Exploring natural product chemistry and biology with multicomponent reactions. 5. Discovery of a novel tubulin-targeting scaffold derived from the rigidin family of marine alkaloids

Article abstract of DOI:10.1021/jm400711t

We developed synthetic chemistry to access the marine alkaloid rigidins and over 40 synthetic analogues based on the 7-deazaxanthine, 7-deazaadenine, 7-deazapurine, and 7-deazahypoxanthine skeletons. Analogues based on the 7-deazahypoxanthine skeleton exh

Full text of DOI:10.1021/jm400711t

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Article
Exploring Natural Product Chemistry and Biology with
Multicomponent Reactions. 5. Discovery of a Novel Tubulin-
Targeting Scaffold Derived from the Rigidin Family of Marine Alkaloids
Liliya V Frolova, Igor V. Magedov, Anntherese E Romero, Menuka Karki, Isaiah Otero, Kathryn Hayden,
Nikolai M Evdokimov, Laetitia Moreno Y Banuls, Shiva K Rastogi, W Ross Smith, Shi-Long Lu, Robert
Kiss, Charles Bradley Shuster, Ernest Hamel, Tania Betancourt, Snezna Rogelj, and Alexander Kornienko
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400711t • Publication Date (Web): 08 Aug 2013
Downloaded from http://pubs.acs.org on August 16, 2013
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Exploring Natural Product Chemistry and Biology with
Multicomponent Reactions. 5. Discovery of a Novel Tubulin-
Targeting Scaffold Derived from the Rigidin Family of Marine
Alkaloids
Liliya V. Frolova,†,* Igor V. Magedov,† Anntherese E. Romero,‡ Menuka Karki,§ Isaiah
Otero,† Kathryn Hayden,‡ Nikolai M. Evdokimov,† Laetitia Moreno Y. Banuls,# Shiva K.
¶
¶
∆
#
§
⊥
Rastogi, W. Ross Smith, Shi-Long Lu, Robert Kiss, Charles B. Shuster, Ernest Hamel,
Tania Betancourt,¶ Snezna Rogelj,‡ and Alexander Kornienko¶,*
Departments of Chemistry and Biology, New Mexico Institute of Mining and Technology, Socorro, NM
87801, USA, Department of Biology, New Mexico State University, Las Cruces, NM 88003, USA,
Laboratoire de Toxicologie and Laboratoire de Chimie Analytique, Toxicologie et Chimie Physique
Appliquée, Institut de Pharmacie, Université Libre de Bruxelles, Brussels, Belgium, Departments of
Otolaryngology, Dermatology and Pathology, University of Colorado, Anschutz Medical Campus,
Aurora, CO 80045, USA, Screening Technologies Branch, Developmental Therapeutics Program,
Division of Cancer Treatment and Diagnosis, National Cancer Institute, Frederick National Laboratory
of Cancer Research, National Institutes of Health, Frederick, MD 21702, USA, Department of Chemistry
and Biochemistry, Texas State University – San Marcos, San Marcos, TX 78666, USA.
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† Department of Chemistry, New Mexico Institute of Mining and Technology
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‡ Department of Biology, New Mexico Institute of Mining and Technology
§ New Mexico State University
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# Université Libre de Bruxelles
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∆ University of Colorado
⊥
National Cancer Institute
¶ Texas State University
Abstract: We developed synthetic chemistry to access the marine alkaloid rigidins and over forty
synthetic analogues based on the 7-deazaxanthine, 7-deazaadenine, 7-deazapurine and 7-
deazahypoxanthine skeletons. Analogues based on the 7-deazahypoxanthine skeleton exhibited nanomolar
potencies against cell lines representing cancers with dismal prognoses, tumor metastases and multidrug
resistant cells. Studies aimed at elucidating the mode(s) of action of the 7-deazahypoxanthines in cancer
cells revealed that they inhibited in vitro tubulin polymerization and disorganized microtubules in live
HeLa cells. Experiments evaluating the effects of the 7-deazahypoxanthines on the binding of
[3H]colchicine to tubulin identified the colchicine site on tubulin as the most likely target for these
compounds in cancer cells. Because many microtubule-targeting compounds are successfully used to
fight cancer in the clinic, we believe the new chemical class of antitubulin agents represented by the 7-
deazahypoxanthine rigidin analogues have significant potential as new anticancer agents.
Introduction
In light of their fascinating novel structures, intriguing biological properties and the difficulty in
obtaining large quantities from natural sources, marine pyrrole-derived alkaloids have attracted
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considerable attention from organic and medicinal chemists.1-8 At present, several marine natural products
are in clinical trials, and one drug, trabectedin, was recently approved in Europe as the first ever marine-
derived anticancer agent.9 In this context, the marine alkaloid rigidins A, B, C, D (Figure 1) and E isolated
from the tunicate Eudistoma cf. rigida found near Okinawa and New Guinea have remained largely
unexplored despite initial reports of promising biological activities.10-12
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OH
OH
O
O
HN
HN
OH
OH
O
O
N
H
N
H
N
H
N
H
OMe
O
O
Rigidin A
Rigidin B
MeO
MeO
OH
OH
O
O
HN
HN
OH
OH
O
O
N
H
N
H
N
H
N
H
OMe
O
O
Rigidin D
Rigidin C
Figure 1. Structures of rigidins A, B, C and D
These compounds were shown to exhibit cytotoxicity against murine leukemia L1210 cells,11 while
rigidin A was also found to possess anticalmodulin activity.10 Further work in this area was hampered by
the miniscule isolation yields of these natural products (0.0015 % for A, 0.00031 % for B, 0.00008 % for
C, 0.00004 % for D, and 0.018 % for E based on dry weight). Up to 2011, the synthetic accomplishments
in the rigidin area included three total syntheses of rigidin A13-16 and one of rigidin E.16 These approaches
involved lengthy synthetic sequences and/or low overall yields and did not offer a solution to the natural
product supply problem. More importantly, however, the lack of synthetic flexibility impeded their use
for the preparation of analogues for structure-activity relationship (SAR)a studies. Because our research
program aims to develop multicomponent reactions (MCR) to access “privileged medicinal scaffolds,”17-20
we became interested in the rigidins, which incorporate the purine-like pyrrolo[2,3-d]pyrimidine ring
system, a common motif in natural products (e.g., tubercidin, toyocamycin, sangivamycin21) and
medicinal agents with diverse bioactivities (e.g., anti-inflammatory,22,23 anticancer,24-29 antiviral,30,31
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adenosine A1 and A3 receptor inhibition,32 adenosine kinase inhibition,33,34 and dihydrofolate reductase
inhibition35).
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7
8
In 2011, we reported in a communication format a general total synthesis of rigidins A, B, C and D,
which involved only four steps from commercially available materials and was amenable to the
production of synthetic rigidin analogues.36 This work has led to further developments and discoveries,
which we describe here. More specifically, first, we detail the synthesis of first-generation synthetic
analogues based on the 7-deazaxanthine skeleton. Second, we describe an anticancer evaluation of
synthetically prepared rigidins A, B, C and D that, together with the first generation analogues, fail to
display any significant effects on cancer cell proliferation. Third, we disclose novel MCRs leading to the
preparation of the second-generation rigidin analogues based on the 7-deazaadenine, 7-deazapurine and 7-
deazahypoxanthine (Figure 2) skeletons. Fourth, we show that the 7-deazahypoxanthines display potent
antiproliferative activities. Fifth, we demonstrate that these compounds maintain their activity against
tumor cells derived from cancers associated with dismal prognoses, metastatic tissues as well as multidrug
resistant cells. Finally, we show that the anticancer properties of these hypoxanthine-like compounds are
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most likely caused by their pronounced effects on microtubule organization in cancer cells.
O
NH2
Ar1
Ar1
HN
6
Ar2
O
1
6
N1
7
7
Ar2
3
O
3
N
N
N
N
H
H
H
O
7-deazaadenine
7-deazaxanthine
Ar3
O
Ar1
Ar1
Ar2
Ar2
6
6
N 1
N1
7
7
3
3
N
H
N
O
O
N
H
N
H
7-deazapurine
7-deazahypoxanthine
Figure 2. Structures of pyrrolo[2,3-d]pyrimidines prepared in this work
Results and Discussion
Chemistry
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The synthesis of natural rigidins A, B, C and D, as well as synthetic 7-deazaxanthine analogues, is
1
2
based on the chemistry shown in Table 1. Sulfonamides 6-11, prepared by reacting commercially
available aminoacetophenones with methanesulfonyl chloride, are utilized in a three-component reaction
(3-CR) with various aromatic aldehydes 1-5 and cyanoacetamide in the presence of potassium carbonate
in refluxing ethanol to give aminopyrroles 12-21 (Table 1). The synthesis of the 7-deazaxanthine
framework is completed by carbonylation with oxalyl chloride in diglyme to give analogues 22a-j
possessing diversely substituted aromatic moieties Ar1 and Ar2. Compounds 22a-d are further
hydrogenolyzed to yield natural rigidins with overall yields of 61% for A, 58% for B, 60% for C and 53%
for D from commercially available aminoacetophenones. This sequence constitutes the first synthesis of
rigidins B, C and D, while rigidin A is accessed via a significantly improved method compared with the
previously described routes.13-16 In addition, our reaction sequence leads to an expedient preparation of
what we call the first-generation library of rigidin analogues based on the 7-deazaxanthine skeleton and
possessing variable substituents in the aromatic moieties. Finally, in addition to these synthetic 7-
deazaxanthines 22a-j, we prepared a dithia variant 22k, which is obtained by treating pyrrole 21 with
carbon disulfide in pyridine (Figure 3).
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Table 1. MCR-based total general synthesis of rigidins A-D and their first-generation 7-deazaxanthine
synthetic analogues 22a-j
Ar2
Ar1
O
O
CHO
1-5
Ar1
K2CO3
H2N
H2N
(COCl)2
diglyme
+
Ar2
NH
EtOH
reflux
CONH2
O
S
O
N
H
O
Me
CN
12-21
6-11
Y
OH
O
Ar1
O
HN
H2 , Pd/C
Ar2
HN
O
MeOH-DMF
N
OH
N
O
H
H
O
N
H
N
H
22a-j
X
O
rigidin A (95% from 22a) X = Y = H
rigidin B (94% from 22b) X = OMe, Y = H
rigidin C (97% from 22c) X = H, Y = OMe
rigidin D (93% from 22d) X = Y = OMe
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aldehyde
methanes pyrrole
ulfonyl
amide
7-deaza
Ar1
Ar2
overall
yield %
1
2
xanthine
3
4
5
6
1
1
6
7
12
13
22a
22b
OBn
64
62
OBn
OMe
OBn
OBn
7
8
9
OMe
OBn
2
2
3
6
7
8
14
15
16
22c
22d
22e
62
57
74
OBn
OMe
OBn
OMe
10
11
12
13
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OBn
OMe
OMe
OMe
OMe
OMe
F
3
3
9
7
17
18
22f
64
59
Br
OMe
OMe
OMe
22g
OBn
OMe
OMe
OMe
4
4
5
10
11
11
19
20
21
22h
22i
22j
57
62
43
OMe
Ph
Ph
OMe
Br
Br
Br
Br
S
CS2
Br
Br
NC
HN
py
S
H2N
57%
N
H
N
H
N
H
O
O
22k
21
Figure 3. Preparation of an additional 7-deazapurine 22k
Our further studies of the above-described three-component reaction led to the discovery of its four-
component (4-CR) variants. Thus, we found that if the reaction is conducted in formamide as a solvent
and cyanoacetamide is replaced with malononitrile (Table 2) or cyanomethylketones (Table 3), the entire
pyrrolo[2,3-d]pyrimidine skeleton is assembled in one step giving rise to 7-deazaadenines 23 (Table 2) or
7-deazapurines 24 (Table 3), respectively. The success of these 4-CRs also depends on an optimized
o
temperature regime, in which the reaction is first kept at 90 C to allow for a clean formation of
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intermediate aminopyrroles (similar to 12-21 in Table 1) and then at 150 oC to bring the fourth component
1
2
3
formamide into the process, leading to closure of the pyrimidine portion of the molecule.
4
5
6
7
8
Table 2. 4-CR leading to the formation of the second-generation analogues based on the 7-
9
deazaadenine skeleton.
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Ar2
O
Ar1
NH2
N
Ar1
Ar2
O
CHO
K2CO3
N
NH
O
O
CN
CN
S
1. 90 oC, 24 h
2. 150 oC, 3-6 h
N
Me
H
HCONH2
23a-f
7-
Ar1
Ar2
% yield
deazaadenine
Br
Ph
58
66
23a
Br
Br
Ph
23b
N
Ph
Ph
48
85
54
23c
23d
23e
Ph
S
CN
OBn
Cl
Ph
72
23f
Cl
Table 3. 4-CR leading to the formation of the second-generation analogues based on the 7-deazapurine
skeleton.
Ar2
Ar3
Ar1
Ar1
O
Ar2
O
CHO
K2CO3
N
1. 90 oC, 24 h
2. 150 oC, 3-6 h
O
NH
O
O
N
N
S
Ar3
H
Me
HCONH2
24a-e
CN
6,7-
Ar1
Ar2
Ar3
% yield
dideazaad
enine
24a
63
30
OMe
OMe
OMe
OMe
24b
Ph
Ph
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Br
24c
24d
24e
33
39
22
Ph
Me
1
2
3
4
5
6
7
8
Br
Br
Ph
Ph
Ph
Ph
Br
N
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Additionally, we discovered a 4-CR leading to the formation of a 7-deazahypoxanthine skeleton 25
(Table 4). In this modification, the starting sulfonamides, aldehydes and cyanoacetamide are reacted in a
1:1 mixture of EtOH and triethyl orthoformate under a similar temperature regime, involving reflux at 90
o
oC followed by a gradual increase in temperature to 150 C, with the concomitant slow evaporation of
ethanol.
Table 4. 4-CR leading to the formation of the second-generation analogues based on the 7-
deazahypoxanthine skeleton.
Ar2
Ar1
O
Ar1
O
Ar2
O
CHO
N
K2CO3, EtOH
NH
O
O
CN
CONH2
S
1. 90 oC, 24 h
2. 150 oC, 3-6 h
N
N
H
Me
H
25a-v
HC(OEt3)
7-deaza-
hypo-
Ar1
Ar2
% yield
7-deaza-
Ar1
Ar2
% yield
hypo-
xanthine
25l
xanthine
25a
Cl
Cl
Ph
Ph
Ph
71
Ph
Ph
74
77
Br
I
25b
68
25m
Br
Cl
OH
25c
25d
Ph
Ph
61
54
25n
25o
Ph
Ph
26
47
Br
CN
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Br
F
25e
25f
25g
25h
25i
Ph
Ph
Ph
Ph
Ph
Ph
53
60
88
61
64
37
25p
25q
25r
25s
25t
56
56
60
61
54
68
F
1
2
3
4
5
6
7
8
Br
Br
Br
OMe
OMe
OMe
OBn
Br
N
OMe
OMe
Br
N
9
Br
10
11
12
13
14
15
16
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20
21
22
23
24
25
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Br
N
Br
OMe
OMe
Br
Br
OMe
I
25j
25u
Ph
OBn
OMe
OMe
Br
Br
25k
Ph
51
25v
Ph
71
OMe
OMe
S
The analogues accessed by these 4-CRs (Tables 2, 3 and 4) are generally obtained in good yields given
the complexity of the newly devised reactions. The product pyrrolopyrimidines contain diversely
substituted aromatic moieties, including alkoxy (e.g., 22a-i, 23e, 24a, 25i-k, 25q, etc), halogen (e.g.,
23a,b, 25b-g, 25r-t, etc), cyano (e.g., 25o) and heterocycles (e.g., 23b, 25f-h, 25v, etc). Additionally, the
hydroxy-substituted pyrrolopyrimidines can be obtained by the use of precursors containing benzyl-
protected phenolic oxygens, with subsequent hydrogenolysis (similar to the synthesis of rigidins A-D
from 22a-d in Table 1) or through another modification in the MCR chemistry involving the use of
HCOOH as the fourth component (Table 5). Formic acid plays a dual role, providing the necessary carbon
for pyrimidine ring-closure and promoting in situ debenzylation of the phenolic benzyl ethers, resulting in
analogues 25w-z (Table 5). Interestingly, a search of the literature reveals no examples of metal-free
utilization of formic acid for deprotection of benzyl ethers. To our knowledge the described chemistry
represents the first examples of construction of the pyrrolo[2,3-d]pyrimidine skeleton in a one-step MCR
process.
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1
2
3
Table 5. 4-CR with concomitant debenzylation leading to the formation of hydroxyl-substituted 7-
4
5
6
deazahypoxanthine analogues.
7
8
9
BnO
Ar1
O
OBn
Ar1
CHO
+
H2N
H2N
K2CO3, EtOH
90 0C, 24 h
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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O
CN
N
H
O
NH
CONH2
O
S
O
Me
OH
Ar2
O
HCOOH
100 0C
N
N
O
N
H
H
7-deaza-
hypo-
xanthine
25w
Ar1
Ph
Ar2
% yield
88
70
Ph
Br
Br
Br
Br
25x
OMe
OMe
25y
25z
64
71
OBn
OH
OH
OBn
Pharmacology
(a) Antiproliferative Activities
The synthetically prepared natural products and their synthesized analogues were evaluated for
antiproliferative activities using the HeLa cell line as a model for human cervical adenocarcinoma and
MCF-7 cells as a model for breast adenocarcinoma. The cells were treated with each compound for 48 h,
and cell viability was assessed using the MTT method37 (Table 6). The natural alkaloids and synthetic 7-
deazaxanthine 22e-k analogues were inactive or weakly active, producing double-digit micromolar GI50
values in some instances (e.g., analogue 22i). Further, scaffold modifications, such as those resulting in 7-
deazaadenine analogues 23 and 7-deazapurine compounds 24, led to the discovery of micromolar
potencies associated with selected compounds (e.g., 23c and 24c). An unexpected and most gratifying
observation was, however, the discovery of nanomolar antiproliferative activity in compounds based on
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the 7-deazahypoxanthine skeleton 25. Thus, analogues 25a, 25e, 25g, 25m and 25u all exhibited double-
digit nanomolar potencies. A closer analysis of the SAR data indicates that the favored substitution
pattern is the unsubstituted or para-substituted benzene ring at the sulfonamide-derived C-8 position (Ar2)
and either the unsubstituted or meta-halogen-substituted benzene group at the aldehyde-derived C-7
position (Ar1). These structural features are found in the most potent analogues 25a-g, 25m, 25r-u. The
replacement of the Ph group at C-7 (as in 25a) with the pyridine moiety (as in 25h) led to a drop in
potency by a factor of 15. Together, these data indicate that the C-7 and, possibly, C-8 aromatic groups
bind in a pocket where hydrophobic interactions predominate, and introduction of hydrophilic pyridine
nitrogen (25h) or hydroxyl groups (25n, 25y,z) leads to an unfavorable loss of solvation energy.
1
2
3
4
5
6
7
8
9
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22
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25
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45
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53
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59
60
Table 6. Antiproliferative activities of the synthesized 7-deazapurines
cell viabilitya
cell viabilitya
cell viabilitya
7-deaza-
purine
7-deaza-
purine
7-deaza-
purine
GI50, µM
GI50, µM
GI50, µM
HeLa
MCF-7
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
14 ± 1
47 ± 5
> 100
> 100
> 100
3.9 ± 0.5
15 ± 0
> 100
HeLa
MCF-7
> 100
HeLa
MCF-7
10 ± 5
rigidin A
rigidin B
rigidin C
rigidin D
22e
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
80 ± 10
71 ± 12
> 100
> 100
> 100
4.4 ± 0.4
28 ± 4
> 100
23f
24a
24b
24c
24d
24e
25a
25b
25c
25d
25e
25f
25g
25h
25i
> 100
20 ± 1
70 ± 15
9.0 ± 1
18 ± 10
45 ± 9
25k
25l
7.1 ± 1
8.0 ± 1
75 ± 11
7.0 ± 1
10 ± 2
51 ± 2
4.9 ± 0.3
2.2 ± 0.1
25m
25n
25o
25p
25q
25r
25s
25t
0.095 ±
0.040
0.86 ± 0.06
0.095 ±
0.010
1.09 ± 0.12
0.19 ± 0.01
6.3 ± 1
0.25 ± 0.02
0.80 ± 0.01
7.0 ± 1
22f
22g
0.035 ±
0.007
0.2 ± 0.1
0.040 ±
0.024
0.150 ±
0.060
0.06 ± 0.02
>100
22h
0.30 ± 0.07
0.40 ± 0.01
2.2 ± 1
0.11 ± 0.035
0.31 ± 0.01
0.6 ± 0.1
22i
0.10 ± 0.07
0.13± 0.05
22j
0.06 ± 0.02
22k
0.065 ±
0.010
0.15 ± 0.05
0.070 ±
0.025
0.135 ±
0.075
0.087 ±
0.020
1.26 ± 0.18
25u
25v
25w
25x
25y
25z
0.045 ±
0.004
43 ± 6
0.053 ±
0.005
56 ± 2
23a
23b
0.080 ±
0.025
0.88 ± 0.06
0.4 ± 0.1
0.30 ± 0.01
5.0 ± 0.6
4.0 ± 0.6
0.3 ± 0.07
0.26 ± 0.05
3.0 ± 0.6
2.8 ± 0.8
23c
23d
48 ± 8
8.2 ± 1
33 ± 4
40 ± 4
23e
25j
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1
2
3
a Concentration required to reduce the viability of cells by 50% after a 48 h treatment with the indicated compounds relative to
a DMSO control ± SD from two independent experiments, each performed in 4 replicates, as determined by the MTT assay.
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7
8
(b) Cell Cycle Analysis
9
The structural similarities between the rigidin analogues and previously described podophyllotoxin
analogues,19 as well as characteristic cellular morphological changes induced by the 7-
deazahypoxanthines, led us to hypothesize that these compounds exert their antiproliferative properties
through inhibition of tubulin dynamics, thus inducing mitiotic arrest in cancer cells. To test this
hypothesis, cell cycle analysis was performed using the cell permeable DNA binding dye Vybrant
Orange. In a normal population, cells are distributed among the three major phases of the cell cycle,
G0/G1, in which there is one set of paired chromosomes per cell (2n), followed by the S or synthesis
phase, in which DNA is synthesized in preparation for division, and finally the G2/M phase, in which the
chromosomes of each cell have now been duplicated (4n). The DNA content can be measured by flow
cytometry using fluorescent DNA selective probes such as Vybrant Orange that produce signals that are
proportional to DNA mass per cell. Cell cycle analysis performed on HeLa cells using 25g and 25m with
0.1% dimethyl sulfoxide (DMSO) as a control revealed a pronounced cell cycle arrest in the G2/M phase
(Figure 4), and this effect is characteristic of antimitotic agents disrupting microtubule assembly.38
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Figure 4. Flow cytometric cell cycle analysis using HeLa cells treated for 32 h with 25g or 25m. Error
bars represent the standard error between triplicate experiments.
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1
2
(c) Inhibition of tubulin assembly and colchicine binding to tubulin
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7
8
9
To gain further insight into the possible antitubulin mechanism of action of the 7-deazahypoxanthines,
the effects of these compounds on in vitro tubulin polymerization were assessed.39 In our initial
experiment, polymerization was followed by fluorescence enhancement due to the incorporation of a
fluorescent reporter, in this case 4’,6-diamidino-2-phenylindole (DAPI), into microtubules as
polymerization occurs. The test compounds were compared to three different controls, a known
microtubule stabilizer (taxol), a known microtubule destabilizer (colchicine) as well as a solvent control
sample. Whereas taxol induced potent enhancement of microtubule formation relative to the effect of the
DMSO control, 7-deazahypoxanthines 25a and 25m displayed potent inhibition of microtubule assembly
in a manner similar to the known tubulin polymerization inhibitor colchicine (Figure 5).
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Figure 5. Effect of 25a and 25m on in vitro tubulin polymerization. Taxol (3 µM) promotes microtubule
formation relative to 0.1% DMSO control. 25a (25 µM), 25m (25 µM) and colchicine (25 µM)
completely suppress tubulin polymerization.
We next examined 25a and 25m in a quantitative turbidimetric assay,40 in comparison with
combretastatin A-4, a well-described potent inhibitor of the reaction (Table 7).41 While 25a differed little
from combretastatin A-4 in its inhibitory effect, 25m was distinctly less active. We also examined 25a
and 25m for potential inhibitory effects on the binding of [3H]colchicine to tubulin (Table 7).42 We found
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25a to be almost as active as combretastatin A-4, while 25m was less active, in accord with their relative
1
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3
activities in the assembly assay.
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5
6
7
Table 7. Inhibition of tubulin assembly and colchicine binding to β-tubulin
8
9
compound
inhibition of tubulin assemblyb
inhibition of colchicine bindingc
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IC50 (µM) ± SD
% inhibition ± SD
5 µM inhibitor
1 µM inhibitor
CA-4a
25a
0.96 ± 0.07
1.2 ± 0.1
99 ± 0.4
88 ± 0.7
71 ± 0.5
89 ± 0.6
58 ± 4
ND
25m
1.6 ± 0.04
a CA-4 = combretastatin A-4. b Inhibition of tubulin polymerization by selected compounds. Tubulin was at 10 µM. c % Inhibition of
[3H]colchicine (5 µM) binding to tubulin (1 µM) by selected compounds.
There are three well-documented binding domains on tubulin, the first two of which include the pocket
for taxanes and epothilones43,44 and the pocket for vinca alkaloids.45 FDA approved tubulin inhibitors
binding to these two domains are all derivatives of complex natural products and are characterized by
high bulkiness, hydrophobicity, and complicated structure. In addition, many tend to be easily recognized
by P-glycoprotein (P-gp) and pumped out of cancer cells, which causes drug resistance.46 The third
binding domain of tubulin is the pocket for colchicine and podophyllotoxin, small natural products with
relatively simple structures. So far, there are no FDA approved anticancer drugs targeting this domain,47
although many colchicine site agents inhibit the growth of multidrug resistant (MDR) tumor cell lines and
have reduced neurotoxicity and good oral bioavailability. These properties should be advantageous
compared to the currently FDA approved tubulin inhibitors.48-49 The results shown in Table 7 indicate that
the 7-deazahypoxanthines, as represented by compounds 25a and 25m, constitute a new class of
colchicine site agents.
(d) Microtubule organization in cells
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To evaluate the effects of 7-deazahypoxanthines on the microtubule cytoskeleton in cells, we cultured
HeLa cells in the presence of compounds 25a and 25m at a range of concentrations and examined
microtubule morphology (Figure 6). At concentrations between 1 and 10 nM, interphase microtubule
organization (Figure 6, panels A-C and M-O) was indistinguishable from DMSO controls (not shown).
Similarly, mitotic spindle morphology and spindle length was unaffected at concentrations at or below 10
nM for both analogues. However, there was a pronounced change in both interphase and mitotic
microtubule organization at concentrations between 10 and 100 nM, where both compounds caused a
complete collapse of mitotic microtubule organization (Figure 6, Panel J-L and V-X). This was also the
concentration range that encompassed the antiproliferative GI50 values for both compounds (GI50 (25a) =
35 nM, GI50 (25m) = 95 nM, Table 6). Additionally, while both compounds dramatically affected the
polymerization and organization of dynamic mitotic microtubules, 25a had a more drastic effect on
interphase microtubule organization (Figure 6, panels D-F) in comparison with 25m (Figure 6, Panels P-
R), suggesting that 25a was capable of affecting both dynamic and stable microtubule arrays. Together,
these results lend further evidence indicating that tubulin-targeting is likely the main mechanism
responsible for the antiproliferative effects of the 7-deazahypoxanthines.
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Figure 6. Microtubule organization in HeLa cells during interphase and mitosis. HeLa cells were treated
for 3 h with compound 25a (panels A-L) and 25m (M-X) at a range of concentrations. Following drug
treatment, cells were probed for microtubules (green), the centrosome marker pericentrin (red) and DNA
(blue). Bar, 10 μm.
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(e) Aqueous Solubility
Because low aqueous solubility is an intrinsic property of many natural and synthetic drug candidates
and is usually associated with poor absorption and bioavailability,50,51 the water solubilities of the most
potent nanomolar compounds 25a, 25e, 25g and 25m were determined with a UV/vis spectrophotometer
at a wavelength of 335-336 nm. The solubility of compounds 25e, 25g and 25m were 65 μM, 30 μM and
40 μM, respectively, while under the same conditions 25a (10 μM) was less soluble. It appears that the
meta-halogen substitution present in compounds 25e, 25g and 25m is beneficial for the enhanced water
solubility and will be considered for further development of this series of compounds. As a whole, these
numbers fall in the range of water solubilities of the other reported colchicine-site targeting anti-tubulin
compounds.52
(f) Activity against MDR cells
Because it is known that colchicine site agents are often insensitive to the action of P-gp,53 we tested
potent 7-deazahypoxanthines against MDR cells. The MDR uterine sarcoma cell line MES-SA/Dx5 was
utilized for this experiment.54 This cell line was established from the parent uterine sarcoma MES-SA,
grown in the presence of increasing concentrations of doxorubicin and is known to be resistant to a
number of P-gp substrates. Both taxol and vinblastine displayed more than a thousand fold drop in
potency when tested for antiproliferative activity against the MDR cell line as compared with the parent
line (Table 8). In contrast, there was only a small variation in the sensitivities of the two cell lines towards
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7-deazahypoxanthines 25a and 25m. Of note, the variation in sensitivity was slightly larger for the more
lipophilic 25m than that for 25a, underscoring the idea that smaller, less lipophilic agents are poorer P-gp
substrates.
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9
Table 8. Antiproliferative effect of selected compounds against MDR cells
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GI50 in vitro Values (nM)a
MES-SA
MES-SA/Dx5
Taxol
Vinblastine
25m
7 ± 1
6 ± 1
9800 ± 283
5000 ± 1414
394 ± 10
81 ± 6
30 ± 4
25a
70 ± 4
a Concentration required to reduce the viability of cells by 50% after a 48 h treatment with the indicated compounds relative
to a DMSO control ± SD from two independent experiments, each performed in 4 replicates, as determined by the MTT assay.
(g) Activity against cancer cell lines representing cancers with dismal prognoses or derived from tumor
metastases
Table 9. Antiproliferative properties of potent 7-deazahypoxanthines 25 against cancer cell lines
representing cancers with dismal prognoses or derived from tumor metastases
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GI50 in vitro values (nM)a
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4
5
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7
8
9
head and neck
metastases
compound
glioma
melanoma
NSCLC
A549
head and neck cancer
UM-
UM-
UM-
UM-
SKMEL-
U373
Hs683
B16F10
SCC10A SCC22A
SCC10B
100
SCC22B
28
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90
90
110
120
20
20
25m
25g
25a
30
30
20
20
10
20
20
50
40
20
30
20
10
130
<10
<10
ND
ND
ND
ND
a Concentration required to reduce the viability of cells by 50% after a 48 h treatment with the indicated compounds relative to
a DMSO control, as determined by the MTT assay
To study the antiproliferative effects of our potent 7-deazahypoxanthines 25 further, we investigated
whether they retained their potency against a panel of cancer cell lines, representing cancers known to be
associated with dismal prognoses (Table 9). These include glioma (U373 and Hs683), melanoma
(SKMEL-28 and B16F10), non-small-cell lung (NSCLC, A549) and head-and-neck cancers (UM-
SCC10A and UM-SCC22A). In addition, because more than 90% of cancer deaths are caused by tumor
metastases, two head-and-neck cancer cell lines derived from lymph node metastases (UM-SCC10B and
UM-SCC22B) were added to the panel. 7-Deazahypoxanthines 25a, 25g and 25m were found to retain
their nanomolar potency against cells in this challenging in vitro panel (Table 9). Especially noteworthy
are antiproliferative effects of 25a against human glioma (U373) and melanoma (SKMEL-28) cells,
which had single digit nanomolar GI50 values.
Conclusion
The research reported in this paper involves the utilization of the pyrrolo[2,3-d]pyrimidine-based
natural alkaloid rigidins to discover novel anticancer agents. We described the development of novel
synthetic chemistry allowing rapid access to various structural variants of this heterocyclic scaffold,
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which led to the generation of a library of diverse synthetic rigidin analogues. Although the natural
products themselves were found to have little if any anticancer activity, synthetic analogues based on the
7-deazahypoxanthine skeleton were found to have promising double to single digit nanomolar
antiproliferative potencies in human cancer cells. These compounds maintain potency in a panel of human
cancer cells including those representing cancers with dismal prognoses as well as cells derived from
metastatic lymph node tumors. All evidence obtained to date indicated that these rigidin analogues exert
their antiproliferative action by inhibiting microtubule dynamics in cancer cells. Because many
microtubule-targeting compounds are successfully used to fight cancer in the clinic, we believe the new
chemical class of antitubulin agents represented by the 7-deazahypoxanthine rigidin analogues have
significant potential as new anticancer agents.
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Experimental Section
General Synthetic Methods
All reagents, solvents and catalysts were purchased from commercial sources (Acros Organics and
Sigma-Aldrich) and used without purification. All reactions were performed in oven-dried flasks open to
the atmosphere or under nitrogen and monitored by thin layer chromatography (TLC) on TLC precoated
(250 μm) silica gel 60 F254 glass-backed plates (EMD Chemicals Inc.). Visualization was accomplished
with UV light. Flash column chromatography was performed on silica gel (32-63 μm, 60 Å pore size). 1H
13
and C NMR spectra were recorded on Jeol 300 or Bruker 400 spectrometers. Chemical shifts (δ) are
reported in ppm relative to the TMS internal standard. Abbreviations are as follows: s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet). HRMS analyses were performed at the Mass Spectrometry
Facility, University of New Mexico or using Waters Synapt G2 LCMS. The synthesized compounds are
at least 95% pure according to UPLC/MS analysis.
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General Procedure for the Synthesis of 7-Deazaxanthines (22a-j). A required pyrrole36 (0.3478 mmol)
was co-evaporated with 5 mL of toluene and dissolved in 3 mL of diglyme. Nitrogen gas was bubbled
through the mixture for 5 min, and 0.20 mL (0.3825 mmol) of oxalyl chloride (2 M in CH2Cl2) was added
dropwise while vigorously stirring the mixture. The mixture was refluxed for 10 h under nitrogen. After
that time, the solvent was evaporated under reduced pressure, and 5 mL of 50% MeOH in CH2Cl2 was
added to the solid in the flask. The precipitate was removed by filtration and rinsed sequentially with 2
mL of MeOH and 5 mL of diethyl ether. The mother liquor was chromatographed on silica gel using 30/1
to 30/5 CH2Cl2/MeOH gradient. The material from filtration and column chromatography was combined
to give 7-deazaxanthines 22a-j.
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6-[4-(Benzyloxy)benzoyl]-5-[4-(benzyloxy)phenyl]-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dione
(22a). 81% as a brown powder, mp > 300 oC decomp. 1H NMR (DMSO-d6) δ: 5.02 (s, 2H), 5.06 (s, 2H),
6.69 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.7 Hz, 2H), 7.29-7.40 (m, 12H), 10.71 (bs,
1H), 11.31 (bs, 1H), 11.94 (bs, 1H). 13C NMR (DMSO-d6) δ: 69.8, 70.0, 98.9, 113.8, 114.3, 128.0, 128.1,
128.3, 128.5, 128.9, 129.0, 131.7, 132.8, 136.8, 137.6, 151.3, 158.1, 160.3, 161.2, 185.7. HRMS m/z
(ESI+) calc’d for C33H26N3O5 (M+H+) 542.1721, found 542.1714.
6-[4-(Benzyloxy)-3-methoxybenzoyl]-5-[4-(benzyloxy)phenyl]-1H-pyrrolo[2,3-d]pyrimidine-
2,4(3H,7H)-dione (22b). 74% as a brown powder, mp > 300 oC decomp. 1H NMR (DMSO-d6) δ: 3.57 (s,
3H), 5.01 (s, 2H), 5.05 (s, 2H), 6.72 (s, 1H), 6.87-7.10 (m, 7H), 7.37 (m, 10H), 10.72 (bs, 1H), 11.27 (bs,
1H), 11.94 (bs, 1H). 13C NMR (DMSO-d6) δ: 56.3, 70.3, 71.2, 99.1, 114.0, 114.1, 123.5, 125.4, 125.6,
127.9, 128.0, 128.2, 128.3, 128.9, 131.9, 132.8, 137.4, 137.8, 142.0, 149.2, 150.9, 151.7, 158.5, 160.1,
185.7. HRMS m/z (ESI+) calc’d for C34H27N3NaO6 (M+Na+) 596.1798, found 596.1792.
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6-[4-(Benzyloxy)benzoyl]-5-[4-(benzyloxy)-3-methoxyphenyl]-1H-pyrrolo[2,3-d]pyrimidine-
2,4(3H,7H)-dione (22c). 76% as a yellow powder, mp > 300 oC decomp. 1H NMR (DMSO-d6) δ: 3.60 (s,
3H), 4.97 (s, 2H), 5.03 (s, 2H), 6.66-6.74 (m, 5H), 7.34-7.38 (m, 12H), 10.70 (bs, 1H), 11.48 (bs, 1H),
11.96 (bs, 1H). 13C NMR (DMSO-d6) δ: 55.9, 70.0, 70.6, 98.9, 113.1, 114.2, 116.2, 124.4, 125.5, 125.8,
128.1, 128.3, 128.5, 128.9, 129.0, 129.1, 131.2, 131.6, 137.0, 137.7, 151.3, 160.3, 161.5, 185.8. HRMS
m/z (ESI+) calc’d for C34H27N3NaO6 (M+Na+) 596.1798, found 596.1804.
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6-[4-(Benzyloxy)-3-methoxybenzoyl]-5-[4-(benzyloxy)-3-(methoxy)phenyl]-1H-pyrrolo[2,3-
o
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d]pyrimidine-2,4(3H,7H)-dione (22d). 72% as a yellow powder, mp > 300 C decomp. H NMR
(DMSO-d6) δ: 3.52 (s, 3H), 3.55 (s, 3H), 4.98 (s, 2H), 5.04 (s, 2H), 6.74 (s, 1H), 6.78 (s, 1H), 6.88 (d, J =
8.4 Hz, 1H), 6.93 (d, J = 1.5 Hz, 1H), 7.10 (dd, J = 1.6 Hz, J = 8.3 Hz, 1H), 7.31-7.39 (m, 10H), 10.73
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(bs, 1H), 11.27 (bs, 1H), 11.96 (bs, 1H). C NMR (DMSO-d6) δ: 55.6, 55.7, 70.5, 70.7, 98.8, 112.7,
113.2, 123.3, 124.3, 125.5, 125.6, 128.0, 128.1, 128.2, 128.4, 128.8, 128.9, 131.4, 137.0, 137.6, 141.9,
147.7, 148.1, 148.3, 151.1, 151.2, 160.2, 185.7. HRMS m/z (ESI+) calc’d for C35H29N3NaO7 (M+Na+)
626.1903, found 626.1908.
6-(4-Fluorobenzoyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dione
(22e): 74%, mp > 300 oC decomp. 1H NMR (DMSO-d6) δ: 3.56 (s, 9H), 6.38 (s, 2H), 6.92 (m, 2H), 7.41
(m, 2H), 10.76 (bs, 1H), 11.32 (bs, 1H), 12.13 (bs, 1H). 13C NMR (DMSO-d6) δ: 55.5, 60.3, 99.2, 109.2,
113.8, 114.9, 126.1, 127.6, 130.0, 131.5, 137.2, 151.1, 160.5, 156.3, 185.5. HRMS m/z (ESI+) calc’d for
C22H18FNaN3O6 (M+Na+) 462.1077, found 462.1064.
6-(4-Bromobenzoyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dione
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(22f): 64%, mp > 300 C decomp. H NMR (DMSO-d6) δ: 3.50 (s, 9H), 6.33 (s, 2H), 7.23 (m, 4H). C
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NMR (DMSO-d6) δ: 56.2, 60.7, 99.4, 109.7, 125.1, 125.7, 127.5, 130.7, 130.8, 130.9, 137.7, 138.0, 142.4,
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151.1, 151.9, 160.0, 185.6. HRMS m/z (ESI+) calc’d for C22H18BrNaN3O6 (M+Na+) 524.0256, found
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524.0259.
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6-(4-Benzyloxy-3-methoxybenzoyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrrolo[2,3-d]pyrimidine-
2,4(3H,7H)-dione (22g): 59%, mp > 300 oC decomp. 1H NMR (DMSO-d6) δ: 3.52 (s, 12H), 4.98 (s, 2H),
6.46 (m, 2H), 6.86 (m, 2H), 7.04 (m, 1H), 7.35 (m, 5H), 10.72 (bs, 1H), 11.25 (bs, 1H), 11.99 (bs, 1H).
13C NMR (DMSO-d6) δ: 55.5, 56.0, 70.4, 98.8, 109.6, 112.6, 112.7, 122.9, 125.8, 127.7, 128.0, 128.4,
128.9, 129.1, 131.2, 131.6, 137.0, 137.3, 148.1, 151.0, 151.7, 160.5, 185.8. HRMS m/z (ESI+) calc’d for
C30H27N3NaO8 (M+Na+) 580.1696, found 580.1697.
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6-(4-Methoxybenzoyl)-5-(4-methoxyphenyl)-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dione (22h).
76% as a yellow powder, mp > 300 oC decomp.1H NMR (DMSO-d6) δ: 3.66 (s, 3H), 3.70 (s, 3H), 6.60 (d,
J = 8.8 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H), 10.70 (bs,
1H), 11.32 (bs, 1H), 11.93 (bs, 1H). 13C NMR (DMSO-d6) δ: 55.7, 55.9, 70.0, 99.1, 113.1, 113.6, 125.0,
125.7, 128.9, 130.9, 131.6, 141.9, 151.1, 159.1, 160.2, 162.5, 185.8. HRMS m/z (ESI+) calc’d for
C21H18N3O5 (M+H+) 392.1246, found 392.1245.
6-Benzoyl-5-(4-methoxyphenyl)-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dione (22i). 81% as a
brown powder, mp > 300 oC decomp. 1H NMR (DMSO-d6) δ: 3.63 (s, 3H), 6.57 (d, J = 8.8 Hz, 2H), 7.01-
7.13 (m, 5H), 10.73 (bs, 1H), 11.30 (bs, 1H), 11.99 (bs, 1H). 13C NMR (DMSO-d6) δ: 55.6, 99.4, 113.0,
124.7, 125.6, 128.1, 129.3, 130.1, 131.6, 132.8, 138.6, 142.3, 151.2, 159.1, 160.2, 186.9. HRMS m/z
(ESI+) calc’d for C20H15N3NaO4 (M+Na+) 362.1141, found 362.1141.
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6-Benzoyl-5-(3,5-dibromophenyl)-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dione (22j): 43%, mp >
300 oC decomp. 1H NMR (DMSO-d6) δ 7.14-7.35 (m, 8H), 10.8 (bs, 1H), 11.48 (bs, 1H), 12.37 (bs, 1H).
13C NMR (DMSO-d6) δ: 99.3, 121.2, 126.3, 126.7, 128.2, 129.0, 132.1, 133.2, 136.5, 138.4, 142.2, 151.2,
160.1, 186.6. HRMS m/z (ESI+) calc’d for C19H11Br2N3NaO3 (M+Na+) 511.9044, found 511.9052.
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6-Benzoyl-5-(3,5-dibromophenyl)-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dithione (22k): 57%, mp
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> 300 C decomp. H NMR (DMSO-d6) δ: 7.30-7.47 (m, 10H), 8.63 (bs, 1H). 13C NMR (DMSO-d6) δ:
112.3, 120.8, 126.6, 128.1, 129.0, 132.1, 132.4, 133.9, 136.3, 138.1, 171.6, 187.1, 191.5. HRMS m/z
(ESI+) calc’d for C19H12Br2N3OS2 (M+H+) 519.8789, found 519.8788.
Synthesis of Rigidins A, B, C and D. A selected benzyl-protected 7-deazaxanthine 22a-d (0.0735 mmol)
and Pd/C (20 mg, 10%) were suspended in DMF (0.3 mL) and MeOH (1 mL). The suspension was
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degassed three times by the freeze-pump-thaw method at -196 C and then stirred under a hydrogen
balloon for 3 h. After that time, the solvent was removed from the frozen sample by lyophilization, and
the crude residue was dissolved in MeOH/CH Cl and subjected to silica gel column chromatography
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(MeOH/CH Cl /AcOH=10/50/1) to afford pure alkaloids.
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6-(4-Hydroxybenzoyl)-5-(4-hydroxyphenyl)-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dione
(Rigidin A). 94.7% as a yellow solid, mp > 300 oC decomp. 1H NMR (DMSO-d6) δ: 6.46 (d, J = 8.5 Hz,
2H), 6.47 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 8.6 Hz, 2H), 9.28 (bs, 1H), 10.02 (bs,
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1H), 10.63 (bs, 1H), 11.32 (bs, 1H), 11.84 (bs, 1H). C NMR (CD3OD) δ: 98.5, 113.6, 114.1, 123.9,
128.5, 129.3, 131.5, 132.3, 156.0, 160.2, 160.8, 162.9, 185.4. HRMS m/z (ESI+) calc’d for C19H13N3NaO5
(M+Na+) 386.0753, found 386.0742.
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6-(4-Hydroxy-3-methoxybenzoyl)-5-(4-hydroxyphenyl)-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-
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dione (Rigidin B). 95% as a brown solid, mp > 300 C decomp. H NMR (DMSO-d6) δ: 3.56 (s, 3H),
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6.47 (d, J = 8.5 Hz, 2H), 6.57 (d, J = 8.2 Hz, 1H), 6.93 (d, J = 1.6 Hz, 1H), 6.99-7.01 (m, 3H), 9.30 (bs,
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1H), 10.54 (bs, 1H), 11.99 (bs, 1H). C NMR (DMSO-d6) δ: 55.7, 98.8, 113.7, 114.3, 115.0, 123.6,
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124.2, 125.3, 128.8, 129.6, 133.0, 147.0, 150.7, 152.7, 156.9, 160.7, 185.8. HRMS m/z (ESI+) calc’d for
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C20H16N3O6 (M+H+) 394.1039, found 394.1032.
6-(4-Hydroxybenzoyl)-5-(4-hydroxy-3-methoxyphenyl)-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-
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dione (Rigidin C). 96% as a dark yellow solid, mp > 300 C decomp. H NMR (DMSO-d6) δ: 3.58 (s,
3H), 6.53 (m, 3H), 6.69-6.76 (m, 2H), 7.33 (d, J = 8.5 Hz, 2H), 8.69 (bs, 1H), 9.66 (bs, 1H), 10.21 (bs,
1H). 13C NMR (DMSO-d6) δ: 56.2, 98.7, 114.8, 115.6, 116.4, 123.5, 125.5, 129.3, 131.7, 132.1, 135.4,
141.8, 147.1, 148.1, 151.1, 161.1, 185.7. HRMS m/z (ESI+) calc’d for C20H16N3O6 (M+H+) 394.1039,
found 394.1028.
6-(4-Hydroxy-3-methoxybenzoyl)-5-(4-hydroxy-3-methoxyphenyl)-1H-pyrrolo[2,3-d]pyrimidine-
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2,4(3H,7H)-dione (Rigidin D). 93% as a maroon solid, mp > 300 C decomp. H NMR (DMSO-d6) δ:
3.52 (s, 3H), 3.54 (s, 3H), 6.49 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 8.2 Hz, 1H), 6.66 (dd, J = 1.7 Hz, J = 8.1
Hz, 1H), 6.73 (d, J = 1.6 Hz, 1H), 6.93 (d, J = 1.6 Hz, 1H), 7.05 (dd, J = 1.6 Hz, J = 8.2 Hz, 1H), 8.89 (bs,
1H), 9.65 (bs, 1H), 10.60 (bs, 1H), 11.83 (bs, 1H). 13C NMR (DMSO-d6) δ: 55.8, 55.8, 98.8, 113.7, 114.7,
116.5, 123.7, 124.0, 124.8, 125.3, 128.8, 129.7, 146.3, 146.6, 146.9, 150.8, 160.6, 185.8. HRMS m/z
(ESI+) calc’d for C14H7N3NaO7 (M+Na+) 446.0964, found 446.0964.
General procedure for the synthesis of 4-amino-7(H)pyrrolo[2,3-d]pyrimidines (7-deazaadenines
23a-f). To a solution of N-(2-oxo-2-arylethyl)methanesulfonamide (2 mmol), the selected aldehyde (2.6
mmol) and malononitrile (2.6 mmol) in formamide (2 mL) was added anhydrous granulated K2CO3 (1.2
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o
mmol) in one portion. The mixture was purged with nitrogen for 5 min and then heated at 100 C for 24
hours under the nitrogen atmosphere. The formation of the intermediate pyrrole was monitored by TLC.
After that, the reaction temperature was increased to 150 oC, and the reaction mixture was heated for 3-6
h. The mixture was cooled to room temperature and poured into water (30 mL). The formed precipitate
was collected by filtration and washed with EtOH (2 mL) and diethyl ether (2 mL) to give the desired 7-
deazaadenine 23a-f.
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4-Amino-6-benzoyl-5-(3,5-dibromophenyl)-7(H)pyrrolo[2,3-d]pyrimidine (23a): 58%, mp > 300 C
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decomp. H NMR (DMSO-d6) δ: 7.23 (t, J = 7.41, 2H), 7.39 (m, 5H), 7.45 (d, J = 7.2 Hz, 2H), 8.24 (s,
1H), 12.63 (s, 1H). 13C NMR (DMSO-d6) δ: 101.4, 119.7, 121.9, 127.5, 128.6, 129.2, 131.9, 132.2, 132.3,
137.1, 137.7, 150.9, 155.0, 158.8, 187.7. HRMS m/z (ESI+) calcd for C19H13Br2N4O (M+H+) 470.9456,
found 470.9455.
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4-Amino-6-benzoyl-5-(6-bromopyridin-2-yl)-7(H)pyrrolo[2,3-d]pyrimidine (23b): 66%, mp > 300 C
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decomp. H NMR (DMSO-d6) δ: 6.37 (bs, 2H), 7.20 (m, 2H), 7.38 (m, 3H), 7.79 (s, 1H), 8.23 (s, 1H),
8.32 (s, 1H), 8.42 (s, 1H), 12.66 (s, 1H). 13C NMR (DMSO-d6) δ: 102.2, 118.0, 120.0, 128.3, 129.3, 130.1,
131.5, 132.4, 138.1, 140.5, 149.1, 149.2, 151.6, 155.5, 159.5, 188.1. HRMS m/z (ESI+) calcd for
C18H13BrN5O(M+H+) 394.0303, found 394.0312.
4-Amino-6-benzoyl-5-phenyl-7(H)pyrrolo[2,3-d]pyrimidine (23c): 48%, mp 284 oC. 1H NMR (DMSO-
d6) δ: 7.13 (m, 2H), 7.29 (m, 5H), 7.31 (m, 1H), 7.45 (d, J = 6.4 Hz, 2H), 7.97 (d, J = 8.8 Hz, 2H), 8.23 (s,
1H), 12.52 (s, 1H). 13C NMR (DMSO-d6) δ: 101.7, 122.5, 127.5, 127.6, 128.1, 128.9, 130.3, 131.7, 133.5,
137.5, 150.7, 154.9, 158.9, 162.9, 187.8. HRMS m/z (ESI+) calcd for C19H15N4O (M+H+) 315.1246, found
315.1238.
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4-Amino-6-benzoyl-5-(thiophen-2-yl)-7(H)pyrrolo[2,3-d]pyrimidine (23d): 85%, mp 260 C. 1H NMR
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(DMSO-d6) δ: 6.92 (m, 1H), 7.03 (s, 1H), 7.25 (t, J = 6.8 Hz, 2H), 7.4 (m, 3H), 7.58 (d, J = 7.4 Hz, 2H),
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7.98 (d, J = 8.9 Hz, 1H), 8.24 (s, 1H), 12.65 (s, 1H). 13C NMR (DMSO-d6) δ: 102.0, 113.2, 127.5, 127.9,
128.2, 128.8, 129.5, 132.2, 133.6, 137.4, 150.6, 154.9, 158.8, 162.8, 187.7. HRMS m/z (ESI) calcd for
C17H13N4OS(M+H+) 321.0810, found 321.0801.
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4-Amino-6-(3-cyanobenzoyl)-5-(4-benzyloxyphenyl)-7(H)pyrrolo[2,3-d]pyrimidine (23e): 54%, mp
252 oC. 1H NMR (DMSO-d6) δ: 5.12 (s, 2H), 6.82 (d, J = 8.08 Hz, 2H), 7.4 (m, 7H), 7.47 (d, J = 7.3 Hz,
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2H), 7.67 (d, J = 6.5 Hz, 2H), 8.24 (s, 1H), 12.60 (s, 1H). C NMR (DMSO-d6) δ: 69.5, 69.5, 109.8,
114.1, 118.8, 119.6, 127.6, 128.0, 128.5, 129.4, 130.1, 131.3, 131.6, 131.9, 136.4, 138.7, 150.9, 154.5,
158.6, 161.6, 186.3. HRMS m/z (ESI+) calcd for C27H20N5O2 (M+H+) 446.1617, found 446.1609.
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4-Amino-6-benzoyl-5-(2,6-dichlorophenyl)-7(H)pyrrolo[2,3-d]pyrimidine (23f): 72%, mp 262 C. H
NMR (DMSO-d6) δ: 7.22 (m, 3H), 7.35 (m, 3H), 7.49 (d, J = 6.7 Hz, 2H), 8.22 (s, 1H), 12.69 (s, 1H).13C
NMR (DMSO-d6) δ: 102.5, 116.5, 128.1, 128.4, 128.8, 129.5, 131.4, 131.9, 132.4, 135.7, 138.3, 151.9,
154.3, 155.8, 159.4, 187.7. HRMS m/z (ESI+) calcd for C19H13Cl2N4O(M+H+) 383.0466, found 383.0474.
General procedure for the synthesis of 4-aryl-7(H)pyrrolo[2,3-d]pyrimidines (7-deazapurines 24a-
e). To a solution of the selected N-(2-oxo-2-arylethyl)methanesulfonamide (3 mmol), the selected
arylcyanomethyl ketone (3.9 mmol), the selected aldehyde (3.9 mmol) in formamide (2 mL) was added
anhydrous granulated K2CO3 (1.8 mmol). The mixture was purged with nitrogen and stirred overnight at
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110 C. The TLC monitoring of the reaction indicated the disappearance of the sulfonamide and the
formation of the intermediate pyrrole (bright yellow spot on TLC). The temperature was then raised to
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160 C and the reaction was stirred for 3-6 h under nitrogen. After the pyrrole disappeared (TLC), the
reaction mixture was cooled to room temperature and poured in a beaker with water (40 mL). The
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precipitate was collected by filtration and purified by column chromatography (CH2Cl2/EtOAc, 10:1) to
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6-(4-Methoxybenzoyl)-4-(4-methoxyphenyl)-5-(4-Methoxyphenyl)-7(H)pyrrolo[2,3-d]pyrimidine
(24a): 63%, mp 224 oC. 1H NMR (DMSO-d6) δ: 3.64 (s, 3H), 3.78 (s, 3H), 3.83 (s, 3H), 6.65 (s, 1H), 6.84
(d, J = 7.9 Hz, 2H), 6.96 (d, J = 8.0 Hz, 2H), 7.03 (d, J = 7.9 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 7.82 (d, J
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= 7.9 Hz, 2H), 8.13 (d, J = 7.9 Hz, 2H), 8.80 (s, 1H); C NMR (DMSO-d6) δ: 54.9, 55.1, 55.3, 113.7,
114.1, 115.4, 125.8, 126.9, 127.8, 129.1, 131.2, 133.3, 134.8, 146.8, 157.0, 158.6, 161.2, 173.7; HRMS
m/z (ESI+) calc’d for C28H24N3O4 (M+H+) 466.1767, found 466.1761.
(5-(4-Methoxyphenyl)-4-phenyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)(phenyl)methanone (24b): 30%,
mp 208 oC. 1H NMR (DMSO-d6) δ: 3.56 (s, 3H), 6.37 (d, J = 6.8 Hz, 1H), 6.66 (d, J = 6.8 Hz, 1H), 7.06
(dd, J = 8.2 Hz, 2H), 7.23 (m, 5H), 7.39 (t, J = 7.4 Hz, 1H), 7.53 (dd, J = 7.4 Hz, 2H), 8.99 (s, 1H); 13.03
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(s, 1H). C NMR (DMSO-d6) δ: 55.4, 113.1, 114.5, 120.8, 125.4, 127.5, 128.4, 129.1, 129.7, 129.8,
132.1, 133.1, 137.1, 137.3, 152.14, 153.5, 158.5, 162.3, 189.8. HRMS m/z (ESI+) calc’d for C26H19N3O2
(M+H+) 406.1556, found 406.1557.
(5-(3,5-Dibromophenyl)-4-(p-tolyl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)(phenyl)methanone
(24c):
33%, mp 250 oC. 1H NMR (DMSO-d6) δ: 2.27 (s, 3H), 6.90 (d, J = 1.7 Hz, 2H), 6.93 (d, J = 7.8 Hz, 1H),
7.10 (dd, J = 8.0 Hz, 2H), 7.25 (dd, J = 8.0 Hz, 2H), 7.37 (t, J = 1.7 Hz, 1H), 7.45 (t, J = 7.4 Hz, 1H),
7.55 (dd, J = 8.1 Hz, 2H), 9.01 (s, 1H); 13.25 (s, 1H). 13C NMR (DMSO-d6) δ: 20.9, 114.0, 118.0, 120.8,
127.8, 128.0, 128.9, 129.2, 131.1, 132.6, 132.9, 133.9, 136.6, 137.0, 138.9, 151.5, 151.5, 162.1, 188.8.
HRMS m/z (ESI+) calc’d for C26H18Br2N3O (M+H+) 545.9817, found 545.9819.
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(5-(3,5-Dibromophenyl)-4-phenyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)(phenyl)methanone (24d): 39%,
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mp 257 C. H NMR (DMSO-d6) δ: 6.94 (d, J = 1.8 Hz, 2H), 7.13 (dd, J = 8.0 Hz, 2H), 7.25 (m, 5H),
7.35 (t, J = 1.7 Hz, 1H), 7.45 (t, J = 7.4 Hz, 1H), 7.55, (dd, J = 8.2 Hz, 2H), 9.03 (s, 1H); 13.29 (s, 1H).
13C NMR (DMSO-d6) δ: 114.4, 118.4, 121.3, 127.7, 128.4, 129.4, 129.6, 130.0, 131.7, 133.0, 133.3,
133.4, 137.1, 137.4, 152.0, 153.9, 162.4, 189.2. HRMS m/z (ESI+) calc’d for C25H16Br2N3O (M+H+)
531.9660, found 531.9653.
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Phenyl(4-phenyl-5-(pyridin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)methanone (24e): 22%, mp 232
oC. H NMR (DMSO-d6) δ: 6.82 (m, 1H), 7.03 (dd, J = 8.0 Hz, 2H), 7.20 (m, 6H), 7.42 (t, J = 7.4 Hz,
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1H), 7.55 (dd, J = 8.2 Hz, 2H), 7.96, (d, = 1.08 Hz, 1H), 8.10, (dd, J = 4.8, Hz, 2H), 9.04 (s, 1H); 13.28
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(s, 1H). C NMR (DMSO-d6) δ: 114.3, 117.2, 121.9, 127.3, 128.1, 129.0, 129.1, 129.2, 129.5, 132.9,
133.2, 132.9, 133.2, 136.6, 136.7, 137.5, 147.3, 150.5, 151.8, 153.4, 162.0, 188.9. HRMS m/z (ESI+)
calc’d for C24H17N4O (M+H+) 377.1402, found 377.1403.
General procedure for the synthesis of 1H-pyrrolo[2,3-d]pyrimidin-4(7H)-ones (7-
deazahypoxanthines 25a-v). To a solution of N-(2-oxo-2-arylethyl)methanesulfonamide (0.676 mmol),
the selected aldehyde (0.879 mmol) and cyanoacetamide (0.072 g, 0.879 mmol) in a mixture of EtOH (2.5
mL) and HC(OEt)3 (2.5 mL) was added anhydrous granulated K2CO3 (0.052g, 0.372 mmol) in one
portion. The mixture was purged with nitrogen for 5 min and then heated at 90 oC for 24 hours under the
nitrogen atmosphere. The formation of the intermediate pyrrole was monitored by TLC. After that the
reaction temperature was increased to 150 oC, and the reaction mixture was heated for 3-6 h. The mixture
was cooled to room temperature, and the formed precipitate was collected by filtration and washed with
EtOH (2 mL) and diethyl ether (2 mL) to give the desired 7-deazahypoxanthine 25a-v. An additional
amount of the product was obtained by the evaporation of the mother liquor and purification of the
residue by column chromatography with MeOH/CH2Cl2=1/40 to 1/20 gradient.
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Journal of Medicinal Chemistry
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6-Benzoyl-5-phenyl-1H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (25a): 71%, mp > 300 C decomp. H
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NMR (DMSO-d6) δ: 7.04 (m, 3H), 7.15 (m, 4H), 7.33 (t, J = 6.8 Hz, 1H), 7.43 (d, J = 7.12 Hz, 2H), 8.02
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(s, 1H), 12.01 (s, 1H), 12.79 (s, 1H). C NMR (DMSO-d6) δ: 106.3, 126.6, 126.7, 126.8, 127.5, 127.7,
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129.0, 131.1, 131.8, 132.4, 137.4, 146.7, 149.3, 158.6, 187.7. HRMS m/z (ESI+) calcd for C19H14N3O2
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(M+H+) 316.1086, found 316.1087.
6-Benzoyl-5-(3-iodophenyl)-1H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (25b): 68%, mp > 300 oC decomp.
1H NMR (DMSO-d6) δ: 6.87 (t, J = 7.52 Hz, 1H), 7.19 (t, J = 7.44 Hz, 2H), 7.25 (d, J = 7.52 Hz, 1H),
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7.42 (m, 5H), 8.04 (s, 1H), 12.90 (s, 1H), 12.09 (s, 1H). C NMR (DMSO-d6) δ: 93.0, 106.3, 125.0,
127.8, 128.7, 128.8, 130.4, 131.9, 134.6, 135.2, 137.4, 139.5, 146.9, 149.4, 158.5, 187.6. HRMS m/z
(ESI+) calcd for C19H13IN3O2 (M+H+) 443.0086, found 443.0080.
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6-Benzoyl-5-(3-chlorophenyl)-1H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (25c): 61%, mp > 300 C
decomp. 1H NMR (DMSO-d6) δ: 7.04 (t, J = 7.48 Hz, 1H), 7.11 (d, J = 7.52 Hz, 2H), 7.19 (m, 3H), 7.38
(t, J = 6.2 Hz, 1H), 7.46 (d, J = 7.4 Hz, 2H), 8.04 (s, 1H), 12.05 (s, 1H), 13.00 (s, 1H). 13C NMR (DMSO-
d6) δ: 106.4, 125.0, 126.5, 127.7, 128.5, 128.9, 129.7, 130.7, 131.5, 132.0, 134.6, 137.5, 146.8, 149.4,
158.5, 187.6. HRMS m/z (ESI+) calcd for C19H13ClN3O2 (M+H+) 350.0696, found 350.0694.
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6-Benzoyl-5-(3-bromophenyl)-1H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (25d): 54%, mp > 300 C
decomp. 1H NMR (DMSO-d6) δ: 7.00 (t, J = 7.08 Hz, 1H), 7.19 (m, 3H), 7.25 (d, J = 7.76 Hz, 1H), 7.34
(s, 1H), 7.38 (t, J = 6.96 Hz, 1H), 7.45 (d, J = 7.32 Hz, 2H), 8.04 (s, 1H), 12.05 (s, 1H), 12.90 (s, 1H). 13C
NMR (DMSO-d6) δ: 106.4, 120.1, 124.9, 127.7, 127.8, 128.8, 128.9, 129.4, 132.0, 130.1, 133.6, 134.8,
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