6-Oxo and 6-thio purine analogs as antimycobacterial agents

Article abstract of DOI:10.1016/j.bmc.2013.01.054

6-Oxo and 6-thio analogs of purine were prepared based on the initial activity screening of a small, diverse purine library against Mycobacterium tuberculosis (Mtb). Certain 6-oxo and 6-thio-substituted purine analogs described herein showed moderate to good inhibitory activity. N ⁹-substitution apparently enhances the anti-mycobacterial activity in the purine series described herein. Several 2-amino and 2-chloro purine analogs were also synthesized that showed moderate inhibitory activity against Mtb.

Full text of DOI:10.1016/j.bmc.2013.01.054

Bioorganic & Medicinal Chemistry 21 (2013) 1685–1695
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
6-Oxo and 6-thio purine analogs as antimycobacterial agents
⇑
Ashish K. Pathak , Vibha Pathak, Lainne E. Seitz, William J. Suling, Robert C. Reynolds
Drug Discovery Division, Southern Research Institute, 2000 9th Avenue South, Birmingham, AL 35205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
6-Oxo and 6-thio analogs of purine were prepared based on the initial activity screening of a small,
diverse purine library against Mycobacterium tuberculosis (Mtb). Certain 6-oxo and 6-thio-substituted
purine analogs described herein showed moderate to good inhibitory activity. N9-substitution apparently
enhances the anti-mycobacterial activity in the purine series described herein. Several 2-amino and
2-chloro purine analogs were also synthesized that showed moderate inhibitory activity against Mtb.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 3 December 2012
Revised 17 January 2013
Accepted 24 January 2013
Available online 4 February 2013
Keywords:
Mycobacterium tuberculosis
Purines
Antimycobacterial agents
1. Introduction
tions are available. Although newer drugs are now in clinical trials,
these issues critically underscore the need for continued emphasis
In spite of the availability of highly active anti-tubercular
agents, tuberculosis has remained one of the primary causes of hu-
man death and suffering worldwide. It is estimated that approxi-
mately one third of the world’s population is infected with the
bacteria that causes tuberculosis, 2–3 million people die world-
wide each year from the disease, and an additional 8 million people
become ill with tuberculosis annually.1 Mycobacterium tuberculosis
(Mtb) is a facultative intracellular pathogen that persists primarily
within macrophages in the human host, and these cells are in-
volved in propagation of infection.2 Intracellularly sequestered ba-
cilli are considered more resistant to treatment and clearance due
to limited access of drugs and the immune system to bacteria with-
in macrophages, necessitating chronic treatment with high thera-
peutic doses for effective control and treatment of the disease.3
Additionally, AIDS patients and others with compromised immune
systems are susceptible to other opportunistic mycobacteria
including Mycobacterium avium and Mycobacterium kansaii, result-
ing in further morbidity and a high mortality.4 It is not surprising
that drug resistance, from single drug resistant (SDR) up to totally
drug resistant (TDR) strains,5 is now becoming commonplace con-
sidering the fact that virtually the same drug regimens have been
in place and poorly deployed worldwide for over half a century.
Treatment of highly resistant forms of Mtb is both difficult and
expensive, and for these more intractable forms, few treatment op-
on the discovery of newer drugs with novel mechanisms of action.6
Phenotypic screening of diverse drug-like compound libraries
against Mtb has been more recently implemented in order to re-
spond to this need and discover compounds that are active against
whole Mtb bacilli,7 potentially circumventing issues with antibac-
terial drug discovery using specific target based screens.8
Through similar screens of the Southern Research proprietary li-
brary, it has been found that several 9-benzylpurines with a variety
of substitutions in the 2-, 6- and/or 8-positions exhibit inhibitory
activity against Mtb.9 High inhibitory activity was found for 9-ben-
zylpurines containing a phenylethynyl-, trans-styryl or aryl substi-
tuent in the 6-position, and generally chlorine in the 2-position
tends to increase activity (compounds 1–4, Fig. 1). Several 6-arylp-
urines carrying a variety of substituents in the 9-position were
prepared by Stille coupling between appropriately substituted
6-chloropurines and aryl(tributyl)tin, and the compounds were
screened for antibacterial activity against Mtb H37Rv.10 One of
the derivatives, 9-benzyl-2-chloro-6-(2-furyl)purine (2), showed
a MIC value of 0.78 lg/mL and also showed relatively low cytotox-
icity against several singly drug-resistant strains. A series of 9-sulf-
onylated/sulfenylated-6-mercapto purines (3 and 4) has been
prepared by reaction of 6-mercaptopurine with sulfonyl/sulfenyl
halides.11 These compounds constitute a new class of potent anti-
mycobacterial agents, possessing excellent MIC values against Mtb
H37Rv, as well as appreciable activity against M. avium. A few com-
pounds in this series have exhibited activity against several drug
resistant strains of Mtb (e.g., compound 4). Currently, beyond the
broad phenotypic activity, no target(s) has/have been identified.
We have also synthesized a small library of 6-thioalkyl/aryl/
benzyl purine analogs to generate a modest structure–activity
⇑
Corresponding author at present address: Department of Chemistry, University
of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham, AL 35294, USA.
Tel.: +1 205 934 8276; fax: +1 205 934 2543.
E-mail addresses: pathaka@southernresearch.org (A.K. Pathak), rcr12lkt@uab.
edu (R.C. Reynolds).
Tel.: +1 205 581 2542.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.054

1686
A. K. Pathak et al. / Bioorg. Med. Chem. 21 (2013) 1685–1695
2. Results and discussion
S
N
3.
4.
R = NMe2
1.
2.
R = H
O
MIC = 0.39 µg/mL
SI > 513
MIC = 3.13 µg/mL
SI = 2.7
N
N
HN
2.1. Chemistry
N
N
N
R
R = NHC6H4-4-Ac
MIC = 0.78 µg/mL
SI > 256
R = Cl
O
S
In the first target set, synthesis of several new 6-oxoaryl/benzyl/
aryl purines and their 2-amino or 2-chloro purine analogs (8–32)
was carried out as shown in Scheme 1 by reacting a suitable
6-chloropurine analog with alcohols in the presence of sodium me-
tal. Neutralization with acetic acid followed by the standard workup
and column chromatographic purification on silica gel G produced
pure products. A total of six analogs of 6-oxopurine, 10 analogs of
2-amino-6-oxopurine and nine analogs of 2-chloro-6-oxopurine
were prepared in the initial phase to determine their antibacterial
activity. A total of nine analogs have shown >50% inhibitory activ-
ity against Mtb H37Rv and are discussed herein (Table 1).
O
N
MIC = 0.78 µg/mL
SI = 10.4
R
Ph
Figure. 1.
relationship (SAR). All the purine derivatives synthesized were
screened for their activity against two strains of Mtb (H37Rv &
37Ra) and three strains of M. avium (MAC NJ211, NJ168,
H
NJ3404). Most of these derivatives were inactive against M. avium.
In particular, (6-decylsulfanyl-purin-9-yl)-acetic acid ethyl ester 5
and the dodecyl derivative 6, and (6-decylsulfanyl-purin-9-yl)-ace-
tic acid tert-butyl ester 7 exhibited MIC values of 1.56, 0.78 and
As previously described by us,12 substitution at the N9-position
enhances antimycobacterial activity, and we have synthesized and
screened four analogs 33–36 starting from active 6-oxopurine ana-
logs 9, 10, 15 and 16 (Scheme 2). The synthesis of these analogs
was carried out by reacting the appropriate commercially available
purine analog with ethylbromoacetate and K2CO3 in anhydrous
DMSO at room temperature.
3.13 l
g/mL respectively against the Mtb H37Rv strain (Fig. 2).12
Concurrent with the determination of MICs, analogs 5–7 were also
tested for cytotoxicity (CC50) in a cell proliferation assay for VERO
cells at concentrations less than or equal to 62.5 lg/mL or 10 times
the MIC for Mtb H37Rv: selectivity is assigned by calculating the
Further analoging of compounds 34 and 35 was achieved by
selectivity index (SI) ratio CC50/MIC.
treating these with different
D- and L-amino acids. These com-
Very little is known about the mechanism of action of these
purine analogs. Purine salvage pathways are predicted to be pres-
ent from the genome sequence of Mtb, and the metabolism in
mycobacteria is similar to that in humans and other organisms.13
Information about the substrate preferences of the mycobacterial
enzymes involved with purine metabolism is still unknown. The
Mtb deoD gene encodes a presumptive purine nucleoside phos-
phorylase (PNP) and the gene was cloned, expressed, purified,
and found to exhibit PNP activity.14 Modest biochemical work
has been pursued on purine nucleosides with anti-mycobacterial
activity, especially 2-methyladenosine that showed potent activity
pounds can utilize bacterial dipeptide transporters to enhance
transport via membrane amino acids beyond the fact that adding
amino acids at the end increases diversity for probing activity. It
is sometimes the case that adding specific chirality can improve
activity and only one stereoisomer shows activity. This result is,
however, a gross generality as that is not always the case and
sometimes both enantiomers are inactive or can show similar
activity. Compounds 40–50 (Scheme 3) which contain D- or L-ami-
no acid chains were synthesized from commercially available
starting material, 2-amino-6-chloro-9H-purine-9-acetic acid (37).
The 6-decyloxy derivative 38 was synthesized by reacting 37 with
1-decanol in the presence of sodium metal. The 6-decylthio deriv-
ative 39 was synthesized from 37 by the reaction with 1-decylthiol
and (CH3)3COK as previously described.12 Analoging of 38 and 39
(99% inhibition, MIC = 3 lg/mL, IC50 (VERO Cells = 1000 lg/mL, SI
>1000).15 2-Methyladenosine has demonstrated selective activity
against Mtb, suggesting differences in the substrate preferences
between mycobacterial and human adenosine kinases that might
be exploited to develop novel nucleoside-based drugs for the treat-
ment of mycobacterial diseases. Beyond the purine salvage path-
ways, ATP binding proteins and kinases are also being
interrogated as new drug targets in Mtb.16
was achieved by treating with different D- or L-amino acids using
the coupling reagent benzotriazole-1-yl-oxy-trispyrrolidino-phos-
phonium hexafluorophosphate (PyBOP) in the presence of base
Et3N.
Based upon our previously synthesized purine series,12 we
planned to generate more diversified 6-substituted mercaptopu-
rines analogs with substitutions at C-2 position along with 6-oxo
substituted series for antimycobacterial screening. Compounds
were prepared from a diversity perspective in order to further
probe specific active scaffolds, but also to explore other purines
such as the 2-amino and 2-chloropurine scaffolds.
Alternatively, for diversity point of view, compounds 51–69
were prepared from easily accessible starting materials. 6-Decyl-
mercaptopurine analogs 51–54 were prepared starting from
6-chloropurine analogs by the reaction with 1-decylthiol and
(CH3)3COK under reflux as previously described (Scheme 4A).12,17
However, 6-decylmercapto purine analogs 55–69 were prepared
OR
Cl
N
N
N
ROH
Na
N
N
N
H
N
H
N
X
X
8 - 32
SR1
8. X=H; R = CH(CH3)2
9. X=H; R = (CH2)9CH3
10. X=H; R = (CH2)11CH3
11. X=H; R = C6H4-4-Cl
12. X=H; R = CH2-C6H11
21. X=NH2; R = CH2-C6H11
22. X=NH2; R = CH2-C6H5
23. X=NH2; R = CH2-C6H3-3-Cl,4-Cl
24. X=Cl; R = CH3
N
N
N
N
OR2
25. X=Cl; R = CH(CH3)2
26. X=Cl; R = (CH2)9CH3
27. X=Cl; R = (CH2)11CH3
28. X=Cl; R = C6H4-4-Cl
29. X=Cl; R = C6H4-4-CH3
30. X=Cl; R = C6H4-3-CH3
31. X=Cl; R = CH2-C6H11
32. X=Cl; R = CH2-C6H3-3-Cl,4-Cl
13. X=H; R = CH2-C6H3-3-Cl,4-Cl
14. X=NH2; R = CH(CH3)2
15. X=NH2; R = (CH2)9CH3
16. X=NH2; R = (CH2)11CH3
17. X= NH2; R = C6H5
O
5.
R1 = C10H21, R2 = Et; MIC = 1.56 µg/mL,
CC50 VERO Cells = >62.5 µg/mL; SI = >40
6. R1 = C12H25, R2 = Et; MIC = 0.78 µg/mL,
CC50 VERO Cells = >10 µg/mL; SI = >12.8
7. R1 = C10H21, R2 = C(CH3)3; MIC = 3.13 µg/mL,
CC50 VERO Cells = >10 µg/mL; SI = >3.19
18. X= NH2; R = C6H4-4-Cl
19. X= NH2; R = C6H4-4-CH3
20. X= NH2; R = C6H4-3-CH3
Figure 2.
Scheme 1.

A. K. Pathak et al. / Bioorg. Med. Chem. 21 (2013) 1685–1695
1687
Table 1
Antimycobacterial activity against Mtb and MAC of substituted purines
Compd no.
% Inhib. Mtb H37Rv
MIC90 Mtb H37Rv
MIC99 Mtb H37Ra
MIC99 MAC NJ211
CC50 VERO cells
Selectivity index (SI) Mtb H37Rv
8
9
23
96
56
6
>6.25
<6.25
>6.25
>6.25
>6.25
>6.25
3.13
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>6.4
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8(P)
32
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>6.4
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>128
>12.8
>32
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
>10
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
>62.5
>10
n.d.
>10
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
12.32
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
8.04
n.d.
n.d.
n.d.
n.d.
—
—
—
—
—
—
—
>12.8
—
—
—
—
—
—
—
—
—
>40.1
>12.8
—
>1.6
—
—
—
—
—
—
2.0
—
—
—
—
—
—
—
10
12
13
14
15
16
18
21
23
24
25
26
27
28
32
33
34
35
36
44
47
48
51
52
53
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
21
23
91
94
20
93
24
24
23
55
59
8
35
100
98
14
100
7
0.78
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
1.56
0.78
>6.25
6.25
>12.8
16
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
6.25
>6.25
>6.25
6.25
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
16
5
35
49
82
97
84
81
93
96
97
74
89
86
90
92
98
96
92
85
6.25
1.56
>6.25
>6.25
>6.25
>6.25
6.25
6.25
6.25
>6.25
>6.25
—
—
1.3
—
—
—
—
% Inhibition was performed at 6.25
mL; SI (Mtb H37Rv) = CC50/MIC90
lg/mL concentration; compounds not listed here showed no inihibition at 6.25
lg/mL; MIC’s are in
lg/mL; CC50 was determined in
l
g/
.
from their corresponding 6-purinethiones by treatment with
1-chlorodecane/anhydrous K2CO3 as reported earlier (Scheme 4B).18
7-Deaza compound 70 was synthesized in three steps starting
from commercially available 6-chloro-7-deazapurine 71 as shown
in Scheme 5. The compound 71 was first converted to 6-thio-
methyl-7-deaza purine 72 in excellent yield. Compound 72 was
then reacted with ethylbromoacetate/K2CO3 followed by ester
hydrolysis using 1 N NaOH gave compound 73. Finally, coupling
of 73 with 3,4-diemthylaniline, HATU and DIEA produced the de-
sired product 70.
OR
N
OR
N
N
N
BrCH2CO2CH2CH3
K2CO3, DMSO
N
N
H
N
X
N
X
CH2CO2CH2CH3
29 - 32
33. X=H; R = (CH2)9CH3
34. X=H; R = (CH2)11CH3
35. X=NH2; R = (CH2)9CH3
36. X=NH2; R = (CH2)11CH3
Scheme 2.
2.2. Anti-mycobacterial activity
Mtb H37Ra were determined using a colorimetric (Alamar blue)
microdilution broth assay reported previously.19 All the com-
pounds were also screened against Mtb H37Rv to determine the
MIC90 (the minimum concentration that inhibits 90% of growth).
Out of 59 purine analogs synthesized, 26 analogs gave >50% inhibi-
tion against H37Rv and are shown in Table 1 (compounds not inc-
uded in Table 1 showed no inhibition against Mtb H37Rv).
The antimycobacterial purine analogs 16, 33, 34, 36, 55 and 65
were examined for toxicity against mammalian cells (VERO
cells),20 and their cytotoxicity (CC50) values are reported in Table 1.
Their selectivity is assigned by calculating the selectivity index (SI)
ratio CC50/MIC. One of the more promising inhibitors of Mtb
growth, compound 33, gave a SI >40.
All new compounds were screened for their in vitro activity
against strains Mtb H37Ra and H37Rv as well as one strain of M. avi-
um (NJ211) and are described below. Compounds demonstrating at
least 90% inhibition against H37Rv strain at 6.25 lg/mL were re-
tested to determine the MIC90 in a BACTEC assay. Active com-
pounds were then screened for cytotoxicity against mammalian
VERO cells to determine their selectivity towards bacteria.
2.2.1. In vitro cell studies
The minimum inhibitory concentration (MIC99, the lowest con-
centration that completely inhibits growth) of all compounds for

1688
A. K. Pathak et al. / Bioorg. Med. Chem. 21 (2013) 1685–1695
C10H21
Cl
SMe
SMe
Cl
N
X
C10H21OH,
Na, DMSO
b
N
N
a
N
N
N
N
N
N
N
N
H
N
H
or
N
N
N
N
H2N
O
H2N
O
N
C
10H21SH
O
(CH3)3COK
71
72
73
37
OH
i-PrOH
OH
38
39
. X = O
OH
. X = S
c
SMe
N
O
O
R
R'
N
PyBOP, Et3N
Pyridine
N
H2N
*
O
C10H21
X
HN
70
N
N
Scheme 5. Reagents and conditions: (a) NaSCH3, MeOH, rt, overnight, 90%; (b) (i)
BrCH2CO2Et, K2CO3, DMF, rt, overnight, (ii) 1 N NaOH, MeOH, rt, overnight, 82% in
two steps; (c) 3,4-dimethylaniline, HATU, DIEA, DMF, rt, 6 h, 54%.
N
N
H2N
O
O
R
40 - 50
O
HN
*
R'
46.
(D)- X = S, R = CH3, R' = Ph
SCH3
N
SCH3
N
40.
(L)- X = O, R = CH3, R' = H
N
N
N
N
41. (L)- X = O, R = CH3, R' = Ph 47. (L)- X = S, R = C2H5, R' = Ph
42. (L)- X = O, R = C2H5, R' = Ph 48. (L)- X = S, R = CH3, R' = OH
N
43.
44.
49.
50.
(L)- X = S, R = CH3, R' = H
(D)- X = S, R = CH3, R' = H
(D)- X = S, R = CH3, R' = OH
(L)- X = S, R = C2H5, R' = OH
O
O
45. (L)- X = S, R = CH3, R' = Ph
70
74
HN
HN
Scheme 3.
Figure 3.
SC10H21
Cl
N
N
N
N
N
C10H21SH
activity. Purine analogs modified in the five-membered ring have
been synthesized and examined for antibacterial activity against
Mtb H37Rv in vitro.21 The 9-deaza analogs were only found to be
weak inhibitors, but the 8-aza-, 7-deaza- and 8-aza-7-deazapurine
analogs studied displayed excellent antimycobacterial activities,
some even substantially better than the parent purine. Hence,
the 7-deazapurine analog 70 of purine 74 was prepared and
screened against Mtb. Interestingly, this modest alteration led to
complete abolition of the whole cell antitubercular activity (IC90
N
A.
(CH3)3COK
i-PrOH, Reflux
N
R2
R1
N
R1
R2
51. R1 = NHCOCH2, R2 = H
52.
53.
54.
55.
R1 = NH2, R2 = CH2CH(OH)CH2OH
R1 = NH2, R2 = CH2Ph
R1 = SC10H21, R2 = CH2OCH2CH2OH
R1 = NH2, R2 = C2H5
SC10H21
S
N
>100 lM) in our case for compound 70. It is also notable that 70
N
N
N
N
C10H21Cl
N
B.
(PubChem
(IC90 = 7.31
CID
M) of Mtb H37Rv in the MLSCN screen (Assay ID
M (non-lethal, sub-
44144249)
showed
modest
inhibition
K2CO3, DMAc
r.t.
N
R2
R1
N
R1
l
R2
485340) when screened in the presence of 6.5
l
56.
57.
R1 = CH2CH2, R2 = H
inhibitory dose to bacteria) of the b-lactam antibiotic Meropenem.
These data suggest that damage to the cell wall may allow uptake
of 70 and activity against the/a molecular target. Speculatively, the
purine analog 74 may be actively uptaken and inhibit a target or
targets, but the 7-deaza analog 70 may lose the ability to penetrate
the mycobacterial cell wall until the peptidoglycan is compromised
by the b-lactam. This hypothesis, however, remains unproven.
The two active purines 5 and 6 synthesized previously12 were
examined against a panel of Mtb strains resistant to currently used
anti-TB drugs; CIP (ciprofloxacin), INH (isoniazid), KM (kanamy-
cin), EMB (ethambutol), RMP (rifampin), PAS (p-aminosalicylic
acid), and TAC (thiacetazone) (Table 2). All compounds examined
retained their activity against all the drug-resistant strains applied
in this study.
R1 = H, R2 = Cycloheptyl
58. R1 = H, R2 = CH CH=CH2
59. R1 = H, R2 = CH2CH OCOCH
60. R1 = H, R2 = CH2CN2
3
2
61.
62.
63.
64.
R1 = H, R2 = N=CHPh
R1 = H, R2 = C2H5
R1 = H, R2 = CH2Ph
R1 = H, R2 = 2-Cyclohexanol
65. R1 = H, R2 = Cyclopentyl
66. R1 = H, R2 = Butyl
67. R1 = H, R2 = 2-Cyclopentanol
68.
69.
R1 = H, R2 = NHCOCH3
R1 = H, R2 = 2-Cyclohexyl
Scheme 4.
A 2-thiomethylpurine analog 74 (PubChem ID 726045, Fig. 3)
commercially available from ChemBridge showed very potent
activity (IC90 <0.1
lg/mL) with a selectivity index (SI) >150 against
2.2.2. In vivo animal studies22
Mtb H37Rv (PubChem Assay ID 1949). Unfortunately, this analog as
well as the general class show only relatively poor bioavailablity
and antitubercular activity in vivo.12 Purines are potential sub-
strates for a number of metabolic enzymes in vivo that include
adenosine deaminase and xanthine oxidase that can alter biologi-
cal properties and activity; a typical alteration that can modify
metabolism involves preparation of 7-deaza analogs that can
potentially circumvent metabolism and retain potent biological
Concurrently with the SAR studies, both compounds 5 and 6
were selected for further evaluation in vivo in order to determine
cytotoxicity and efficacy in a murine Mtb model to evaluate these
scaffolds as further candidates for antitubercular drug discovery.
The lead compounds of the series, compounds 6 and 16, were ini-
tially evaluated for in vivo toxicity using an acute toxicity mouse
model. These compounds did not show any lethality at 100, 300
and 500 mg/kg. In the short term GKO mouse model, 6 showed

A. K. Pathak et al. / Bioorg. Med. Chem. 21 (2013) 1685–1695
1689
Table 2
MIC of compounds 5 and 6 with H37Rv, Erdman and single-drug resistant strains of Mtb in the Alamar blue assay
Compd
MIC90
CIP
12.5
1.56
(lg/mL) against drug resistance strains in Alamar blue assay
H
37Rv
Erdman
INH
KM
EMB
RMP
PAS
TAC
5
6
6.25
3.13
12.5
3.13
12.5
1.56
6.25
—
6.25
3.13
6.25
3.13
—
3.13
—
3.13
MIC of 5 and 6 were observed 1.56 and 0.78 lg/mL, respectively against H37Rv in Bactec assay. CIP (ciprofloxacin), INH (isoniazid), KM (kanamycin), EMB (ethambutol), RMP
(rifampin), PAS (p-aminosalicylic acid), and TAC (thiacetazone).
poor efficacy in the lungs by reducing the bacterial load by
0.46Log10 CFU (mean CFU = 8.16) whereas the compound did not
show any significant activity in the spleen (0.2Log10 CFU reduction,
mean CFU = 6.83). For compound 16, there was no in vivo activity
observed in the lungs or spleens in the GKO mouse model
[ꢀ0.141Log10 CFU (mean CFU = 8.82) in lungs, and ꢀ0.05Log10 CFU
(mean CFU = 7.11)].
an argon atmosphere. The reaction mixture was stirred at reflux
for 2–6 h and then allowed to stir at room temperature overnight
under argon. Deionized water (10–20 mL) was added and the reac-
tion mixture was neutralized by addition of acetic acid. The neu-
tralized solution was then extracted with diethyl ether. The
ethereal layers were pooled, dried over Na2SO4, concentrated,
and dried in vacuum over P2O5 at 25 °C. In most cases, the crude
material was purified by column chromatography (CC) over silica
gel G.
2.3. Conclusions
In the 6-oxo series, compound 9, 15 and 16 showed good activ-
ity against Mtb H37Rv and possess a decyl/dodecyl chain at the C-6
position of purine. Based on their activity, compounds 29–34 pos-
sessing N9 substitution were synthesized and screened. Out of
these four compounds, however, compound 35 significantly lost
antimycobacterial activity over the comparator compound, 15.
3.1.1.1. 6-[(Isopropyl)oxy]purine (8).
CHCl3–MeOH (99:1)
with 0.5% NH4OH was used as eluent for column chromatography
(CC), off-white solid, yield 45%. 1H NMR: d 13.37 (1H, br s, NH),
8.46 (1H, s, H-2), 8.34 (1H, s, H-8), 5.58 (1H, m, OCH), 1.40 (6H,
d, J = 6.2 Hz, 2ꢁCH3). FABMS m/z: 179 (M+H)+. ESI-MS: Found
179.0932, calcd for C8H10N4O 179.0927. Anal. (C8H10N4O) Found:
C, 54.24; H, 5.77; N, 30.35. Calcd C, 53.92; H, 5.66; N, 31.44.
Structural diversity using D- and L-amino acids in compounds 34
and 35 did not increase the activity significantly. The diversity ana-
logs 51–69 showed significant high % inhibition but also produced
high MIC90 values in the Mtb H37Rv screen. In conclusion, small
sets of diverse 6-substituted purine analogs were synthesized
and screened against Mtb strains. Certain purine analogs synthe-
sized showed interesting antimycobacterial activity against Mtb
3.1.1.2. 6-(Decyloxy)purine (9).
CHCl3 was used as eluent for
CC, off-white solid, yield 51%. 1H NMR: d 13.50 (1H, br s, NH), 8.47
1
1
(1H, s, JCH = 203.0 Hz, H-2), 8.36 (1H, s, JCH = 210.9 Hz, H-8), 4.52
(2H, t, J = 6.6 Hz, OCH2), 1.80 (2H, m, CH2), 1.34 (14H, m, 7ꢁ CH2),
0.85 (3H, m, CH3). FABMS m/z: 277 (M+H)+. Anal. (C15H24N4O)
Found: C, 64.86; H, 9.01; N, 19.90. Calcd C, 65.19; H, 8.75; N, 20.27.
H37Rv. At this point, however, no definitive structure–activity rela-
tionship has been determined.
3.1.1.3. 6-(Dodecyloxy)purine (10).
CHCl3–MeOH (98:2) was
used for CC, off-white solid, yield 63%. 1H NMR: d 13.40 (1H, br s,
NH), 8.47 (1H, s, H-2), 8.36 (1H, s, H-8), 4.52 (2H, dd, J = 6.6,
6.7 Hz, OCH2), 1.79 (2H, m, CH2), 1.29 (18H, m, 9ꢁ CH2), 0.85
(3H, m, CH3). FABMS m/z: 305 (M+H)+. Anal. (C17H28N4O) Found:
C, 67.26; H, 9.02; N, 18.02. Calcd C, 67.07; H, 9.27; N, 18.40.
3. Experimental
3.1. Synthesis
Purifications by column chromatography were done with silica
gel (Merck, 60 A, 230–400 mesh). Target compounds were dried in
vacuo at 25 °C and 78 °C over P2O5. Analytical results indicated by
element symbols were within 0.4% of the theoretical values, un-
less otherwise noted. In such noted cases, a high-resolution mass
spectrum was obtained and the result listed. Where solvents are
indicated in the formula, their presence was confirmed by 1H
NMR. The 1H NMR spectra were determined with a Nicolet NMC
300 NB spectrometer using TMS as internal reference and chemical
shifts (d) are in ppm. 1H NMR spectra were recorded in DMSO-d6 as
a solvent. NOE’s were performed on certain intermediates and tar-
get compounds to confirm structures. Mass spectra were recorded
on a Varian MAT 311A mass spectrometer in the fast-atom-bom-
bardment (FAB) mode. High-resolution mass spectra were ob-
tained on a Bruker BIOTOF II spectrometer. Microanalyses were
performed on a Perkin–Elmer 2400 CHN analyzer. All starting
materials and reagents were purchased from commercial sources
and used as such. Chemical yields have not been optimized and
represent the result of a single preparation. All target compounds
were stored under argon at ꢀ20 °C until used for screening. Gen-
eral synthetic procedures are described as below.
3.1.1.4. 6-[(4-Chlorophenyl)oxy]purine (11).
CHCl3–MeOH
(98:2) was used for CC, off-white solid, yield 84%. 1H NMR: d
13.61 (1H, br s, NH), 8.53 (1H, s, H-2); 8.44 (1H, s, H-8), 7.54
(2H, d, J = 8.9 Hz, H-30, H-50), 7.36 (2H, d, J = 8.9 Hz, H-20, H-60). FAB-
MS m/z: 247 (M+H)+. Anal. (C11H7N4ClO) Found: C, 53.51; H, 2.83;
N 22.51. Calcd C, 53.56; H, 2.86; N, 22.71.
3.1.1.5. 6-[(Cyclohexylmethyl)oxy]purine (12)23
.
CHCl3–
MeOH (98:2) was used for CC, off-white solid, yield 32%. 1H
NMR: d 13.39 (1H, br s, NH), 8.43 (1H, s, H-2); 8.33 (1H, s, H-8),
4.31 (2H, d, J = 6.3 Hz, OCH2), 1.50 (11H, m, C6H11). FABMS m/z:
233 (M+H)+. Anal. (C12H16N4Oꢂ0.1H2O) Found: C, 61.59; H, 7.01;
N, 23.60. Calcd C, 61.57; H, 6.98; N, 23.93.
3.1.1.6. 6-[(3,4-Dichlorobenzyl)oxy]purine (13).
CHCl3–
MeOH (99:1) with 0.5% NH4OH was used for CC, off-white solid,
yield 35%. 1H NMR: d 13.51 (1H, br s, NH), 8.52 (1H, s, H-2); 8.42
(1H, s, H-8), 7.82 (1H, d, J = 1.8 Hz, H-20), 7.69 (1H, d, J = 8.2 Hz,
H-50), 7.53 (1H, dd, J = 1.8, 2.1 Hz, H-60), 5.62 (2H, s, OCH2). FABMS
m/z: 295 (M+H)+. Anal. (C12H8N4Cl2Oꢂ0.45CH3OH) Found: C, 48.63;
H, 2.94; N, 17.73. Calcd C, 48.31; H, 3.19; N, 18.10.
3.1.1. General procedure for synthesis of 6-oxyalkyl/aryl/benzyl
purines (8–32)
3.1.1.7. 2-Amino-6-[(isopropyl)oxy]purine (14).
Off-white
To a solution of Na (1.36 equiv) in the appropriate alcohol
(20 equiv) was added the appropriately substituted purine under
solid, yield 59%. 1H NMR: d 7.78 (1H, s, H-8), 6.14 (2H, br s, NH2),

1690
A. K. Pathak et al. / Bioorg. Med. Chem. 21 (2013) 1685–1695
5.48 (1H, ddd, J = 6.2, 6.3, 6.3 Hz, OCH), 1.34 (6H, d, J = 6.2 Hz, 2ꢁ
CH3). FABMS m/z: 194 (M+H)+. Anal. (C8H11N5O) Found: C, 49.91;
H, 5.83; N, 35.86. Calcd C, 49.73; H, 5.74; N, 36.25.
off-white solid, yield 57%. 1H NMR: d 12.5 (1H, br s, NH), 7.85
(1H, br s, H-8), 7.79 (1H, d, J = 1.9 Hz, H-20), 7.67 (1H, d,
J = 8.4 Hz, H-50), 7.51 (1H, dd, J = 2.1, 2.1 Hz, H-60), 6.32 (2H, br s,
NH2), 5.48 (2H, s, OCH2). FABMS m/z: 310 (M+H)+. Anal.
(C12H9Cl2N5O) Found: C, 46.48; H, 3.19; N, 22.34. Calcd C, 46.47;
H, 2.92; N, 22.58.
3.1.1.8. 2-Amino-6-(decyloxy)purine (15).
CHCl3–MeOH
(20:1) was used for CC, off-white solid, yield 62%. 1H NMR: d
12.4 (1H, br s, NH), 7.81 (1H, s, H-8), 6.19 (2H, br s, NH2), 4.37
(2H, dd, J = 6.6, 6.7 Hz, OCH2), 1.74 (2H, m, CH2), 1.33 (14H, m,
7ꢁ CH2), 0.85 (3H, s, CH3). FABMS m/z: 292 (M+H)+. Anal.
(C15H25N5O) Found: C, 61.73; H, 8.58; N, 23.81. Calcd C, 61.83; H,
8.65; N, 24.03.
3.1.1.17. 2-Chloro-6-(methoxy)purine (24).
CHCl3–MeOH
(98:2) was used for CC, off-white solid, yield 49%. 1H NMR: d
13.67 (1H, br s, NH), 8.41 (1H, br s, H-8), 4.10 (3H, s, CH3). FABMS
m/z: 185 (M+H)+. ESI-MS: Found 185.0221, Calcd for C6H5ClN4O
185.0225. Anal. (C6H5ClN4O) Found: C, 37.36; H, 2.51; N, 28.77.
Calcd C, 37.23; H, 3.12; N, 28.94.
3.1.1.9. 2-Amino-6-(dodecyloxy)purine (16).
CHCl3–MeOH
(20:1) was used for CC, off-white solid, yield 59%. 1H NMR: d
12.4 (1H, br s, NH), 7.80 (1H, s, H-8), 6.19 (2H, br s, NH2), 4.37
(2H, dd, J = 6.6, 6.7 Hz, OCH2), 1.73 (2H, m, CH2), 1.30 (18H, m,
9ꢁ CH2), 0.85 (3H, m, CH3). FABMS m/z: 320 (M+H)+. Anal.
(C17H29N5Oꢂ0.5H2O) Found: C, 61.92; H, 8.97; N, 21.24. Calcd C,
62.17; H, 9.21; N, 21.32.
3.1.1.18. 2-Chloro-6-[(isopropyl)oxy]purine (25).
CHCl3–
MeOH (99:1) with 0.5% NH4OH was used for CC, white solid, yield
62%. 1H NMR: d 8.40 (1H, s, H-8), 5.52 (1H, septet, J = 6.2 Hz, OCH),
1.40 (6H, d, J = 6.2 Hz, 2ꢁ CH3). FABMS m/z: 213 (M+H)+. Anal.
(C8H9ClN4O) Found: C, 45.15; H, 4.34; N, 26.31. Calcd C, 45.19; H,
4.27; N, 26.35.
3.1.1.10. 2-Amino-6-(benzyloxy)purine (17).
CHCl3–MeOH
(20:1) was used for CC, off-white solid, yield 45%. 1H NMR: d
12.6 (1H, br s, NH), 7.95 (1H, s, H-8), 7.44 (2H, m, H-20, H-60),
7.24 (3H, m, H-30, H-40, H-50), 6.25 (2H, br s, NH2). FABMS m/z:
228 (M+H)+. Anal. (C11H9N5Oꢂ0.2CH3OH); Found: C, 57.74; H,
4.36; N, 29.83. Calcd C, 57.58; H, 4.23; N, 29.98.
3.1.1.19. 2-Chloro-6-(decyloxy)purine (26).
Cyclohexane–
EtOAc (5:1) was used for CC, white solid, yield 62%. 1H NMR: d
8.41 (1H, s, H-8), 4.51 (2H, t, OCH2), 1.81 (2H, m, CH2), 1.21
(14H, m, 7ꢁ CH2), 0.86 (3H, m, CH3). FABMS m/z: 311 (M+H)+. Anal.
(C15H23ClN4O) Found: C, 57.85; H, 7.41; N, 17.98. Calcd C, 57.96; H,
7.46; N, 18.02.
3.1.1.11.
(18).
2-Amino-6-[(4-chlorophenyl)oxy]purine
CHCl3–MeOH (20:1) was used for CC, off-white solid,
3.1.1.20. 2-Chloro-6-(dodecyloxy)purine (27).
Cyclohexane–
yield 83%. 1H NMR: d 12.6 (1H, br s, NH), 7.94 (1H, s, H-8), 7.49
(2H, m, H-20, H-60), 7.29 (2H, m, H-30, H-50), 6.30 (2H, br s, NH2).
FABMS m/z: 262 (M+H)+. Anal. (C11H8N5ClOꢂ0.4H2O) Found: C,
49.12; H, 3.66; N, 25.76. Calcd C, 49.14; H, 3.30; N, 26.05.
EtOAc (5:1) was used for CC, white solid, yield 71%. 1H NMR: d
13.62 (1H, br s, NH), 8.41 (1H, s, H-8), 4.51 (2H, t, J = 6.6 Hz,
OCH2), 1.79 (2H, m, CH2), 1.28 (18H, m, 9ꢁ CH2), 0.85 (3H, m,
CH3). FABMS m/z: 339 (M+H)+. Anal. (C17H27ClN4O) Found: C,
60.28; H, 8.09; N, 16.17. Calcd C, 60.25; H, 8.03; N, 16.53.
3.1.1.12.
(19).
2-Amino-6-[(4-methylphenyl)oxy]purine
CHCl3–MeOH (20:1) was used for CC, yellow solid, yield
3.1.1.21.
(28).
2-Chloro-6-[(4-chlorophenyl)oxy]purine
83%. 1H NMR: d 12.5 (1H, br s, NH), 7.93 (1H, br s, H-8), 7.22 (2H, d,
J = 8.2 Hz, H-30, H-50), 7.10 (2H, d, J = 8.4 Hz, H-20, H-60), 6.22 (2H, br
s, NH2), 2.32 (3H, s, CH3). FABMS m/z: 242 (M+H)+. Anal.
(C12H11N5Oꢂ0.4H2O) Found: C, 58.03; H, 4.91; N, 28.26. Calcd C,
58.01; H, 4.79; N, 28.19.
CHCl3–MeOH (98:2) was used for CC, off-white solid,
yield 56%. 1H NMR: d 13.86 (1H, br s, NH), 8.58 (1H, s, H-8), 7.57
(2H, m, H-20, H-60), 7.40 (2H, m, H-30, H-50). FABMS m/z: 281
(M+H)+. Anal. (C11H6Cl2N4O) Found: C, 47.34; H, 2.32; N, 19.80.
Calcd C, 47.00; H, 2.15; N, 19.93.
3.1.1.13.
(20).
2-Amino-6-[(3-methylphenyl)oxy]purine
CHCl3–MeOH (20:1) was used for CC, light brown solid,
3.1.1.22.
(29).
2-Chloro-6-[(4-methylphenyl)oxy]purine
CHCl3–MeOH (98:2) was used for CC, off-white solid,
yield 85%. 1H NMR: d 12.56 (1H, br s, NH), 7.94 (1H, br s, H-8), 7.31
(1H, dd, J = 7.9, 8.2 Hz, H-50), 7.03 (3H, m, H-20, H-40, H-60), 6.26
(2H, br s, NH2), 2.33 (3H, s, CH3). FABMS m/z: 242 (M+H)+. Anal.
(C12H11N5Oꢂ0.45H2O) Found: C, 58.03; H, 4.93; N, 27.82. Calcd C,
57.80; H, 4.81; N, 28.09.
yield 91%. 1H NMR: d 13.82 (1H, br s, NH), 8.55 (1H, s, H-8), 7.29
(2H, d, J = 8.2 Hz, H-30, H-50), 7.20 (2H, dd, J = 2.3, 8.6 Hz, H-20, H-
60), 2.36 (3H, s, CH3). FABMS m/z: 261 (M+H)+. Anal. (C12H9ClN4O)
Found: C, 55.15; H, 3.56; N, 21.36. Calcd C, 55.29; H, 3.48; N, 21.49.
3.1.1.23.
(30).
2-Chloro-6-[(3-methylphenyl)oxy]purine
3.1.1.14.
(21)23
2-Amino-6-[(cyclohexylmethyl)oxy]purine
CHCl3–MeOH (98:2) was used for CC, off-white solid,
.
CHCl3–MeOH (20:1) was used for CC, off-white solid,
yield 58%. 1H NMR: d 13.8 (1H, br s, NH), 8.56 (1H, s, H-8), 7.38
(1H, dd, J = 7.9, 8.1 Hz, H-50), 7.14 (3H, m, H-20, H-40, H-60), 2.36
(3H, s, CH3). FABMS m/z: 261 (M+H)+. Anal. (C12H9ClN4O) Found:
C, 55.47; H, 3.74; N, 21.14. Calcd C, 55.29; H, 3.48; N, 21.49.
yield 66%. 1H NMR: d 12.38 (1H, br s, NH), 7.79 (1H, s, H-8), 6.21
(2H, br s, NH2), 4.20 (2H, d, J = 6.2 Hz, OCH2), 1.50 (11H, m,
C6H11). FABMS m/z: 248 (M+H)+. Anal. (C12H17N5Oꢂ0.3CH3OH)
Found: C, 57.25; H, 7.03; N, 27.09. Calcd C, 57.50; H, 7.14; N, 27.26.
3.1.1.24.
(31).
2-Chloro-6-[(cyclohexylmethyl)oxy]purine
3.1.1.15.
2-Amino-6-(benzyloxy)purine
(22)24
.
CHCl3–
CHCl3–MeOH (98:2) used for CC, off-white solid, yield
MeOH (9:1) was used for CC, off-white solid, yield 41%. 1H NMR:
d 12.44 (1H, br s, NH), 7.83 (1H, br s, H-8), 7.50 (2H, m, H-20,
H-60), 7.38 (3H, m, H-30, H-40, H-50), 6.29 (2H, br s, NH2), 5.48 (2H,
s, OCH2). FABMS m/z: 242 (M+H)+. Anal. (C12H11N5Oꢂ0.4CH3OH)
Found: C, 58.84; H, 4.89; N, 27.27. Calcd C, 58.62; H, 5.00; N, 27.56.
76%. 1H NMR: d 13.64 (1H, br s, NH), 8.43 (1H, s, H-8), 4.33 (2H,
d, J = 6.3 Hz, OCH2), 1.5 (11H, m, C6H11). FABMS m/z: 267 (M+H)+.
Anal. (C12H15ClN4O) Found: C, 53.91; H, 5.70; N, 20.94. Calcd C,
54.04; H, 5.67; N, 21.01.
3.1.1.25.
(32).
2-Chloro-6-[(3,4-dichlorobenzyl)oxy]purine
3.1.1.16.
(23).
2-Amino-6-[(3,4-dichlorobenzyl)oxy]purine
CHCl3–MeOH (99:1) with 0.5% NH4OH was used for CC,
Cyclohexane–EtOAc (5:1) was used for CC, off-white so-
lid, yield 84%. 1H NMR: d 13.73 (1H, br s, NH), 8.47 (1H, s, H-8), 7.85

A. K. Pathak et al. / Bioorg. Med. Chem. 21 (2013) 1685–1695
1691
(1H, d, J = 1.8 Hz, H-20), 7.71 (1H, d, J = 8.2 Hz, H-50), 7.50 (1H, ddd,
J = 1.9, 2.1, 8.2 Hz, H-60), 5.59 (2H, s, OCH2). FABMS m/z: 329
(M+H)+. Anal. (C12H7Cl3N4Oꢂ0.15C4H8O2) Found: C, 44.32; H, 2.49;
N, 16.17. Calcd C, 44.15; H, 2.41; N, 16.34.
(2H, t, J = 6.6 Hz, OCH2), 4.28 (2H, s, NCH2), 1.74 (2H, m, CH2),
1.37 (14H, m, 7ꢁ CH2), 0.85 (3H, m, CH3). ESI-MS m/z: Found
350.2204 (M+H)+, Calcd for C17H27N5O3 350.2187.
3.1.3.2.
(39).
2-Amino-6-decylthio-9H-purine-9-acetic
CHCl3–MeOH (7:1) was used for CC, off-white solid,
acid
3.1.2. General procedure for synthesis of N9-substituted purines
(33–36)
yield 64%, 1H NMR: d 7.81 (1H, s, H-8), 6.31 (2H, br s, NH2), 4.40
(2H, br s, NCH2), 3.26 (2H, t, J = 7.1 Hz, SCH2), 1.65 (2H, m, CH2),
1.37 (14H, m, 7ꢁ CH2), 0.85 (3H, m, CH3). ESI-MS m/z: Found
366.1959 (M+H)+, Calcd for C17H27N5O2S 366.1958. Anal.
(C17H27N5O2Sꢂ2.25H2O) Found: C, 50.68; H, 6.54; N, 16.86. Calcd
C, 50.29; H, 7.82; N, 16.86.
To a suspension of the appropriate substituted purine, dry
K2CO3 (1 equiv), and anhydrous DMSO, was added ethylbromoace-
tate (1.04 equiv) dropwise at 0 °C under an argon atmosphere. The
reaction mixture was allowed to stir at 0 °C for 4 to 6 h and then
allowed to stir at room temperature overnight under argon. Deion-
ized water (10–20 mL) was added and the reaction mixture was
extracted with diethyl ether. The ethereal layers were pooled, dried
over Na2SO4, concentrated, and dried in vacuo over P2O5 at 25 °C.
The crude material was purified by column chromatography (CC)
over silica gel G (230–400 mesh).
3.1.4. General procedure for synthesis of 6-decyloxy or 6-
decylthio-9-L-Phe/or Ala-substituted purine derivatives (39–50)
To a solution of the appropriate intermediate (38 or 39) in anhy-
drous pyridine (5 mL) under argon, was added the appropriate
amino acid ester HCl (1.5 equiv) followed by triethylamine
(1.5 equiv). The resulting solution was chilled slightly and PyBOP
(1.5 equiv) was added. The reaction solution was allowed to stir
under argon overnight at room temperature. Deionized water
(20 mL) was added and extracted with CHCl3 (2 ꢁ 350 mL). The
combined organic layers were washed with 1 N HCl (50 mL), d.
H2O (50 mL), dried over Na2SO4, concentrated, re-dissolved in tol-
uene and co-evaporated to remove excess pyridine. The resulting
residue was dried in vacuo over P2O5 at 25 °C. The crude material
was then purified by column chromatography (CC) over silica gel
G (230–400 mesh).
3.1.2.1. 9-Purine acetic acid, 6-(decyloxy) ethyl ester
(33).
CHCl3 (100%) was used for CC, off-white solid, yield
42%. 1H NMR: d 8.50 (1H, s, H-2), 8.35 (1H, s, H-8), 5.18 (2H, s,
NCH2), 4.54 (2H, dd, J = 6.6, 6.7 Hz, OCH2), 4.17 (2H, ddd, J = 7.0,
7.1, 7.2 Hz, OCH2), 1.80 (2H, m, CH2), 1.40 (2H, m, CH2), 1.22
(15H, m, 6ꢁ CH2, CH3), 0.85 (3H, m, CH3). FABMS m/z: 363
(M+H)+. Anal. (C19H30N4O3) Found: C, 62.72; H, 8.11; N, 15.16.
Calcd. C, 62.96; H, 8.34; N, 15.46.
3.1.2.2. 9-Purine acetic acid, 6-(dodecyloxy) ethyl ester
(34).
CHCl3 (100%) was used for CC, off-white solid, yield
77%. 1H NMR: d 8.50 (1H, s, H-2 or H-8), 8.35 (1H, s, H-2 or H-8),
5.18 (2H, s, NCH2), 4.54 (2H, dd, J = 6.5, 6.8 Hz, OCH2), 4.17 (2H,
ddd, J = 7.0, 7.1, 7.1 Hz, OCH2), 1.80 (2H, m, CH2), 1.3 (21H, m, 9ꢁ
CH2, CH3), 0.85 (3H, m, CH3). FABMS m/z: 391 (M+H)+. ESI-MS:
3.1.4.1.
L
-Alanine, N-[2-(2-amino-6-decyloxy-9H-purin-9-yl)-1-
CHCl3–MeOH (95:5) was used
oxoethyl]-, methyl ester (40).
for CC, off-white solid, yield 57%. 1H NMR: d 8.74 (1H, d, J = 7.0 Hz,
NHCH), 7.76 (1H, s, H-8), 6.36 (2H, br s, NH2), 4.74 (2H, br s, NCH2),
4.39 (2H, t, J = 6.6 Hz, OCH2), 4.29 (1H, m, NHCH), 3.63 (3H, m,
CO2CH3), 1.74 (2H, m, CH2), 1.25 (17H, m, CH3, 7ꢁ CH2), 0.85
(3H, m, CH3). ESI-MS m/z: Found 435.2721 (M+H)+, Calcd for
Found 391.2700, Calcd for
C21H34N4O3 391.2704. Anal.
(C21H34N4O3ꢂ0.5H2O) Found: C, 63.16; H, 8.46; N, 12.98. Calcd C,
63.13; H, 8.83; N, 14.02.
C21H34N6O4 435.2714. Anal. (C21H34N6O4) Found: C, 53.87; H,
3.1.2.3. 9-Purine acetic acid, 2-amino-6-(decyloxy) ethyl ester
7.23; N, 16.49.
(35).
CHCl3 (100%) was used for CC, off-white solid, yield 51%.
1H NMR: d 7.82 (1H, s, H-8), 6.43 (2H, br s, NH2), 4.91 (2H, s, NCH2),
4.39 (2H, dd, J = 6.6, 6.7 Hz, OCH2), 4.15 (2H, ddd, J = 7.1, 7.1, 7.1 Hz,
OCH2), 1.76 (2H, m, CH2), 1.24 (17H, m, 7ꢁ CH2, CH3), 0.85 (3H, m,
CH3). FABMS m/z: 378 (M+H)+. ESI-MS: Found 378.2491, Calcd for
3.1.4.2.
L-Phenylalanine, N-[2-(2-amino-6-decyloxy-9H-purin-9-
yl)-1-oxoethyl]-, methyl ester (41).
CHCl3–MeOH (99:1) was
used for CC, off-white solid, yield 51%. 1H NMR: d 8.76 (1H, d,
J = 7.5 Hz, NHCH), 7.69 (1H, s, H-8), 7.25 (5H, m, Ar), 6.33 (2H, br
s, NH2), 4.71 (2H, dd, NCH2), 4.48 (1H, m, NHCH), 4.38 (2H, t,
J = 6.5 Hz, OCH2), 3.59 (3H, s, CO2CH3), 2.98 (2H, m, CH2Ph), 1.73
(2H, m, CH2), 1.25 (14H, m, 7ꢁ CH2), 0.85 (3H, m, CH3). FABMS
m/z: 511 (M+H)+. Anal. (C27H38N6O4) Found: C, 63.41; H, 7.23; N,
16.44. Calcd C, 63.51; H, 7.50; N, 16.46.
C
19H31N5O3 378.2500.Anal. (C19H31N5O3ꢂ0.3H2O) Found: C, 59.39;
H, 7.81; N, 17.95. Calcd C, 59.60; H, 8.32; N, 18.29.
3.1.2.4. 9-Purine acetic acid, 2-amino-6-(dodecyloxy) ethyl ester
(36).
CHCl3 (100%) was used for CC, off-white solid, yield 31%.
1H NMR: d 7.82 (1H, s, H-8), 6.43 (2H, br s, NH2), 4.91 (2H, s, NCH2),
4.39 (2H, dd, J = 6.6, 6.8 Hz, OCH2), 4.15 (2H, ddd, J = 7.0, 7.1, 7.1 Hz,
OCH2), 1.74 (2H, m, CH2), 1.24 (21H, m, 5ꢁ CH2, CH3), 0.85 (3H, m,
CH3). FABMS m/z: 406 (M+H)+. Anal. (C21H35N5O3) Found: C, 61.82;
H, 8.42; N, 16.99. Calcd. C, 62.20; H, 8.70; N, 17.27.
3.1.4.3.
L-Phenylalanine, N-[2-(2-amino-6-decyloxy-9H-purin-9-
yl)-1-oxoethyl]-, ethyl ester (42).
CHCl3–MeOH (99:1) was
used for CC, off-white solid, yield 59%. 1H NMR: d 8.76 (1H, d,
J = 7.5 Hz, NHCH), 7.70 (1H, s, H-8), 7.25 (5H, m, Ar), 6.33 (2H, br
s, NH2), 4.72 (2H, dd, NCH2), 4.48 (1H, m, NHCH), 4.39 (2H, t,
J = 6.4 Hz, OCH2), 4.03 (2H, dd, CO2CH2), 2.98 (2H, m, CH2Ph),
1.74 (2H, m, CH2), 1.24 (14H, m, 7ꢁ CH2), 1.07 (3H, t, J = 7.0 Hz,
CH3), 0.85 (3H, m, CH3). FABMS m/z: 525 (M+H)+. Anal.
(C28H40N6O4ꢂ0.8H2O) Found: C, 62.63; H, 7.38; N, 15.30. Calcd C,
62.39; H, 7.78; N, 15.59. 20982.
3.1.3. General procedure for synthesis of 6-decyloxy (38) and 6-
decylthio (39) substituted purine intermediates
The starting material, 2-amino-6-chloro-9H-purine-9-acetic
acid (37), for the synthesis of 38 and 39 was purchased commer-
cially and used as such. The 6-decyloxy derivative 38 was synthe-
sized in the same manner as described above for compound 8. The
6-decylthio derivative 39 was synthesized by the method previ-
ously described by us.12
3.1.4.4.
L-Alanine, N-[2-(2-amino-6-decylthio-9H-purin-9-yl)-1-
oxoethyl]-, methyl ester (43).
Petroleum ether–EtOAc (1:4)
was used for CC, off-white solid, yield 66%. 1H NMR: d 8.76 (1H,
d, J = 7.0 Hz, NHCH), 7.84 (1H, s, H-8), 6.44 (2H, br s, NH2), 4.75
(2H, s, NCH2), 4.31 (1H, m, NHCH), 3.63 (3H, s, CO2CH3), 3.26
(2H, t, J = 7.0 Hz, SCH2), 1.64 (2H, m, CH2), 1.40 (2H, m, CH2), 1.29
3.1.3.1.
(38).
2-Amino-6-decyloxy-9H-purine-9-acetic
acid
CHCl3–MeOH (3:1) was used for CC, off-white solid,
yield 65%. 1H NMR: d 7.69 (1H, s, H-8), 6.20 (2H, br s, NH2), 4.38

1692
A. K. Pathak et al. / Bioorg. Med. Chem. 21 (2013) 1685–1695
(15H, m, CH3, 6ꢁ CH2), 0.85 (3H, m, CH3). ESI-MS m/z: Found
451.2487 (M+H)+, Calcd for C21H34N6O3S 451.2486. Anal.
(C21H34N6O3S) Found: C, 56.98; H, 7.64; N, 17.34. Calcd. C, 55.98;
H, 7.61; N, 18.65.
m, SCH2), 1.65 (2H, m, CH2), 1.40 (2H, m. CH2), 1.24 (12H, m, 6ꢁ
CH2), 0.85 (3H, m, CH3). FABMS m/z: 467 (M+H)+. Anal.
(C21H34N6O4S) Found: C, 54.04; H, 7.09; N, 18.01. Calcd C, 54.06;
H, 7.34; N, 18.00.
3.1.4.5.
D
-Alanine, N-[2-(2-amino-6-decylthio-9H-purin-9-yl)-1-
CHCl3–MeOH (98:2) was used
3.1.4.11.
oxoethyl]-, ethyl ester (50).
L
-Serine, N-[2-(2-amino-6-decylthio-9H-purin-9-yl)-1-
oxoethyl]-, methyl ester (44).
CHCl3–MeOH (95:5) was used
for CC, off-white solid, yield 57% yield. 1H NMR: d 8.75 (1H, d,
J = 6.9 Hz, NH), 7.84 (1H, s, H-8), 6.45 (2H, s, NH2), 4.76 (2H, m,
NCH2), 4.31 (1H, m, CH), 3.63 (3H, s, CH3), 3.27 (2H, m, SCH2),
1.64 (2H, m, CH2), 1.37 (2H, m, CH2), 1.31 (3H, d, J = 7.2 Hz, CH3),
1.24 (12H, m, 6ꢁ CH2), 0.85 (3H, m, CH3). ESI-MS m/z: found
451.2481 (M+H)+, Calcd 451.2485 for C21H34N6O3S. Anal.
(C21H34N6O3S) Found: C, 56.98; H, 7.64; N, 17.34. Calcd C, 55.98;
H, 7.61; N, 18.65.
for CC, off-white solid, yield 83%. 1H NMR: d 8.70 (1H, d,
J = 7.8 Hz, NHCH), 7.84 (1H, s, H-8), 6.44 (2H, br s, NH2), 5.15
(1H, t, J = 5.6 Hz, OH), 4.80 (2H, m, NCH2), 4.35 (1H, m, NHCH),
4.10 (2H, dd, J = 7.1, 14.2 Hz, OCH2), 3.73 (1H, m, CH2OH), 3.63
(1H, m, CH2OH), 3.26 (2H, t, J = 7.1 Hz, SCH2), 1.18 (3H, t,
J = 7.1 Hz, CH3), 1.65 (2H, m, CH2), 1.40 (2H, m, CH2), 1.24 (12H,
m, 6ꢁ CH2), 0.85 (3H, m, CH3). FABMS m/z: 481 (M+H)+. Anal.
(C22H36N6O4S) Found: C, 55.39; H, 7.43; N, 17.25. Calcd C, 54.98;
H, 7.55; N, 17.49.
3.1.4.6.
L
-Phenylalanine, N-[2-(2-amino-6-decylthio-9H-purin-
CHCl3–MeOH (99:1)
9-yl)-1-oxoethyl]-, methyl ester (45).
3.1.5. General procedure for synthesis of purine analogs 51–54
The 6-decylmercapto purine analogs 51–54 were prepared
starting from 6-chloropurine analogs by treating overnight with
1-decanethiol in presence of (CH3)3CO–K+ in isopropanol (3 mL)
under reflux condition. The reactions were cooled to room temper-
ature, 2 mL of deionized water were added and the mixture acidi-
fied with acetic acid (0.5 mL). Solid was collected by filtration and
washed with water followed by petroleum ether. The resulting
material was dried under vacuum at 78 °C overnight and analyzed.
was used for CC, off-white solid, yield 69%. 1H NMR: d 8.77 (1H,
d, J = 7.5 Hz, NHCH), 7.77 (1H, s, H-8), 7.22 (5H, m, Ar), 6.40 (2H,
br s, NH2), 4.72 (2H, dd, NCH2), 4.48 (1H, m, NHCH), 3.59 (3H, s,
CO2CH3), 3.26 (2H, m, SCH2), 2.99 (2H, m, CH2Ph), 1.65 (2H, m,
CH2), 1.24 (14H, m, 7ꢁ CH2), 0.85 (3H, m, CH3). FABMS m/z: 527
(M+H)+. Anal. (C27H38N6O3S) Found: C, 61.39; H, 7.23; N, 15.76.
Calcd C, 61.57; H, 7.26; N, 15.96.
3.1.4.7.
D
-Phenylalanine, N-[2-(2-amino-6-decylthio-9H-purin-
CHCl3–MeOH (98:2)
9-yl)-1-oxoethyl]-, methyl ester (46).
3.1.5.1.
(51).
N-(6-(Decylthio)-9H-purin-2-yl)acetamide
Starting from N-(6-chloro-6,9-dihydro-1H-purin-2-
was used for CC, off-white solid, yield 87%. 1H NMR: d 8.79 (1H,
d, J = 7.5 Hz, NHCH), 7.77 (1H, s, H-8), 7.22 (5H, m, Ar), 6.42 (2H,
br s, NH2), 4.73 (2H, m, NCH2), 4.48 (1H, m, NHCH), 3.59 (3H, s,
CO2CH3), 3.26 (2H, t, J = 7.1 Hz, SCH2), 3.03 (1H, dd, J = 5.9,
13.6 Hz, CH2Ph), 2.93 (1H, dd, J = 8.4, 13.6 Hz, CH2Ph), 1.65 (2H,
m, CH2), 1.39 (2H, m, CH2), 1.24 (12H, m, 6ꢁ CH2), 0.85 (3H, m,
CH3). FABMS m/z: 527 (M+H)+. Anal. (C27H38N6O3S) Found: C,
61.51; H, 7.24; N, 15.88. Calcd C, 61.57; H, 7.26; N, 15.96.
yl)acetamide17 (100 mg, 0.47 mmol), 106 mg (yield 64%). 1H
NMR: d 10.33 (1H, s, NH), 8.25 (1H, s, H-8), 3.33 (2H, m, SCH2),
2.20 (3H, s, COCH3), 1.69 (2H, m, CH2), 1.42 (2H, m, CH2), 1.24
(12H, m, 6ꢁ CH2), 0.85 (3H, m, CH3). ESI-MS m/z: 350 (M+H)+. Anal.
(C17H27N5OS); Found: C, 58.26; H, 7.97; N, 19.87. Calcd C, 58.42; H,
7.79; N, 20.04.
3.1.5.2. 3-(2-Amino-6-(decylthio)-9H-purin-9-yl)propane-1,2-
3.1.4.8.
L
-Phenylalanine, N-[2-(2-amino-6-decylthio-9H-purin-
Petroleum ether–EtOAc
diol (52).
Starting from 3-(2-amino-6-chloro-1H-purin-
9-yl)-1-oxoethyl]-, ethyl ester (47).
9(6H)-yl)propane-1,2-diol17 (100 mg, 0.41 mmol), 82 mg (yield
52%). 1H NMR: d 7.82 (1H, s, H-8), 6.44 (2H, br s, NH2), 5.07 (1H,
d, J = 5.1 Hz, CHOH), 4.16 (1H, dd, J = 3.3, 13.5 Hz, NCH2), 3.85
(1H, dd, J = 7.9, 13.5 Hz, NCH2), 3.77 (1H, m, CHOH) 3.37 (2H, m,
CH2OH), 3.26 (2H, dd, J = 6.9, 7.2 Hz, SCH2), 1.65 (2H, m, CH2),
1.38 (2H, m, CH2), 1.24 (12H, m, 6ꢁ CH2), 0.85 (3H, m, CH3). ESI-
MS m/z: 382 (M+H)+. Anal. (C18H31N5O2S); Found: C, 56.59; H,
8.04; N, 18.39. Calcd C, 56.66; H, 8.19; N, 18.36.
(1:2) was used for CC, off-white solid, yield 60%. 1H NMR: d 8.78
(1H, d, J = 7.5 Hz, NHCH), 7.78 (1H, s, H-8), 7.23 (5H, m, Ar), 6.41
(2H, br s, NH2), 4.75 (1H, d, NCH2), 4.72 (1H, d, NCH2), 4.48 (1H,
m, NHCH), 4.02 (2H, m, OCH2CH3), 3.26 (2H, m, SCH2), 2.98 (2H,
m, CH2Ph), 1.65 (2H, m, CH2), 1.29 (14H, m, 7ꢁ CH2), 1.07 (3H, t,
J = 7.0 Hz, CO2CH2CH3), 0.85 (3H, m, CH3). ESI-MS m/z: Found
541.2976 (M+H)+, Calcd for C28H40N6O3S 541.2955. Anal.
(C28H40N6O3S ꢂ0.5H2O) Found: C, 62.80; H, 7.72; N, 15.03. Calcd
C, 61.18; H, 7.52; N, 15.29.
3.1.5.3.
(53).
9-Benzyl-6-(decylthio)-9H-purin-2-amine
Starting from 9-benzyl-6-chloro-6,9-dihydro-1H-purin-
3.1.4.9.
L
-Serine, N-[2-(2-amino-6-decylthio-9H-purin-9-yl)-1-
2-amine17 (100 mg, 0.38 mmol), 135 mg (yield 88%). 1H NMR: d
8.02 (1H, s, H-8), 7.31 (3H, m, Ar), 7.22 (2H, m, Ar), 6.48 (2H, br
s, NH2), 5.25 (2H, s, NCH2), 3.26 (2H, dd, J = 6.9, 7.2 Hz, SCH2),
1.65 (2H, m, CH2), 1.38 (2H, m, CH2), 1.24 (12H, m, 6ꢁ CH2), 0.85
(3H, m, CH3). ESI-MS m/z: 398 (M+H)+. Anal. (C22H31N5S); Found:
C, 66.50 H, 7.89; N, 17.42. Calcd C, 66.46; H, 7.86; N, 17.61.
oxoethyl]-, methyl ester (48).
CHCl3–MeOH (9:1) was used
for CC, off-white solid, yield 53%. 1H NMR: d 8.74 (1H, d,
J = 7.8 Hz, NHCH), 7.84 (1H, s, H-8), 6.44 (2H, br s, NH2), 5.16
(1H, t, J = 5.4 Hz, OH), 4.81 (2H, m, NCH2), 4.38 (1H, m, NHCH),
3.73 (1H, m, CH2), 3.64 (3H, s, CH3), 3.62 (1H, m, CH2), 3.26 (2H,
t, J = 7.1 Hz, SCH2), 1.65 (2H, m, CH2), 1.40 (2H, m, CH2), 1.24
(12H, m, 6ꢁ CH2), 0.85 (3H, m, CH3). FABMS m/z: 467 (M+H)+. Anal.
(C21H34N4O4Sꢂ0.15H2O) Found: C, 53.80; H, 7.10; N, 17.51. Calcd C,
53.75; H, 7.37; N, 17.91.
3.1.5.4. 2-((2,6-Bis(decylthio)-9H-purin-9-yl)methoxy)ethanol
(54).
Starting
from
2-((2,6-dichloro-1H-purin-9(6H)-
yl)methoxy)ethanol17 (100 mg, 0.38 mmol), 40 mg (yield 26%). 1H
NMR: d 8.43 (1H, s, H-8), 5.57 (2H, s, NCH2), 4.66 (1H, t,
J = 5.3 Hz, OH), 3.51 (2H, m, OCH2), 3.45 (2H, m, CH2OH), 3.27
(2H, dd, J = 4.5, 7.8 Hz, SCH2), 3.18 (2H, dd, J = 7.2, 7.5 Hz, SCH2),
1.70 (4H, m, 2ꢁ CH2), 1.41 (4H, m, 2ꢁ CH2), 1.24 (24H, m, 12ꢁ
CH2), 0.84 (6H, m, 2ꢁ CH3). ESI-MS m/z: 539 (M+H)+. Anal.
(C28H50N4O2S2); Found: C, 62.53; H, 9.46; N, 10.22. Calcd C,
62.41; H, 9.35; N, 10.40.
3.1.4.10.
oxoethyl]-, methyl ester (49).
D-Serine, N-[2-(2-amino-6-decylthio-9H-purin-9-yl)-1-
CHCl3–MeOH (9:1) was used
for CC, off-white solid, yield 59%. 1H NMR: d 8.74 (1H, d,
J = 7.8 Hz, NHCH), 7.84 (1H, s, H-8), 6.44 (2H, br s, NH2), 5.17
(1H, t, J = 5.4 Hz, OH), 4.80 (2H, m, NCH2), 4.38 (1H, m, NHCH),
3.73 (1H, m, CH2), 3.64 (3H, s, CH3), 3.62 (1H, m, CH2), 3.26 (2H,

A. K. Pathak et al. / Bioorg. Med. Chem. 21 (2013) 1685–1695
1693
3.1.6. General procedure for synthesis of purine analogs 55–69
The 6-decylmercapto purine analogs 55–69 were prepared
starting from 6-purinethiones which were treated overnight with
1-chlorodecane/anhydrous K2CO3 in DMAc (3 mL) at room temper-
ature. Deionized water (2 mL) was added and the mixture was
acidified with acetic acid (0.2 mL). Solid was collected by filtration
and washed with water followed by petroleum ether. The material
was dried under vacuum at 78 °C overnight and analyzed.
d 9.78 (1H, s, CH), 8.90 (1H, s, H-2), 8.82 (1H, s, H-8), 7.95 (2H,
m, Ar), 7.60 (3H, m, Ar), 3.37 (2H, dd, J = 7.2, 7.5 Hz, SCH2), 1.71
(2H, m, CH2), 1.41 (2H, m, CH2), 1.24 (12H, m, 6ꢁ CH2), 0.85 (3H,
m, CH3). ESI-MS m/z: 396 (M+H)+. Anal. (C22H29N5S.0.1H2O);
Found: C, 66.52; H, 7.07; N, 17.62. Calcd C, 66.50; H, 7.36; N, 17.62.
3.1.6.8. 6-(Decylthio)-9-ethyl-9H-purine (62).
Starting from
9-ethyl-1H-purine-6(9H)-thione29 (100 mg, 0.55 mmol), 160 mg
(yield 90%). 1H NMR: d 8.70 (1H, s, H-2), 8.48 (1H, s, H-8), 4.27
(2H, dd, J = 7.2, 14.4 Hz, NCH2), 3.34 (2H, dd, J = 7.2, 7.5 Hz,
SCH2), 1.70 (2H, m, CH2), 1.43 (3H, t, J = 7.2 Hz, CH3), 1.40 (2H, m,
CH2), 1.24 (12H, m, 6ꢁ CH2), 0.85 (3H, m, CH3). ESI-MS m/z: 321
(M+H)+. Anal. (C17H28N4S); Found: C, 63.72; H, 8.94; N, 17.44. Calcd
C, 63.71; H, 8.81; N, 17.48.
3.1.6.1. 6-(Decylthio)-9-ethyl-9H-purin-2-amine (55).
Start-
ing from 2-amino-9-ethyl-1H-purine-6(9H)-thione25 (100 mg,
0.51 mmol), 80 mg (yield 47%). 1H NMR: d 7.94 (1H, s, H-8), 6.42
(2H, br s, NH2), 4.03 (2H, dd, J = 7.2, 14.4 Hz, NCH2), 3.26 (2H, t,
J = 7.2 Hz, SCH2), 1.64 (2H, m, CH2), 1.34 (3H, t, J = 7.2 Hz, CH3),
1.40 (2H, m, CH2), 1.24 (12H, m, 6ꢁ CH2), 0.85 (3H, m, CH3). ESI-
MS m/z: 336 (M+H)+. Anal. (C17H29N5S.0.1H2O); Found: C, 60.47;
H, 8.75; N, 20.54. Calcd C, 60.53; H, 8.73; N, 20.76.
3.1.6.9. 9-Benzyl-6-(decylthio)-9H-purine (63).
Starting
from 9-benzyl-1H-purine-6(9H)-thione17 (100 mg, 0.41 mmol),
59 mg (yield 37%). 1H NMR: d 8.71 (1H, s, H-2), 8.59 (1H, s, H-8),
7.33 (5H, m, Ar), 5.47 (2H, s, NCH2), 3.35 (2H, m, SCH2), 1.70 (2H,
m, CH2), 1.40 (2H, m, CH2), 1.24 (12H, m, 6ꢁ CH2), 0.85 (3H, m,
CH3). ESI-MS m/z: 383 (M+H)+. Anal. (C22H30N4S); Found: C,
69.05; H, 7.83; N, 14.66. Calcd C, 69.07; H, 7.90; N, 14.65.
3.1.6.2. 6-(Decylthio)-2-ethyl-9H-purine (56).
Starting from
2-ethyl-1H-purine-6(9H)-thione26 (100 mg, 0.55 mmol), 56 mg
(yield 31%). 1H NMR: d 13.2 (1H, br s, NH), 8.31 (1H, s, H-8), 3.33
(2H, dd, J = 6.9, 7.2 Hz, SCH2), 2.89 (2H, dd, J = 7.5, 15 Hz, CH2),
1.70 (2H, m, CH2), 1.41 (2H, m, CH2), 1.31 (3H, t, J = 7.5 Hz, CH3),
1.23 (12H, m, 6ꢁ CH2), 0.84 (3H, m, CH3). ESI-MS m/z: 321
(M+H)+. Anal. (C17H28N4S.0.2H2O); Found: C, 63.14; H, 8.70; N,
17.29. Calcd C, 63.00; H, 8.83; N, 17.28.
3.1.6.10.
(64).
2-(6-(Decylthio)-9H-purin-9-yl)cyclohexanol
from 9-(2-hydroxycyclohexyl)-1H-purine-
Starting
6(9H)-thione18d (100 mg, 0.40 mmol), 80 mg (yield 51%). 1H
NMR: d 8.68 (1H, s, H-2), 8.42 (1H, s, H-8), 4.99 (1H, d, J = 4.2 Hz,
OH), 4.55 (1H, m, NCH), 3.97 (1H, br s, CHOH), 3.34 (2H, m,
SCH2), 2.30 (1H, m, CH2), 1.74 (6H, m, CH2), 1.42 (5H, m, CH2),
1.24 (12H, m, 6ꢁ CH2), 0.85 (3H, m, CH3). ESI-MS m/z: 391
(M+H)+. Anal. (C21H34N4OS); Found: C, 64.45; H, 8.83; N, 14.34.
Calcd C, 64.58; H, 8.77; N, 14.34.
3.1.6.3. 9-Cycloheptyl-6-(decylthio)-9H-purine (57).
Start-
ing from 9-cycloheptyl-1H-purine-6(9H)-thione27 (100 mg,
0.40 mmol), 124 mg (yield 79%). 1H NMR: d 8.68 (1H, s, H-2),
8.54 (1H, s, H-8), 4.64 (1H, m, NCH), 3.32 (2H, dd, J = 7.2, 7.5 Hz,
SCH2), 2.08 (6H, m, 3ꢁ CH2), 1.65 (8H, m, 4ꢁ CH2), 1.41 (2H, m,
CH2), 1.23 (12H, m, 6ꢁ CH2), 0.84 (3H, m, CH3). ESI-MS m/z: 389
(M+H)+. Anal. (C22H36N4S.0.1H2O); Found: C, 67.77; H, 9.37; N,
14.34. Calcd C, 67.68; H, 9.35; N, 14.35.
3.1.6.11. 9-Cyclopentyl-6-(decylthio)-9H-purine (65).
Start-
ing from 9-cyclopentyl-1H-purine-6(9H)-thione18a (100 mg,
0.48 mmol), 134 mg (yield 82%). 1H NMR: d 8.69 (1H, s, H-2),
8.52 (1H, s, H-8), 4.93 (1H, m, NCH), 3.33 (2H, m, SCH2), 2.16
(2H, m, CH2), 2.03 (2H, m, CH2), 1.89 (2H, m, CH2), 1.70 (2H, m,
CH2), 1.40 (2H, m, CH2), 1.23 (12H, m, 6ꢁ CH2), 0.84 (3H, m,
CH3). ESI-MS m/z: 361 (M+H)+. Anal. (C30H32N4S.0.1H2O); Found:
C, 66.06; H, 9.10; N, 15.89. Calcd C, 66.29; H, 8.96; N, 15.46.
3.1.6.4. 9-Allyl-6-(decylthio)-9H-purine (58).
Starting from
9-allyl-1H-purine-6(9H)-thione (100 mg, 0.52 mmol), 72 mg (yield
42%). 1H NMR: d 8.70 (1H, s, H-2), 8.44 (1H, s, H-8), 6.08 (1H, m,
CH), 5.21 (1H, m, CH2a), 5.06 (1H, m, CH2b), 4.89 (2H, m, NCH2),
3.35 (2H, dd, J = 7.2, 7.5 Hz, SCH2), 1.70 (2H, m, CH2), 1.41 (2H,
m, CH2), 1.23 (12H, m, 6ꢁ CH2), 0.84 (3H, m, CH3). ESI-MS m/z:
333 (M+H)+. Anal. (C18H28N4S.0.1H2O); Found: C, 64.74; H, 8.55;
N, 16.72. Calcd C, 64.67; H, 8.50; N, 16.76.
3.1.6.12. 9-Butyl-6-(decylthio)-9H-purine (66).
Starting
from 9-butyl-1H-purine-6(9H)-thione18a (100 mg, 0.48 mmol),
124 mg (yield 74%). 1H NMR: d 8.69 (1H, s, H-2), 8.47 (1H, s, H-
8), 4.24 (2H, dd, J = 6.9, 7.2 Hz, NCH2), 3.34 (2H, t, J = 7.2 Hz,
SCH2), 1.81 (2H, m, CH2), 1.69 (2H, m, CH2), 1.39 (2H, m, CH2),
1.23 (14H, m, 7ꢁ CH2), 0.89 (3H, t, J = 7.2 Hz, CH3), 0.84 (3H, m,
CH3). ESI-MS m/z: 349 (M+H)+. Anal. (C19H32N4S.0.2H2O); Found:
C, 64.88; H, 9.28; N, 15.89. Calcd C, 64.80; H, 9.27; N, 15.90.
3.1.6.5.
(59).
2-(6-(Decylthio)-9H-purin-9-yl)ethyl
Starting from 2-(6-thioxo-1H-purin-9(6H)-yl)ethyl ace-
acetate
tate (100 mg, 0.42 mmol), 146 mg (yield 92%). 1H NMR: d 8.71
(1H, s, H-2), 8.47 (1H, s, H-8), 4.50 (2H, m, NCH2), 4.41 (2H, m,
OCH2), 3.33 (2H, dd, J = 7.2, 7.5 Hz, SCH2), 2.93 (3H, s, CH3), 1.70
(2H, m, CH2), 1.41 (2H, m, CH2), 1.24 (12H, m, 6ꢁ CH2), 0.85 (3H,
m, CH3). ESI-MS m/z: 379 (M+H)+. Anal. (C19H30N4O2S.0.15H2O);
Found: C, 59.69; H, 7.88; N, 14.61. Calcd C, 59.86; H, 8.01; N, 14.69.
3.1.6.13.
(67).
2-(6-(Decylthio)-9H-purin-9-yl)cyclopentanol
Starting from 9-(2-hydroxycyclopentyl)-1H-purine-
6(9H)-thione18c (100 mg, 0.42 mmol), 94 mg (yield 59%). 1H
NMR: d 8.68 (1H, s, H-2), 8.45 (1H, s, H-8), 4.92 (1H, d, J = 4.2 Hz,
OH), 4.78 (1H, m, NCH), 4.18 (1H, m, CHOH), 3.34 (2H, m, SCH2),
2.29 (2H, m, CH2), 2.11 (2H, m, CH2), 1.94 (2H, m, CH2), 1.70 (2H,
m, CH2), 1.40 (2H, m, CH2), 1.24 (12H, m, 6ꢁ CH2), 0.85 (3H, m,
CH3). ESI-MS m/z: 377 (M+H)+. Anal. (C20H32N4OS); Found: C,
63.88; H, 8.56; N, 14.75. Calcd C, 63.79; H, 8.57; N, 14.88.
3.1.6.6.
(60).
2-(6-(Decylthio)-1H-purin-9(6H)-yl)acetonitrile
Starting from 2-(6-thioxo-1H-purin-9(6H)-yl)acetoni-
trile (100 mg, 0.52 mmol), 75 mg (yield 43%). 1H NMR: d 8.79
(1H, s, H-2), 8.51 (1H, s, H-8), 5.55 (2H, m, NCH2), 3.36 (2H, dd,
J = 7.2, 7.5 Hz, SCH2), 1.71 (2H, m, CH2), 1.41 (2H, m, CH2), 1.24
(12H, m, 6ꢁ CH2), 0.85 (3H, m, CH3). ESI-MS m/z: 332 (M+H)+. Anal.
(C17H25N5S); Found: C, 61.48; H, 7.36; N, 20.99. Calcd C, 61.60; H,
7.60; N, 21.13.
3.1.6.14.
(68).
N-(6-(Decylthio)-9H-purin-9-yl)acetamide
Starting from N-(6-thioxo-1H-purin-9(6H)-yl)acetam-
3.1.6.7.
(61).
(E)-N-Benzylidene-6-(decylthio)-9H-purin-9-amine
Starting from (E)-9-(benzylideneamino)-1H-purine-
ide18b (100 mg, 0.48 mmol), 40 mg (yield 24%). 1H NMR: d 8.69
(1H, s, H-2), 8.48 (1H, s, H-8), 3.35 (2H, m, SCH2), 2.10 (3H, s,
CH3), 1.71 (2H, m, CH2), 1.40 (2H, m, CH2), 1.24 (12H, m, 6ꢁ
6(9H)-thione28 (100 mg, 0.39 mmol), 82 mg (yield 53%). 1H NMR:

1694
A. K. Pathak et al. / Bioorg. Med. Chem. 21 (2013) 1685–1695
CH2), 0.85 (3H, m, CH3). ESI-MS m/z: 350 (M+H)+. Anal.
(C17H27N5OS.0.1H2O); Found: C, 58.01; H, 8.03; N, 19.80. Calcd C,
58.12; H, 7.80; N, 19.93.
supplemented with 0.2% glycerol and ADC enrichment using a col-
orimetric (Alamar blue) microdilution broth assay.30 The active
compounds (612.8 lg/mL) were re-tested using twofold dilutions
to obtain the actual MIC99. The MIC99 was recorded as the lowest
drug concentration that inhibited the growth completely.
3.1.6.15. 9-Cyclohexyl-6-(decylthio)-9H-purine (69).
Start-
ing from 9-cyclohexyl-1H-purine-6(9H)-thione18a (100 mg,
0.43 mmol), 93 mg (yield 58%). 1H NMR: d 8.68 (1H, s, H-2), 8.54
(1H, s, H-8), 4.45 (1H, m, NCH), 3.33 (2H, m, SCH2), 1.91 (8H, m,
4ꢁ CH2), 1.69 (2H, m, CH2), 1.41 (4H, m, 2ꢁ CH2), 1.24 (12H, m,
6ꢁ CH2), 0.85 (3H, m, CH3). ESI-MS m/z: 375 (M+H)+. Anal.
(C21H34N4S.0.1H2O); Found: C, 66.97; H, 9.19; N, 14.85. Calcd C,
67.01; H, 9.16; N, 14.89.
3.2.2. Activity against H37Rv20
The primary screen was conducted at 6.25
lg/mL against Mtb.
H37Rv (ATCC 27294) using radiometric BACTEC assay in a 12B
medium by Tuberculosis Antimicrobial Coordinating Facility
(TAACF). Compounds demonstrating at least 90% inhibition at
6.25 lg/mL were re-tested to determine the MIC90, defined as the
lowest concentrating inhibiting growth by 90% or higher.
3.1.7. N-(3,4-Dimethylphenyl)-2-(4-(methylthio)-7H-
pyrrolo[2,3-d]pyrimidin-7-yl)acetamide (70)
3.2.3. Cytotoxicity against VERO cells and selectivity index (SI)20
Concurrent with the determination of MIC90’s, compounds 16,
33, 34, 36, 55 and 65 were tested for cytotoxicity (CC50) in VERO
cells using Promega non-radioactive cell proliferation assay kits
by TAACF. The selectivity index is defined as the ratio of the mea-
sured CC50 in VERO cells to the MIC90 (against Mtb H37Rv).
Compound 73 (110 mg, 0.58 mmol) was dissolved in dry DMF
(5 mL) under argon and 3,4-dimethylaniline (106 mg, 0.87 mmol)
was added followed by the addition of HATU (265 mg, 0.70 mmol)
and DIEA (0.16 mL, 0.16 mmol). The reaction mixture was stirred at
room temperature overnight. The resulting solid was filtered and
washed with ether to give pure 70 (86 mg, yield 54%) after drying
overnight under vacuum over P2O5 at 78 °C. Mp: 203–205 °C. 1H
NMR: d 10.26 (1H, s, CONH), 8.60 (1H, s), 7.54 (1H, d, J = 3.6 Hz),
7.35 (1H, s), 7.27 (1H, d, J = 2.0, 11.0 Hz), 7.05 (1H, d, J = 8.1 Hz),
6.54 (1H, d, J = 3.3 Hz), 5.11 (2H, s), 2.66 (3H, s), 2.17 (3H, s), 2.16
(3H, s). ESI-MS: m/z calcd for C17H18N4OS (M+H)+: 327.1274,
found: 327.1278.
Acknowledgments
The authors are thankful to the TAACF, a research and develop-
ment contract with the NIAID, NIH (Contract No. NO1-AI45246) for
screening against Mtb H37Rv. This work was supported by NIH/
NIAID Grant R01AI45317. The M. avium strains were kindly pro-
vided by Dr. Leonid Heifts, National Jewish Center for Immunology
and Respiratory Diseases, Denver, CO, USA.
3.1.8. 4-(Methylthio)-7H-pyrrolo[2,3-d]pyrimidine (72)
To a methanol (15 mL) solution of commercially available
4-chloro-7H-pyrrolo[2,3-d]pyrimidine (400 mg, 2.60 mmol) was
added NaSCH3 (272 mg, 3.90 mmol), and the reaction mixture
was stirred overnight at room temperature. The mixture was con-
centrated and the crude material was purified by column chroma-
tography using 5% CHCl3–MeOH to yield pure product 68 (386 mg,
yield 90%). 1H NMR: d 12.08 (1H, br s, NH), 8.58 (1H, s, H-2), 7.45
(1H, d, J = 3.4 Hz, H-8), 6.47 (1H, d, J = 3.5 Hz, H-7), 2.64 (3H, s,
SCH3). ESI-MS: m/z calcd for C7H7N3S (M+H)+: 166.0433, found:
166.0432.
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