si as a reference standard. Mass
4
spectra were recorded on a VG Trio-2 GC-MS and 6460 Triple Quad LC/MS.
1
7

4
.1.1. Sulfamoylation
o
To a solution of starting material (1 eq) in anhydrous THF was added NaH (3 eq) slowly at 0 C.
After stirring for 30 min, freshly prepared sulfamoyl chloride (3 eq) was added. The reaction
mixture was gradually warmed to room temperature and stirred for several hours. After the starting
material disappeared, the reaction was quenched with MeOH and then, the solvent was evaporated.
The crude residue was partitioned between EtOAc and H O and the organic layer was dried over
2
MgSO and concentrated under reduced pressure. The residue was purified by column
4
chromatography over silica gel (EtOAc:MeOH=10:1) to give the sulfamoylated product.
4
.1.2. Amide coupling
To a solution of 5’-O-sulfamoylated intermediate (1 eq) in anhydrous CH Cl was added the
2
2
corresponding acid (1.5 eq) and DMAP (0.05 eq) at room temperature. 1M DCC in CH Cl (1.5 eq)
2
2
was added dropwise and the reaction mixture was stirred for 12 h at room temperature. The reaction
mixture was filtered through celite pad, washed with EtOAc and concentrated under reduced
pressure. The residue was purified by column chromatography over silica gel (EtOAc:MeOH=10:1)
to give the desired compound.
4
.1.3. Acetyl deprotection
The starting material was dissolved in 0.02 M sodium methoxide solution in MeOH and stirred for
h at room temperature. After the reaction was completed, DOWEX 50WX8 hydrogen form resin
2
was added in portions until pH of the reaction mixture reached ca. 6~7. The reaction mixture was
filtered and concentrated under reduced pressure to give the pure final compound.
4
4
.2. Chemical spectra
.2.1. ((2R,3S,4R,5R)-3,4-Dihydroxy-5-(6-(methylamino)-9H-purin-9-yl)tetrahydrofuran-2-
1
8

yl)methyl ((2R,3S)-2,3-dihydroxy-4-methylpentanoyl)sulfamate (14)
1
Yield = 56%, white solid; H NMR (300 MHz, CDCl ) δ 8.49 (s, 1H), 8.24 (s, 1H), 6.07 (d, J = 5.50
3
Hz, 1H), 4.61(t, J = 5.12 Hz, 1H), 4.39-4.26 (m, 3H), 4.08 (s, 1H), 3.49 (d, J = 8.21 Hz, 1H), 3.10
(m, 2H), 1.00 (d, J = 6.62 Hz, 3H), 0.95 (d, J = 6.62 Hz, 3H); HRMS (ESI) m/z calcd for
+
C H N O S [M + H] 490.1482, found: 491.1563.
17
26
6
9
4
.2.2. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
((2R,3S)-2,3-dihydroxy-4-methylpentanoyl)sulfamate (15)
1
Yield = 61%, white solid; H-NMR (300 MHz, CD OD) δ 8.52 (s, 1H), 8.18 (s, 1H), 6.07 (d, J =
3
5
1
.51 Hz, 1H), 4.60 (t, J = 5.32 Hz, 1H), 4.38 (t, J = 5.0 Hz, 1H), 4.32 (m, 2H), 4.28 (m, 2H), 4.13 (s,
H), 3.48 (d, J = 9.21 Hz, 1H), 0.99 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.42, 3H); HRMS (ESI) m/z
+
calcd for C H N O S [M + H] 477.1325, found: 277.1380.
1
6
24
6
9
4
.2.3.
((2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yl)methyl
((2S,3R)-2,3-dihydroxy-4-methylpentanoyl)sulfamate (16)
1
Yield = 63%, white solid; H NMR (500 MHz, CD OD) δ 8.49 (s, 1H), 8.17 (s, 1H), 6.49 (t, J =
3
6
3
1
.80 Hz, 1H), 4.63 (t, J = 2.75 Hz, 1H), 4.30-4.24 (m, 2H), 4.19 (q, J = 2.80 Hz, 1H), 4.10 (s, 1H),
.48 (d, J = 11.60 Hz, 1H), 2.76 (dd, J = 6.30, 0.75 Hz, 1H), 2.47 (dd, J = 6.10, 3.05 Hz, 1H), 1.90-
.83 (m, 1H), 1.01 (d, J = 6.60 Hz, 3H), 0.94 (d, J = 6.75 Hz, 3H); HRMS (ESI) m/z calcd for
+
C H N O S [M + H] 461.1454, found: 461.1443.
16
24
6
8
4
.2.4. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
((S)-2-hydroxy-4-methylpentanoyl)sulfamate (17)
1
Yield = 88%, white solid; H NMR (500 MHz, CD OD) δ 8.50 (s, 1H), 8.19 (s, 1H), 6.08 (d, J =
3
5
.35 Hz, 1H), 4.64 (t, J = 7.90 Hz, 1H), 4.38 (t, J = 3.80 Hz, 1H), 4.35-4.25 (m, 3H), 4.03 (dd, J =
1
9

9
.15, 3.25 Hz, 1H), 1.65-1.43 (m, 3H), 0.91 (t, J = 6.75 Hz, 6H); HRMS (ESI) m/z calcd for
+
C H N O S [M + H] 461.1454, found: 461.1445.
16
24
6
8
4
.2.5.
((2R,3S,4R,5R)-5-(6-Amino-2-iodo-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-
yl)methyl ((2S,3R)-2,3-dihydroxy-4-methylpentanoyl)sulfamate (18)
1
Yield = 72%, white solid; H NMR (300 MHz, CD OD) δ 8.41 (s, 1H), 6.01 (d, J = 5.35 Hz, 1H),
3
4
1
.56 (t, J = 5.12 Hz, 1H), 4.39-4.36 (m, 3H), 4.07 (m, 1H), 3.50 (d, J = 9.00 Hz, 1H), 1.87 (m, 1H),
.00 (d, J = 6.75 Hz, 3H), 0.94 (d, J = 6.75 Hz, 3H); HRMS (ESI) m/z calcd for C H IN O S [M +
1
6
23
6
9
+
H] 603.0292, found: 603.0281.
4
.2.6.
((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methyl
((2S,3R)-2,3-dihydroxy-4-methylpentanoyl)sulfamate (19)
1
Yield = 52%, white solid; H NMR (500 MHz, CD OD) δ 8.46 (s, 1H), 8.18 (s, 1H), 5.99 (d, J =
3
1
.55 Hz, 1H), 4.70-4.66 (m, 1H), 4.65-4.64 (m, 1H), 4.43 (dd, J = 11.25, 2.35 Hz, 1H), 4.26 (dd, J =
1.25, 3.25 Hz, 1H), 4.11 (s, 1H), 3.51 (d, J = 8.10 Hz, 1H), 2.40 (dd, J = 9.00, 5.65 Hz, 1H), 2.11
dd, J = 7.15, 4.45 Hz, H ), 1.00 (d, J = 6.65 Hz, 3H), 0.94 (d, J = 6.75 Hz, 3H); HRMS (ESI) m/z
1
(
3
”
+
calcd for C H N O S [M + H] 461.1454, found: 461.1452.
1
6
24
6
8
4
.2.7. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
((E)-4-methylpent-2-enoyl)sulfamate (20)
1
Yield = 90%, white solid; H NMR (300 MHz, CD OD) δ 8.36 (s, 1H), 8.20 (s, 1H), 6.91(dd, J =
3
1
1
5.4 Hz, 6.4 Hz, 1H), 6.04 (m, 1H), 5.81 (d, J = 15.4 Hz, 1H), 4.65 (m, 1H), 4.51 (m, 2H), 4.37 (m,
H), 4.29 (m, 1H), 2.43 (m, 1H), 1.04 (d, J = 2.02 Hz, 3H), 1.01(d, J = 2.02 Hz, 3H); HRMS (ESI)
+
m/z calcd for C H N O S [M + H] 443.1271, found: 443.1342.
1
6
22
6
7
2
0

4
.2.8. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
((2S,3R)-2,3-dihydroxy-4-methylpentanoyl)sulfamate (21)
1
Yield = 77%, white solid; H NMR (300 MHz, CD OD) δ 8.04 (s, 1H), 7.83 (s, 1H), 6.07 (d, J =
3
4
.45 Hz, 1H), 4.08-3.92 (m, 4H), 3.79 (m, 1H), 3.70 (d, J = 2.00 Hz, 1H), 2.40 (dd, J = 9.00, 5.65
Hz, 1H), 2.11 (dd, J = 7.15, 4.45 Hz, 1H), 1.00 (d, J = 6.65 Hz, 3H), 0.94 (d, J = 6.75 Hz, 3H);
+
HRMS (ESI) m/z calcd for C H N O S [M + H] 477.1325, found: 477.1311.
1
6
24
6
9
4
.2.9. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
((S)-2-hydroxy-4-methylpentanoyl)sulfamate (22)
1
Yield = 87%, white solid; H NMR (300 MHz, CD OD) δ 8.04 (s, 1H), 7.83 (s, 1H), 6.07 (d, J =
3
4
1
4
.45 Hz, 1H), 4.48 (m, 1H), 4.43 (m, 2H), 4.36 (t 1H), 4.28 (q, 1H), 4.02 (dd, 1H), 1.81 (m, 1H),
,
+
.48 (m, 2H), 0.92 (s, 3H), 0.90 (s, 3H); HRMS (ESI) m/z calcd for C H N O S [M + H]
1
6
24
6
8
61.1376, found: 461.1365.
4
.2.10. ((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-
yl)methyl ((S)-2-hydroxy-4-methylpentanoyl)sulfamate (23)
1
Yield = 68%, white solid; H NMR (500 MHz, CD OD) δ 8.37 (s, 1H), 5.99 (d, J = 5.35 Hz, 1H),
3
4
1
.58 (t, J = 3.80 Hz, 1H), 4.43 (m, 2H), 4.36 (m, 1H), 4.28 (q, 1H), 4.02 (dd, J = 9.15, 3.25 Hz, 1H),
.81 (m, 1H), 1.48 (m, 2H), 0.92 (s, 3H), 0.90 (s, 3H); HRMS (ESI) m/z calcd for C H ClN O S
1
6
23
6
8
+
[
M + H] 495.0987, found: 495.1068.
4
.2.11. ((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-
yl)methyl ((2S,3R)-3-hydroxy-2-methoxy-4-methylpentanoyl)sulfamate (24)
1
Yield = 99%, white solid; H NMR (500 MHz, CD OD) δ 8.48 (s, 1H), 6.01 (d, J = 5.35 Hz, 1H),
3
4
.59 (t, J = 5.10 Hz, 1H), 4.39 (t, J = 3.90 Hz, 1H), 4.32 (m, 2H), 4.28 (m, 1H), 3.66 (d, J = 3.55 Hz,
2
1

1
H), 3.47 (m, 1H), 3.36 (s, 3H), 1.85 (m, 1H), 0.97 (d, J = 6.65 Hz, 3H), 0.90 (d, J = 6.75 Hz, 3H);
+
HRMS (ESI) m/z calcd for C H ClN O S [M + H] 525.1165, found: 525.1147.
1
7
25
6
9
4
.2.12. ((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-
yl)methyl ((2S,3R)-2,3-dimethoxy-4-methylpentanoyl)sulfamate (25)
1
Yield = 43%, white solid; H NMR (500 MHz, CD OD) δ 8.49 (s, 1H), 6.01 (d, 1H), 4.61 (t, 1H),
3
4
1
5
.38 (m, 1H), 4.34 (m, 2H), 4.28 (m, 1H), 3.67 (d, 1H), 3.40 (s, 3H), 3.33 (s, 3H), 3.21 (m, 1H),
+
.91 (m, 1H), 0.97 (d, 3H), 0.89 (s, 3H); HRMS (ESI) m/z calcd for C H ClN O S [M + H]
1
8
27
6
9
39.1322, found 539.1287.
4
.2.13. ((2R,3S,4R,5R)-3,4-Dihydroxy-5-(6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-
yl)methyl ((2S,3R)-2,3-dihydroxy-4-methylpentanoyl)sulfamate (26)
1
Yield = 95%, white solid; H NMR (300 MHz, CD OD) δ 8.33 (s, 1H), 7.95 (s, 1H), 5.95 (d, J =
3
5
.31 Hz, 1H), 4.51 (t, J = 5.31 Hz, 1H), 4.29 (t, J = 3.48 Hz, 1H), 4.18 (m, 2H), 4.28 (m, 1H), 3.97
(s, 1H), 3.40 (d, J = 8.79 Hz, 1H), 1.85 (m, 1H), 0.92 (d, J = 6.57 Hz, 3H), 0.85 (d, J = 6.78 Hz, 3H);
+
HRMS (ESI) m/z calcd for C H N O S [M + H] 478.1238, found 478.1230.
1
6
23
5
9
4
.2.14. ((2R,3S,4R,5R)-3,4-Dihydroxy-5-(6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-
yl)methyl ((S)-2-hydroxy-4-methylpentanoyl)sulfamate (27)
1
Yield = 92%, white solid; H NMR (300 MHz, CD OD) δ 8.44 (s, 1H), 8.05 (s, 1H), 6.06 (d, J =
3
5
1
+
.67 Hz, 1H), 4.65 (t, J = 5.31 Hz, 1H), 4.37 (m, 1H), 4.28 (m, 4H), 3.98 (dd, J = 3.66 Hz, 1H),
.82 (m, 1H), 1.54 (m, 2H), 0.91 (t, J = 6.42 Hz, 6H); HRMS (ESI) m/z calcd for C H N O S [M
1
6
23
5
9
+
H] 462.1289, found 462.1266.
4
.2.15.
((2R,3S,4R,5R)-5-(6-Amino-2-iodo-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-
2
2

yl)methyl ((2S,3R)-3-cyclopropyl-2,3-dihydroxypropanoyl)sulfamate (28)
1
Yield = 75%, white solid; H NMR (300 MHz, CD OD) δ 8.06 (s, 1H), 5.65 (d, J = 5.50 Hz, 1H),
3
4
0
6
.21 (t, J = 5.10 Hz, 1H), 4.02-3.91 (m, 4H), 3.66 (d, J = 2.21 Hz, 1H), 2.77 (d, J = 9.11 Hz, 1H),
+
.82 (m, 1H), 0.91 (m, 4H); HRMS (ESI) m/z calcd for C H IN O S [M + H] 601.0292, found
16
21
6
9
01.0147.
4
.2.16. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
((2S,3R)-3-cyclopropyl-2,3-dihydroxypropanoyl)sulfamate (29)
1
Yield = 65 %, white solid; H NMR (300 MHz, CD OD) δ 8.04 (s, 1H), 7.83 (s, 1H), 6.07 (d, J =
3
4
1
.42 Hz, 1H), 4.08-3.92 (m, 4H), 3.79 (m, 1H), 3.70 (d, J = 2.01 Hz, 1H), 2.78 (m, 1H), 0.84 (m,
+
H), 0.20 (m, 4H); HRMS (ESI) m/z calcd for C H IN O S [M + H] 601.0135, found 601.0121.
16
21
6
9
4
.2.17. ((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-
yl)methyl ((S)-2-chloro-4-methylpentanoyl)sulfamate (30)
1
Yield = 50%, white solid; H NMR (500 MHz, CD OD) δ 8.46 (s, 1H), 6.01 (d, J = 5.95 Hz, 1H),
3
4
6
.62 (t, J = 5.25 Hz, 1H), 4.36 (dd, J = 4.7, 3.15 Hz), 4.30 (m, 2H), 4.28 (m, 1H), 4.22 (dd, J = 8.3,
.05 Hz), 1.78 (m, 2H), 1.71 (m, 1H), 0.91 (dd, J = 23.55, 6.3 Hz); HRMS (ESI) m/z calcd for
+
C H Cl N O S [M + H] 513.0804, found 513.0705.
16
22
2
6
7
4
.2.18.
((2R,3S,4R,5R)-5-(6-Amino-2-iodo-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-
yl)methyl (3-isopropyl-1H-pyrazole-5-carbonyl)sulfamate (31)
1
Yield = 69%, white solid; H NMR (500 MHz, CD OD) δ 8.49 (s, 1H), 6.55 (s, 1H), 6.01 (d, 1H),
3
4
.61 (t, 1H), 4.38 (m, 1H), 4.32 (m, 2H), 4.18 (m, 1H), 3.67 (d, 1H), 3.40 (m, 4H), 0.89 (m, 6H);
+
HRMS (ESI) m/z calcd for C H IN O S [M + H] 609.0455, found 609.0435.
1
7
21
8
7
2
3

4
.2.19. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
(3-isopropyl-1-methyl-1H-pyrazole-5-carbonyl)sulfamate (32)
1
Yield = 69%, white solid; H NMR (500 MHz, CD OD) δ 8.49 (s, 1H), 6.01 (d, 1H), 4.61 (t, 1H),
3
4
1
4
.38 (m, 1H), 4.34 (m, 1H), 4.28 (m, 1H), 3.67 (d, 1H), 3.40 (s, 2H), 3.33 (s, 3H), 3.21 (m, 1H),
+
.91 (m, 1H), 0.89 (s, 6H); HRMS (ESI) m/z calcd for C H N O S [M + H] 497.1322, found
1
8
24
8
7
97.1287.
4
.2.20.
((2R,3S,4R,5R)-5-(6-Amino-2-iodo-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-
yl)methyl (3-isopropyl-1-methyl-1H-pyrazole-5-carbonyl)sulfamate (33)
1
Yield = 72%, white solid; H NMR (500 MHz, CD OD) δ 8.49 (s, 1H), 6.01 (d, 1H), 4.38 (m, 1H),
3
4
0
6
.34 (m, 2H), 4.28 (m, 1H), 3.67 (d, 1H), 3.40 (s, 3H), 3.33 (s, 3H), 3.21 (m, 1H), 1.91 (m, 1H),
+
.97 (d, 3H), 0.89 (s, 3H); HRMS (ESI) m/z calcd for C H IN O S [M + H] 623.0455, found
1
8
23
8
7
23.0441.
4
.2.21.
((3aR,4S,6R,6aR)-6-(6-Amino-9H-purin-9-yl)-2-iminohexahydrofuro[3,4-d]oxazol-4-
yl)methyl ((2S,3R)-3-cyclopropyl-2,3-dihydroxypropanoyl)sulfamate (34)
1
Yield = 68%, white solid; H NMR (300 MHz, CD OD) δ 8.68 (s, 1H), 8.58 (s, 1H), 6.34 (s, 1H),
3
4
1
.48 (t, J = 3.50 Hz, 1H), 4.43 (d, J = 2.32 Hz, 1H), 4.26 (m, 2H), 4.20 (dd, J = 11.15, 3.75 Hz,
H), 4.00 (m, 1H), 3.09 (d, J = 8.80 Hz, 1H), 2.71 (dd, J = 8.97, 2.55 Hz, 1H), 1.14 (m, 1H), 0.57
(
m, 1H), 0.49 (m, 1H), 0.38 (m, 1H), 0.30 (m, 1H); HRMS (ESI) m/z calcd for C H N O S [M +
17 21 7 9
+
H] 500.1278, found 500.1266.
4
.2.22. ((2R,3S,4R,5R)-3,4-Dihydroxy-5-(6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-
yl)methyl ((R)-2-hydroxy-4-methylpentanoyl)sulfamate (35)
1
Yield = 81%, white solid; H NMR (500 MHz, CD OD) δ 8.31 (s, 1H), 8.05 (s, 1H), 6.27 (s, 1H),
3
2
4

4
5
.80 (s, 1H), 4.71 (t, J = 1.61 Hz, 1H), 4.56 (dd, J = 13.90, 2.35 Hz, 1H), 4.36 (m, 2H), 4.05 (d, J =
.45 Hz, 1H), 1.85 (m, 1H), 1.52 (m, 2H), 0.93 (dd, J = 11.95, 5.65 Hz, 6H); HRMS (ESI) m/z
+
calcd for C H N O S [M + H] 462.1373, found 462.1305.
1
6
23
5
9
4
.2.23. ((2R,3S,4R,5R)-3,4-Dihydroxy-5-(6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-
yl)methyl ((R)-2-mercapto-4-methylpentanoyl)sulfamate (36)
1
Yield = 99%, white solid; H NMR (300 MHz, CD OD) δ 8.36 (s, 1H), 8.06 (s, 1H), 6.06 (s, 1H),
3
4
1
4
.43 (m, 1H), 4.40 (m, 1H), 4.33 (m, 2H), 4.29 (m, 1H), 3.63 (m, 1H), 1.78 (m, 1H), 1.68 (m, 1H),
+
.49 (m, 1H), 0.90 (m, 6H); HRMS (ESI) m/z calcd for C H N O S [M + H] 478.0988, found
16
23
5
8 2
78.1056.
4
.2.24. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
((2S,3R)-3-cyclopropyl-2,3-dihydroxypropanoyl)sulfamate (37)
1
Yield = 82%, white solid; H NMR (500 MHz, CD OD) δ 8.51 (s, 1H), 8.18 (s, 1H), 6.08 (d, J =
3
5
.60 Hz, 1H), 4.64 (t, J = 5.30 Hz, 1H), 4.38 (dd, J = 8.20, 3.50 Hz, 1H), 4.35-4.27 (m, 3H), 4.00 (d,
J = 2.10 Hz, 1H), 3.11 (dd, J = 9.10, 2.00 Hz, 1H), 1.17-1.11 (m, 1H), 0.56-0.29 (m, 4H); HRMS
+
(
ESI) m/z calcd for C H N O S [M + H] 475.1247, found: 475.1245.
1
6
22
5
9
4
.2.25. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
(3-isopropyl-1H-pyrazole-5-carbonyl)sulfamate (38)
1
Yield = 72%, white solid; H NMR (300 MHz, CD OD) δ 8.51 (s, 1H), 8.18 (s, 1H), 6.55 (s, 1H),
3
6
1
.08 (d, J = 5.60 Hz, 1H), 4.64 (t, J = 5.30 Hz, 1H), 4.38 (dd, J = 8.20, 3.50 Hz, 1H), 4.35-4.27 (m,
H), 4.00 (d, J = 2.10 Hz, 1H), 3.11 (dd, J = 9.10, 2.00 Hz, 1H),0.89 (m, 6H); HRMS (ESI) m/z
+
calcd for C H N O S [M + H] 483.1332, found: 483.1320.
1
7
22
8
7
2
5

4
.2.26. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
(3-(tert-butyl)-1H-pyrazole-5-carbonyl)sulfamate (39)
1
Yield = 72%, white solid; H NMR (300 MHz, CD OD) δ 8.50 (s, 1H), 8.17 (s, 1H), 6.54 (s, 1H),
3
6
1
4
.08 (d, J = 5.60 Hz, 1H), 4.64 (t, J = 5.30 Hz, 1H), 4.38 (dd, J = 8.20, 3.50 Hz, 1H), 4.35-4.27 (m,
+
H), 3.99 (m, 1H), 3.11 (m, 1H),0.89 (m, 9H); HRMS (ESI) m/z calcd for C H N O S [M + H]
1
8
24
8
7
97.1489, found: 487.1478.
4
.2.27. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
(3-cyclopropyl-1H-pyrazole-5-carbonyl)sulfamate (40)
1
Yield = 65%, white solid; H NMR (300 MHz, CD OD) δ 8.50 (s, 1H), 8.17 (s, 1H), 6.54 (s, 1H),
3
6
2
.08 (d, J = 5.60 Hz, 1H), 4.64 (t, J = 5.30 Hz, 1H), 4.38 (dd, J = 8.20, 3.50 Hz, 1H), 4.35-4.27 (m,
H), 3.99 (m, 1H), 3.11 (m, 1H), 0.56-0.29 (m, 4H); HRMS (ESI) m/z calcd for C H N O S [M +
1
7
20
8
7
+
H] 481.1176, found: 481.1165.
4
.2.28. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
((R)-2-hydroxy-4-methylpentanoyl)sulfamate (41)
1
Yield = 72%, white solid; H NMR (300 MHz, CD OD) δ 8.56 (s, 1H) 8.31 (s, 1H), 6.37 (s, 1H),
3
5
1
+
.02 (d, J = 13.6, 1H), 4.75 (m, 2H), 4.38 (t, J = 4.8, 1H), 4.11 (d, J = 5.41 Hz, 1H), 4.01 (m, 1H),
.84 (m, 1H), 1.51 (m, 1H), 0.90 (t, J = 5.60 Hz, 6H); HRMS (ESI) m/z calcd for C H N O S [M
1
6
24
6
8
+
H] 461.1533, found 461.1520.
4
.2.29. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
((S)-2-mercapto-4-methylpentanoyl)sulfamate (42)
1
Yield = 77%, white solid; H-NMR (300 MHz, CD OD) δ 8.44 (s, 1H), 8.18 (s, 1H), 6.09 (d, 1H),
3
4
.67 (m, 1H), 4.49 (s, 1H), 4.49 (s, 1H), 4.41 (s, 2H), 4.33 (m, 3H), 3.63 (s, 1H), 0.88-0.94 (m, 6H);
2
6

+
HRMS (ESI) m/z calcd for C H N O S [M + H] 477.1221, found 477.1209.
1
6
26
6
7 2
4
.2.30. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
(
methyl-L-leucyl)sulfamate (43)
1
Yield = 99%, white solid; H NMR (500 MHz, CD OD) δ 8.51 (s, 1H, Ar, 1H), 8.19 (s, 1H, Ar, 1H),
3
6
1
1
.08 (d, J = 5.70, 1H), 4.65 (t, J = 5.32 Hz, 1H), 4.39 (t, J = 5.30 Hz, 1H), 4.35 (d, J = 3.75 Hz,m
H), 4.33 (d, J = 3.75 Hz, 1H), 4.28 (m, 2H), 3.12 (t, J = 7.71 Hz, 1H), 2.39 (s, 3H), 1.73 (m, 1H),
.56 (m, 1H), 1.38 (m, 1H), 0.93 (d, J = 6.50 Hz, 3H), 0.86 (d, J = 6.60 Hz, 3H); HRMS (ESI) m/z
+
calcd for C H N O S [M + H] 474.1349, found 474.1289.
1
7
27
7
7
4
.2.31.
((2S,3S,4R,5R)-3-Amino-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-
yl)methyl (L-leucyl)sulfamate (44)
1
Yield = 78%, white solid; H-NMR (300 MHz, CD OD) δ 8.64 (s, 1H), 8.59 (s, 1H), 6.35 (s, 1H),
3
4
.50 (t, J = 3.51 Hz, 1H), 4.45 (d, J = 2.62 Hz, 1H), 4.27 (d, J = 2.40 Hz, 1H), 4.22 (m, 2H), 3.39
(m, 1H), 1.73 (m, 1H), 1.62 (m, 1H), 1.41 (m, 1H), 0.92 (m, 6H); HRMS (ESI) m/z calcd for
+
C H N O S [M + H] 485.1645, found 485.1633.
17
24
8
7
4
.2.32. ((2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yl)methyl (L-
leucyl)sulfamate (45)
Yield = 60%, white solid; H-NMR (300 MHz, D O) δ 7.75 (s, 1H), 7.69 (s, 1H), 3.46 (m, 1H), 3.15
1
2
(
s, 2H), 3.05 (s, 2H), 1.06 (m, 6H), 0.27 (m, 6H); HRMS (ESI) m/z calcd for C H N O S [M +
16 25 7 6
+
H] 444.1645, found 444.1633.
4
.2.33.
((2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yl)methyl
((2R,3S)-2,3-dihydroxy-4-methylpentanoyl)sulfamate (46)
2
7

1
Yield = 71%, white solid; H NMR (500 MHz, CD OD) δ 8.51 (s, 1H), 8.18 (s, 1H), 6.49 (t, J =
3
6
.85 Hz, 1H), 4.63-4.62 (m, 1H), 4.30-4.24 (m, 2H), 4.19 (d, J = 3.70 Hz, 1H), 4.06 (m, 1H), 3.5
(dd, J = 8.85, 1.25 Hz, 1H), 2.75 (qd, J = 7.25, 3.60 Hz, 1H), 2.46 (qd, J = 6.05, 3.10 Hz, 1H), 1.88-
1
.81 (m, H), 0.99 (d, J = 6.70 Hz, 3H), 0.94 (d, J = 6.70 Hz, 3H); HRMS (ESI) m/z calcd for
+
C H N O S [M + H] 461.1454, found: 461.1408.
16
24
6
8
4
.2.34. ((2R,3S,4R,5R)-3,4-Dihydroxy-5-(6-(methylamino)-9H-purin-9-yl)tetrahydrofuran-2-
yl)methyl ((S)-2-methoxy-4-methylpentanoyl)sulfamate (47)
1
Yield = 80%, white solid; H NMR (300 MHz, CD OD) δ 8.45 (s, 1H), 8.18 (s, 1H), 5.99 (d, J =
3
1
4
1
.83 Hz, 1H), 4.71 (m, 2H), 4.42 (dd, J = 11.34, 2.55 Hz, 1H), 4.23 (dd, J = 11.19, 3.30 Hz, 1H),
.08 (dd, J = 9.15, 3.66 Hz, 1H), 2.40 (d, J = 2.49 Hz, 1H), 2.11 (qd, J = 6.03, 2.88 Hz, 1H), 1.82-
.80 (m, 1H), 0.91 (d, J = 2.58 Hz, 3H), 0.89 (d, J = 2.73 Hz, 3H); HRMS (ESI) m/z calcd for
+
C H N O S [M + H] 475.1689, found: 475.1672.
17
26
6
8
4
.2.35. ((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methyl ((S)-2-
hydroxy-4-methylpentanoyl)sulfamate (48)
1
Yield = 83%, white solid; H NMR (300 MHz, CD OD) δ 8.45 (s, 1H), 8.18 (s, 1H), 5.99 (d, J =
3
1
1
1
.83 Hz, 1H), 4.71-4.65 (m, 2H), 4.44 (dd, J = 11.34, 2.55 Hz, 1H), 4.27 (dd, J = 11.19, 3.30 Hz,
H), 4.08 (dd, J = 9.15, 3.66 Hz, 1H), 2.41 (qd, J = 8.97, 2.49 Hz, 1H), 2.11 (qd, J = 6.03, 2.88 Hz,
H), 1.82-1.80 (m, 1H), 1.60-1.42 (m, 2H), 0.91 (d, J = 2.58 Hz, 3H), 0.89 (d, J = 2.73 Hz, 3H);
+
HRMS (ESI) m/z calcd for C H N O S [M + H] 445.1584, found: 445.1571.
1
6
24
6
7
4
.2.36. ((2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yl)methyl ((S)-2-
hydroxy-4-methylpentanoyl)sulfamate (49)
1
Yield = 76%, white solid; H NMR (600 MHz, CD OD) δ 8.47 (s, 1H), 8.18 (s, 1H), 6.49 (dd, J =
3
2
8

7
.32, 5.94 Hz, 1H), 4.62 (q, J = 2.76 Hz, 1H), 4.28-4.23 (m, 2H), 4.20 (q, J = 3.66 Hz, 1H), 3.99
(dd, J = 9.60, 3.18 Hz, 1H), 2.77 (dd, J = 7.80, 6.00 Hz, 1H), 2.46 (dd, J = 5.94, 0.60 Hz, 1H), 1.86-
1
.80 (m, 1H), 1.57 (dd, J = 9.18, 3.66 Hz, 1H), 1.45 (dd, J = 9.60, 5.04 Hz, 1H), 0.92 (d, J = 6.36
+
Hz, 3H), 0.90 (d, J = 6.90 Hz, 3H); HRMS (ESI) m/z calcd for C H N O S [M + H] 445.1505,
1
6
24
6
7
found: 445.1473.
4
.2.37. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
((2S,3R)-3-hydroxy-2-methoxy-4-methylpentanoyl)sulfamate (50)
1
Yield = 79%, white solid; H NMR (300 MHz, CD OD) δ 8.46 (s, 1H), 8.19 (s, 1H), 6.08 (d, J =
3
5
1
.31 Hz, 1H), 4.66 (t, J = 5.49 Hz, 1H), 4.40-4.28 (m, 4H), 3.70 (d, J = 3.48 Hz, 1H), 3.42-3.41 (m,
H), 3.33 (s, 3H), 1.88-1.81 (m, 1H), 0.98 (d, J = 6.78 Hz, 3H), 0.91 (d, J = 6.78 Hz, 3H); HRMS
+
(
ESI) m/z calcd for C H N O S [M + H] 491.1638, found: 491.1622.
1
7
26
6
9
4
.2.38.
((2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yl)methyl
((2S,3R)-3-hydroxy-2-methoxy-4-methylpentanoyl)sulfamate (51)
1
Yield = 68%, white solid; H-NMR (300 MHz, CD OD) δ 8.55 (s, 1H), 8.22 (s, 1H), 6.66 (t, J =
3
6
.24 Hz, 1H), 6.50 (s, 1H), 4.56-4.68 (m, 4H), 4.20-4.29 (m, 3H), 3.43 (s, 3H), 2.43 (m, 1H) 2.03
+
(
m, 1H), 0.95(m, 6H); HRMS (ESI) m/z calcd for C H N O S [M + H] 475.1689, found:
1
7
26
6
8
4
75.1678.
4
.2.39. ((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl
((S)-2-chloro-4-methylpentanoyl)sulfamate (52)
1
Yield = 70%, white solid; H-NMR (300 MHz, CD OD) δ 8.51 (s, 1H), 8,20 (s, 1H), 6.09 (d, J =
3
6
.21 Hz, 1H), 4.69 (t, J = 5.82 Hz, 1H), 4.38-4.20 (m, 5H), 1.78 (m, 3H), 0.96-0.87 (m, 6H); HRMS
+
(
ESI) m/z calcd for C H ClN O S [M + H] 479.1134, found: 479.1123.
1
6
23
6
7
2
9