Modifications of flexible nonyl chain and nucleobase head group of (+)-erythro-9-(2′s-hydroxy-3′s-nonyl)adenine [(+)-EHNA] as adenosine deaminase inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.09.015

A series of terminal nonyl chain and nucleobase modified analogues of (+)-EHNA (III) were synthesized and evaluated for their ability to inhibit adenosine deaminase (ADA). The constrained carbon analogues of (+)-EHNA, 7a-7h, 10a-c, 12, 13, 14 and 17a-c appeared very potent with Ki values in the low nanomolar range. Thio-analogues of (+)-EHNA 24a-e wherein 5′C of nonyl chain replaced by sulfur atom found to be less potent compared to (+)-EHNA. Docking of the representative compounds into the active site of ADA was performed to understand structure-activity relationships. Compounds 7a (Ki: 1.1 nM) 7b (Ki: 5.2 nM) and 26a (Ki: 5.9 nM) showed suitable balance of potency, microsomal stability and demonstrated better pharmacokinetic properties as compared to (+)-EHNA and therefore may have therapeutic potential for various inflammatory diseases, hypertension and cancer.

Full text of DOI:10.1016/j.bmc.2017.09.015

Accepted Manuscript
Modifications of flexible nonyl chain and nucleobase head group of (+)-eryth-
ro-9-(2’s-hydroxy-3’s-nonyl)adenine [(+)-EHNA] as adenosine deaminase in-
hibitors
Sachin R. Kandalkar, Parimi Atchuta Ramaiah, Manoj Joshi, Atul Wavhal,
Yogesh Waman, Amol Raje, Ashwini Tambe, Shariq Ansari, Siddhartha De,
Venkata P. Palle, Kasim A. Mookhtiar, Anil M. Deshpande, DineshA. Barawkar
PII:
S0968-0896(17)30836-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.09.015
BMC 13975
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
24 April 2017
7 September 2017
11 September 2017
Please cite this article as: Kandalkar, S.R., Ramaiah, P.A., Joshi, M., Wavhal, A., Waman, Y., Raje, A., Tambe, A.,
Ansari, S., De, S., Palle, V.P., Mookhtiar, K.A., Deshpande, A.M., Barawkar, D.A., Modifications of flexible nonyl
chain and nucleobase head group of (+)-erythro-9-(2’s-hydroxy-3’s-nonyl)adenine [(+)-EHNA] as adenosine
deaminase inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.09.015
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Modifications of flexible nonyl chain and nucleobase head group of (+)-
erythro-9-(2ʹs-hydroxy-3ʹs-nonyl)adenine [(+)-EHNA] as adenosine
deaminase inhibitors
a, b
b
a
a
Sachin R. Kandalkar , Parimi Atchuta Ramaiah , Manoj Joshi , Atul Wavhal , Yogesh
a
a
a
a
a
a
Waman , Amol Raje , Ashwini Tambe , Shariq Ansari , Siddhartha De , Venkata P. Palle ,
a
a,
a,
Kasim A. Mookhtiar , Anil M. Deshpande * and Dinesh A. Barawkar *
a
Advinus Therapeutics Ltd., Drug Discovery Facility, Quantum Towers, Plot-9, Phase-I,
Rajiv Gandhi Infotech Park, Hinjewadi, Pune 411 057, India
b
Department of Organic Chemistry, Andhra university, Visakhapatnam 530003, India
*
Corresponding Authors
Tel.: 91-20-66539600 (AMD); 91-20-24371304 (DAB); Fax: 91-20-66539620;
E-mail: anil.deshpande@advinus.com (AMD); dinesh.b@advinus.com (DAB)
Abstract:
A series of terminal nonyl chain and nucleobase modified analogues of (+)-EHNA (III) were
synthesized and evaluated for their ability to inhibit adenosine deaminase (ADA). The
constrained carbon analogues of (+)-EHNA, 7a-7h, 10a-c, 12, 13, 14 and 17a-c appeared
very potent with Ki values in the low nanomolar range. Thio-analogues of (+)-EHNA 24a-e
wherein 5ʹC of nonyl chain replaced by sulfur atom found to be less potent compared to (+)-
EHNA. Docking of the representative compounds into the active site of ADA was performed
to understand structure-activity relationships. Compounds 7a (Ki: 1.1 nM) 7b (Ki: 5.2 nM)
and 26a (Ki: 5.9 nM) showed suitable balance of potency, microsomal stability and
demonstrated better pharmacokinetic properties as compared to (+)-EHNA and therefore may
have therapeutic potential for various inflammatory diseases, hypertension and cancer.
Keywords: ADA; EHNA; Nonyl chain modification; Constrained analogues; Docking,
Pharmacokinetics

1
. Introduction
Hydrolytic irreversible deamination of adenosine and 2-deoxyadenosine to inosine and 2-
deoxyinosine by adenosine deaminase (adenosine aminohydrolase, ADA, EC3.5.4.4) is an
1
important enzymatic reaction in purine salvage pathway. Both adenosine and 2-
deoxyadenosine are biologically active purines having profound effect on cellular physiology.
The enzymatic activity of ADA helps in the regulation of extracellular and intracellular
concentration of these substrates. Extracellular levels of adenosine regulate many
physiological responses by activating four G-protein coupled adenosine receptors namely A1,
2
A , A and A . Adenosine also acts as a sensor and provides information to the immune
2
A
2B
3
system about the tissue damage or acute inflammatory changes occurring in the vicinity of
3
the immune system. While 2ʹ-deoxyadenosine behaves as a cytotoxic metabolite and is
generally considered the primary cause of lymphotoxicity in ADA deficient severe combined
immunodeficiency (SCID) by impairment of the development and function of both T and B
4
cells. Several lines of evidence support a complex regulatory role played by ADA in
different immune cell functions as well as a significant involvement of this catabolic enzyme
in the pathophysiology of several inflammatory diseases. Indeed, abnormalities in ADA
5
activity have also been reported in a variety of other diseases such as AIDS, rheumatoid
6
7
8
9
arthritis, coronary artery disease, anemia, lymphomas and leukemia. Therefore inhibition
of ADA activity has special indication against inflammatory diseases, hypertension, and
ischemic injury. In addition ADA inhibition potentiates 2ʹ-deoxyadenosine mediated
1
0
lymphotoxicity in the treatment of some leukemias and could block the inactivation of
cytotoxic and antiviral adenosine analogues that are substrates of ADA and thereby
1
1
increasing their potency.
The involvement of ADA in various health disorders triggered numerous attempts to
develop ADA inhibitors as a potential therapeutic agent. Several structurally unrelated natural
and synthetic compounds with various degree of potency have been reported as ADA
12
inhibitors. Among them 2ʹ-deoxycoformycin (I, dcf or pentostatin) and coformycin (II) are
1
3-15
naturally occurring transition state ADA inhibitors with pico-molar affinity.
The very
potent inhibitory activity of transition state analogue inhibitors (dcf and analogues) make its
1
6
binding to enzyme almost irreversible resulting into severe toxicity, which limits their use
as a chemotherapeutic agents.

I R = H, 2'-Deoxycoformycin, Ki = 2.5 pM
II R = OH, Coformycin, Ki = 10 pM
III (+)-EHNA, Ki = 1.13 nM
Figure 1. Known inhibitors of Adenosine deaminase
Inhibitors like (+)-EHNA, III (Ki: 1–3 nM) and its analogues are semi-tight
17
competitive inhibitor of ADA, categorized in the class of ground state inhibitors. The use of
EHNA as a therapeutic agent is limited due to low in vivo metabolic stability (poor
pharmacokinetic properties) as it is rapidly metabolized to several hydroxylated derivatives
1
8
and excreted out of body. Palle et. al. reported synthesis and biological evaluation of 8ʹ-
hydroxy, 9ʹ-hydroxy and 8ʹ, 9ʹ-dihydroxy derivatives of (+)-EHNA believed to be putative
19
metabolites in (+)-EHNA metabolism. These hydroxylated analogues were less potent than
+)-EHNA confirming the importance of hydrophobic nature of nonyl chain. Subsequently
same group have studied introduction of unsaturation by having double and triple bond at the
(
2
0
5
’,6’-position in nonyl chain. The potency data revealed that cis-isomer maintains the
potency where as trans-isomer and acetylene derivatives are less active compared to (+)-
EHNA. Further SAR was expanded wherein cis-geometry was maintained by introducing
phenyl ring in terminal nonyl side chain and these confimationally restricted analogues
2
0
showed similar potency to (+)-EHNA. However, very limited SAR was explored and all
reported analogues were profiled only for potency. Hence ADA inhibitors with reduced
toxicity and improved pharmacokinetic profile are eagerly craved.
In the present study, it was aimed to identify a novel EHNA analogue with improved
in vitro and in vivo metabolic stability while retaining nanomolar potency. Recently the
crystal structure of EHNA-ADA complex is reported wherein (+)-EHNA tightly binds in the
2
1
active site of ADA by several hydrogen bonds, van der Walls and hydrophobic interactions.
The critical hydrogen bond interactions of (+)-EHNA with ADA resulting from the
nucleoside base and C-2ʹ hydroxy group where as nonyl chain is responsible for hydrophobic
interactions. Hence we thought of modifying EHNA structure to impart desired properties
with minimal impact on key interactions at active sites. Accordingly, EHNA structure was
fragmented into three main segments P1, P2 and P3 based on their binding position in active

site of ADA (Figure 2). Our SAR investigation employed a strategy of point modification in
these segments which may help to improve metabolic stability while retaining potency. The
chemistry and biological results of this series of compounds are presented in the following
sections.
P2
P1: i) Insertion of sulfur atom at C-5ʹ in nonyl chain
ii) Replacing terminal carbon unit (4 carbon) of nonyl
chain with substituted phenyl ring
P2: Replacement of adenine by other heterocyclic
head group.
P3: C-2ʹ modifications to understand the importance of C-
1'P3
7'
P1
5'
3'
9'
2
ʹ-OH and chirality
Figure 2. Modification strategy of EHNA analogues.
2
. Results and Discussion
2
.1 Chemistry
i
ii
iii
1
2
3a-h
Ethyl-L-lactate
iv
v
vi
vii
4
a-h
5a-h
7
a-h
R = a, -Ph-(m-CF3); b, -Ph-(p-CF3),
c, -Ph-(o-CF3); d, -Ph-(m-F),
6a-h
e, -Ph-(m-Br); f, -5-Ethyl-thiophene,
g, -cyclohexane; h, -Ph-(m-COOMe),
Scheme 1. Reagents and conditions: (i) TBDMSCl, DMAP, triethylamine, THF, rt, 15 h; (ii) (MeO) P(O)CH ,
2
3
n-BuLi, THF, –78 °C, 2.5 h; (iii) R-CHO, n-BuLi, –50°C to rt, 16 h; or 3-bromobenzaldehyde, NaH, THF, 0 °C,
h; (iv) CeCl .7H O, NaBH , MeOH, –78°C, 1 h; (v) H /Pd-C EtOAc, rt, 1 h or RhCl(PPh ) , H , THF/tert-
3
3
2
4
2
3 3
2
butyl alcohol, (1:1), rt, 16 h; (vi) N6,N6-Diboc adenine, DEAD, PPh , THF, 0 °C to rt, 16 h (vii) Methanolic
3
HCl, Methanol, rt 5 h.
To probe the role of nonyl chain on potency and metabolic stability (P1 segment
modification, Figure 2), various meta- substituted phenyl analogues were synthesized via the
general route outlined in Scheme 1. Various hydrophobic, hydrophilic and heteroaryls groups
were selected on meta- position of phenyl group in anticipation of gaining additional H-
bonding or van-der Walls contact to retain potency of these analogues. The synthesis started
from commercially available ethyl-L-lactate where α-hydroxy group was protected with tert-

2
2
butyl dimethyl silane to afford 1. Condensation of 1 with lithium methyldimethyl
phosphonate in THF furnished the phosphonate 2 which was then subjected to Wittig-Horner
2
3
reaction with appropriate aldehyde to provide olefine derivatives 3a-h. Stereoselective
reduction of ketone (3a-h) using cerium chloride and sodium borohydride in methanol
2
4
furnished alcohol (4a-h) followed by reduction of double bond with H (g) and Pd/C or
2
Wilkinson’s catalyst afforded compounds 5a-h, These modified nonyl analogues were
2
5a
coupled with nucleobase, N6,N6-Diboc adenine using Mitsunobu reaction to provide
2
5b
compounds 6a-h . Deprotection of 6a-h using methanolic HCl afforded the (+)-EHNA
analogues 7a-h having desired 2ʹS, 3ʹR configuration.
i
ii
6e
8
9a-c
iii
R = a,
c,
b,
10a-c
Scheme 2. Reagents and conditions: (i) Bis(pinacolato)diboron, Pd(dppf)Cl .CH Cl , CH COOK, dioxane, 80
2
2
2
3
°
C, 3h; (ii) Heteroarylhalides, Pd(dppf)Cl .CH Cl , K CO , dioxane, 80 °C, 2–4 h; (iii) 4M HCl in dioxane,
2 2 2 2 3
dioxane, rt, 5–10 h.
Synthesis of biaryl derivatives (10a-c) wherein heteroaryl ring substituted at meta-
position on phenyl ring, is outlined in Scheme 2. Intermediate 6e was converted to boronate
ester 8, which was treated with commercially available heteroaryl halides under typical
Suzuki reaction condition to obtain corresponding biaryl derivatives 9a–c in good yields.
Finally, deprotection of 9a–c with 4M HCl in dioxane at rt provided target compounds 10a–c
in 50–70% yields.
Ade =
iii
iv
i
1
1
3
4
11
12
ii
7
h

Scheme 3. Reagents and conditions: (i) LiOH.H O, THF:MeOH:H O (3:1:1), rt, 5h; (ii) LAH, THF, Reflux,
2
2
1
6h; (iii) Mg N , MeOH, 80 °C, 48h; (iv) 40 % aqueous Methylamine, 80 °C, 10h
3 2
Next, we synthesized analogues having negatively charged or neutral polar
substitution on phenyl ring as decribed in Scheme 3 and Scheme 4. Methyl ester of
intermediate 7h was converted to acid 11, alcohol 12, amides 13 and 14. Additional amide
derivatives 17a–c were synthesized by converting the ester 6h to acid 15, followed by
reacting with respective amines under amide coupling condition and subsequent deprotection.
i
ii
6h
15
16a-c
iii
R = a,
b,
c,
1
7a-c
Scheme 4. Reagents and conditions: (i) LiOH.H O, THF:MeOH:H O (3:1:1), rt, 16 h; (ii) Respective amine,
2
2
EDCI.HCl, HOBt, Et N, DCM, 25 °C, 16 h; (iii) 4M HCl in dioxane, dioxane, rt, 5–10 h.
3
iv, v
i, ii
iii
18
19
20a-e
vi
vii
viii, ix
21a-e
22a-e
23a-e
24a-e
R = a, -C4H9; b, -Ph; c, -Ph-(o-CH3); d, -Ph-(m-CH3); e, -Ph-(m-CF3);
0
Scheme 5. Reagents and conditions: (i) NaBH , THF, 0 C, rt, overnight; (ii) tosyl choride, triethylamine, DCM,
4
0
rt, 6 h; (iii) sodium salt of butanethiol/thiophenols, dioxane, rt, 5-8 h; (iv) methanolic HCl, 40 C, 3 h; (v) 5-
amino 4,6-dichloro purine, 1-butanol, triethylamine, reflux, 21 h; (vi) triethylorthoformate, conc. HCl 6 h; (vii)
0
methanolic ammonia, 60 C, 16 h; (viii) p-nitrobenzoic acid, triphenyl phosphine, DEAD, THF, rt, 16 h; (ix)
NaOH, THF:MeOH:H O (3:1:1), rt, 5 h.
2

The synthesis of thio-derivatives of (+)-EHNA is outlined in scheme 5. Readily
available boc protected L-threonine methyl ester 18 was subjected to ester reduction using
NaBH followed by tosylation gave compound 19. Nucleophilic substitution of 19 with
4
sodium salt of butane thiol and various thiophenols yielded aryl sulphides 20a-e respectively.
Deprotection of 20a-e with methanolic HCl followed by condensation with 5-amino-4,6-
2
6
dichloropyrimidine provided pyrimidine derivatives 21a-e in moderate yields. Treatment of
2
6
2
1a-e with triethylorthoformate afforded cyclized chloropurine derivatives 22a-e ,
subsequent amination with liquid ammonia provided thio- analogues of EHNA 23a-e with
2
7
2
ʹR, 3ʹS threo configuration. Finally inversion of C-2ʹ hydroxy with Mitsunobu reaction
followed by hydrolysis afforded desired thio-analogues of (+)-EHNA 24a-e with 2ʹS, 3ʹS
erythro configuration.
To understand the significance of adenine group towards potency contribution (P2
segment modification, Figure 2), we replaced adenine group by 4-carboxamide-imidazole
(
26a-b), 4-amino-pyrazolo[3,4-d]pyrimidine (26c-d) and 4-amino-pyrolo[3,4-d]pyrimidine
26e-f). The desired analogues were synthesized using intermediate 5a and 5b as described
(
in Scheme 6. In two steps 3-hydroxy group of 5a/5b was converted to mesylate and
displacement by S 2 reaction with 4-imidazolecarboxamide in the presence of NaH in DMF
N
2
8
afforded 25a-b. Whereas 25c-f were synthesized by reacting 4-Diboc-aminopyrazolo[3,4-
2
5a
d]pyrimidine /4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 5a/5b under Mitsunobu condition,
Finally deprotection of 25a-f afforded desired compounds 26a-f.
i or ii
iii or iv
1
5
5
a, R = m-CF3
1
1
2
2
5a/c/e, R = m-CF3
2
2
6a/c/e, R = m-CF3
1
b, R = p-CF3
1
1
5b/d/f, R = p-CF3
6b/d/f, R = p-CF3
2
6a
26b
p-CF3
26c
m-CF3
26d
p-CF3
26e
m-CF3
26f
p-CF3
1
2
m-CF3
R
R
Scheme 6. Reagents and conditions: (i) (a) MsCl, Et N, CH Cl , 0 °C, 1 h; (b) mesylate added to 4-
3
2
2
imidazolecarboxamide, NaH, DMF, 80 °C, 36 - 48 h; (ii) 4-Diboc-aminopyrazolo[3,4-d]pyrimidine or 4-chloro-

7
H-pyrrolo[2,3-d]pyrimidine, DEAD, PPh , THF, 0 °C to rt, 16 h; (iii) 4M HCl in Dioxane, Dioxane, rt, 5–10 h.
3
(
iv) NaN , PPh , DMSO, 110 °C, 1N HCl in water, 5 h.
3 3
We probed the role of C-2ʹ hydroxyl and chirality on potency (P3 segment
modification, Figure 2) by converting secondary hydroxyl of 7b to ketone 29 and tertiary
hydroxyl group 31. As described in Scheme 7 selective deprotection of TDBMS group of
intermediate 6b yielded alcohol 27b which was converted to ketone 28 by using pyridinium
chlorochromate. Subsequent boc deprotection of 28 using 4M HCl in dioxane gave desired
ketone derivative 29 in good yield. Intermediate 28 was treated with methyl magnesium
bromide, which afforded the conversion of ketone to tert-hydroxy derivative along with
deprotection of one boc group to get monoboc-adenine derivative 30. The second boc group
was deprotected using 4M HCl in dioxane yielded tert-alcohol derivative 31.
i
ii
6b
27b
28
iv
iii
iii
31
30
29
Scheme 7. Reagents and conditions: (i) TBAF, THF, 0 °C, 1 h.; (ii) PCC, DCM, rt, 4 h; (iii) 4M HCl in dioxane,
dioxane, rt, 5–10 h; (iv) CH MgBr (3.0M in diethyl ether), THF, 0 °C to rt, 2 h.
3
2
.2 Structure Activity Relationship
All analogues of (+)-EHNA were evaluated for their adenosine deaminase inhibitory
2
9
potencies against bovine ADA using recombinant enzyme (Table 1). Inhibitory potency of
compounds was expressed as inhibitory constant (K ). The (+)-EHNA showed potency with
i
3
0
Ki: 2.2 nM which is comparable with reported data and it is considered as a reference
standard in this study.
Table 1. Inhibitory potency of (+)-EHNA analogues with bovine ADA

Compound
Number
Ki
nM)
Compound
No.
Ki
(nM)
X
R
X
R
(
(
+)-EHNA -CH
2
2
2
-
-
-
-C
4
H
9
2.2
1.1
5.2
11
12
13
-CH
-CH
-CH
2
-
-
-
53
4.6
2.9
7
a
-CH
-CH
2
2
7
b
7
c
-CH
2
2
-
-
6.2
1.6
14
-CH
2
2
-
-
2.7
1.8
7
d
-CH
17a
-CH
7
e
-CH
-CH
-CH
2
-
-
-
0.6
2.1
1.9
17b
17c
24a
-CH
-CH
-S-
2
-
-
1.8
2.2
54
7
f
2
2
2
7
g
-(CH ) CH
2 3 3
7
h
-CH
2
2
-
-
2.2
2.6
24b
24c
-S-
-S-
135
66
1
0a
-CH
1
0b
-CH
2
2
-
-
2.3
1.9
24d
24e
-S-
-S-
49
25
1
0c
-CH

Initially we focused our attention on the modification of segment P1 due to its
1
8, 19
involvement in potential metabolism of (+) EHNA.
To avoid observed ω-oxidation of
nonyl chain as well as to impart rigidity it was planned to replace C-6’ to C-9’ fragment of
nonyl chain in EHNA by appropriately substituted phenyl, thiophene and cyclohexyl group.
First we were interested to explore trifluoromethyl substitution on phenyl ring which may
bring pharmacokinetic and pharmacodynamic advantages. Trifluoromethyl group enhances
metabolic stability as well as increased lipophilicity may improve absorption, distribution or
enhanced interaction with a hydrophobic binding region. We were pleased to find that meta-
trifluoromethyl analogue (7a, Ki: 1.1 nM) appeared 2-fold more potent compared to (+)-
EHNA (Ki: 2.2 nM) and ~5-fold better in potency compared to para- and ortho-
trifluoromethyl analogues (7b, Ki: 5.2 nM & 7c, Ki: 6.2 nM). These results suggested meta-
trifluoromethyl is slightly better compared to ortho- and para- and moreover 7a offered a
good microsomal stability across species (refer Table 3).
Next, we explored various hydrophobic substituents by replacing meta-CF of 7a.
3
Small hydrophobic substituent like fluorine 7d (Ki: 1.6 nM) and bromine 7e (Ki: 0.6 nM)
maintained good potency but had detrimental effect on metabolic stability (data not
captured). Thiophene (7f, Ki: 1.1 nM), cyclohexyl (7g, Ki: 1.1 nM) and meta- methyl
benzoate (7h, Ki: 2.2 nM) analogues showed comparable potency to (+)-EHNA but showed
moderate to poor metabolic stability across species. Further analogues with bulkier
substituent more specifically heterocycles like 2-thiazole 10a (Ki: 2.6 nM), 2-pyridine 10b
(Ki: 2.3 nM), and 2-pyrazine 10c (Ki: 1.9 nM) exhibited similar activity relative to 7a and
showed moderate to good metabolic stability.
Further, it was hypothesized that analogues appended with polar substitution on
phenyl would lead an improvement in potency via additional hydrogen bond interaction with
backbone residues. Also it was thought that the polar substitution may contribute towards
improving drug like features. Encouragingly, most of the analogues synthesized in this series
1
2-14 and 17a-c showed the potency in the range of 2–5 nM, exception of compound 11,
having carboxylic acid moiety showed a significant loss of the activity (Ki: 53 nM).
We were interested to explore thio-EHNA analogues, wherein C-5ʹ was replaced by
sulfur. Previously described oxo-EHNA analogues (C-5ʹ replaced by oxygen) resulted in
decreased potency, presumably due to loss of hydrophobic feature of alkyl chain. Hence it
was rationalized the sulfur being less hydrophilic compare to oxygen thio-EHNA analogues
may help to maintain the hydrophobicity of alkyl chain. Additionally, sulfur mimic C=C

bond, and will help to reduce the degree of freedom which in turn may improve
pharmacokinetic properties. We examined focused thio-analogues 24a-e. Thio-EHNA
analogue 24a (Ki: 54 nM) and constrained analogues 24c-d showed 25-fold loss of potency
compared to (+)-EHNA, whereas constrained analogue 24b (Ki: 135 nM) without any
substitution on phenyl showed significant loss in potency. Thio- analogue of 7a (24e, Ki: 25
nM), exhibited 2-fold gain in the potency compared to thio-EHNA, highlighting the
significance of meta- trifluoromethyl group.
Next, we examined the significance of adenine ring towards potency contribution by
replacing adenine with appropriately substituted heterocyclic ring such as 4-carboxamide-
imidazole (26a-b), 4-amino-pyrazolo[3,4-d]pyrimidine (26c-d) and 4-amino-pyrolo[3,4-
d]pyrimidine (26e-f). Imidazole 4-carboxamide derivatives 26a is equipotent to 7a whereas
2
6b appeared 5- fold less potent indicating the importance of meta-CF substitution on
3
phenyl. All other analogues 26c–f led complete loss in the potency. Thus, it supports the
2
1
earlier finding that N7 nitrogen is involved in hydrogen bonding with Asp-296 and is
3
1
critical for the inhibitory activity of (+)-EHNA.
In order to validate the role of secondary hydroxyl in P3 segment, we thought of
converting C-2ʹ-hydroxy of 6b to ketone and tertiary hydroxyl group and its influence on the
potency. The affinity of keto compound 29 to the ADA was completely lost where as
compound with tert-hydroxyl at C-2ʹ 31 (Ki: 69 nM) appeared less potent compared to (+)-
EHNA and 7b. This revealed that sec-hydroxyl group is necessary for the activity. The
possible explanation for loss of activity in 31 may be due to extra methyl causing steric
interaction as well as devoid of chirality at C-2ʹposition

Table 2. Inhibitory potency of (+)-EHNA analogues with bovine ADA
2
2
6a, 26c, 26e (meta-CF3)
6b, 26d, 26f (para-CF3)
29, 31
Compound
Number
R
K (nM)
i
2
6a
6b
6c
6d
6e
6f
5.9
29
2
2
Inactive
Inactive
2
2
Inactive
Inactive
2
2
9
1
Inactive
69
3
2
.3 Docking studies in active site of ADA-(+)-EHNA co-crystal structure
To account for strong ADA inhibitory activity of 7a, we build a docking model of
ADA/7a complex based on X-ray crystal structure of ADA complex with (+)-EHNA (pdb id
2
1
2
Z7G) followed by energy minimization refinement using GOLD and MOE. Initially to
confirm the reliability of docking procedure, (+)-EHNA was extracted from and re-docked to
the crystal structure of ADA. As demonstrated in Figure 3a compound 7a occupied an
active site of ADA in a similar manner as observed in the complex of ADA/(+)-EHNA. The
edited phenyl group of 7a maintained most of the hydrophobic van der Walls interactions
with Leu 58, Phe 61, Leu 62, Phe 65, Leu 106, Trp 117, Met 155 and Gly 184 amino acids of
ADA. The appended trifluoromethyl group on phenyl appears to fit more favorably in ADA

active site pocket. The structural modification of 7a does not disturb the hydrophobic
interaction of C-2ʹ methyl and hydrogen bond interaction of adenine with Glu 217 and Asp
2
96 as well as C-2ʹ-hydroxyl with His 17 and Asp 19.
HIS-238
ASP-295
GLU-217
ASP-296
Zn
PHE-61
LEU-58
HIS-17
LEU-62
PHE-65
ASP-19
a
b
c
Figure 3. (a) The docked pose of compound 7a (Yellow) overlayed with cocrystal structure of (+)-
EHNA (Magenta) from pdb id 2Z7G, (b) compound 24e (cyans) overlayed with cocrystal structure of
(
+)-EHNA (Magenta) from pdb id 2Z7G and (C) compound 11 (White) overlayed with cocrystal
structure of (+)-EHNA (Magenta) from pdb id 2Z7G. The oxygen (O), nitrogen (N), fluorine (F) and
sulfur (S) atoms are colored in Red, Blue, Pale cyans and Olive respectively. Docking was done using
program GOLD. The docked poses of compounds were further minimized using program MOE. The
putative hydrogen bonds are shown by dotted line. The atomic numbering used here is from pdb id
2Z7G.
As shown in Figure 3b constrained thio-EHNA analogue 24e oriented slightly in
different way to that of terminal alkyl chain in (+)-EHNA. The possible explanation could be
o
the smaller bond angle of C-S-C (~90 °) in 24e as compared to bond angle of C-C-C (110 )
in (+)-EHNA. The change in orientation of terminal phenyl ring might have lost few
hydrophobic interactions and thus affecting the potency of 24e.
Another remarkable binding feature associated with compound 7a and 24e is that the
trifluoromethyl group substituted on meta-position of terminal phenyl ring appears to fit more
favorably and might allow van der Walls interaction between one of the fluorine and the
carbonyl from backbone located in hydrophobic pocket. This additional interaction resulted
in gaining two and five fold higher inhibitory activity for 24e compared to 24d and 24b
respectively.
Docking study of compound 11 exhibited that C-2ʹ-hydroxy group maintaining its hydrogen
bonding interactions with Asp-19 and His-17. On the other hand adenine ring of 11 showed

slight misalignment with respect to adenine ring of (+)-EHNA as shown in Figure 3c. This
might have affected water mediated hydrogen bonding interactions of C6-amino group with
Glu-217 as C6-amino group moving slightly away from Glu-217. Another interesting
observation from docking pose of 11 clearly suggests the different orientation of terminal
phenyl ring which results in loss of some hydrophobic interactions with amino acid residues
like Phe-61, Phe-65, Leu-58 and Trp-117 leading the reduction in potency. The possible
explanation is repulsive forces due to charge carboxylic group. This was further supported by
molecular docking of compounds 7h and 14 wherein carboxylic acid group is converted to
methyl ester and N-methyl amide respectively. Both compounds docked well in ADA
binding site. This modeling hypothesis was validated as compound 11 (Gold Score 17.01) has
lost the potency whereas compounds 7h (Gold Score 13.42) and 14 (Gold Score 13.14)
showed good inhibitory potencies.
2
.4 In vitro metabolic stability and intravenous pharmacokinetic in male Wistar rats
The metabolic stability results of all potent compounds from both series against
mouse, rat and human liver microsomes are summarized in Table 3. In constrained carbon
analogues of (+)-EHNA compounds 7a, 7b, 7h and 14 were found to be stable whereas
compounds 7f and 7g showed moderate stability. Compounds with bulkier hydrophobic
substituent (10a-c) showed moderate metabolic stability across species. Thio-EHNA
analogue 24e showed good stability in rat and human but moderately stable in mouse.
Table 3. Metabolic Stability of selected compounds.
MR (nmol/min/mg)
% R at 30min
( +NADPH)
Compound
Number
t½
min)
% R at 30min
(-NADPH)
@0.125mg/ml
Matrix
(
protein
0
0
0
0
.08
.04
.06
.04
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
13
>30
>30
>30
>30
>30
74
86
80
85
93
99
64
58
76
77
19
65
88
96
68
100
89
91
95
94
93
95
90
95
95
92
94
83
100
99
65
96
MLM
RLM
HLM
MLM
RLM
HLM
MLM
RLM
HLM
MLM
RLM
HLM
MLM
RLM
HLM
MLM
7
a
7
b
<
<
0.04
0.04
0
0
.11
.15
.07
.07
.44
.11
.04
7
f
0
0
0
0
0
7
g
7
h
<0.04
0.10
<0.04
1
4

<
<
0
0.09
0
0
0.12
0
0.04
0.04
.10
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
25
91
90
65
76
84
58
64
82
57
90
71
100
86
94
43
83
83
89
87
79
100
92
92
RLM
HLM
MLM
RLM
HLM
MLM
RLM
HLM
MLM
RLM
HLM
MLM
RLM
HLM
MLM
RLM
HLM
MLM
RLM
HLM
1
0a
0b
0c
4a
4e
6a
91
.05
.15
100
100
100
91
94
85
94
94
97
85
100
100
89
93
92
1
.06
.16
0
1
<
<
<
0.04
0
0.04
0
.09
2
.04
0.04
0
0
0
0
0
0
.22
.05
.05
.04
.04
.06
2
>100
>100
>30
>30
>30
2
94
Plasma Concentration Vs time profile of
+)-EHNA and 26a
Plasma Concentration Vs time profile
of 7a and 7b
(
Comp. 7a
Comp. 7b
Comp. 26a
+)-EHNA
(
Compound
Number
IV Dose
AUC0-t
V
ss
CL (mL/min/Kg)
t
1/2
(
mg/kg)
(μM.h)
(L/Kg)
(h)
#
#
#
(
+)-EHNA
6a
1
1
0.35
1.42
NR
NR
NR
2
2
33.3
85
0.94
7
a
3
3
1.61
2.21
1.88
7.01
<0.5
2.21
7
b
59
#
Formulation vehicle: NMP-10%, PEG-300-15%, 0.1M Ammonium acetate buffer q.s.(pH 3); NR: not reportable due to very fast
elimination. Route of
Figure 4. Intravenous pharmacokinetic profile of (+)-EHNA, 26a, 7a and 7b in male Wistar rat

As the primary objective of this study was identification of ADA inhibitors with
improved pharmacokinetics to that of (+)-EHNA, potent and stable compounds 26a, 7a and
7
b were evaluated for intravenous pharmacokinetic study in male wistar rat (Figure 4). (+)-
EHNA showed very rapid plasma clearance and concentrations were found below
quantification level beyond 1 h post dose administration. However, 26a, 7a and 7b
demonstrated better pharmacokinetic properties than (+)-EHNA with respect clearance, half
life and plasma exposures.
3
. Conclusion
In summary, we explored SAR on three segments of (+)-EHNA by carrying out point
modifications with aim to improve pharmacokinetic properties. All synthesized novel
analogues were initially evaluated for their ability to inhibit ADA and subsequent profiling of
potent compounds for microsomal stability and pharmacokinetics. Majority of modifications
attempted on n-hexyl sidechain of (+)-EHNA were able to retain the potency with good
microsomal stability. The constrained carbon analogues (7a–h, 10a–c, 12, 13, 14 and 17a–c)
exhibited low nanomolar potency whereas thio- analogues (24a–e) were found to be less
potent. SAR supported by docking study also revealed that the hydrophobic binding pocket in
the active site has much more tolerant of structural deviance and substitution on terminal
phenyl ring allows polar as well as non-polar groups. Compound 7a, 7b and 26a showed
good balance of potency and microsomal stability and demonstrated better pharmacokinetic
properties in rat compared to (+)-EHNA and therefore may have therapeutic potential for
various inflammatory diseases, hypertension and cancer.
4
. Experimental Section
4
.1
General methods for chemistry.
All reagents, starting materials, and solvents (including dry solvents) were obtained from
commercial suppliers and used as such without further purification. Reactions were carried
out in oven-dried glassware under a positive pressure of argon/nitrogen unless otherwise
mentioned. Reaction progress was determined by thin layer chromatography (TLC) analysis
on Merck Kieselgel 60 F254 plates. Column chromatography was performed on silica gel
(Rankem, 60-120 mesh). Deuterated solvents (Cambridge Isotope Laboratories) for NMR
spectroscopic analysis were used as received. All NMR spectra were recorded on Varian 400
MHz spectrometer. Coupling constants are measured in Hertz. All chemical shifts are quoted
in ppm, relative to tetramethylsilane, using the residual solvent peak as a reference standard.

Low-resolution mass spectra (MS) were determined on an Agilent 1200 series LC/MSD/VL.
All MS experiments were performed using electrospray ionization (ESI) in positive ion mode.
Analytical HPLC was performed on an Agilent 1200 series instrument equipped with Diode
Array Detector Reagents and solvents were obtained from commercial sources and used
without further purification. (Method: Column Agilent Eclipse XDB C-18, 5µM, 250mm X
4
.6mm, at a flow of 1.5 ml/min, mobile phase: 0.05% formic acid in water and acetonitrile)
Abbreviations of the solvents are used as follows: AcOEt, ethyl acetate; THF,
tetrahydrofuran; EtOH, ethanol; DMF, N,N-dimethylformamide; Et O, diethyl ether; MeOH,
2
methanol; CH CN, acetonitrile; K CO , potassium carbonate;
3
2
3
4
.1.1. Ethyl (2S)-2-[tert-butyl(dimethyl)silyl]oxypropanoate (1)
To a stirred solution of ethyl-L-lactate (25 g, 211 mmol) in DMF (500 mL) was added
tert-butyl dimethylsilyl chloride (33.52 g, 222 mmol), 4-dimethylamino pyridine (2.58 g, 21
mmol) followed by triethylamine (103 mL, 741mmol ) at room temperature. The reaction
mixture was stirred for 15 h (progress of reaction was monitored by TLC). Upon reaction
completion, the mixture was diluted with water (300 mL) and the product was extracted with
dichloromethane (250 mL x 3). Combined organic layer was washed with brine (200 mL),
dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the
crude product. The crude product was purified by column chromatography using 4% EtOAc–
1
Hexane as solvent system to afford pure 1 (39.8 g, 81%). H NMR (400 MHz; CDCl ) : δ
3
0
4
1
4
.07 (s, 3H), 0.10 (s, 3H), 0.90 (s, 9H), 1.28 (t, J = 6.9 Hz, 3H), 1.39 (d, J = 6.9 Hz, 3H),
1
3
.11-4.24 (m, 2H), 4.31 (q, J = 6.9 Hz, 1H); C-NMR (100 MHz; CDCl ) : δ −5.16, −4.83,
3
+
4.29, 18.42, 21.40, 25.83 (3C), 60.81, 68.58, 174.20; ESI-MS: m/z: 233.3 [M + 1].
.1.2. (3S)-3-[tert-butyl(dimethyl)silyl]oxy-1-dimethoxyphosphoryl-butan-2-one (2)
To a stirred solution of dimethylmethylphosphonate (32.03 g, 258 mmol) in THF (75
mL) maintained at −78 °C was added n-BuLi (103.3 mL, 258 mmol, 2.5 M soln. in Hexane)
in dropwise manner under argon atmosphere. The reaction mixture was continued to stirr at
−
78 °C for 1 h followed by slow addition of solution of (S)-2(tert-butyl-methyl-silanyloxy)-
propionic acid ethyl ester (1, 15g, 64.6 mmol) in THF (10 mL) and was stirred for additional
0 min.. After reaction completion, the mixture was quenched by the slow addition of
9
aqueous ammonium chloride (250 mL). The aqueous layer was extracted with EtOAc (250
mL x 2), the combined organic layer was washed with water (200 mL), brine (100 mL), dried
over anhydrous sodium sulfate and conc. under reduced pressure to give crude product as
yellow oil. The crude product was purified by column chromatography using 35% EtOAc–
1
Hexane as solvent system to afford pure 2 (14.3 g, 71%). H NMR (400 MHz; CDCl ): δ
3

2
0
.09 (s, 3H), 0.10 (s, 3H), 0.92 (s, 9H), 1.32 (d, J = 6.8 Hz, 3H), 3.26 (dd, JHH = 14.7 Hz, JHP
2
3
=
22 Hz, 1H), 3.38 (dd, JHH = 15.2 Hz, J = 21 Hz, 1H), 3.78 (d, J = 3.4 Hz, 3H), 3.80 (d,
HP HP
3
13
J = 3.5 Hz, 3H), 4.24 (q, J = 6.4 Hz, 1H). C-NMR (100 MHz; (CD ) SO): δ −5.00
HP
3 2
1
2
(
SiCH ), −4.62 (SiCH ), 18.05, 20.25, 25.76 (3C), 34.73 (d, J = 135.4 Hz), 52.88 (d, J =
3 3 PC PC
2
3
2
6
.2 Hz, OCH ), 52.97 (d, J = 6.3 Hz, OCH ), 74.83 (d, J = 3.1 Hz), 205.10 (d, J = 7.0
3
PC
3
PC
PC
+
Hz); ESI-MS: m/z: 311.2 [M + 1].
4
.1.3 General method for Wittig-Horner reaction to get 3a-d and 3f-h
To a stirred solution of 3-(tert-butyl-dimethyl-silanyloxy)-2-oxo-butyl]phosphonic
acid dimethyl ester (2, 11.27 mmol) in THF (21 mL) maintained at −78 °C under argon
atomosphere was added n-BuLi (11.27 mmol, 1.6 M in hexane) in a dropwise manner. After
1
0 minutes, to this reaction mixture corresponding aldehyde (11.27 mmol) in THF (28 mL)
was added and the reaction temperature allowed to come to room temperature gradually. This
reaction mixture was stirred for 18 h. The progress of reaction was monitored by TLC, upon
completion the reaction was quenched with aq. ammonium chloride (20 mL). The reaction
mixture was extracted with EtOAc (20 mL x 3). The combined organic layer was washed
with brine, dried over anhy. sodium sulfate and concentrated under reduced pressure to obtain
a crude product. The crude product was purified by column chromatography.
4
.1.3.1.
(E, 4S)-4-[tert-butyl(dimethyl)silyl]oxy-1-[3-(trifluoromethyl) phenyl] pent-1-
1
en-3-one (3a). Yield: 82%; H NMR (400 MHz; CDCl ): δ 0.01 (s, 3H), 0.11 (s, 3H), 0.95 (s,
3
9
7
H), 1.38 (d, J = 6.9 Hz, 3H), 4.35 (q, J = 6.9 Hz, 1H), 7.36 (d, J = 16.1 Hz, 1H), 7.54 (t, J =
+
.8 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.68-7.78 (m, 2H), 7.82 (s, 1H); ESI-MS m/z: 359. [M
1].
.1.3.2.
+
4
(E, 4S)-4-[tert-butyl(dimethyl)silyl]oxy-1-[4-(trifluoromethyl) phenyl] pent-1-
1
en-3-one (3b). Yield: 79%; H NMR (400 MHz; CDCl ): δ 0.09 (s, 3H), 0.11 (s, 3H), 0.94 (s,
3
9
H), 1.37 (d, J = 6.8 Hz, 3H), 4.34 (q, J = 6.8 Hz, 1H), 7.35 (d, J = 16.1 Hz, 1H), 7.65-7.68
1
3
(
m, 4H), 7.71 (d, J = 16.2 Hz, 1H). C-NMR (100 MHz; CDCl ): δ −4.82, –4.58, 18.30,
3
1
3
2
1
4
1.30, 25.90 (3C), 74.82, 122.59, 123.98 (q, J = 270.9 Hz), 126.04 (q, J = 3.9 Hz, 2C),
FC FC
2
+
28.62 (2C), 132.02 (q, J = 32.5 Hz), 138.40, 141.95, 201.98; ESI-MS m/z: 359.3 [M + 1].
FC
.1.3.3.
(E, 4S)-4-[tert-butyl(dimethyl)silyl]oxy-1-[2-(trifluoromethyl) phenyl] pent-1-
1
en-3-one. (3c). Yield: 69%; H NMR (400 MHz; CDCl ): δ 0.09 (s, 3H), 0.11 (s, 3H), 0.93 (s,
3
9
7
1
H), 1.38 (d, J = 6.9 Hz, 3H), 4.37 (q, J = 6.9 Hz, 1H), 7.21 (d, J = 15.9 Hz, 1H), 7.49 (t, J =
.9 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.74 (dd, J = 7.9 Hz, 17.4 Hz, 2H), 8.10 (dd, J = 2.0 Hz,
1
3
5.9 Hz, 1H); C-NMR (100 MHz; CDCl ): δ −4.84, –4.64, 18.29, 21.28, 25.89(3C), 74.79,
3

1
3
2
1
24.10 (q, J = 274.1 Hz), 124.64, 126.37 (q, J = 5.4 Hz), 127.85, 129.49 (q, J = 30.2
FC FC FC
4
+
Hz), 129.78, 132.21, 134.12, 139.22 (q, J = 1.5 Hz), 201.44; ESI-MS m/z: 359.2 [M + 1].
FC
4
.1.3.4.
(E, 4S)-4-[tert-butyl(dimethyl)silyl]oxy-1-(3-fluorophenyl) pent-1-en-3-one
1
(
3d). Yield: 75%; H NMR (400 MHz; CDCl ): δ 0.09 (s, 3H), 0.11 (s, 3H), 0.94 (s, 9H),
3
1
7
1
1
=
.37 (d, J = 6.8 Hz, 3H), 4.33 (q, J = 6.8 Hz, 1H), 7.06-7.13 (m, 1H), 7.23-7.31 (m, 2H),
1
3
.32-7.41 (m, 2H), 7.66 (d, J = 16.2 Hz, 1H). C-NMR (100 MHz; CDCl ): δ −4.84, −4.64,
3
2
2
8.27, 21.29, 25.87 (3C), 74.78, 114.58 (d J = 21.8 Hz), 117.42 (d, J = 21.0 Hz), 121.53,
FC
FC
4
3
3
4
24.57 (d, J = 3.1 Hz), 130.57 (d, J = 7.8 Hz), 137.27 (d, J = 7.7 Hz), 142.44 (d, J
FC
FC
FC
FC
1
+
2.4 Hz), 163.14 (d, J = 246.8 Hz), 201.97; ESI-MS m/z: 309.1 [M + 1].
FC
4
.1.3.5.
(E, 4S)-4-[tert-butyl(dimethyl)silyl]oxy-1-(5-ethyl-2-thienyl)pent -1-en-3-one
1
(
3f). Yield: 71%; H NMR (400 MHz; CDCl ): δ 0.09 (s, 6H), 0.94 (s, 9H), 1.30-1.36 (m,
3
6
1
H), 2.85 (q, J = 7.6 Hz, 2H), 4.28 (q, J = 6.8 Hz, 1H), 6.75 (d, J = 3.6 Hz, 1H), 6.96 (d, J =
+
5.6 Hz, 1H), 7.13 (d, J = 4 Hz, 1H), 7.76 (d, J = 15.6 Hz, 1H); ESI-MS m/z: 325.2 [M + 1]
.1.3.6. (E, 4S)-4-[tert-butyl(dimethyl)silyl]oxy-1-cyclohexyl-pent-1-en-3-one (3g).
Yield: 61%; H NMR (400 MHz; CDCl ): δ 0.05 (s, 3H), 0.06 (s, 3H), 0.90 (s, 9H), 1.12-1.32
4
1
3
(m, 8H), 1.64-1.80 (m, 5H), 2.11-2.18 (m, 1H), 4.24 (q, J = 6.8 Hz, 1H), 6.54 (dd, J = 1.6 Hz,
+
1
6.4 Hz, 1H), 6.95 (dd, J = 6.8 Hz, 15.6Hz, 1H); ESI-MS m/z: 297.4 [M + 1].
4
.1.3.7.
3-[(E, 4S)-4-[tert-butyl(dimethyl)silyl]oxy-3-oxo-pent-1-enyl] benzoate (3h).
1
Yield: 76%; H NMR (400 MHz; CDCl ): δ 0.10 (s, 3H), 0.11 (s, 3H), 0.95 (s, 9H), 1.38 (d, J
3
=
6.9 Hz, 3H), 3.95 (s, 3H), 4.35 (q, J = 6.9 Hz, 1H), 7.35 (d, J = 16.2 Hz, 1H), 7.49 (t, J =
1
3
7
.9 Hz, 1H), 7.70-7.78 (m, 2H), 8.06 (d, J = 7.9 Hz, 1H), 8.27 (s, 1H). C-NMR (100 MHz;
(
CDCl ): δ −4.97, −4.75, 18.17, 21.16, 25.76, 52.32, 74.66, 121.36, 129.07, 129.21, 130.94,
3
+
1
31.22, 132.59, 135.20, 142.47, 166.45, 201.89. ESI-MS m/z: 349.3 [M + 1].
4
.1.3.8.
(E, 4S)-1-(3-bromophenyl)-4-[tert-butyl(dimethyl)silyl]oxy-pent-1-en-3-one
(3e). To a stirred suspension of sodium hydride (1.13 g, 28.35 mmol, 60 % in oil) in THF (25
mL) maintained at 0 °C under argon atomosphere was slowly added solution of 3-(tert-butyl-
dimethyl-silanyloxy)-2-oxo-butyl] phosphonic acid dimethyl ester (2, 8 g, 25.77 mmol) in
THF (48 mL). The reaction mixture was stirred for 20 min. and then 3-bromobenzaldehyde
(4.77 g, 25.77 mmol) in THF (64 mL) was added in a dropwise manner. The reaction
temperature was allowed to come to room temperature gradually and then stirred for
additional 4 h. The progress of reaction was monitored by TLC. Upon completion, the
reaction mixture was slowly diluted with water (100 mL) and extracted with EtOAc (70 mL x
3
). Combined organic layer was washed with brine, dried over anhydrous sodium sulfate and
concentrated under reduced pressure to give a crude product which was purified by column

chromatography using 5% EtOAc–Hexane as solvent system to afford pure 3e (6.4 g, 67%).
1
H NMR (400 MHz; CDCl ): δ 0.09 (s, 3H), 0.11 (s, 3H), 0.94 (s, 9H), 1.37 (d, J = 6.8 Hz,
3
3
H), 4.33 (q, J = 6.8 Hz, 1H), 7.25 (d, J = 15.6 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 7.47-7.54
+
79
(
m, 2H), 7.62 (d, J = 16.1 Hz, 1H), 7.71 (t, J = 1.5 Hz, 1H). ESI-MS m/z: 369.2 [M ( Br) +
+
81
1
4
], 371.1 [M ( Br) + 1].
.1.4. General method for stereoselective reduction of ketone to get 4a-f
To a stirred solution of ketone (4.18 mmol) in methanol (50 mL) was added cerium
chloride heptahydrate (4.60 mmol) and was cooled to −78 °C. To this suspension, sodium
borohydride (4.60 mmol) in methanol (12 mL) was added dropwise manner. The reaction
temperature was allowed to come to room temperature gradually and then stirred for
additional 1 h. The reaction mixture was quenched by the addition of sat. aq. NH Cl (60 mL)
4
and extracted with EtOAc (50 mL x 3). The combined organic layer was washed with water
(
50 mL), brine (50 mL), dried over anhydrous Na SO and concentrated under reduced
2 4
pressure to afford the crude product. The crude product was purified by column
chromatography.
4
.1.4.1.
(E, 3S, 4S)-4-(tert-butyl(dimethyl)silyl)oxy-1-[3-(trifluoro methyl)
1
phenyl]pent-1-en-3-ol (4a). Yield: 78%; H NMR (400 MHz, CDCl ): δ 0.09 (s, 3H), 0.10 (s,
3
3
4
3
4
H), 0.91 (s, 9H), 1.22 (d, J = 5.9 Hz, 3H), 2.73 (d, J = 2.9 Hz, 1H), 3.77 (quint. 1H), 3.98-
.05 (m, 1H), 6.25 (dd, J = 6.4 Hz, 16.1 Hz, 1H), 6.70 (d, J = 16.1 Hz, 1H), 7.39-7.57 (m,
+
+
H), 7.61 (s, 1H); ESI-MS m/z: 229.2 [M – 131] base peak, 383.1 [M + Na].
.1.4.2.
Synthesize of (E, 3S, 4S)-4-[tert-butyl(dimethyl)silyl]oxy-1-[4-
1
(
trifluoromethyl) phenyl]pent-1-en-3-ol (4b). Yield: 81% yield; H NMR (400 MHz, CDCl ):
3
δ 0.08 (s, 3H), 0.10 (s, 3H), 0.91 (s, 9H), 1.22 (d, J = 6.4 Hz, 3H), 2.76 (d, J = 4.9 Hz, 1H),
3
1
.79 (quint. 1H), 4.03 (q, J = 5.2 Hz, 1H), 6.28 (dd, J = 6.1 Hz, 15.9 Hz, 1H), 6.70 (d, J =
5.8 Hz, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.56 (d, J = 8.3 Hz, 2H); ESI-MS m/z
: 343.0
+
[
M – H O].
2
4
.1.4.3.
(E, 3S, 4S)-4-(tert-butyl(dimethyl)silyl)oxy-1-[2-(trifluoro methyl)
1
phenyl]pent-1-en-3-ol (4c). Yield: 89%; H NMR (400 MHz; CDCl ): δ 0.09 (s, 3H), 0.11 (s,
3
3
3
7
H), 0.91 (s, 9H), 1.21 (d, J = 6.2 Hz, 3H), 2.72 (d, J = 3.9 Hz, 1H, -OH), 3.78 (quint., 1H),
.98-4.07 (m, 1H), 6.14 (dd, J = 6.6 Hz, 15.9 Hz, 1H), 7.02 (d, J = 15.7 Hz, 1H), 7.34 (t, J =
+
.3 Hz, 1H), 7.49 (d, J = 7.3 Hz, 1H), 7.57-7.68 (m, 2H); ESI-MS m/z: 343.3 [M – 17] base
+
peak, 378.3 [M + H O].
2

4
.1.4.4.
(E, 3S, 4S)-4-(tert-butyl(dimethyl)silyl)oxy-1-(3-fluoro phenyl)pent-1-en-3-ol
1
(
4d). Yield: 88%; H NMR (400 MHz; CDCl ): δ 0.09 (s, 3H), 0.10 (s, 3H), 0.91 (s, 9H), 1.21
3
(d, J = 6.2 Hz, 3H), 2.71 (d, J = 4.7 Hz, 1H), 3.76 (quint. 1H), 4.00 (app. q, J = 4.8 Hz, 1H),
6
1
.18 (dd, J = 6.1 Hz, 15.9 Hz, 1H), 6.63 (d, J = 15.9 Hz, 1H), 6.92 (td, J = 2.4 Hz, 8.3 Hz,
1
3
H), 7.07 (d, J = 10.3 Hz, 1H), 7.13 (d, J = 7.5 Hz, 1H), 7.23-7.30 (m, 1H); C-NMR (100
2
MHz; CDCl ): δ −4.69, −4.15, 18.13, 20.19, 25.90 (3C), 72.03, 76.80, 112.93 (d, J = 21.8
3
FC
2
4
3
Hz), 114.44 (d, J = 21.0 Hz), 122.45 (d, J = 3.1 Hz), 130.07 (d, J = 7.8 Hz), 130.64
FC
FC
FC
4
3
1
(
d, J = 2.4 Hz), 130.70, 139.33 (d, J = 7.8 Hz), 163.19 (d, J = 244.5 Hz); ESI-MS
FC FC FC
+
+
m/z: 156.1 [½M + 1] base peak, 293.1 [M – H O].
2
4
.1.4.5.
(E, 3S, 4S)-1-(3-bromophenyl)-4-[tert-butyl(dimethyl)silyl] oxy-pent-1-en-3-ol
1
(
4e). Yield: 71%; H NMR (400 MHz; CDCl ): δ 0.09 (s, 3H), 0.10 (s, 3H), 0.91 (s, 9H), 1.20
3
(
d, J = 6.0 Hz, 3H), 2.71 (d, J = 4 Hz, 1H, -OH), 3.75 (quint., 1H), 3.76-4.03 (m, 1H), 6.17
dd, J = 6.1 Hz, 16.0 Hz, 1H), 6.60 (d, J = 15.8 Hz, 1H), 7.18 (t, J = 7.7 Hz, 1H), 7.29 (d, J =
(
+
79
7
.6 Hz, 1H), 7.36 (d, J = 7.7 Hz, 1H), 7.52 (s, 1H); ESI-MS m/z: 371.0 [M ( Br) + 1] , 373.0
+
81
[
M ( Br) + 1].
.1.4.6.
4
(E, 3S, 4S)-4-(tert-butyl(dimethyl)silyl)oxy-1-(5-ethyl-2-thienyl) pent-1-en-3-
1
ol (4f). Yield: 83%; H NMR (400 MHz; CDCl ): δ 0.09 (s, 6H), 0.91 (s, 9H), 1.19 (d, J = 5.8
3
Hz, 3H), 1.29 (t, J = 7.8 Hz, 3H), 2.65 (d, J = 4.4 Hz, 1H, -OH), 2.80 (q, J = 7.8 Hz, 2H),
3
1
.72 (quint., 1H), 3.88-3.95 (m, 1H), 5.87 (dd, J = 6.8 Hz, 15.6 Hz, 1H), 6.62 (d, J = 3.4 Hz,
1
3
H), 6.71 (d, J = 15.6 Hz, 1H), 7.74 (d, J = 3.4 Hz, 1H); C-NMR (100 MHz; CDCl ) : δ
3
−4.71, −4.20, 15.82, 18.10, 20.18, 23.68, 25.88 (3C), 72.15, 76.91, 123.61, 125.51, 125.87,
+
1
27.39, 139.61, 146.79; ESI-MS m/z: 309.2 [M – 17].
4
.1.4.7.
(E, 3S, 4S)-4-(tert-butyl(dimethyl)silyl)oxy-1-cyclohexyl-pent-1-en-3-ol (4g).
1
Yield: 94%; H NMR (400 MHz; CDCl ): δ 0.08 (s, 6H), 0.89 (s, 9H), 1.0-1.33 (m, 5H), 1.11
3
(d, J = 6.1 Hz, 3H), 1.60-1.77 (m, 5H), 1.90-2.0 (m, 1H), 2.59 (br. s, 1H), 3.62 (quint., 1H),
3
1
.72 (t, J = 6.6 Hz, 1H), 5.33 ( ddd, J = 1.2 Hz, 7.1 Hz, 15.4 Hz, 1H), 5.67 (dd, J = 6.3 Hz,
1
3
5.4 Hz, 1H); C-NMR (100 MHz; CDCl ) : δ −4.68, −4.09, 18.14, 20.10, 25.93 (3C), 26.13
3
+
(
2C), 26.28, 32.79, 32.84, 40.53, 72.32, 77.59, 126.79, 139.94; ESI-MS m/z: 321.4 [M +
Na] .
.1.4.8.
4
Methyl 3-[(E, 3S, 4S)-4-(tert-butyl(dimethyl)silyl)oxy-3-hydroxy-pent-1-enyl]
1
benzoate (4h). Yield: 87%; H NMR (400 MHz; CDCl ): δ 0.09 (s, 3H), 0.11 (s, 3H), 0.91 (s,
3
9
3
7
H), 1.21 (d, J = 6.0 Hz, 3H), 2.775 (d, J = 4 Hz, 1H, -OH), 3.78 (quint., 1H), 3.92 (s, 3H),
.99-4.05 (m, 1H), 6.26 (dd, J = 6.2 Hz, 15.9 Hz, 1H), 6.70 (d, J = 15.9 Hz, 1H), 7.38 (t, J =
13
.8 Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H), 7.90 (d, J = 7.5 Hz, 1H), 8.06 (s, 1H); C-NMR (100

MHz; (CDCl ): δ −4.68, −4.12, 18.15, 20.24, 25.90 (3C), 52.28, 72.08, 76.97, 127.59, 128.68,
3
+
1
4
5
28.73, 130.54, 130.56, 130.87, 130.92, 137.24, 167.12. ESI-MS m/z: 368.3 [M + H O] .
2
.1.5. General method for double bond reduction of olefin (4a-d and 4f-h) to get 5a-d and
f-h
To a solution of olefin (4a-d and 4f-h, 3.33 mmol) in EtOAc (72 mL) was added 10%
palladium on carbon (20% w/w) and stirred at RT under hydrogen gas maintained at 1
atmospheric pressure for 30 min. The progress of reaction was monitored by LCMS. After
completion of reaction, Pd–C was removed by filtration and the filtrate was evaporated to
give the crude product. The crude product was purified by column chromatography.
4
3
1
2
1
3
.1.5.1.
(3S, 4S)-4-(tert-butyl(dimethyl)silyl)oxy-1-[3-(trifluoromethyl) phenyl] pentan-
1
-ol (5a). Yield: 92%; H NMR (400 MHz, CDCl ): δ 0.08 (s, 3H), 0.09 (s, 3H), 0.90 (s, 9H),
3
.14 (d, J = 6.1 Hz, 3H), 1.68-1.75 (m, 2H), 2.45 (d, J = 5.1 Hz, 1H), 2.68-2.80 (m, 1H),
.88-2.98 (m, 1H), 3.28 (quint., 1H), 3.64 (quint., 1H), 7.37-7.42 (m, 2H), 7.44 (d, J = 3.6 Hz,
1
3
H), 7.46 (s, 1H). C-NMR (100 MHz; CDCl ): δ −4.79, −4.06, 18.09, 20.28, 25.89 (3C),
3
1
3
2.01, 35.10, 71.90, 74.85, 124.46 (q, J = 270.1 Hz), 125.34 (q, J = 3.1 Hz, 2C), 128.22
FC
FC
2
+
(
q, J = 32.5 Hz), 128.88 (2C), 146.52; ESI-MS m/z: 363.3 [M + 1].
FC
4
.1.5.2.
(3S, 4S)-4-(tert-butyl(dimethyl)silyl)oxy-1-[4-(trifluoromethyl) phenyl] pentan-
1
3
9
1
7
-ol (5b). Yield: 94%; H NMR (400 MHz; CDCl ): δ 0.097 (s, 3H), 0.103 (s, 3H), 0.92 (s,
3
H), 1.15 (d, J = 6.1 Hz, 3H), 1.72 (app. q, J = 7.4 Hz, 2H), 2.46 (br s, 1H), 2.71-2.82 (m,
H), 2.94 (quint., 1H) 3.29 (d, J = 5.4 Hz, 1H), 3.65 (quint., 1H), 7.33 (d, J = 7.8 Hz, 2H),
1
3
.55 (d, J = 8.1 Hz, 2H); C-NMR (100 MHz; CDCl ): δ −4.79, −4.06, 18.09, 20.28, 25.89
3
1
3
(
3C), 32.01, 35.10, 71.90, 74.85, 124.46 (q, J = 270.1 Hz), 125.34 (q, J = 3.1 Hz, 2C),
FC FC
2
+
1
4
28.22 (q, J = 32.5 Hz), 128.88 (2C), 146.52. ESI-MS m/z: 363.3 [M + 1].
FC
.1.5.3.
(3S, 4S)-4-(tert-butyl(dimethyl)silyl)oxy-1-[2-(trifluoro methyl) phenyl]
1
pentan-3-ol (5c). Yield: 76% ; H NMR (400 MHz; CDCl ): δ 0.07 (s, 3H), 0.08 (s, 3H), 0.90
3
(s, 9H), 1.15 (d, J = 6.4 Hz, 3H), 1.66-1.75 (m, 2H), 2.47 (d, J = 3.4 Hz, 1H), 2.79-2.89 (m,
1
H), 3.0-3.10 (m, 1H), 3.31-3.39 (m, 1H), 3.65 (quint., 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.37 (d,
1
3
J = 7.6 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H); C-NMR (100 MHz;
1
CDCl ): δ −4.79, −4.03, 18.11, 20.27, 25.91(3C), 29.23, 35.65, 71.83, 75.42, 124.75 (q, J
3
FC
3
2
=
274.0 Hz), 126.05 (q, J = 5.5 Hz, 2C), 128.56 (q, J = 29.6 Hz), 131.39, 131.84,
FC FC
+
1
41.35 ESI-MS m/z: 345.1 [M – H O] .
2
4
.1.5.4.
(3S, 4S)-4-(tert-butyl(dimethyl)silyl)oxy-1-(3-fluorophenyl) pentan-3-ol (5d).
1
Yield: 83%; H NMR (400 MHz; CDCl ): δ 0.08 (s, 3H), 0.09 (s, 3H), 0.90 (s, 9H), 1.14 (d, J
3
=
6.1 Hz, 3H), 1.65-1.74 (m, 2H), 2.43 (d, J = 3.7 Hz, 1H), 2.64-2.74 (m, 1H), 2.86 (quint.,

1
7
4
H), 3.24-3.32 (m, 1H), 3.63 (quint., 1H), 6.84-6.94 (m, 2H), 6.98 (d, J = 7.6 Hz, 1H), 7.19-
+
.25 (m, 1H); ESI-MS m/z: 335.1 [M + Na] .
.1.5.5.
(3S, 4S)-4-(tert-butyl(dimethyl)silyl)oxy-1-(5-ethyl-2-thienyl) pentan-3-ol (5f).
1
Yield: 85%; H NMR (400 MHz; CDCl ): δ 0.09 (s, 6H), 0.90 (s, 9H), 1.15 (d, J = 6.1 Hz,
3
3
2
1
2
H), 1.28 (t, J = 7.3 Hz, 3H), 1.70-1.78 (m, 2H), 2.39 (br s, 1H), 2.78 (q, J = 7.3 Hz, 2H),
.82-2.91 (m, 1H), 2.99 (quint., 1H), 3.31-3.38 (m, 1H), 3.65 (quint., 1H), 6.57 (d, J = 3.4 Hz,
1
3
H), 6.60 (d, J = 3.4 Hz, 1H); C-NMR (100 MHz; CDCl ): δ −4.71, −4.03, 16.04, 18.15,
3
0.34, 23.56, 25.96 (3C), 26.59, 35.73, 71.89, 74.92, 122.83, 123.82, 142.58, 145.15. ESI-MS
+
m/z: 329.2 [M + 1].
4
.1.5.6.
(3S, 4S)-4-(tert-butyl(dimethyl)silyl)oxy-1-cyclohexyl-pentan-3-ol (5g).
1
Yield: 87%; H NMR (400 MHz; CDCl ): δ 0.08 (s, 3H), 0.08 (s, 3H), 0.89 (s, 9H), 1.14 (d, J
3
=
6.1 Hz, 3H), 1.15-1.48 (m, 10H), 1.58-1.74 (m, 5H), 2.37 (br s, 1H), 3.20-3.27 (m, 1H),
1
3
3
2
.62 (quint., 1H); C-NMR (100 MHz; CDCl ): δ −4.70, −3.99, 18.16, 20.44, 25.97 (3C),
3
+
6.53, 26.56, 26.86, 30.94, 33.34, 33.60, 33.69, 37.89, 71.83, 76.28. ESI-MS m/z: 301.3 [M
+
1].
4
.1.5.7.
Methyl 3-[(3S, 4S)-4-(tert-butyl(dimethyl)silyl)oxy-3-hydroxy-pentyl] benzoate
1
(
5h). Yield: 72%; H NMR (400 MHz, CDCl ): δ 0.09 (s, 6H), 0.90 (s, 9H), 1.14 (d, J = 5.9
3
Hz, 3H), 1.68-1.78 (m, 2H), 2.47 (d, J = 4.6 Hz, 1H), 2.69-2.81 (m, 1H), 2.87-2.97 (m, 1H),
3
1
4
.28 (quint., 1H), 3.64 (quint., 1H), 3.91 (s, 3H), 7.35 (t, J = 7.4 Hz, 1H), 7.42 (d, J = 7.3 Hz,
+
H), 7.87 (d, J = 7.8 Hz, 1H), 7.90 (s, 1H); ESI-MS m/z: 370.3 [M + H O].
2
.1.5.8.
(3S, 4S)-1-(3-bromophenyl)-4-[tert-butyl(dimethyl)silyl]oxy-pentan-3-ol (5e).
To a solution of (E, 3S, 4S)-1-(3-bromophenyl)-4-[tert-butyl(dimethyl)silyl]oxy-pent-1-en-3-
ol (4e, 3.0 g, 8.07 mmol) in degassed solvent THF:tert-butyl alcohol (1: 1, 58 mL)
Wilkinson’s catalyst (0.45 g, 15 % w/w) was added. The solution was stirred at RT under
hydrogen gas maintained at 1 atm. pressure for 16 h. After reaction completion the mixture
was filtered through a short pad of alumina and washed thoroughly with ethyl acetate. The
filtrate was evaporated to give crude product. The crude product was purified by column
chromatography using 10–30% EtOAc–Hexane as solvent system to afford pure 5e (2.5 g,
1
8
3
3
3% yield); H NMR (400 MHz; CDCl ): δ 0.08 (s, 6H), 0.90 (s, 9H), 1.13 (d, J = 6.3 Hz,
3
H), 1.62-1.74 (m, 2H), 2.44 (d, J = 4.3 Hz, 1H -OH), 2.61-2.72 (m, 1H), 2.84 (quint., 1H)
1
3
.27 (quint., 1H), 3.63 (quint., 1H), 7.11-7.19 (m, 2H), 7.28-7.35 (m, 1H), 7.36 (s, 1H); C-
NMR (100 MHz; CDCl ): δ −4.71, −3.99, 18.15, 20.36, 25.95 (3C), 31.88, 35.21, 71.97,
3
+
79
7
3
4.92, 122.54, 127.33, 129.01, 130.04, 131.67, 144.80. ESI-MS m/z: 373.0 [M ( Br) + 1],
+
81
75.0 [M ( Br) + 1].

4
.1.6. General method for Mitsunobu reaction to get 6a-f
To a solution of corresponding alcohol (0.55 mmol) in THF (15 mL) was added tert-
butyl N-tert-butoxycarbonyl-N-(9H-purin-6-yl) carbamate (0.66 mmol) followed by
triphenylphosphine (1.1 mmol). This reaction mixture was cooled to 0 °C and DEAD (1.1
mmol) was added dropwise manner. The reaction mixture was stirred at room temperature
for 16 h. The reaction mixture was quenched by the addition of water (30 mL). Aqueous layer
was extracted with EtOAc (35 mL x 3). The combined organic layer was washed with brine
(
30 mL), dried over anhydrous Na SO , and concentrated under reduced pressure to afford
2 4
crude product which was purified by column chromatography
.1.6.1. tert-butyl N-tert-butoxycarbonyl-N-[9-[(1R, 2S)-2-(tert-butyl
4
(dimethyl)silyl)oxy-1-[2-[3-(trifluoromethyl)phenyl]ethyl] propyl]purin-6-yl]carbamate (6a).
1
Yield: 52%; H NMR (400 MHz; CDCl ): δ −0.13 (s, 3H), 0.01 (s, 3H), 0.87 (s, 9H), 1.13 (d,
3
J = 6.1 Hz, 3H), 1.43 (s, 18H), 2.36-2.64 (m, 4H), 4.10-4.16 (m, 1H), 4.43-4.50 (m, 1H), 7.18
(d, J = 7.6 Hz, 1H), 7.23 (s, 1H), 7.35 (t, J = 7.6 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H), 8.17 (s,
+
1
H), 8.72 (s, 1H); ESI-MS m/z: 680.4 [M + 1].
4
.1.5.2.
tert-butyl N-tert-butoxycarbonyl-N-[9-[(1R, 2S)-2-(tert-butyl
(dimethyl)silyl)oxy-1-[2-[4-(trifluoromethyl)phenyl]ethyl]propyl]purin-6-yl]carbamate (6b).
1
Yield: 66%; H NMR (400 MHz; CDCl ): δ −0.12 (s, 3H), 0.02 (s, 3H), 0.88 (s, 9H), 1.12 (d,
3
J = 6.4 Hz, 3H), 1.44 (s, 18H), 2.35-2.64 (m, 4H), 4.20-4.30 (m, 1H), 4.43-4.51 (m, 1H), 7.13
(d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.1 Hz, 2H), 8.14 (s, 1H), 8.86 (s, 1H). ESI-MS m/z: 680.5
+
[
M + 1].
4
.1.6.3.
tert-butyl N-tert-butoxycarbonyl-N-[9-[(1R,2S)-2-(tert-
butyl(dimethyl)silyl)oxy-1-[2-[2-(trifluoromethyl)phenyl]ethyl] propyl]purin-6-yl]carbamate
1
(
6c). Yield: 58%; H NMR (400 MHz; CDCl ): δ −0.16 (s, 3H), 0.01 (s, 3H), 0.86 (s, 9H),
3
1
1
7
.15 (d, J = 6.2 Hz, 3H), 1.41 (s, 18H), 2.32-2.45 (m, 1H), 2.47-2.59 (m, 2H), 2.62-2.74 (m,
H), 4.15 (quint., 1H), 4.56-4.64 (m, 1H), 7.16 (d, J = 7.9 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H),
13
.44 (t, J = 7.5 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 8.19 (s, 1H), 8.87 (s, 1H); C-NMR (100
MHz; CDCl ): δ −4.85, −4.20, 18.02, 20.62, 25.92 (3C), 27.91 (6C), 29.79, 30.03, 62.20,
3
1
3
7
0.25, 83.62 (2C), 124.65 (q, J = 274.1 Hz), 126.39 (q, J = 5.4 Hz), 126.69, 128.58 (q,
FC FC
2
4
J = 29.5 Hz), 129.32, 131.19, 132.14, 139.38 (q, J = 1.5 Hz), 144.39, 150.35, 150.67,
FC
FC
+
1
52.04, 153.84 (2C); ESI-MS m/z: 680.2 [M + 1].
.1.6.4. tert-butyl N-tert-butoxycarbonyl-N-[9-[(1R,2S)-2-(tert-butyl (di
4
methyl)silyl)oxy-1-[2-(3-fluorophenyl)ethyl]propyl]purin-6-yl]carbamate (6d). Yield:

1
5
4%; H NMR (400 MHz; CDCl ): δ −0.14 (s, 3H), 0.01 (s, 3H), 0.87 (s, 9H), 1.11 (d, J = 6.2
3
Hz, 3H), 1.42 (s, 18H), 2.30-2.43 (m, 2H), 2.45-2.62 (m, 2H), 4.15 (quint., 1H), 4.42-4.48 (m,
1
H), 6.73 (t, J = 8.4 Hz, 1H), 6.74 (d, J = 7.1 Hz, 1H), 6.88 (td, J = 2.2 Hz, 8.8 Hz, 1H), 7.19
+
(
dd, J = 6.2 Hz, 8.0 Hz, 1H), 8.12 (s, 1H), 8.84 (s, 1H); ESI-MS m/z: 630.2 [M + 1].
4
.1.6.5.
tert-butyl N-[9-[(1R,2S)-1-[2-(3-bromophenyl)ethyl]-2-[tert-
butyl(dimethyl)silyl]oxy -propyl]purin-6-yl]-N-tert-butoxycarbonyl-carbamate (6e). Yield:
1
6
6
3%; H NMR (400 MHz; CDCl ): δ −0.14 (s, 3H), −0.01 (s, 3H), 0.87 (s, 9H), 1.11 (d, J =
3
.2 Hz, 3H), 1.42 (s, 18H), 2.26-2.60 (m, 4H), 4.14 (quint., 1H), 4.41-4.48 (m, 1H), 6.91 (d, J
=
7.9 Hz, 1H), 7.10 (t, J = 8.0 Hz, 1H), 7.13 (s, 1H), 7.34 (d, J = 7.9 Hz, 1H), 8.13 (s, 1H),
1
3
8
.86 (s, 1H); C-NMR (100 MHz; CDCl ): δ −5.01, −4.38, 17.85, 20.47, 25.76 (3C), 27.74
3
(6C), 29.13, 31.71, 61.14, 69.94, 83.56 (2C), 122.61, 126.86, 128.98, 129.52, 130.11, 131.35,
+
79
1
1
42.39, 144.31, 150.22, 150.37, 151.91, 153.53 (C=O, 2C); ESI-MS m/z: 690.1 [M ( Br) +
+
+ 81
] , 692.1 [M ( Br) + 1].
.1.6.6. tert-butyl N-tert-butoxycarbonyl-N-[9-[(1R,2S)-2-(tert-butyl
4
(dimethyl)silyl)oxy-1-[2-(5-ethyl-2-thienyl)ethyl]propyl] purin-6-yl]carbamate (6f). Yield:
1
7
6
4
8
4
1; H NMR (400 MHz, CDCl ): δ −0.133 (s, 3H), −0.01 (s, 3H), 0.88 (s, 9H), 1.12 (d, J =
3
.4 Hz, 3H), 1.31 (t, J = 7.1 Hz, 3H), 1.42 (s, 18H), 2.35-2.68 (m, 4H), 4.14-4.22 (m, 1H),
.32 (q J = 7.1 Hz, 2H), 4.48-4.54 (m, 1H), 6.37 (d, J = 3.1 Hz, 1H), 6.53 (d, J = 3.4 Hz, 1H),
+
.11 (s, 1H), 8.84 (s, 1H); ESI-MS m/z: 646.5 [M + 1].
.1.6.7.
tert-butyl N-tert-butoxycarbonyl-N-[9-[(1R,2S)-2-(tert-
butyl(dimethyl)silyl)oxy-1-(2-cyclohexylethyl)propyl]purin-6-yl]carbamate (6g). Yield: 63% ;
1
H NMR (400 MHz; CDCl ): δ −0.15 (s, 3H), −0.01 (s, 3H), 0.70-0.85 (m, 3H), 0.88 (s, 9H),
3
0
2
1
4
.98-1.11 (m, 3H), 1.16 (d, J = 6.4 Hz, 3H), 1.41 (s, 18H), 1.52-1.68 (m, 7H), 2.02-2.15 (m,
+
H), 4.14 (quint., 1H), 4.47 (quint., 1H), 8.10 (s, 1H), 8.84 (s, 1H); ESI-MS m/z: 618.6 [M +
].
.1.6.8.
Methyl 3-[(3R,4S)-3-[6-[bis(tert-butoxycarbonyl)amino] purin-9-yl]-4-[tert-
1
butyl (dimethyl)silyl]oxy-pentyl]benzoate (6h). Yield: 59%; H NMR (400 MHz; CDCl ): δ
3
−0.13 (s, 3H), −0.01 (s, 3H), 0.87 (s, 9H), 1.12 (d, J = 6.3 Hz, 3H), 1.44 (s, 18H), 2.35-2.47
(m, 2H), 2.48-2.63 (m, 2H), 3.90 (s, 3H), 4.15 (quint., 1H), 4.43-4.51 (m, 1H), 7.17 (d, J =
7
.6 Hz, 1H), 7.32 (t, J = 7.5 Hz, 1H), 7.70 (s, 1H), 7.88 (d, J = 7.9 Hz, 1H), 8.15 (s, 1H), 8.85
+
(
s, 1H); ESI-MS m/z: 670.3 [M + 1].
4
.1.7. General method for Diboc and TBDMS deprotection to get 7a-f
To a solution of protected compound 6a-h (0.17 mmol) in 1, 4-dioxane (2 mL) was
added slowly 4M HCl in dioxane (1.76 mmol) at room temperature. The reaction mixture was

stirred for 16 h at RT. After completion of reaction, the volatiles were evaporated under
reduced pressure and residue was netrualized with sat. aq. NaHCO solution. Aquous layer
3
was extracted with EtOAc (15 mL x 3). The combined organic layer was washed with water
(
20 mL), brine (20 mL), dried over anhydrous Na SO , and concentrated under reduced
2 4
pressure to afford crude product. The crude product was purified by column chromatography.
4
.1.7.1.
(2S, 3R)-3-(6-aminopurin-9-yl)-5-[3-(trifluoromethyl)phenyl] pentan-2-ol
1
(
7a). Yield: 90%; H NMR (400 MHz; CD OD): δ 1.07 (d, J = 6.4 Hz, 3H), 2.42-2.67 (m,
3
1
3
4
H), 4.07 (quint., 1H), 4.33-4.42 (m, 1H), 7.24-7.42 (m, 4H), 8.17 (s, 1H), 8.19 (s, 1H); C-
3
NMR (100 MHz; (CD OD): δ 20.26, 31.06, 33.26, 62.42, 70.19, 119.99, 123.88 (q, J = 3.9
3
FC
1
3
2
Hz), 125.64 (q, J = 271.7 Hz), 126.0 (q, J = 3.9 Hz), 130.92, 131.52 (q, J = 31.9 Hz),
FC
FC
FC
+
1
33.17, 141.89, 143.27, 150.89, 153.54, 157.27; ESI-MS m/z: 366.2 [M + 1].
.1.7.2. (2S, 3R)-3-(6-aminopurin-9-yl)-5-[4-(trifluoromethyl)phenyl] pentan-2-ol (7b).
4
1
Yield: 93 %; H NMR (400 MHz; (CD ) SO): δ 0.87 (d, J = 6.1 Hz, 3H), 2.32-2.48 (m, 4H),
3
2
4
.00 (q, J = 6.2 Hz, 1H), 4.20-4.28 (m, 1H), 5.20 (d, J = 5.3 Hz, 1H), 7.22 (br s, 2H), 7.32 (d,
1
3
J = 7.9 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 8.12 (s, 1H), 8.22 (s, 1H); C-NMR (100 MHz;
1
(
CD ) SO): δ 20.22, 30.34, 31.63, 60.52, 67.85, 118.80, 124.24 (q, J = 271.7 Hz,), 125.03
3
2
Fc
3
2
(
q, J = 3.8 Hz, 2C), 126.68 (q, J = 32.0 Hz), 129.08 (2C), 140.21, 146.05, 149.86,
Fc Fc
+
1
4
52.28, 155.96; ESI-MS m/z : 366.2 [M + 1]; HPLC purity: 98 %
.1.7.3.
(2S, 3R)-3-(6-aminopurin-9-yl)-5-[2-(trifluoromethyl)phenyl] pentan-2-ol
1
(
7c). Yield: 91%; H NMR (400 MHz; CD OD): δ 1.11 (d, J = 6.4 Hz, 3H), 2.38-2.52 (m,
3
3
7
H), 2.65-2.78 (m, 1H), 4.13 (quint., 1H), 4.48 (q, J = 6.4 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H),
.33 (d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H), 7.57 (d, J = 8.1 Hz, 1H),8.21 (s, 1H), 8.25
1
3
(
s, 1H); C-NMR (100 MHz; CD OD): δ 20.39, 30.47, 32.08, 62.93, 70.03, 120.06, 125.98
3
1
3
2
(
q, J = 271.7 Hz, 1C), 126.88 (q, J = 5.4 Hz, 1C), 127.57, 129.13 (q, J = 28.7 Hz, 1C),
Fc
Fc
Fc
+
23
1
32.44, 133.29, 140.95, 141.91, 151.01, 153.59,157.34; ESI-MS m/z: 366.2 [M + 1]; [α] :
D
+
2.8 (c 0.25, CH OH); HPLC purity: 99 %
3
4
.1.7.4.
(2S, 3R)-3-(6-aminopurin-9-yl)-5-(3-fluorophenyl)pentan-2-ol (7d). Yield:
1
8
5 %; H NMR (400 MHz; CD OD): δ 1.07 (d, J = 6.4 Hz, 3H), 2.40-2.58 (m, 4H), 4.09
3
(quint., 1H), 4.33-4.40 (m, 1H), 6.76 (dd, J =1.8 Hz, 10.0 Hz, 1H), 6.79-6.85 (m, 2H), 7.16 (q,
1
3
J = 7.9 Hz, 1H), 8.18 (s, 1H), 8.19 (s, 1H). C-NMR (100 MHz; CD OD): δ 20.26, 31.07,
3
2
2
3
3.15, 62.49, 70.17, 113.69 (d, J = 20.9 Hz), 115.97 (d, J = 21.7 Hz), 120.37, 125.17 (d,
FC FC
4
3
3
J = 2.3 Hz), 130.94 (d, J = 8.5 Hz), 141.96, 144.76 (d, J = 7.0 Hz), 150.93, 153.48,
FC
FC
FC

1
+
23
1
57.26, 164.19 (d, J = 243.1 Hz); ESI-MS m/z: 316.3 [M + 1]; [α] : +25.2 (c 0.25,
FC
D
CH OH); HPLC purity: 95 %.
3
4
.1.7.5.
(2S, 3R)-3-(6-aminopurin-9-yl)-5-(3-bromophenyl)pentan-2-ol (7e). Yield:
1
7
5 %; H NMR (400 MHz, CD OD): δ 1.07 (d, J = 6.3 Hz, 3H), 2.40-2.58 (m, 4H), 4.07
3
(quint., 1H), 4.33-4.40 (m, 1H), 6.99 (d, J = 7.8 Hz, 1H), 7.07 (t, J = 7.8 Hz, 1H), 7.11 (s,
1
3
1
2
1
H), 7.23 (d, J = 8.3 Hz, 1H), 8.16 (s, 1H), 8.19 (s, 1H); C-NMR (100 MHz; CD OD): δ
3
0.22, 30.98, 33.13, 62.40, 70.20, 119.98, 123.23, 128.16, 130.10, 131.05, 132.38, 141.89,
+
79
+ 81
44.48, 150.85, 153.52, 157.24; ESI-MS m/z: 376.2 [M ( Br) + 1] , 378.1 [M ( Br) + 1];
HPLC purity: 95 %.
4
8
2
2
1
1
1
.1.7.6.
(2S, 3R)-3-(6-aminopurin-9-yl)-5-(5-ethyl-2-thienyl)pentan-2-ol (7f). Yield:
1
7 %; H NMR (400 MHz; CDCl3): δ 1.28 (t, J = 7.6 Hz, 3H), 1.30 (d, J = 4.8 Hz, 3H), 2.23-
.33 (m, 1H), 2.42-2.58 (m, 2H), 2.67-2.74 (m, 1H), 2.78 (q, J = 7.6 Hz, 2H), 4.21-4.32 (m,
H), 5.79 (br s, 2H), 6.53 (d, J = 3.3 Hz, 1H), 6.58 (d, J = 3.3 Hz, 1H), 7.72 (s, 1H), 8.32 (s,
1
3
H); C-NMR (100 MHz; CDCl3): δ 16.17, 20.49, 23.69, 26.73, 29.08, 62.32, 69.71, 120.07,
+
23.14, 124.68, 140.39, 140.96, 146.13, 149.74, 152.58, 156.15; ESI-MS m/z: 332.2 [M +
]; HPLC purity: 93 %
4
.1.7.7.
(2S, 3R)-3-(6-aminopurin-9-yl)-5-cyclohexyl-pentan-2-ol (7g). Yield: 86 %;
1
H NMR (400 MHz; CDCl3): δ 0.68-0.94 (m, 3H), 1.02-1.22 (m, 5H), 1.30 (d, J = 6.4 Hz,
H), 1.56-1.70 (m, 2H), 1.86-2.25 (m, 5H), 4.15-4.32 (m, 2H), 5.79 (br s, 2H), 7.77 (s, 1H),
3
1
3
8
3
.31 (s, 1H); C-NMR (100 MHz; CDCl ): δ 20.34, 25.09, 26.35, 26.42, 26.67, 30.09, 33.56,
3
+
4.06, 37.54, 63.36, 69.58, 119.75, 140.53, 149.88, 152.52, 156.08; ESI-MS m/z: 304.2 [M
+
1]; HPLC purity: 98 %.
.1.7.7.1. Methyl 3-[(3R, 4S)-3-(6-aminopurin-9-yl)-4-hydroxy-pentyl] benzoate (7h).
Yield: 81%; H NMR (400 MHz, CDCl3): δ 1.28 (d, J = 6.2 Hz, 3H), 2.26-2.40 (m, 1H),
.45-2.62 (m, 3H), 3.91 (s, 3H), 4.19-4.31 (m, 2H), 4.95 (br s, 1H), 5.72 (br s, 2H), 7.23-7.27
4
1
2
(m, 1H), 7.33 (t, J = 7.5 Hz, 1H), 7.72-7.78 (m, 2H), 7.86 (d, J = 7.5 Hz, 1H), 8.33 (s, 1H);
1
3
C-NMR (100 MHz; CDCl ): δ 20.29, 28.95, 32.19, 52.17, 62.19, 69.56, 119.78, 127.64,
3
1
28.63, 129.33, 130.45, 132.95, 140.35, 140.69, 149.84, 152.60, 155.97, 167.11; ESI-MS
+
m/z: 356.3 [M + 1]. HPLC purity: 99 %.
4
.1.8. tert-butyl N-tert-butoxycarbonyl-N-[9-[(1R,2S)-2-[tert-butyl(dimethyl) silyl]oxy-1-[2-
3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]ethyl]propyl]purin-6-yl] carbamate
8).
[
(

A mixture of intermediate 6e (1.9 g, 2.75 mmol), bis(pinacolato)diboron (1.04 g, 4.12
mmol) and potassium acetate (0.67 g, 6.87 mmol) in dioxane (35 mL) was degassed with
Argon gas for 30 min. [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex
with dichloromethane (0.22 g, 0.27 mmol) was added and the degassing continued again for
further 15 min. Reaction mixture was refluxed for 3 h. Reaction progress was monitored by
LCMS. After completion of the reaction, this mixture was passed through a short celite pad to
remove insoluble material. The solvent was then removed under reduced pressure to give
crude product. The crude product was purified by column chromatography using 20 % ethyl
1
acetate–hexane as solvent system to afford pure 8 (1.8 g, 88 %). H NMR (400 MHz; CDCl ):
3
δ –0.14 (s, 3H), –0.02 (s, 3H), 0.87 (s, 9H), 1.12 (d, J = 6.2 Hz, 3H), 1.34 (s, 12H), 1.42 (s,
1
8H), 2.30-2.61 (m, 4H), 4.16 (quint., 1H), 4.43-4.52 (m, 1H), 7.09 (d, J = 7.7 Hz, 1H), 7.27
1
3
(
t, J = 7.3 Hz, 1H), 7.49 (s, 1H), 7.65 (d, J = 7.3 Hz, 1H), 8.15 (s, 1H), 8.85 (s, 1H); C-
NMR (100 MHz; CDCl ): δ –5.02, –4.42, 17.86, 20.42, 24.84 (4C), 25.79 (3C), 27.75 (6C),
3
2
1
4
9.38, 32.04, 61.36, 69.92, 83.49 (2C), 83.79 (2C), 127.99, 128.57, 129.07, 131.17, 132.79,
+
34.57, 139.40, 144.53, 150.17, 150.29, 151.84, 153.63 (2C); ESI-MS m/z: 738.2 [M + 1].
.1.9. General method for Suzuki reaction to get 9a-c
A mixture of boronate ester 8 (0.33 mmol), heteroaryl halide (0.44 mmol) and
potassium carbonate (0.84 mmol) in dioxane:water (4:1, 6.25 mL) was degassed with Argon
gas for 10 min. [1,1′-Bis(diphenylphosphino)ferrocene]dichloro palladium(II), complex with
dichloromethane (0.05 mmol) was added and the degassing continued again for further 5 min.
Reaction mixture was heated at 90 °C for 3-5 h. Reaction progress was monitored by TLC.
After reaction completion the mixture was passed through celite pad to remove insoluble
material. The solvent was then removed under reduced pressure to give crude product. The
crude product was purified by column chromatography.
4
.1.9.1.
tert-butyl N-tert-butoxycarbonyl-N-[9-[(1R,2S)-2-[tert-
butyl(dimethyl)silyl]oxy-1-[2-(3-thiazol-2-ylphenyl)ethyl]propyl]purin-6-yl]carbamate (9a).
1
Yield: 54%; H NMR (400 MHz; CDCl ): δ –0.13 (s, 3H), –0.02 (s, 3H), 0.87 (s, 9H), 1.12 (d,
3
J = 6.4 Hz, 3H), 1.43 (s, 18H), 2.37-2.68 (m, 4H), 4.15 (quint., 1H), 4.48-4.56 (m, 1H), 7.05
(d, J = 7.3 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.33 (d, J = 3.4 Hz, 1H), 7.67 (s, 1H), 7.79 (d, J
+
=
7.8 Hz, 1H), 7.86 (d, J = 3.0 Hz, 1H), 8.17 (s, 1H), 8.84 (s, 1H); ESI-MS m/z: 695.1 [M +
1
].
.1.9.2.
4
tert-butyl N-tert-butoxycarbonyl-N-[9-[(1R, 2S)-2-[tert-butyl
(dimethyl)silyl]oxy-1-[2-[3-(2-pyridyl)phenyl]ethyl]propyl] purin-6-yl]carbamate (9b). Yeld:

1
5
6
6%; H NMR (400 MHz; CDCl ): δ –0.14 (s, 3H), –0.02 (s, 3H), 0.86 (s, 9H), 1.12 (d, J =
3
.2 Hz, 3H), 1.43 (s, 18H), 2.39-2.50 (m, 2H), 2.52-2.69 (m, 2H), 4.16 (quint., 1H), 4.49-4.56
(m, 1H), 7.06 (d, J = 7.9 Hz, 1H), 7.32 (ddd, J = 1.1 Hz, 4.9 Hz, 7.5 Hz, 1H), 7.36 (t, J = 7.7
Hz, 1H), 7.63-7.68 (m, 2H), 7.74 (td, J = 2.0 Hz, 8.0 Hz, 1H), 7.81 (dt, J = 1.6 Hz, 8.2 Hz,
1
3
1
4
1
1
4
H), 8.18 (s, 1H), 8.66-8.69 (m, 1H), 8.82 (s, 1H); C-NMR (100 MHz; CDCl ): δ –5.02, –
3
.41, 17.87, 20.45, 25.77 (3C), 27.76 (6C), 29.67, 32.18, 61.26, 69.96, 83.55 (2C), 120.61,
22.19, 125.01, 126.98, 128.86, 129.01, 129.07, 136.78, 139.67, 140.65, 144.58, 149.60,
+
50.24, 150.31, 151.85, 153.63 (2C), 157.19; ESI-MS m/z: 689.2 [M + 1].
.1.9.3.
tert-butyl N-tert-butoxycarbonyl-N-[9-[(1R, 2S)-2-[tert-butyl
(dimethyl)silyl]oxy-1-[2-(3-pyrazin-2-ylphenyl)ethyl]propyl] purin-6-yl]carbamate (9c).
1
Yield: 61%; H NMR (400 MHz; CDCl ): δ –0.13 (s, 3H), –0.01 (s, 3H), 0.87 (s, 9H), 1.12 (d,
3
J = 6.2 Hz, 3H), 1.43 (s, 18H), 2.40-2.53 (m, 2H), 2.55-2.68 (m, 2H), 4.17 (quint., 1H), 4.48-
4
.56 (m, 1H), 7.12 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.72 (s, 1H), 7.83 (d, J = 7.8
Hz, 1H), 8.18 (s, 1H), 8.52 (d, J = 2.4 Hz, 1H), 8.62 (dd, J = 1.7 Hz, 2.4 Hz, 1H), 8.83 (s,
1
3
1
2
1
1
4
H), 9.00 (d, J = 1.5 Hz, 1H); C-NMR (100 MHz; CDCl ): δ –4.99, –4.38, 17.87, 20.48,
3
5.77 (3C), 27.76 (6C), 29.67, 32.15, 61.26, 69.99, 83.59 (2C), 124.95, 126.99, 129.02,
29.30, 129.86, 136.64, 141.13, 142.21, 142.97, 144.14, 144.44, 150.27, 150.34, 151.89,
+
52.60, 153.60 (2C); ESI-MS m/z: 690.2 [M + 1].
.1.10. Synthesis of the biaryl derivatives 10a-c
Diboc and TBDMS deprotection of 9a-c was carried employing general method
described for synthesis of 7a-f. All crude compounds were purified by column
chromatography.
4
.1.10.1.
(2S, 3R)-3-(6-aminopurin-9-yl)-5-(3-thiazol-2-ylphenyl) pentan-2-ol (10a).
1
Yield: 77 %; H NMR (400 MHz; CD OD): δ 1.07 (d, J = 6.4 Hz, 3H), 2.46-2.66 (m, 4H),
3
4
1
8
1
1
4
.07 (quint., 1H), 4.36-4.44 (m, 1H), 7.16 (d, J = 7.8 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.49 (s,
H), 7.56 (d, J = 3.4 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.82 (d, J = 2.9 Hz, 1H), 8.11 (s, 1H),
1
3
.18 (s, 1H); C-NMR (100 MHz; CD OD): δ 20.16, 30.85, 33.36, 62.36, 70.27, 119.90,
3
20.80, 125.31, 127.48, 130.23, 131.37, 134.27, 141.91, 142.95, 144.19, 150.77, 153.43,
+
57.07, 170.12; ESI-MS m/z : 381.2 [M + 1]; HPLC purity: 98 %.
.1.10.2.
(2S, 3R)-3-(6-aminopurin-9-yl)-5-[3-(2-pyridyl)phenyl]pentan-2-ol (10b).
1
Yield: 75%; H NMR (400 MHz; CD OD): δ 1.07 (d, J = 6.3 Hz, 3H), 2.48-2.65 (m, 4H),
3
4
7
1
.07 (quint., 1H), 4.38-4.45 (m, 1H), 7.14 (d, J = 7.6 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.33-
.36 (m, 1H), 7.51 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.87 (td, J =
1
3
.9 Hz,7.8 Hz, 1H), 8.09 (s, 1H), 8.19 (s, 1H), 8.55 (dd, J = 1.0 Hz, 5.9Hz, 1H); C-NMR