Synthetic and biological studies of tubulin targeting C2-substituted 7-deazahypoxanthines derived from marine alkaloid rigidins

Article abstract of DOI:10.1002/cmdc.201300532

C2-aryl- and C2-alkyl-7-deazahypoxanthines as analogues of marine alkaloid rigidins were prepared utilizing novel synthetic methods developed for the construction of the pyrrolo[2,3-d]pyrimidine ring system. The new compounds exhibited sub-micromolar to nanomolar antiproliferative potencies against a panel of cell lines including in vitro models for drug-resistant tumors, such as glioblastoma, melanoma and non-small-cell lung cancer. A selected representative C2-methyl-7-deazahypoxanthine was found to inhibit microtubule dynamics in cancer cells, lending evidence for tubulin targeting as a mode of action for these compounds in cancer cells. The results of the docking studies utilizing the colchicine site on β-tubulin were consistent with the observed structure-activity relationship data, including an important finding that derivatization at C2 with linear alkyl groups leads to the retention of activity, thus permitting the attachment of a biotin-containing linker for the subsequent proteomics assays. Because many microtubule-targeting compounds are successfully used to fight cancer in the clinic, the reported antitubulin rigidin analogues have significant potential as new anticancer agents. Rigid(in) development! Novel analogues of marine alkaloid rigidins were prepared utilizing new synthetic methods developed for the construction of the pyrrolo[2,3-d]pyrimidine ring system. The compounds exhibited sub-micromolar to nanomolar antiproliferative potencies against drug-resistant cells, such as glioblastoma, melanoma and non-small-cell lung cancer.

Full text of DOI:10.1002/cmdc.201300532

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DOI: 10.1002/cmdc.201300532
Synthetic and Biological Studies of Tubulin Targeting C2-
Substituted 7-Deazahypoxanthines Derived from Marine
Alkaloid Rigidins
Robert Scott,[a] Menuka Karki,[b] Mary R. Reisenauer,[c] Roberta Rodrigues,[a] Ramesh Dasari,[a]
W. Ross Smith,[a] Stephen C. Pelly,[d] Willem A. L. van Otterlo,[d] Charles B. Shuster,[b]
Snezna Rogelj,[c] Igor V. Magedov,[c] Liliya V. Frolova,*[c] and Alexander Kornienko*[a]
C2-aryl- and C2-alkyl-7-deazahypoxanthines as analogues of
marine alkaloid rigidins were prepared utilizing novel synthetic
methods developed for the construction of the pyrrolo[2,3-
d]pyrimidine ring system. The new compounds exhibited sub-
micromolar to nanomolar antiproliferative potencies against
a panel of cell lines including in vitro models for drug-resistant
tumors, such as glioblastoma, melanoma and non-small-cell
lung cancer. A selected representative C2-methyl-7-deazahy-
poxanthine was found to inhibit microtubule dynamics in
cancer cells, lending evidence for tubulin targeting as a mode
of action for these compounds in cancer cells. The results of
the docking studies utilizing the colchicine site on b-tubulin
were consistent with the observed structure–activity relation-
ship data, including an important finding that derivatization at
C2 with linear alkyl groups leads to the retention of activity,
thus permitting the attachment of a biotin-containing linker
for the subsequent proteomics assays. Because many microtu-
bule-targeting compounds are successfully used to fight
cancer in the clinic, the reported antitubulin rigidin analogues
have significant potential as new anticancer agents.
Introduction
Due to their useful biological properties, marine pyrrole-de-
rived alkaloids have been the subject of numerous studies by
organic and medicinal chemistry research groups.[1–9] One such
investigation conducted in our laboratories is aimed at the de-
velopment of synthetic chemistry and elucidation of biological
properties, associated with the marine alkaloid rigidins. This
group of natural products, which includes rigidins A, B, C, D
(Figure 1) and E, was isolated from the tunicate Eudistoma cf.
rigida found near Okinawa and New Guinea and has remained
scantly explored despite initial reports of promising biological
activities.[10–12] The first general total synthesis of rigidins A, B, C
[a] R. Scott,+ R. Rodrigues, Dr. R. Dasari, W. R. Smith, Prof. Dr. A. Kornienko
Department of Chemistry and Biochemistry, Texas State University
San Marcos, TX, 78666 (USA)
E-mail: a_k76@txstate.edu
[b] Dr. M. Karki,+ Prof. Dr. C. B. Shuster
Department of Biology, New Mexico State University
Las Cruces, NM 88003 (USA)
[c] M. R. Reisenauer, Prof. Dr. S. Rogelj, Prof. Dr. I. V. Magedov,
Prof. Dr. L. V. Frolova
Figure 1. Structures of rigidins A, B, C, D, their synthetic analogues based on
7-deazahypoxanthine (A), 7-deazaadenine (B) and 7-deazapurine (C) skele-
tons, and proposed C2-substituted 7-deazahypoxanthines (D).
Departments of Chemistry and Biology
New Mexico Institute of Mining and Technology
Socorro, NM 87801 (USA)
E-mail: lfrolova@nmt.edu
and D, which involved only four steps from commercially avail-
able materials, was reported by our group in 2011[13] and al-
lowed for a thorough investigation of their biological proper-
ties. However, despite the earlier reports of promising antiproli-
ferative activity against murine leukemia L1210 cells,[11] the ri-
gidins were found to have very little, if any, activity against cul-
tured human cancer cells.[14] Further synthetic investigations
[d] Prof. Dr. S. C. Pelly, Prof. Dr. W. A. L. van Otterlo
Department of Chemistry and Polymer Science, Stellenbosch University
Stellenbosch, Private Bag X1
Matieland, 7602 (South Africa)
[+] These authors contribute equally.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300532.
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led to the development of approaches allowing for the modifi-
cation of the 7-deazaxanthine skeleton present in the rigidins
to obtain the corresponding 7-deazahypoxanthine (A), 7-dea-
zaadenine (B) and 7-deazapurine (C) frameworks (Figure 1).
These studies culminated in the discovery of potent antiproli-
ferative activities associated with the synthesized 7-deazahy-
poxanthines (A) and their ability to disrupt microtubule organi-
zation in cancer cells by binding to the colchicine site of b-tu-
bulin.[14]
ble Taft steric parameters of the CF3 and iPr groups.[16,17] Be-
cause the CF3 and iPr groups differ significantly in their elec-
tronic properties, it is thus conceivable that steric rather than
electronic properties of the C2 substituent govern the activity
in this series of compounds.
MCR-based synthesis and SAR of C2-methyl-7-deazahypo-
xanthines
Because the only difference between the rigidins’ 7-deazax-
anthine skeleton and the newly discovered 7-deazahypoxan-
thines A is the absence of the carbonyl group at C2 in the
structure of the latter, modifications at this position seemed
crucial for activity. Thus, it was further decided to prepare
Potent activity of the C2-methyl analogue 13 led to further ex-
ploration of C2-methyl compounds by varying the aldehyde-
derived C7 aromatic group. Because the previous work with 7-
deazahypoxanthines A indicated that the best substitution pat-
tern at the C7 and C8 positions (Figure 1) involved the use of
the benzoyl group at C8 (Ar2 =Ph) and a meta-halogen-substi-
tuted phenyl and 3-pyridinyl substituent (Ar1) at C7, a small
group of derivatives containing such structural features was
prepared (Table 2).[14] To facilitate the synthesis of these com-
pounds, a new four-component cyclocondensation of methyl-
sulfonamidoacetophenone, cyanoacetamide and triethyl or-
thoacetate with various aldehydes was developed (Table 2). In
this reaction, the entire pyrrolo[2,3-d]pyrimidine skeleton is as-
sembled in one step, and its success depended on an opti-
mized temperature regime, in which the reaction was first kept
at 908C to allow for a clean formation of intermediate amino-
pyrroles (similar to 1 in Table 1), and then at 1508C to bring
the fourth component orthoacetate into the process, leading
to closure of the pyrimidine portion of the molecule.
a
series of C2-aryl- and C2-alkyl-deazahypoxanthines (D,
Figure 1) and evaluate these compounds for anticancer activi-
ties. Such a study was also warranted by the results of the
computer modeling experiments revealing a favorable accom-
modation of the lipophilic C2 side chain at the colchicine bind-
ing site, as described below.
Results and Discussion
Synthesis and SAR of C2-aryl- and C2-alkyl-7-deazahypo-
xanthines
To develop synthetic access to the C2-substituted 7-deazahy-
poxanthines D, a previously discovered[13] multicomponent re-
action (MCR) of sulfonamidoacetophenones with aldehydes
and cyanoacetamide was utilized to prepare pyrrole
1 (Table 1). This was followed by the pyrimidine ring closure
using the reaction of 1 with a variety of aromatic and aliphatic
esters catalyzed by sodium ethoxide in ethanol, which was re-
ported for a related purine system.[15] The desired 7-deazahy-
poxanthines 2–16 were obtained in variable but acceptable
yields (Table 1), given that in most cases these products pre-
cipitated after the acidification of the reaction mixtures and re-
quired no further purification.
The antiproliferative effects of compounds 13 and 17–22
were studied in a more challenging cell line panel that, in addi-
tion to the previously utilized HeLa and MCF-7, contained in
vitro models for cancers known to be associated with poor
prognoses, such as the U87 human glioblastoma and A549
human non-small-cell lung (NSCLC) carcinoma. Although the
newly synthesized compounds 17–22 were found to be inferi-
or to the original C7-phenyl analogue 13, in general the sub-
micromolar potencies were retained with the exception of the
pyridine-containing variant 20 exhibiting single-digit micromo-
lar potencies. The drop in activity of this analogue could be
also explained by its polar character, impairing its cell permea-
bility.
The obtained compounds were evaluated for antiprolifera-
tive 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 3-(4,5-dime-
Microtubule organization in cells
thylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT)
method (Table 1). The results revealed that in comparison with
rigidin-related 7-deazaxanthine 2, containing the carbonyl
group at C2, most of the 7-deazahypoxanthines showed anti-
proliferative activity. Although the activity was found to be
only double- to single-digit micromolar for C2-aryl (5, 6), C2-
OEt (8) and branched C2-alkyl (9), the linear C2-alkyl deriva-
tives 10–12 showed potency at sub-micromolar, and the C2-
methyl compound 13 at nanomolar concentrations. Of note,
when fluorine was incorporated into the C2-Me group, the ac-
tivity progressively dropped along with increasing steric size at
the C2 position, that is, CH3 (13)!CH2F (14)!CHF2 (15)!CF3
(16).[16] The activity of the C2-CF3 derivative 16 is similar to that
of C2-iPr compound 9, which is consistent with the compara-
Because the original 7-deazahypoxanthines were found to
have strong effects on the microtubule cytoskeleton in cells,[14]
it was important to confirm that these properties remained un-
changed in these novel analogues. To this end, cultured HeLa
cells were treated with 13 over a range of concentrations, and
microtubule morphology was examined (Figure 2a). Results re-
vealed a pronounced change in mitotic microtubule organiza-
tion at concentrations between 1 mm and 2 mm, where 13
caused a complete collapse of the mitotic spindle (panels J
and K). The interphase microtubules were affected to a lesser
extent at these concentrations (panels D and E), suggesting
that 13 was primarily affecting dynamic microtubules. While
there appeared to be little effect on stable interphase or spin-
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Table 1. Synthesis and antiproliferative activities of C2-aryl and C2-alkyl-7-deazahypoxanthines 2–16.
Compd
Yield
[%]
Cell Viability, GI50 [mm][a]
Compd
Yield
[%]
Cell Viability, GI50 [mm][a]
HeLa
MCF-7
HeLa
MCF-7
2
3
NA[b]
>50
>50
>50
10
11
60
42
0.78ꢀ0.05
0.34ꢀ0.01
0.93ꢀ0.07
41
>50
>50
0.23ꢀ0.01
1.1ꢀ0.1
4
33
>50
12
72
0.92ꢀ0.11
5
6
18
58
2.7ꢀ0.2
2.5ꢀ0.3
13
14
49
35
0.029ꢀ0.001
3.0ꢀ0.2
0.035ꢀ0.003
3.2ꢀ0.2
18.4ꢀ1.8
23.1ꢀ0.5
7
8
9
41
NA[c]
21
>50
>50
15
16
47
66
5.1ꢀ0.2
4.2ꢀ0.5
11.1ꢀ0.4
9.1ꢀ0.3
10.4ꢀ0.8
8.3ꢀ0.8
21.5ꢀ0.4
21.1ꢀ1.1
[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. Data
represent the meanꢀSD from two independent experiments performed in four replicates, as determined by the MTT assay. [b] Synthesized using the
method described in ref. [13]. [c] Obtained as a side product of the reaction of 1 with CO(OEt)2.
Figure 2. Microtubule organization is altered with increasing concentrations of 13. a) HeLa cells were treated with 0.1% DMSO or 13 at different concentra-
tions for 3 h, fixed and probed for the presence of microtubules (green), F-actin (red) and DNA (blue). Scale bar, 10 mm. b) Line graph showing an increase in
the eccentric spindles with the increasing concentrations of 13, 150 mitotic cells scored per condition.
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lend further evidence for tubu-
lin-targeting being a likely mech-
anism responsible for the anti-
proliferative effects of the 7-dea-
zahypoxanthines.
Table 2. MCR-based synthesis and antiproliferative activities of C2-methyl-7-deazahypoxanthines 13 and 17–
22.
Molecular modeling
In our previous work, 7-deazahy-
poxanthines were proposed to
target to the colchicine site on
b-tubulin based on their potent
inhibitory effects on the binding
of [3H]colchicine to tubulin.[14] In
the current study, molecular
docking simulations were per-
formed on this series of com-
pounds to understand the con-
tribution of the C2 substituent
toward the binding at this site.
Figure 3 shows the proposed
binding mode of the 7-deazahy-
poxanthines in the colchicine
pocket.[19] Of particular interest,
is the accommodation of alkyl
substituents at the C2 position.
A small channel in the region of
Asn258 and Lys352 accommo-
dates the colchicine methoxy
group protruding from the
seven-membered C-ring system.
Docking results of 7-deazahy-
poxanthine 13 suggest that the
C2-methyl group is located in
a nearly identical position to the
above-mentioned colchicine me-
thoxy group (Figure 3a). More-
over, this narrow channel is able
to accommodate linear alkyl
substituents at C2, such as the
butyl group in 11 (Figure 3b).
Sterically demanding substitu-
ents at C2 are, however, too
bulky for the channel, perhaps
explaining the lack of activity of
3–6, 9 and 16. A superimposi-
Compd
Yield
[%]
in vitro GI50 [mm][a]
HeLa
MCF-7
U-87
A549
13
17
40
61
0.029ꢀ0.001
0.035ꢀ0.003
0.23ꢀ0.00
0.077ꢀ0.002
0.90ꢀ0.16
0.25ꢀ0.01
0.27ꢀ0.01
0.29ꢀ0.01
0.29ꢀ0.03
3.1ꢀ0.1
0.60ꢀ0.23
0.65ꢀ0.19
1.0ꢀ0.9
18
19
20
21
40
43
65
63
0.27ꢀ0.03
0.36ꢀ0.02
4.7ꢀ0.8
0.94ꢀ0.12
1.7ꢀ0.1
9.23ꢀ2.13
2.4ꢀ0.6
12.0ꢀ1.4
1.1ꢀ0.4
0.49ꢀ0.05
0.61ꢀ0.01
22
58
0.29ꢀ0.01
0.27ꢀ0.01
0.72ꢀ0.06
0.38ꢀ0.08
[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. Data represent the meanꢀSD from two independent experiments per-
formed in four replicates, as determined by the MTT assay.
dle microtubules at lower concentrations, a distinct displace-
ment of the mitotic spindle could be observed (panels H
and I), suggesting a defect in astral microtubules. The central
localization of the mitotic spindle is made possible by interac-
tions between dynamic astral microtubules emanating from
the spindle poles and the cell cortex, often described as Hert-
wig’s rule.[18] During mitosis, astral microtubules are the most
dynamic microtubule population, and therefore, they would
be most sensitive to tubulin-targeting drugs such as 13. The
quantification of spindle eccentricity with increasing concen-
trations of 13 is shown in Figure 2b. Together, these results
tion of the co-crystalized colchicine and the docked 7-deazahy-
poxanthine 13 reveals the similar binding modes of the two
compounds, with the C7-aryl substituent on the 7-deazahypox-
anthine scaffold accommodated in a near identical fashion to
that of the trimethoxy-aryl moiety of colchicine (Figure 3c).
Quantitative video microscopy
Poor prognoses of glioblastoma, melanoma and NSCLC pa-
tients are generally attributed to the drug-resistant nature of
these malignancies[20–22] and therefore it was encouraging that
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Figure 3. Docking studies (PDBID: 3UT5) of 13 (a) and 11 (b) reveal that linear C2-alkyl substituents on the 7-deazahypoxanthine scaffold can be accommo-
dated in a small channel in the region of Asn258 and Lys352. Co-crystallized colchicine (blue) and 13 (orange) adopt similar binding poses in the colchicine
pocket (c).
7-deazahypoxanthines 13 and 17–22 were active against the
U87 human glioblastoma and A549 NSCLC carcinoma cell
lines. Since tubulin-targeting agents are generally poorly effec-
tive against cancer cells resistant to the induction of apopto-
sis,[23–25] compound 22 was further evaluated against U373
human glioblastoma and SKMEL human melanoma cells, re-
ported to display apoptosis resistance.[26,27] The obtained MTT-
based GI50 value of 0.3 mm in both of these cell lines indicates
that this compound maintains similar levels of activity in these
cell types (compare with GI50 values in Table 2). Computer-as-
sisted phase-contrast microscopy (quantitative video microsco-
py) was utilized to confirm cell death as the principal mecha-
nism of action associated with this compound’s in vitro growth
inhibitory effects in these cell lines. Figure 4 shows that 22
indeed inhibited cancer cell proliferation by inducing cell
death when assayed at its MTT-based GI50 values in U373 glio-
blastoma and SKMEL melanoma cells.
Drug Administration (FDA)-approved anticancer agents target-
ing the colchicine binding domain on b-tubulin, although
many colchinoids inhibit the growth of multidrug-resistant
(MDR) tumor cell lines and have reduced neurotoxicity and
good oral bioavailability. A number of colchinoids have been
examined in human clinical trials with generally promising re-
sults, but having drawbacks that include narrow therapeutic
windows and unacceptable toxicities.[28] It is thus important to
understand and predict possible causes of potential toxicity in
the clinic by elucidating other intracellular targets of these
agents during their preclinical evaluation. The importance of
identifying additional targets interacting with the 7-deazahy-
poxanthines could also help understand their effectiveness
against drug-resistant cancer cell lines used in the current
study, since sole tubulin targeting may not be sufficient to
induce death of these cancer cells. The current study revealed
that the linear alkyl substitution at C2 of the 7-deazahypoxan-
thine skeleton (such as C2-Bu, see 11 in Table 1) is tolerated,
and thus this position appeared ideal for the probe prepara-
tion to be used in proteomic pull-down assays. To this end,
using the developed chemistry, pyrrole 1 was successfully con-
verted to the C2-alkyne-containing 7-deazahypoxanthine 23,
which was then coupled with a commercially available biotin–
azide reagent utilizing click chemistry (Scheme 1).[29] Mode of
action studies utilizing the prepared biotinylated probe 24 are
in progress and will be reported in due course.
Synthesis of a biotinylated protein pull-down reagent
Microtubules represent an important target for a number of
clinically successful drugs, but so far, there are no US Food and
Conclusions
A synthetic route was developed to access C2-aryl- and C2-
alkyl-deazahypoxanthines as analogues of the marine alkaloid
rigidins containing a carbon group instead of the carbonyl
present in the natural products. The new 7-deazahypoxan-
thines were found to have sub-micromolar to nanomolar anti-
proliferative potencies and to inhibit microtubule dynamics in
cancer cells. This activity was most plausibly explained by bind-
ing of these compounds to the colchicine site on b-tubulin,
a proposal consistent with the experimental findings of their
effects on microtubule organization and a theoretical docking
model developed for these compounds. These agents are also
effective against cancer cells representing drug-resistant can-
cers with poor prognoses, raising the possibility that additional
Figure 4. Cellular imaging of 22 against U373 glioblastoma and SKMEL mela-
noma cells illustrating cell death at MTT colorimetric assay-related GI50 value
of 0.3 mm.
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0.16 mmol) were added to the solution of EtONa in EtOH prepared
by dissolving Na (30 mg, 1.3 mmol) in EtOH (2 mL). The mixture
was held at reflux for 10 h. The reaction mixture was then diluted
with H2O and neutralized with 1m HCl. The formed precipitate was
collected by filtration and dried under vacuum overnight. Although
in most cases the product deazahypoxanthines were >95% pure,
they could be further purified using column chromatography (5%
MeOH in CHCl3).
6-Benzoyl-2-phenyl-5-phenyl-1H-pyrrolo[2,3-d]pyrimidin-4(7H)-
one (3): White powder (27 mg, 41%): 1H NMR ([D6]DMSO): d=
12.21 (brs, 1H), 11.64 (brs, 1H), 8.01–7.96 (m, 2H), 7.74 (t, J=
7.3 Hz, 1H), 7.67 (t, J=7.5 Hz, 2H), 7.37–7.09 ppm (m, 10H);
13C NMR ([D6]DMSO): d=185.8, 167.5, 165.5, 138.4, 133.6, 133.1,
132.6, 131.3, 131.0, 129.8, 129.7, 129.0, 128.7, 128.6, 128.5, 127.9,
127.6, 123.3, 103.4 ppm; HRMS (ESI) m/z [M+NH4]+ calcd for
C25H20DN4O2: 410.1727, found: 410.1722.
Scheme 1. Synthesis of the biotinylated 7-deazahypoxanthine probe 24.
6-Benzoyl-2-(2-ethoxycarbonylethyl)-5-phenyl-1H-pyrrolo[2,3-
d]pyrimidin-4(7H)-one (10): Red powder (41 mg, 60%): 1H NMR
([D6]DMSO): d=12.59 (brs, 1H), 11.91 (brs, 1H), 7.42 (d, J=8.0 Hz,
2H), 7.32 (t, J=8.0 Hz, 1H), 7.20–6.99 (m, 7H), 4.09 (q, J=8.0 Hz,
2H), 2.98–2.75 (m, 4H), 1.15 ppm (t, J=8.0 Hz, 3H); 13C NMR
([D6]DMSO): d=188.1, 173.9, 172.4, 159.7, 159.6, 158.6, 150.1,
138.0, 133.0, 131.6, 129.5, 128.1, 127.3, 104.9, 60.5, 31.1, 30.4,
14.5 ppm; HRMS (ESI) m/z [M+Na]+ calcd for C24H21N3NaO4:
438.1430, found: 438.1431.
intracellular targets may be involved. The current investigation
sets the stage for further mode-of-action studies utilizing bio-
tinylated protein pull-down probes. Since many microtubule-
targeting compounds have been successfully used to fight
cancer in the clinic, the antitubulin agents represented by the
7-deazahypoxanthine rigidin analogues have significant poten-
tial as new anticancer agents.
6-Benzoyl-2-butyl-5-phenyl-1H-pyrrolo[2,3-d]pyrimidin-4(7H)-
1
one (11): Red powder (25 mg, 42%): H NMR ([D6]DMSO): d=12.54
(brs, 1H), 11.86 (brs, 1H), 7.43 (d, J=6.7 Hz, 2H), 7.30 (t, J=6.8 Hz,
1H), 7.23–6.85 (m, 7H), 2.62 (t, J=6.9 Hz, 2H), 1.81–1.60 (m, 2H),
1.36–1.28 (m, 2H), 0.92 ppm (t, J=7.0 Hz, 3H); 13C NMR
([D6]DMSO): d=189.1, 160.7, 138.9, 133.9, 133.0, 132.4, 130.3,
128.9, 128.0, 105.6, 35.0, 30.4, 22.9, 14.9 ppm; HRMS (ESI) m/z [M+
Na]+ calcd for C23H21N3NaO2: 394.1531, found: 394.1528.
Experimental Section
Chemistry
General: 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 mm)
silica gel 60 F254 glass-backed plates (EMD Chemicals Inc.). Visuali-
zation was accomplished with UV light. Flash column chromatogra-
6-Benzoyl-2-methyl-5-phenyl-1H-pyrrolo[2,3-d]pyrimidin-4(7H)-
one (13): White powder (26 mg, 49%): 1H NMR ([D6]DMSO): d=
12.57 (brs, 1H), 11.90 (brs, 1H), 7.42 (dd, J=6.7, 0.4 Hz, 2H), 7.31 (t,
J=7.4 Hz, 1H), 7.17–7.01 (m, 7H), 2.36 ppm (s, 3H); 13C NMR
([D6]DMSO): d=188.1, 159.8, 156.8, 150.5, 138.1, 133.0, 132.1,
131.6, 129.5, 128.1, 127.6, 127.4, 127.2, 127.1, 104.7, 21.5 ppm;
HRMS (ESI) m/z [M+Na]+ calcd for C20H15N3NaO2: 352.1062, found:
352.1068.
1
phy was performed on silica gel (32–63 mm, 60 ꢁ pore size). H and
13C NMR spectra were recorded on a Bruker 400 spectrometer.
Chemical shifts (d) 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
using Waters Synapt G2 LCMS. The >95% purity of the synthe-
sized compounds was ascertained by UPLC–MS analyses.
6-Benzoyl-2-(difluoromethyl)-5-phenyl-1H-pyrrolo[2,3-d]pyrimi-
din-4(7H)-one (15): White powder (28 mg; 47%): 1H NMR
([D6]DMSO): d=13.12 (brs, 1H), 12.80 (brs, 1H), 7.46 (d, J=7.0 Hz,
2H), 7.35 (t, J=7.4 Hz, 1H), 7.24–7.05 (m, 7H); 13C NMR ([D6]DMSO):
d=188.3, 159.0, 148.2, 137.6, 132.6, 132.5, 131.6, 129.6, 129.2,
128.2, 127.4, 127.0, 112.9, 110.5, 108.1, 106.9 ppm; HRMS (ESI) m/z
[M+H]+ calcd for C20H14F2N3O2: 366.1054, found: 366.1054.
2-Amino-5-benzoyl-4-phenyl-1H-pyrrole-3-carboxamide (1): An-
hydrous granulated K2CO3 (0.028 g, 0.2 mmol) was added to
a stirred solution of N-methylsulfonamidoacetophenone[13] (0.084 g,
0.4 mmol), benzaldehyde (0.055 g, 0.52 mmol) and cyanoacetamide
(0.044 g, 0.52 mmol) in EtOH (3 mL). The mixture was held at reflux
for 14 h. The reaction mixture was then cooled to RT and concen-
trated in vacuo. The crude product was purified by flash chroma-
tography on silica gel using CH2Cl2/AcOEt (2:1) as eluent to obtain
pyrrole 1 as a yellow powder (74 mg, 61%): 1H NMR ([D6]DMSO):
d=10.89 (brs, 1H), 7.18–7.05 (m, 8H), 6.97 (t, J=7.8 Hz, 2H), 6.70
(brs, 1H), 6.39 (s, 2H), 4.59 ppm (brs, 1H); 13C NMR ([D6]DMSO): d=
184.3, 167.6, 149.8, 139.7, 134.6, 132.6, 130.9, 129.9, 128.3, 128.2,
128.0, 127.5, 121.5, 99.4 ppm; HRMS (ESI) m/z [M+H]+ calcd for
C18H16N3O2: 306.1243, found 306.1248.
6-Benzoyl-5-phenyl-2-(trifluoromethyl)-1H-pyrrolo[2,3-d]pyrimi-
din-4(7H)-one (16): White/brown powder (63 mg; 66%): 1H NMR
([D6]DMSO): d=13.34 (brs, 1H), 13.25 (brs, 1H), 7.48 (d, J=7.0 Hz,
2H), 7.36 (t, J=7.4 Hz, 1H), 7.22–7.04 ppm (m, 7H); 13C NMR
([D6]DMSO): d=188.4, 137.4, 132.8, 132.4, 131.5, 131.3, 131.1,
130.2, 129.6, 129.2, 128.9, 128.3, 128.1, 128.0, 127.5, 127.2 ppm;
HRMS (ESI) m/z [M+Na]+ calcd for C20H12F3N3NaO2: 406.0779,
found: 406.0777.
6-Benzoyl-2-(pent-4-ynyl)-5-phenyl-1H-pyrrolo[2,3-d]pyrimidin-
4(7H)-one (23): White/brown powder (45 mg 72%): 1H NMR
([D6]DMSO) d=12.57 (brs, 1H), 11.91 (brs, 1H), 7.42 (d, J=7.4 Hz,
2H), 7.30 (t, J=7.4 Hz, 1H), 7.19–6.97 (m, 7H), 2.82 (s, 1H), 2.72 (t,
General procedure for the synthesis of deazahypoxanthines 3–
16, 23: A selected ethyl ester (1.28 mmol) and pyrrole 1 (50 mg,
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J=6.9 Hz, 2H), 2.28 (t, J=6.8 Hz, 2H), 1.98–1.85 ppm (m, 2H);
13C NMR ([D6]DMSO): d=187.6, 159.4, 158.8, 149.8, 137.6, 132.6,
131.7, 131.1, 129.0, 127.6, 127.3, 126.8, 126.6, 104.5, 83.8, 71.7, 32.9,
25.7, 17.3 ppm; HRMS (ESI) m/z [M+Na]+ calcd for C24H19N3NaO2:
404.1375, found: 404.1374.
7.81 (s, 1H), 7.52–7.11 (m, 5H), 2.40 ppm (s, 3H); 13C NMR
([D6]DMSO): d=187.6, 159.9, 157.4, 150.7, 150.0, 148.4, 140.8,
138.1, 132.6, 131.2, 129.6, 128.5, 122.2, 119.1, 105.2, 21.7 ppm;
HRMS (ESI) m/z [M+H]+ calcd for C19H14BrN4O2: 409.0300, found:
409.0287.
General procedure for the synthesis of deazahypoxanthines 13
and 17–22: Anhydrous granulated K2CO3 (0.052 g, 0.372 mmol)
was added in one portion to a solution of N-(2-oxo-2-arylethyl)me-
6-Benzoyl-5-(3,5-dibromophenyl)-2-methyl-1H-pyrrolo[2,3-d]pyri-
midin-4(7H)-one (22): White powder (66 mg, 58%): 1H NMR
([D6]DMSO) d=12.82 (s, 1H), 11.94 (s, 1H), 7.48 (s, 1H), 7.44 (d, J=
6.0 Hz, 2H), 7.37 (t, J=7.2 Hz, 1H), 7.33 (s, 2H), 7.21 (t, J=6.8 Hz,
2H), 2.28 (s, 3H); 13C NMR ([D6]DMSO): d=187.9, 160.0, 157.0,
151.1, 138.2, 137.2, 133.2, 132.5, 131.8, 129.2, 128.2, 124.2, 121.1,
104.8, 21.7 ppm; HRMS (ESI) m/z [M+H]+ calcd for C20H14Br2N3O2:
485.9453, found: 485.9438.
thanesulfonamide
(0.676 mmol),
the
selected
aldehyde
(0.879 mmol) and cyanoacetamide (0.072 g, 0.879 mmol) in a mix-
ture of EtOH (2.5 mL) and MeC(OEt)3 (2.5 mL). The mixture was
purged with N2 for 5 min and then heated at 908C for 24 h under
nitrogen atmosphere. The formation of the intermediate pyrrole
was monitored by TLC. The reaction temperature was then in-
creased to 1508C, and the reaction mixture was heated for 3–6 h.
The mixture was cooled to RT, and the formed precipitate was col-
lected by filtration and washed with EtOH (2 mL) and Et2O (2 mL)
to give the desired 7-deazahypoxanthine 13 and 17–23. An addi-
tional amount of the product was obtained by evaporation of the
mother liquor and purification of the residue by column chroma-
tography with MeOH/CH2Cl2 (1/40!1/20).
6-Benzoyl-5-(3-chlorophenyl)-2-methyl-1H-pyrrolo[2,3-d]pyrimi-
din-4(7H)-one (17): White powder (150 mg, 61%): 1H NMR
([D6]DMSO): d=12.67 (brs, 1H), 11.93 (brs, 1H), 7.44 (d, J=7.2 Hz,
2H), 7.36 (t, J=7.4 Hz, 1H), 7.17 (t, J=7.3 Hz, 2H), 7.13–7.09 (m,
3H), 7.04 (t, J=7.6 Hz, 1H), 2.37 ppm (s, 3H); 13C NMR ([D6]DMSO):
d=187.9, 159.7, 156.9, 150.5, 138.1, 135.2, 132.3, 132.0, 131.2,
130.2, 129.4, 129.0, 128.2, 127.9, 126.9, 125.7, 104.8, 21.5 ppm;
HRMS (ESI) m/z [M+H]+ calcd for C20H15ClN3O2: 364.0853, found:
364.0851.
N-(2-(2-(2-(2-(4-(3-(6-benzoyl-4-oxo-5-phenyl-4,7-dihydro-1H-
pyrrolo[2,3-d]pyrimidin-2-yl)propyl)-1H-1,2,3-triazol-1-yl)ethox-
y)ethoxy)ethoxy)ethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-yl)pentanamide (24): A freshly prepared
aq 1m sodiumascorbate solution (4 mL, 4.0 mmol), and freshly pre-
pared aq 0.2m CuSO4·5H2O solution (4 mL, 0.8 mmol) were added
to a solution of 23 (5 mg, 0.013 mmol) and biotin-PEG3-azide
(5.8 mg, 0.013 mmol) in tBuOH/H2O (1:2, 1.5 mL). The mixture was
stirred at RT for 48 h and concentrated in vacuo. The crude prod-
uct was purified by preparative TLC (1.5:8.5 MeOH/CHCl3) to obtain
24 as a white solid (1.1 mg, 10%): 1H NMR ([D6]DMSO) d=12.58
(brs, 1H), 11.89 (brs, 1H), 7.87 (s, 1H), 7.79 (t, J=5.6 Hz, 1H), 7.41
(dd, J=8.3, 1.3 Hz, 2H), 7.31 (t, J=7.4 Hz, 1H), 7.18–6.99 (m, 7H),
6.36 (d, J=21.2 Hz, 1H), 5.02 (s, 1H), 4.47 (t, J=5.2 Hz, 2H), 4.32–
4.26 (m, 1H), 4.15–4.07 (m, 1H), 3.80 (t, J=5.5 Hz, 2H), 3.55–3.41
(m, 8H), 3.19–3.15 (m, 4H), 3.11–3.06 (m, 3H), 2.82 (dd, J=12.4,
5.1 Hz, 2H), 2.68–2.65 (m, 2H), 2.58–2.52 (m, 2H), 2.46–2.42 (m,
1H), 2.35–2.31 (m, 1H), 1.62–1.41 ppm (m, 6H); HRMS (ESI) m/z
[M+Na]+ calcd for C42H51N9NaO7S: 848.3530, found: 848.3528.
6-Benzoyl-5-(3-bromophenyl)-2-methyl-1H-pyrrolo[2,3-d]pyrimi-
din-4(7H)-one (18): White/brown powder (90 mg, 40%): 1H NMR
([D6]DMSO): d=12.67 (brs, 1H), 11.93 (brs, 1H), 7.43 (d, J=7.0 Hz,
2H), 7.36 (t, J=7.5 Hz, 1H), 7.30 (t, J=1.8 Hz, 1H), 7.25–7.15 (m,
4H), 6.99 (t, J=7.8 Hz, 1H), 2.37 ppm (s, 3H); 13C NMR ([D6]DMSO):
d=187.9, 159.7, 157.0, 150.4, 138.1, 135.4, 134.0, 132.3, 130.6,
129.8, 129.3, 129.2, 128.2, 127.9, 125.6, 120.5, 104.8, 21.6 ppm;
HRMS (ESI) m/z [M+Na]+ calcd for C20H14BrN3NaO2: 430.0167,
found: 430.0168.
Biology
Cell culture: Human cancer cell lines were obtained from the Amer-
ican Type Culture Collection (ATCC, Manassas, VA, USA), the Euro-
pean Collection of Cell Culture (ECACC, Salisbury, UK) and the
Deutsche Sammlung von Mikroorganismen und Zellkulturen
(DSMZ, Braunschweig, Germany). Human cervical adenocarcinoma
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (FBS). Human
mammary carcinoma MCF-7 cells were cultured in RPMI supple-
mented with 10% FBS. U87 and U373 cells were cultured in DMEM
culture medium (Lonza code 12–136F, Vervier, Belgium), while
SKMEL-28 and A549 cells were cultured in RPMI culture medium
(Lonza; code 12–115F) supplemented with 10% heat-inactivated
FBS (Lonza, FBS South America code DE14–801F). Cell culture
media were supplemented with 4 mm glutamine (Lonza code
BE17–605E), 100 mgmLꢁ1 gentamicin (Lonza code 17–5182), and
penicillin-streptomycin (200 unitsmLꢁ1 and 200 mgmLꢁ1) (Lonza
code 17–602E). All cell lines were cultured in T25 flasks, maintained
and grown at 378C, 95% humidity, 5% CO2.
6-Benzoyl-5-(3-fluorophenyl)-2-methyl-1H-pyrrolo[2,3-d]pyrimi-
1
din-4(7H)-one (19): White/brown powder (107 mg, 43%): H NMR
([D6]DMSO): d=12.66 (brs, 1H), 11.93 (brs, 1H), 7.44 (d, J=7.0 Hz,
2H), 7.35 (t, J=7.4 Hz, 1H), 7.17 (t, J=7.8 Hz, 2H), 7.06–6.99 (m,
2H), 6.94 (dt, J=7.8, 1.2 Hz, 1H), 6.91–6.85 (m, 1H), 2.37 ppm (s,
3H); 13C NMR ([D6]DMSO): d=187.3, 162.0, 159.6, 159.1, 156.2,
149.8, 137.5, 134.8, 131.6, 128.8, 128.3, 127.5, 127.2, 125.1, 117.6,
113.3, 104.1, 20.9 ppm; HRMS (ESI) m/z [M+K]+ calcd for
C20H14FKN3O2: 386.0707, found: 386.0708.
6-Benzoyl-2-methyl-5-(pyridin-3-yl)-1H-pyrrolo[2,3-d]pyrimidin-
4(7H)-one (20): White/yellow powder (146 mg, 65%): 1H NMR
([D6]DMSO): d=12.74 (brs, 1H), 11.97 (brs, 1H), 8.30 (dd, J=2.2,
0.7 Hz, 1H), 8.22 (dd, J=4.8, 1.6 Hz, 1H), 7.61–7.57 (m, 1H), 7.47–
7.42 (m, 2H), 7.38–7.33 (m, 1H), 7.20–7.14 (m, 2H), 7.07 (ddd, J=
7.8, 4.8, 0.8 Hz, 1H), 2.37 ppm (s, 3H); 13C NMR ([D6]DMSO): d=
187.2, 159.4, 156.5, 150.8, 150.2, 147.2, 138.0, 137.5, 131.9, 129.1,
128.7, 127.8, 127.6, 123.2, 121.9, 104.6, 21.1 ppm; HRMS (ESI) m/z
[M+Na]+ calcd for C19H14N4NaO2: 353.1014, found: 353.1019.
Antiproliferative properties: To evaluate antiproliferative properties
of the synthesized compounds, the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay was used. The cell lines
were assessed by trypsinizing each cell line and seeding 4ꢂ103
cells per well into 96-well plates. All compounds were dissolved in
dimethyl sulfoxide (DMSO) at a concentration of either 100 mm or
50 mm prior to cell treatment. Cells were grown for 24 h and then
treated with compounds at concentrations ranging from 0.04 mm
to 100 mm and incubated for 48 h in 200 mL media. 20 mL of MTT
6-Benzoyl-5-(5-bromopyridin-3-yl)-2-methyl-1H-pyrrolo[2,3-
1
d]pyrimidin-4(7H)-one (21): White powder (60 mg, 63%): H NMR
([D6]DMSO): d=12.90 (s, 1H), 12.09 (s, 1H), 8.36 (d, J=9.0 Hz, 2H),
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reagent in serum-free medium (5 mgmLꢁ1) was added to each well
and incubated further for 2 h. Media was removed, and the result-
ing formazan crystals were re-solubilized in 200 mL of DMSO. A490
was measured using a Thermomax Molecular Device plate reader.
The experiments were performed in quadruplicate and repeated at
least twice for each compound per cell line. Cells treated with
0.1% DMSO were used as a control, and 1 mm phenyl arsine oxide
(PAO) was used as a positive killing control.
Keywords: 7-deazapurines
privileged structures · pyrrolo[2,3-d]pyrimidines
· alkaloids · drug discovery ·
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Molecular modeling
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the colchicine binding site were downloaded from the Protein
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plete residues. The structure PDBID: 3UT5 was found to be the
most suitable receptor for modeling purposes. From this structure,
chain B was retained, along with the corresponding co-crystallized
colchicine ligand. All other chains, ligands and water molecules
were deleted. A minimization was performed on the receptor
ligand complex using Accelrys Discovery Studio 4.0 (Smart Mini-
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applied to all non-hydrogen atoms, and a GBSW solvent model
was employed. Docking studies were performed using the CDocker
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ligand conformations and 75 structures for refinement per ligand.
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Acknowledgements
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This project was supported by the Texas State University start-up
funding (USA) and National Institute of General Medical Sciences
(USA) (P20GM103451). The modeling studies were made possible
by the Centre for High Performance Computing (Rosebank, Cape
Town), who provided the access to Accelrys Discovery Studio.
S.C.P. and W.A.L.v.O. acknowledge funding from the National Re-
search Foundation (NRF, RSA) and the University of Stellenbosch
(South Africa).
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Received: December 16, 2013
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Rigid(in) development! Novel ana-
logues of marine alkaloid rigidins were
prepared utilizing new synthetic meth-
ods developed for the construction of
the pyrrolo[2,3-d]pyrimidine ring
R. Scott, M. Karki, M. R. Reisenauer,
R. Rodrigues, R. Dasari, W. R. Smith,
S. C. Pelly, W. A. L. van Otterlo,
C. B. Shuster, S. Rogelj, I. V. Magedov,
L. V. Frolova,* A. Kornienko*
system. The compounds exhibited sub-
micromolar to nanomolar antiprolifera-
tive potencies against drug-resistant
cells, such as glioblastoma, melanoma
and non-small-cell lung cancer.
&& – &&
Synthetic and Biological Studies of
Tubulin Targeting C2-Substituted 7-
Deazahypoxanthines Derived from
Marine Alkaloid Rigidins
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 9
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