Synthesis of N4-(substituted phenyl)-N4-alkyl/ desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines and identification of new microtubule disrupting compounds that are effective against multidrug resistant cells

Article abstract of DOI:10.1016/j.bmc.2012.12.010

A series of fourteen N⁴-(substituted phenyl)-N ⁴-alkyl/desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines was synthesized as potential microtubule targeting agents. The synthesis involved a Fisher indole cyclization of 2-amino-6-hydrazin

Full text of DOI:10.1016/j.bmc.2012.12.010

Bioorganic & Medicinal Chemistry 21 (2013) 891–902
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of N4-(substituted phenyl)-N4-alkyl/desalkyl-9H-pyrimi-
do[4,5-b]indole-2,4-diamines and identification of new microtubule
disrupting compounds that are effective against multidrug resistant
cells q
a
a
a
Aleem Gangjee a, , , Nilesh Zaware , Ravi Kumar Vyas Devambatla , Sudhir Raghavan ,
⇑
Cara D. Westbrook b,c, Nicholas F. Dybdal-Hargreaves b,c, Ernest Hamel d, Susan L. Mooberry b,c, ,
⇑
a Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282, USA
b Department of Pharmacology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA
c Cancer Therapy & Research Center, 7979 Wurzback Road, San Antonio, TX 78229, USA
d Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Frederick National Laboratory
for Cancer Research, National Institutes of Health, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
A series of fourteen N4-(substituted phenyl)-N4-alkyl/desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines
was synthesized as potential microtubule targeting agents. The synthesis involved a Fisher indole cycli-
zation of 2-amino-6-hydrazinylpyrimidin-4(3H)-one with cyclohexanone, followed by oxidation, chlori-
nation and displacement with appropriate anilines. Compounds 6, 14 and 15 had low nanomolar potency
against MDA-MB-435 tumor cells and depolymerized microtubules. Compound 6 additionally had nano-
molar GI50 values against 57 of the NCI 60-tumor panel cell lines. Mechanistic studies showed that 6
inhibited tubulin polymerization and [3H]colchicine binding to tubulin. The most potent compounds
were all effective in cells expressing P-glycoprotein or the bIII isotype of tubulin, which have been asso-
ciated with clinical drug resistance. Modeling studies provided the potential interactions of 6, 14 and 15
within the colchicine site.
Article history:
Received 25 July 2012
Revised 30 November 2012
Accepted 8 December 2012
Available online 20 December 2012
Keywords:
Pyrimido[4,5-b]indole-2,4-diamines
Microtubules
Multidrug resistance
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
docetaxel and nab-paclitaxel, are utilized primarily for the man-
agement of adult solid tumors, including breast, ovarian, and pros-
Microtubules play a crucial role in normal cellular metabolism,
in the orchestration of mitotic events and are validated targets for
treating cancer.2,3 Microtubule-targeting compounds as antitumor
agents have met with unprecedented clinical success and are fre-
quently referred to as antimitotic agents. More recently, however,
the idea that disruption of interphase events play a major role in
the anticancer actions of microtubule targeting drugs has gained
new attention.4 Chemotherapeutic regimens often include a taxane
or a vinca alkaloid (Fig. 1). The taxanes, including paclitaxel,
tate, as well as cancers of the head and neck and non-small cell
lung cancer. The vinca alkaloids (vincristine, vinblastine, and vino-
relbine) are most often used for hematological malignancies, such
as lymphomas and leukemias. A simplistic classification of micro-
tubule targeting agents includes microtubule stabilizers (taxanes)
and microtubule destabilizers (vincas).5 The taxanes bind at the
taxane site located on b-tubulin in the interior surface of microtu-
bules where they stabilize cellular microtubules while the vincas
bind within the vinca domain of b-tubulin located on the outer sur-
face of the microtubule and destabilize microtubules causing a net
loss of cellular microtubules. Compounds that bind within another,
non-overlapping binding site on tubulin, the colchicine site, elicit
the same ‘destabilizing’ effect as observed with the vincas.6
Although colchicine, due to its high toxicity, is not used as an anti-
cancer agent, colchicine-site binding drugs, such as combretastatin
A-4 (CA-4), are advancing in clinical trials. CA-4P, the phosphory-
lated prodrug of CA-4, was recently evaluated in ovarian, non-
small cell lung cancer and anaplastic thyroid cancer and additional
Abbreviations: CA-4, combretastatin A-4; MDR, multidrug resistance; Pgp, P-
glycoprotein’; Rr, relative resistance; DAMA colchicine, N-deacetyl-N-(2-
mercaptoacetyl)colchicine.
q
See Ref. 1.
⇑
Corresponding authors. Tel.: +1 412 396 6070; fax: +1 412 396 5593 (A.G.); tel.:
+1 210 567 4788; fax: +1 210 567 4300 (S.L.M.).
E-mail addresses: gangjee@duq.edu (A. Gangjee), mooberry@uthscsa.edu (S.L.
Mooberry).
These two authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.010

892
A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 891–902
R2O
O
OH
N
R1
N
OH
NH
O
N
H
O
H
N
H3COOC
H3CO
HO
N
H
O
H
HO
O
N
H3COOC
H3CO
OH
OAc
O
N
R1
OR3
OH
H
O
N
OCOCH3
R2
R3
H
O
R1
R2
OCH3
R1
COPh
R2
COCH3
H
vinblastine
vincristine
vindesine
vinorelbine
CH3
OCH3 COCH3
CHO OCH3 COCH3
CH3 NH2
paclitaxel
docetaxel COOtBu
H
H3CO
H3CO
H3CO
H3CO
NHCOCH3
O
OCH3
H3CO
OH
OCH3
combretastatin A-4 (CA-4)
Figure 1. Tubulin binding agents.
OCH3
colchicine
studies are planned.7 CA-1P is currently being evaluated in a Phase
I trial in acute myelogenous leukemia (AML) and in myelodysplas-
tic syndrome (MDS).8
cancers is linked with resistance to the taxanes or vinorelbine
and with poor prognosis (reviewed in Ref. 3). Although many other
tubulin-based mechanisms of multidrug resistance have been
identified in cell lines, thus far, only expression of Pgp and the bIII
tubulin isotype have been linked with clinical resistance to diverse
tubulin binding agents.16
Despite the exceptional success of microtubule targeting agents
in cancer chemotherapy, multidrug resistance (MDR) is a major
limitation in their clinical use. One of the mechanisms for tumor
resistance is the inherent expression of MDR proteins like P-glyco-
protein (Pgp), an ATP-binding cassette (ABC) transporter.9 These
transporters can act as drug efflux pumps and reduce the accumu-
lation of Pgp substrates in tumor cells.10 In addition to innate resis-
tance, the administration of the taxanes and other Pgp substrates
has been associated with acquired tumor resistance due to Pgp
overexpression.11 Attempts to reverse drug resistance by combin-
ing microtubule agents with inhibitors of drug efflux proteins have
been disappointing.12 The fact that microtubule-binding agents are
substrates for ABC efflux pumps also limits their transport into the
central nervous system (CNS) and represents an obstacle to their
oral administration, suggesting that new compounds, which are
less susceptible to transport by ABC proteins, could lead to im-
proved efficacy in MDR tumors.13
We17 had previously reported N-(4-methoxyphenyl)-N,2-di-
methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine 1 (Fig. 2) as a micro-
tubule-targeting agent with nanomolar potency (Table 1). We
showed that 1 functions by a mechanism similar to that of colchi-
cine and CA-4 and inhibits tumor cells with equal potency without
regard to their expression of Pgp or bIII-tubulin. In addition, com-
pound 1 inhibits the binding of [3H]colchicine to tubulin, suggest-
ing that it binds within the colchicine site. With 1 as the lead
compound, we designed the tricyclic pyrimido[4,5-b]indole struc-
ture A (Fig. 2), to develop inhibitors of tubulin similar to or more
potent than 1. The additional C-ring introduced in structure A con-
formationally restricts the rotation of the aniline moiety and was
hypothesized to be favorable for additional interactions within
the colchicine site on tubulin. To ascertain the importance of vari-
ous groups on the tricyclic scaffold to biological activity, com-
pounds 2–15 were designed with variations at the 4-nitrogen
and the aniline phenyl group. Compounds 2–5, 8, 9, and 12 did
not have a methylated aniline nitrogen. A methyl group at N4
was anticipated to further restrict rotation of the C4–N4 bond as
Another mechanism for resistance to the taxanes and the vinca
alkaloids is the expression of the bIII isotype of tubulin. In a variety
of cell lines, overexpression of bIII-tubulin is associated with resis-
tance to tubulin-binding antimitotic agents.14,15 Clinically, expres-
sion of bIII-tubulin in ovarian, breast and non-small cell lung
R1
H
R2
H
R3
2
3'-OMe
3'-OMe
4'-OMe
4'-OMe
4'-OMe
4'-OMe
3'-Br
OCH3
3
Piv
H
H
4
H
R3
6
R2
H3C
N
5
Piv
H
H
N
N
N
5
6
Me
Me
H
4
7
3
C
7
Piv
H
N
8
A
B
8
2
R1
9
Piv
H
H
3'-Br
N
H
N
H
N
H
N
H3C
10
Me
3'-OMe
3'-OMe
3',4',5'-triOMe
4'-Me
1
11 Piv Me
2-15
1
12
13
14
15
H
H
H
H
H
A
Me
Et
4'-OMe
4'-OEt
Me
Figure 2. Parent pyrrolo[2,3-d]pyrimidine 1 and target pyrimido[4,5-b]indoles 2–15.

A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 891–902
893
Table 1
NH2 should be more compatible and was the preferred choice in
this study. The 2-NH2 group was protected with a pivaloyl group
in 3, 5, 7, 9 and 11 as intermediates in the synthesis of 2, 4, 6, 8
and 10, respectively, and it was of interest to test these protected
analogs as well to study the importance to biological activity of a
free 2-NH2 as compared with a 2-CH3 and to determine whether
additional binding ability was conferred by the pivaloyl group.
IC50 values for the ability of the compounds to inhibit proliferation of MDA-MB-435
cells and EC50 values for loss of cellular microtubules
Compd
IC50 SD (nM)
(MDA-MB-435)
EC50 for microtubule
depolymerization
in A-10 cells (nM)
EC50/IC50
2
3
4
5
6
7
8
9
NDa
>40
>40
>40
>40
l
l
l
l
M
M
M
M
NDa
NDa
NDa
NDa
7.1
NDa
NDa
NDa
2. Chemistry
14.7 1.5
105 12
NDa
>40
>40
>40
>40
>40
>10
>10
198
83
l
l
l
l
l
l
l
M
M
M
M
M
M
M
8
NDa
NDa
NDa
NDa
NDa
NDa
NDa
8.4
The synthesis of target compounds commenced from
commercially available 2-amino-6-chloropyrimidin-4(3H)-one 16
(Scheme 1) using a reported method18 to obtain 17 in 46% yield.
A thermal Fisher indole cyclization of 17 and cyclohexanone in di-
phenyl ether furnished the tricyclic scaffold 18. The partially satu-
rated ring in 18 was oxidized using 10% Pd/C to provide 19 in 57%
yield. Pivaloyl protection of 19 gave 20, and subsequent chlorina-
tion afforded the common intermediate 21. Compound 21 was
treated with various substituted anilines 22–26 in isopropanol
and a catalytic amount (1–3 drops) of concd HCl at reflux to provide
3, 5, 7, 9, and 11, respectively in 37–80% yield. The pivaloyl group in
3, 5, 7, 9, and 11 was removed by basic hydrolysis of the 2-amide
linkage in these compounds using 1 N NaOH to give compounds
2, 4, 6, 8, and 10, respectively, in yields ranging from 53% to 93%.
To address the water solubility problems associated with paclitaxel
and other antimitotic agents, the water soluble HCl salt of 6 (6ꢀHCl)
was obtained by bubbling anhydrous HCl gas through a solution of
6 (55 mg) in diethyl ether, ethyl acetate and chloroform.
NDa
NDa
10
11
12
13
14
15
1b
CA-4c
NDa
NDa
NDa
NDa
23.5 1.2
14.4 0.5
183 3.4
3.4 0.6
4
6.5
31.7
3.8
5800
13
a
b
c
ND: not determined.
Results previously published.17
Results previously published.23 The IC50 values were obtained in 3–5 inde-
pendent experiments each utilizing triplicate or quadruplicate wells for each con-
centration and are expressed SD. The EC50 values were obtained in 3–4
experiments, each evaluating a large range of concentrations of each compound and
are expressed SD.
well as the N4–C0 bond to also restrict phenyl ring rotation. Activity
of such analogs was anticipated to reflect conformational restric-
tion as well as hydrophobic interactions of the N4-methyl moiety
with the colchicine site on tubulin. Compounds 6, 7, 10, 11, 13,
and 15 represent such conformationally restricted analogs with a
N4-methyl substitution akin to 1. Compound 14 was designed with
a larger ethyl group at the N4-position to determine the effect of
size at the N4-position on biological activity. Molecular modeling
(see below) does indeed predict that the N4-alkyl groups lie in
the C5-C6 region of N-deacetyl-N-(2-mercaptoacetyl)colchicine
(DAMA colchicine) when docked into tubulin.
To determine the importance of the nature and position of the
aniline substituent on activity, compounds with electron donating
40-OMe (4, 5, 6, 7, 14), 30-OMe (2, 3, 10, 11), 40-Me (13), 40-OEt (15)
and electron withdrawing 30-Br (8, 9) substituents were designed.
The 30,40,50-triOMePh group is present in colchicine and CA-4, thus
it was of interest to study the effect of this group when combined
with the pyrimido[4,5-b]indole scaffold in 12. Molecular modeling
(see below) also predicts that such a 30,40,50-triOMePh analog could
mimic colchicine.
Reaction of 21 with appropriate anilines 27–29 (Scheme 2) and
catalytic concd HCl (1–3 drops) in n-butanol at reflux provided 12–
14, respectively, in 31–79% yield. Compound 21 upon treatment
with 30 in isopropanol afforded target compound 15 in 71% yield.
Compounds 2919 and 3020 were synthesized from 31 and 32,
respectively, using reported procedures.
3. Biological evaluations
Compounds 2–15 were designed as microtubule targeting
agents, and they were first tested for their ability to disrupt cellular
microtubules (Table 1). Compounds 2–5 and 7–13 had no effects
on cellular microtubules at concentrations as high as 10 or
40 lM. It should be noted that all the N4-desalkyl analogs includ-
ing 4 (40-OMe) and 12 (30,40,50-triOMe) were inactive. Among the 2-
amino-N4-alkyl analogs, the 40-O-alkyl analogs 6, 14 and 15 were
active whereas the analogs 13 (40-Me) and 10 (30-OMe) were inac-
tive. The inactivity of 13 can be attributed to the lack of hydrogen
bonding ability that is observed with the highly active 6 (Fig. 6),
which had an EC50, the concentration required to cause 50% loss
of cellular microtubules, of 105 nM (Table 1). The pivaloyl
protected analog of 6, compound 7, is inactive indicating the
The 2-CH3 in 1 is isosterically replaced with a 2-NH2 (2, 4, 6, 8,
10, 12–15). The 2-CH3 and 2-NH2 groups are oriented towards the
solvent (see molecular modeling below, Figs. 6–8) and hence the 2-
Vehicle (DMSO)
6 500 nM
CA-4 20 nM
Figure 3. The effects of vehicle, CA-4 or 6 on interphase microtubules. A-10 cells were treated with vehicle, 20 nM CA-4 or 500 nM 6. Following an 18 h incubation, the cells
were fixed with cold methanol and microtubules were visualized by indirect immunofluorescence techniques with a b-tubulin antibody.

894
A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 891–902
Figure 4. The effects of 6 on cell cycle distribution. MDA-MD-435 cells were treated with vehicle (A), 12.5 nM paclitaxel (6.5 ꢁ IC50 for inhibition of proliferation) (B), 44.1 nM
6 (3 ꢁ IC50) (C), or 73.5 nM 6 (5 ꢁ IC50) (D), for 18 h. The cells were harvested and stained with propidium iodide, and cell cycle distribution was evaluated using flow
cytometry.
intolerance of pivaloyl group in the binding site. The data indicates
that N4-alkyl, 40-O-alkyl and a free 2-NH2 group are important for
activity. Compound 6 was 55-times more potent than 1 in depoly-
merizing cellular microtubules and 8-times less potent than CA-4
for this effect. The antiproliferative effects of 6 were evaluated
against the drug sensitive MDA-MB-435 cell line using the sulfo-
rhodamine B assay.21,22 The data (Table 1) indicated that 6 had po-
depolymerizers with EC50 values of 198 and 83 nM, respectively
(Table 1). These compounds were potent inhibitors of tumor cell
proliferation with IC50 values of 23.5 and 14.4 nM against MDA-
MB-435 melanoma cells, respectively. Consistent with the activi-
ties of 6, compounds 14 and 15 had EC50/IC50 ratios of 8.4 and
6.5, again suggesting a much closer linkage of the microtubule
depolymerizing effects and antiproliferative activities than were
observed with 1.
tent antiproliferative effects with an IC50, the concentration
required to cause 50% inhibition of proliferation, of 14.7 nM. Com-
pound 6 was 12-fold more potent than 1 for inhibition of prolifer-
ation, indicating that the addition of the C-ring and isosteric
In the preclinical National Cancer Institute 60 cancer cell line
panel, compound 6 exhibited potent GI50s (Table 2).24
The microtubule disrupting effects of 6 are shown in Figure 3 in
a cell-based immunofluorescence assay. Compound 6 caused
extensive loss of the interphase microtubule network, similar to
the effects of CA-4. Similar effects were noted for 14 and 15 (data
not shown).
Consistent with its ability to disrupt interphase microtubules
(Fig. 3), compounds 6, 14 and 15 caused the formation of aberrant
mitotic spindles (data not shown). The effects of 6 on cell cycle dis-
tribution were evaluated by flow cytometry (Fig. 4), which showed
accumulation of cells in the G2/M phase of the cell cycle.
The ability of 6, 14 and 15 to circumvent Pgp-mediated drug
resistance was evaluated by using an SK-OV-3 isogenic cell line
pair (Table 3). This cell line pair consists of the parental SK-OV-3
ovarian carcinoma cell and a subline, the SK-OV-3 MDR-1-6/6,
which was generated by transduction of MDR-1 gene and clonal
replacement of the 2-CH3 with 2-NH2 was favorable. The EC50
/
IC50 ratio gives an indication of the linkage of the microtubule
depolymerizing effects and the antiproliferative activities. Com-
pound 6 had a substantially lower EC50/IC50 ratio of 7.1 as com-
pared with 1, which had a ratio of 31.7. The high ratio obtained
with 1 suggests that additional mechanisms are implicated in the
ability of this compound to inhibit cell proliferation. A low ratio
provides an indication of a tighter linkage of the microtubule depo-
lymerizing effects and inhibition of cell proliferation. The ratio for
CA-4, at 3.8, was even lower than that of compound 6, suggesting
the possibility that 6 might impact microtubule-independent
mechanisms of growth inhibition.
Two additional microtubule active compounds 14 and 15 were
identified in this series. They were also potent microtubule

A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 891–902
895
Figure 5. Effects of 6 on tubulin assembly. Porcine brain tubulin, 2.5 mg/mL (Cytoskeleton Inc, Denver, CO), was incubated with 6, colchicine or DMSO vehicle in general
tubulin buffer containing 10% glycerol and 1 mM GTP. The effects of these drugs on tubulin polymerization were monitored by measuring the absorbance at 340 nm at37 °C in
a 96 well Spectromax Plus spectrophotometer (molecular devices).
Figure 6. Stereo view. Superimposition of docked poses of 6 (white) and 1 (blue) overlaid with DAMA colchicine (red) in the colchicine site of tubulin.
isolation.23 The ability of compounds to overcome Pgp-mediated
drug resistance was evaluated by examining the sensitivity of the
two cell lines to the compounds. The IC50 values obtained in the
Pgp-expressing MDR-1-6/6 cells were divided by the IC50 obtained
in the parental cells to generate a relative resistance value, desig-
nated Rr. A low Rr indicates that the cell lines have similar sensitiv-
ity to the compound and that the compound is able to overcome
the expression of Pgp and suggests that it is a poor substrate for
transport by Pgp. A high Rr value is found with known Pgp sub-
strates, including paclitaxel. In this cell line pair, the relative resis-
tance (Rr) value of paclitaxel was 590 while the Rr value of 6 was
1.2, comparable with the Rr value obtained with CA-4 of 1.2.
Similar results were obtained with 14 and 15 with Rr values of
1.4 and 1.3, respectively (Table 3). Thus, compounds 6, 14 and 15
are effective in this cell line that overexpresses Pgp and the data
suggest that these compounds are poor substrates for transport
by Pgp and thus have advantages over paclitaxel and the vincas.
A second mechanism of drug resistance that is associated with
treatment failure with tubulin binding agents is the expression of
bIII-tubulin. A HeLa cell line pair was used to study the effects of
bIII-tubulin on the potency of 6, 14 and 15 (Table 3). The WT bIII
cell line was generated from HeLa cells transfected with the gene
for bIII-tubulin.23 The IC50 of each of these compounds were deter-
mined in the two cell lines and then the relative resistance (Rr)

896
A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 891–902
Figure 7. Stereo view. Superimposition of docked poses of 14 (white) overlaid with DAMA colchicine (red) in the colchicine site of tubulin.
Figure 8. Stereo view. Superimposition of docked poses of 15 (white) overlaid with DAMA colchicine (red) in the colchicine site of tubulin.
value was calculated by dividing the IC50 of the bIII expressing cell
line by the IC50 value obtained in the HeLa parental cell line. Com-
pound 6 has an Rr value of 1.0, compound 14 a Rr of 1.4 and com-
pound 15 a Rr of 0.8 in this cell line pair, suggesting that they
overcome drug resistance mediated by bIII-tubulin as compared
with paclitaxel, which has a Rr of 5.8 in these cell lines. Thus, com-
pounds 6, 14 and 15 potently inhibited the proliferation of human
cancer cells without regard to their expression of Pgp or bIII-
tubulin.
assembly in a quantitative study (Table 4). In this assay, com-
pounds 6, 14 and 15 inhibited tubulin assembly about as well as
CA-4. The data in Table 4 also shows that these compounds bind
to the colchicine site on tubulin, since they inhibited [3H]colchicine
binding to the protein. The potency of this inhibition with 6, 14 and
15 was better than with 1 although somewhat less than the inhibi-
tion obtained with CA-4.
Inhibition of tubulin assembly was performed as described pre-
viously,26 with tubulin at 10
lM (1.0 mg/mL) and varying concen-
Studies were conducted to determine if 6, 14 and 15 inhibited
the polymerization of purified bovine brain tubulin,25,26 as would
be predicted from the effects in cells. These biochemical studies
provided an indication of a direct interaction of the compounds
with tubulin. An initial study shown in Figure 5 indicated that 6
was a potent inhibitor of tubulin assembly. Compounds 6, 14 and
15 were therefore compared with CA-4 as inhibitors of tubulin
trations of potential inhibitors. Extent of assembly at 20 min was
the parameter measured. The CA-4 control is performed twice a
year, and IC50 values obtained range from 1 to 1.2
lM. The most re-
cent value is shown in the table. Inhibition of colchicine binding
was performed as described previously,27 with tubulin at 1.0
lM
and both [3H]colchicine and potential inhibitor at 5.0
lM. Incuba-
tion was for 10 min at 37 °C.

A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 891–902
897
Cyclohexanone,
Ph2O, 120 oC, 14 h
and
O
O
N
O
10% Pd/C,
Hydrazine,
water
HN
H2N
Ph2O, reflux
HN
H2N
HN
H2N
3 h
reflux, 3 h
reflux, 3 h
N
H
N
17
NHNH2
N
16
Cl
18
X
Ar
X
N
Ar
N
H
Ar
22, 3 3'-OMePh
23, 5
X
H
H
O
N
Cl
N
22-26
POCl3,
reflux
conc. HCl,
IPA, reflux
4'-OMePh
HN
HN
N
24, 7 4'-OMePh Me
25, 9
3'-BrPh
H
N
H
N
H
N
H
HN
Piv
N
HN
Piv
N
4 h
18-120 h
26, 11 3'-OMePh Me
R
21
3,5,7, 9, 11
Pivalic anhydride,
DMAP, NEt3,
DMF, 60 oC,
40 h
R
19 H
20 Piv
OMe
X
N
Ar
N
1 N NaOH,
IPA, reflux
Ar
X
N
. HCl
2
3'-OMePh
4'-OMePh
H
H
anh. HCl (g)
4
6
8
6
14 h
N
4'-OMePh Me
anh. ether,
N
H
H2N
N
3'-BrPh
H
CHCl3, EtOAc
N
H
6.HCl
10 3'-OMePh Me
H2N
N
2,4,6, 8, 10
Scheme 1. Synthesis of compounds 2–11 and 6ꢀHCl.
Cysb241 of tubulin and the oxygen atom of the 40-OCH3 of 6 (res-
idue number follows the modeling convention used by Ravelli
et al.28 rather than the actual residue numbers in the beta tubulin
sequence). The phenyl ring interacts with Cysb241, Leub248,
Alab250, Leub255, Alab316 and Valb318. The importance of the
N-CH3 group of 6 is that it mimics the C5-C6 atoms of the B-ring
of DAMA colchicine and interacts with Leub248, Alab250, Lysb254,
and Leub255. Additionally, the N-CH3 group aids in maintaining
the relative conformation of the pyrimido[4,5-b]indole and the
phenyl ring. The pyrimido[4,5-b]indole scaffold of 6 overlaps with
the C-ring of DAMA colchicine and is stabilized by hydrophobic
X
N
Ar
Cl
N
N
N
X
Ar
27 29
N
H
-
N
n-butanol, reflux,
1-3 drops conc. HCl,
6 h-72 h
N
H
HN
Piv
N
H
H2N
21
Ar
X
or
27, 12
28, 13
3',4',5'-triOMePh
4'-MePh
H
X
Ar
Me
Et
N
H
30
29, 14 4'-OMePh
30, 15
4'-OEtPh
Me
i-propanol, reflux,
1 drop conc. HCl,
120 h
interactions with Ala
and Alab316. The indole NH forms a hydrogen bond with the back-
bone carbonyl of Thr 179 and mimics the hydrogen bond formed
between the C9-carbonyl of DAMA colchicine and the backbone
NH of Thr 179. The 2-NH2 moiety of 6 is exposed towards the sol-
a180, Vala181, Leub248, Leub255, Metb259,
NHEt
NH2
NEt3, 10%Pd/C
a
toluene, 175 oC
µW, 1.5 h
MeO
EtO
MeO
EtO
29
30
31
a
vent front and was not found to interact with the amino acids of
the active site of tubulin. The inactivity of the pivaloyl protected
6, compound 7, can also be explained by the exposure of the hydro-
phobic pivaloyl moiety of 7 to the solvent. Comparison of the
docked conformations of 1 and 6 indicates that the larger tricyclic
scaffold of 6 better fits the large binding pocket of the colchicine
site in the DAMA colchicine X-ray crystal structure as compared
to the bicyclic scaffold of 1. In addition, the pyrimido[4,5-b]indole
scaffold of 6 not only retains the binding interactions of the pyrrol-
o[2,3-d]pyrimidine scaffold of 1 but also provides additional
hydrophobic interactions with the pocket residues which accounts,
in part, for the improved docking score of 6 compared to 1 in tubu-
lin (Fig. 6).
Comparison of the docked poses of 14 (Fig. 7) and 6 (Fig. 6)
shows that the presence of the N-Et group causes a shift in the
bound conformation of the 40-OMe-phenyl groups of the two mol-
ecules. This change in the bound conformation of the 40-OMe-phe-
nyl group of 14 still retains the binding interactions of the
corresponding 40-OMe-phenyl group of 6 and can be explained
by the large volume of the binding pocket. The N-Et group of 14
forms hydrophobic interactions with Leub248, Alab250, Leub252,
Leub255 and the carbon atoms of the side chain of Lysb254. The
docking score of 14 was ꢂ7.6817 kcal/mol, comparable to that of
6 (ꢂ7.6342 kcal/mol) and 14 and 6 have similar activities.
1. (CH2O)n, MeOH
2. NaOMe, MeOH
NH2
NHMe
32
3. then , rt, argon, 5 h
4. NaBH4, 2.5 h, reflux
32
Scheme 2. Synthesis of compounds 12–15.
4. Molecular modeling
In an attempt to explain the potent activity of 6 against tubulin,
we performed molecular modeling studies in the colchicine bind-
ing site of tubulin (PDB: 1SA0),28 using MOE 2008.1029 and meth-
ods reported previously in the docking studies of 1.17 Multiple low
energy binding poses (within 1 kcal/mol of the lowest energy)
were observed for 6 and can be accounted for by the large volume
of the colchicine binding site.28,30 The docked pose of 6 (Fig. 6) had
a score of ꢂ7.6342 kcal/mol compared to the docked pose of 1,17
which had a score of ꢂ6.0330 kcal/mol. Comparison of the docked
conformation of 6 with the crystal structure conformation of
DAMA colchicine showed an overlap of the 40-OCH3 phenyl group
of 6 with the trimethoxybenzene A-ring of DAMA colchicine, with
the 40-OCH3 moiety overlapping the 20-OCH3 of DAMA colchicine.
This conformation aids the formation of a hydrogen bond between

898
A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 891–902
Table 2
Human cancer cell growth inhibitory activity GI50 (nM) of 6 in NCI 60 cell line panel
Panel/cell line
GI50 (nM) compd 6
Panel/cell line
GI50 (nM) compd 6
Panel/cell line
GI50 (nM) compd 6
Panel/cell line
GI50 (nM) compd 6
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
NSCLC
A549/ATCC
EKVX
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
Melanoma
LOX IMVI
MALME-3 M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-62
Renal cancer
786–0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK10
UO-31
Prostate cancer
PC-3
DU-145
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
41.1
32.2
<10
47.7
41.1
<10
25.8
23.6
29.8
26.1
31.0
13.2
19.0
68.4
304
16.6
<10
19.7
<10
49.0
15.8
89.6
34.0
16.9
80.1
56.2
53.7
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
32.3
27.0
36.4
16.7
40.0
242
133
31.8
64.2
30.0
<10
37.7
26.0
24.2
50.9
12.7
37.3
Ovarian cancer
IGROVI
HOP-62
HOP-92
34.0
12.9
52.7
51.2
42.3
11.5
29.9
44.3
31.4
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
16.4
64.6
11.5
94.5
26.9
MDA-MB-468
Table 3
Compounds 6, 14 and 15 are effective in cell lines expressing Pgp or bIII-tubulin
Compd
Effect of Pgp on drug sensitivityb IC50 SD (nM)
Effect of bIII-tubulin on drug sensitivityc IC50 SD (nM)
SK-OV-3
SK-OV-3 MDR-1-6/6
Rra
HeLa
WT bIII
Rra
6
27.6 1.8
278 19
53.0 1.9
23.0 1.1
4.4 0.6
9.7 0.2
34.4 5.9
435 33
72.0 7.9
29.1 1.7
2596 119
11.5 1.7
1.2
21.3 2.2
270 26
32.8 0.8
23.1 0.8
1.6 0.5
4.7 1.1
21.4 3.5
186 21
45.5 1.0
18.8 0.5
9.2 0.2
5.2 0.4
1.0
0.7
1.4
0.8
5.8
1.1
1d
1.6
1.4
1.3
590
1.2
14
15
Paclitaxel
CA-4
a
Rr: relative resistance.
b
The SRB assay was used to evaluate the antiproliferative effects and determine the IC50 values of the compounds indicated. The SK-OV-3 MDR-1-6/6 cells were transduced
with the MDR-1 gene, and the sensitivity of this Pgp-expressing cell line was compared with the parental SK-OV-3 cells by dividing the IC50 of the Pgp overexpressing cell line
by the IC50 of the parental cell line, yielding the Rr value.
c
WT bIII cells were generated from HeLa cells by transfection with the bIII tubulin gene. The effects of the expression of bIII-tubulin on the sensitivity of the cell line pair
were evaluated by calculating the Rr, by dividing the IC50 of the WT bIII cell line by the IC50 obtained in the parental HeLa cells.
d
Results previously published.17 The values were obtained from 3–5 independent experiments each using triplicate or quadruplicate points.
Table 4
while a N4-C2H5 (14) or a 40-OC2H5 (15) maintains activity. This
study also provided an explanation of the importance of the N-
Inhibition of tubulin assembly and inhibition of colchicine binding
Compd
Inhibition of tubulin
Inhibition of colchicine binding
CH3 moiety of 6 for biological activity via molecular modeling.
Compound 6 is currently undergoing hollow fiber assay at the Na-
tional Cancer Institute, National Institutes of Health. Compound
6ꢀHCl is freely soluble in water and is a water-soluble version of
potent antitubulin agent 6. It is anticipated that this should cir-
cumvent the water solubility problems associated with paclitaxel
and other antimitotic agents. Hence, compound 6 serves as an
important lead compound for the development of further com-
pounds for preclinical evaluations as antimitotic agents that cir-
cumvent some of the major drawbacks of paclitaxel, such as
tumor resistance and lack of water solubility.
assembly IC50 SD (
l
M)
(%inhibition SD) at 5
lM
6
1.4 0.007
1.7 0.2
1.7 0.07
2.6 0.05
1.0 0.09
84 0.5
14
15
1
76
82
70
3
1
2
CA-4
99 0.2
The docked pose of 15 (Fig. 8) retains binding interactions seen
in the docked pose of 6 (Fig. 6) in the tubulin active site. The 40-OEt
substitution of 15 forms hydrophobic interactions with Vala318,
Ileb378, Cysb241 and Leub242 in the binding pocket. Comparison
of the docked poses of 6 and 15 indicates that the shorter 40-
OMe substitution of 6 does not interact with Ileb378. This addi-
tional hydrophobic interaction could account, in part, for the im-
proved activity of 15 compared to 6 and is also reflected in the
slight improvement in the docking score of 15 (ꢂ7.7512 kcal/
mol) compared to 6 (ꢂ7.6342 kcal/mol).
5. Experimental
Analytical samples were dried in vacuo (0.2 mm Hg) in a CHEM-
DRY drying apparatus over P2O5 at 50 °C. Melting points were
determined on a digital MEL-TEMP II melting point apparatus with
FLUKE 51 K/J electronic thermometer and are uncorrected. Nuclear
magnetic resonance spectra for proton (1H NMR) were recorded on
Bruker Avance II 400 (400 MHz) and 500 (500 MHz) NMR systems.
The chemical shift values are expressed in ppm (parts per million)
relative to tetramethylsilane as an internal standard: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; br, broad singlet.
Thin-layer chromatography (TLC) was performed on Whatman Sil
G/UV254 silica gel plates with a fluorescent indicator, and the
In conclusion, we report the synthesis and identification of no-
vel antimitotic agents containing the pyrimido[4,5-b]indole scaf-
fold, albeit with stringent substitution requirements. Tricyclic
compound 6 was several fold more potent against tumor cells in
culture than bicyclic 1, indicating that bulk in the form of the C-
ring and isosteric replacement of the 2-CH3 with 2-NH2 is favored.
The presence of a N4-CH3 and 40-OCH3 (6) is important for activity

A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 891–902
899
spots were visualized under 254 and 366 nm illumination. Propor-
5.4. 2,2-Dimethyl-N-(4-oxo-4,9-dihydro-3H-pyrimido[4,5-
b]indol-2-yl)propanamide (20)
tions of solvents used for TLC are by volume. Column chromatogra-
phy was performed on a 230ꢂ400 mesh silica gel (Fisher Scientific)
column. Elemental analyses were performed by Atlantic Microlab,
Inc., Norcross, GA. Elemental compositions are within 0.4% of the
calculated values and indicate >95% purity. Fractional moles of
water or organic solvents frequently found in some analytical sam-
ples could not be prevented despite 24ꢂ48 h of drying in vacuo
and were confirmed where possible by their presence in the 1H
NMR spectra. Mass spectrum data were acquired on an Agilent
G6220AA TOF LC/MS system using the nano ESI (Agilent chip tube
system with infusion chip). All solvents and chemicals were pur-
chased from Sigma–Aldrich Co. or Fisher Scientific Inc. and were
used as received.
Compound 19 (50 mg, 0.25 mmol), trimethyl acetic anhydride
(6 g, 32 mmol), DMAP (16 mg, 0.13 mmol), and triethylamine
(101 mg, 1 mmol) were dissolved in 8 mL of DMF. The mixture
was heated at 60 °C for 40 h. The DMF and trimethyl acetic anhy-
dride were removed under reduced pressure using an oil pump.
The residue thus obtained was dissolved in methanol, and 3 g of
silica gel was added to the solution. The solvent was removed un-
der reduced pressure to afford a dry plug. The plug was loaded on
top of a silica gel column in chloroform (the silica was twenty
times the weight of the plug), which was eluted with 1% methanol
in chloroform to afford 52 mg of 20 as a yellow solid in 73% yield.
TLC Rf 0.67 (chloroform/methanol 5:1); mp decomposes at 322 °C;
1H NMR (DMSO-d6) d 1.26 (s, 9H, C(CH3)3), 7.17–7.21 (m, 1H, Ar),
7.24–7.28 (m, 1H, Ar), 7.43–7.45 (m, 1H, Ar), 7.86–7.88 (m, 1H,
Ar), 11.12 (s, 1H, NH, exch), 11.85 (s, 1H, NH, exch), 12.03 (s, 1H,
NH, exch). Anal. Calcd for C15H16N4O2ꢀ0.3 H2O: C, 62.18; H, 5.77;
N, 19.33. Found: C, 62.24; H, 5.83; N, 18.98.
5.1. 2-Amino-6-hydrazinopyrimidin-4(3H)-one (17)
To a stirred suspension of 15.0 g (103 mmol) of 16 in 250 mL
water was added 12 g (375 mmol) of anhydrous hydrazine, and
the mixture was heated to reflux for 3 h. The resulting clear solu-
tion was cooled, and the precipitate that separated was collected
by filtration, washed with water followed by ethanol and dried
to give 6.6 g (46%) of 17 as a white solid. mp decomposes at
313 °C (Literature mp 314–315 °C).31
5.5. N-(4-Chloro-9H-pyrimido[4,5-b]indol-2-yl)-2,2-
dimethylpropanamide (21)
In
a 50 mL round bottom flask was placed 20 (56 mg,
5.2. 2-Amino-3,5,6,7,8,9-hexahydro-4H-pyrimido[4,5-b]indol-4-
one (18)
0.17 mmol) and phosphoryl trichloride (15 mL). The mixture was
stirred and heated at reflux for 4 h. The phosphoryl trichloride
was removed by evaporation under reduced pressure using a vac-
uum aspirator. The resulting residue was cooled in an ice and
water mixture and neutralized with ammonium hydroxide solu-
tion to yield a precipitate that was filtered and dried over P2O5.
The filtrate was extracted with chloroform and dried over sodium
sulfate. The dry precipitate and filtrate were combined and dried
under vacuum to provide 35 mg of 21 as a brown solid in 59% yield.
TLC Rf 0.65 (chloroform/methanol 10:1); mp 234 °C; 1H NMR
(DMSO-d6) d 1.24 (s, 9H, C(CH3)3), 7.34–7.38 (m, 1H, Ar), 7.53–
7.55 (m, 2H, Ar), 8.15–8.17 (m, 1H, Ar), 10.25 (s, 1H, 2-NH, exch),
12.55 (s, 1H, 9-NH, exch). Anal. Calcd for C15H15ClN4Oꢀ0.15 H2O:
C, 58.98; H, 5.04; N, 18.34; Cl, 11.60. Found: C, 59.00; H, 5.09; N,
17.97; Cl, 11.83.
A
mixture of 2-amino-6-hydrazinopyrimidin-4(3H)-one 17
(350 mg, 2.5 mmol) and cyclohexanone (245 mg, 2.5 mmol) in di-
phenyl ether (30 ml) was stirred and heated at 120–130 °C in an
oil bath overnight and then under reflux at 250 °C for 3 h. After
cooling to room temperature, hexane (50 mL) was added, and the
precipitated solid was collected by filtration. The solid was dried
over P2O5, dissolved in methanol, and silica gel (three times the
weight of solid) was added, following which the solvent was re-
moved under reduced pressure to obtain a dry plug. The plug
was loaded on top of a column packed with silica in chloroform.
The weight of silica in the column was 30 times the weight of
the plug. Flash chromatography using 20% methanol in chloroform
afforded 18 (82%) as a yellow solid. TLC Rf 0.35 (chloroform/meth-
anol 5:1); mp decomposes at 338 °C; 1H NMR (DMSO-d6) d 1.64–
1.69 (m, 4H, 6-CH2, 7-CH2), 2.42 (m, 2H, 5-CH2), 2.53 (m, 2H, 8-
CH2), 5.88 (s, 2H, 2-NH2, exch), 10.02 (s, 1H, 3-NH, exch), 10.50
(s, 1H, 9-NH, exch). Anal. Calcd for C10H12N4Oꢀ0.4 H2O: C, 56.80;
H, 6.10; N, 26.50. Found: C, 56.90; H, 6.06; N, 26.49.
5.6. General procedure for the synthesis of 3, 5, 7, 9, and 11
Compound 21 (1 equiv) was dissolved in isopropanol, and to
this solution was added a substituted aniline (7.5 equiv). Three
drops of concd HCl were added, and the resulting mixture was stir-
red and heated to reflux for 18–120 h, depending upon the aniline
used. For work up of this reaction mixture, the solvent was re-
moved under reduced pressure, the mixture was basified with
ammonia in methanol, dissolved in methanol, and silica gel was
added, and the solvent was removed by evaporation to yield a
dry plug. The plug was loaded on top of a silica gel column in chlo-
roform (the silica was 50 times the weight of the plug), and the col-
umn was eluted with 0.2% methanol in chloroform to obtain
compounds 3, 5, 7, 9, and 11 in yields of 37–80%.
5.3. 2-Amino-3,9-dihydro-4H-pyrimido[4,5-b]indol-4-one (19)
A mixture of 18 (50 mg, 0.24) and 10% Pd/C (24 mg) in Ph2O
(5 mL) was heated to reflux for 3 h. The reaction mixture was
cooled to room temperature, and DMF (20 mL) was added to dis-
solve the product. The catalyst was removed by filtration through
celite and washed with DMF to give a solution that was evaporated
to yield a solid residue. Hexane (25 mL) was added to the residue,
and the solid was filtered. The solid was dissolved in DMF (5 mL),
and silica gel (1 g) was added. Solvent was removed under reduced
pressure to afford a plug, which was loaded on top of a silica gel
column in chloroform (the silica was thirty times the weight of
the plug). The column was eluted with 20% methanol in chloroform
to afford 28 mg of 19 as a pale white solid in 57% yield. TLC Rf 0.32
(chloroform/methanol 5:1); mp >340 °C; 1H NMR (DMSO-d6) d
6.51 (s, 2H, NH2), 7.03–7.10 (m, 2H, Ar), 7.22–7.25 (m, 1H, Ar),
7.68–7.70 (m, 1H, Ar), 10.47 (s, 1H, 3-NH, exch), 11.35 (s, 1H, 9-
NH, exch). HRMS (ESI) Calcd for C10H8N4O: 200.0698. Found:
200.0693.
5.7. N-{4-[(3-Methoxyphenyl)amino]-9H-pyrimido[4,5-b]indol-
2-yl}-2,2-dimethylpropanamide (3)
Using the general procedure described above, the reaction of 21
(160 mg, 0.52 mmol) and 3-methoxyaniline 22 (488 mg,
3.96 mmol) was run for 36 h, to provide 107 mg of 3 as an off white
solid in 52% yield. TLC Rf 0.64 (chloroform/methanol 15:1); mp
219.4–220.2 °C; 1H NMR (DMSO-d6) d 1.23 (s, 9H, C(CH3)3), 3.81
(s, 3H, OCH3), 6.57–6.59 (m, 1H, Ar), 7.17–7.26 (m, 2H, Ar), 7.36–
7.38 (m, 1H, Ar), 7.45–7.47 (m, 1H, Ar), 7.51–7.53 (m, 1H, Ar), 7.9

900
A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 891–902
(s, 1H, Ar), 8.29–8.31 (m, 1H, Ar), 8.62 (s, 1H, 4-NH, exch), 9.59 (s,
1H, 2-NH, exch), 11.85 (s, 1H, 9-NH, exch). Anal. Calcd for
work up of this reaction mixture, the solvent was removed under
reduced pressure, and the residue was dried over P2O5. The dry res-
idue was dissolved in methanol, and silica gel was added. The sol-
vent was removed by evaporation to yield a dry plug. The plug was
loaded on top of a silica gel column in chloroform (the weight of
the silica was 15 times that of the plug), and the column was eluted
with 1% methanol in chloroform to yield compound 2, 4, 6, 8, or 10.
These compounds were washed with nonpolar solvents (hexane,
diethyl ether) and dried in vacuo. The yields ranged from 53 to 93%.
C
22H23N5O2ꢀ0.45 CH3OH: C, 66.76; H, 6.19; N, 17.34. Found: C,
66.95; H, 6.18; N, 16.96.
5.8. N-{4-[(4-Methoxyphenyl)amino]-9H-pyrimido[4,5-b]indol-
2-yl}-2,2-dimethylpropanamide (5)
Using the general procedure described above, the reaction of 21
(80 mg, 0.26 mmol) and 4-methoxyaniline 23 (244 mg, 1.98 mmol)
was run for 18 h, to provide 52 mg of 5 as a white solid in 51%
yield. TLC Rf 0.55 (chloroform/methanol 15:1); mp 266.8–267 °C;
1H NMR (DMSO-d6) d 1.23 (s, 9H, C(CH3)3), 3.75 (s, 3H, OCH3),
6.88–6.90 (d, 2H, Ar), 7.19–7.22 (m, 1H, Ar), 7.31–7.35 (m, 1H,
Ar), 7.43–7.44 (m, 1H, Ar), 7.90–7.93 (d, 2H, Ar), 8.26–8.28 (m,
1H, Ar), 8.55 (s, 1H, 4-NH, exch), 9.41 (s, 1H, 2-NH, exch), 11.79
(s, 1H, 9-NH, exch). Anal. Calcd for C22H23N5O2ꢀ0.4 H2O: C, 66.62;
H, 6.04; N, 17.65. Found: C, 66.61; H, 6.00; N, 17.37.
5.13. N4-(3-Methoxyphenyl)-9H-pyrimido[4,5-b]indole-2,4-
diamine (2)
Using the general procedure described above, the reaction of 3
(80 mg, 0.20 mmol) and 1 N NaOH provided 40 mg of 2 as an off-
white solid in 64% yield. TLC Rf 0.30 (chloroform/methanol 15:1);
mp 208.9–209.1 °C; 1H NMR (DMSO-d6) d 3.76 (s, 3H, OCH3),
6.19 (s, 2H, NH2, exch), 7.09–7.17 (m, 1H, Ar), 7.18–7.21 (m, 1H,
Ar), 7.28–7.29 (m, 2H, Ar), 7.41–7.42 (m, 1H, Ar), 7.48–7.49 (m,
2H, Ar), 8.03–8.05 (m, 1H, Ar), 8.26 (s, 1H, 4-NH, exch), 11.27 (s,
1H, 9-NH, exch). Anal. Calcd for C17H15N5Oꢀ0.35 CH3OH: C, 65.83;
H, 5.22; N, 22.12. Found: C, 66.08; H, 5.22; N, 21.81.
5.9. N-{4-[(4-Methoxyphenyl)(methyl)amino]-9H-pyrimido[4,5-
b]indol-2-yl}-2,2-dimethyl-propanamide (7)
Using the general procedure described above, the reaction of 21
(180 mg, 0.59 mmol) and 4-methoxy-N-methylaniline 24 (612 mg,
4.46 mmol) was run for 72 h, to provide 109 mg of 7 as brown crys-
tals in 45% yield. TLC Rf 0.62 (chloroform/methanol 15:1); mp
248.2–249 °C; 1H NMR (DMSO-d6) d 1.27 (s, 9H, C(CH3)3), 3.63 (s,
3H, OCH3), 3.75 (s, 3H, NCH3), 5.78–5.80 (m, 1H, Ar), 6.64–6.67
(m, 1H, Ar), 6.93–6.95 (m, 2H, Ar), 7.11–7.15 (m, 1H, Ar), 7.22–
7.31 (m, 3H, Ar), 9.52 (s, 1H, 2-NH, exch), 11.83 (s, 1H, 9-NH, exch).
Anal. Calcd for C23H25N5O2ꢀ0.55 CH3OH: C, 67.16; H, 6.51; N, 16.63.
Found: C, 67.33; H, 6.49; N, 16.28. HRMS (ESI) Calcd for
5.14. N4-(4-Methoxyphenyl)-9H-pyrimido[4,5-b]indole-2,4-
diamine (4)
Using the general procedure described above, the reaction of 5
(90 mg, 0.23 mmol) and 1 N NaOH provided 65 mg of 4 as a trans-
parent solid in 92% yield. TLC Rf 0.34 (chloroform/methanol 15:1);
mp 225.2–225.6 °C; 1H NMR (DMSO-d6) d 3.74 (s, 3H, OCH3), 6.05
(br s, 2H, NH2, exch), 6.88–6.90 (d, 2H, Ar), 7.05–7.09 (m, 1H, Ar),
7.13–7.17 (m, 1H, Ar), 7.26–7.28 (m, 1H, Ar), 7.62–7.64 (d, 2H,
Ar), 8.05–8.07 (m, 1H, Ar), 8.16 (s, 1H, 4-NH, exch), 11.20 (s, 1H,
9-NH, exch). Anal. Calcd for C17H15N5Oꢀ0.15 (C2H5)2O: C, 66.79;
H, 5.25; N, 22.13. Found: C, 66.77; H, 5.08; N, 22.13.
C23H25N5O2: 403.2008. Found: 403.1988.
5.10. N-{4-[(3-Bromophenyl)amino]-9H-pyrimido[4,5-b]indol-
2-yl}-2,2-dimethylpropanamide (9)
5.15. N4-(4-Methoxyphenyl)-N4-methyl-9H-pyrimido[4,5-
b]indole-2,4-diamine (6)
Using the general procedure described above, the reaction of 21
(181 mg, 0.59 mmol) and 3-bromoaniline 25 (771 mg, 4.48 mmol),
was run for 26 h, to provide 209 mg of 9 as a white solid in 80%
yield. TLC Rf 0.28 (chloroform/methanol 20:1); mp 286.7–
287.5 °C; 1H NMR (DMSO-d6) d 1.27 (s, 9H, C(CH3)3), 7.19–7.21
(m, 1H, Ar), 7.25–7.29 (m, 2H, Ar), 7.37–7.40 (m, 1H, Ar), 7.48–
7.49 (m, 1H, Ar), 8.18–8.20 (m, 1H, Ar), 8.37–8.39 (m, 1H, Ar),
8.45–8.46 (m, 1H, Ar), 8.79 (s, 1H, 4-NH, exch), 9.64 (s, 1H, 2-NH,
exch), 11.89 (s, 1H, 9-NH, exch). Anal. Calcd for C21H20BrN5O: C,
57.54; H, 4.59; N, 15.97; Br, 18.22; Found: C, 57.34; H, 4.65; N,
15.80; Br, 17.96.
Using the general procedure described above, the reaction of 7
(90 mg, 0.22 mmol) and 1 N NaOH provided 66 mg of 6 as a white
solid in 93% yield. TLC Rf 0.27 (chloroform/methanol 15:1); mp
245.3–245.7 °C; 1H NMR (DMSO-d6) d 3.48 (s, 3H, OCH3), 3.71 (s,
3H, NCH3), 5.75–5.77 (m, 1H, Ar), 6.20 (s, 2H, NH2, exch), 6.51–
6.55 (m, 1H, Ar), 6.87–6.90 (m, 1H, Ar), 6.95–6.97 (m, 1H, Ar),
7.12–7.15 (m, 4H, Ar), 11.22 (s, 1H, 9-NH, exch). Anal. Calcd for
C
18H17N5Oꢀ0.17 H2O: C, 67.05; H, 5.42; N, 21.72. Found: C, 67.06;
H, 5.37; N, 21.56.
5.16. N4-(3-Bromophenyl)-9H-pyrimido[4,5-b]indole-2,4-
5.11. N-{4-[(3-Methoxyphenyl)(methyl)amino]-9H-
diamine (8)
pyrimido[4,5-b]indol-2-yl}-2,2-dimethyl-propanamide (11)
Using the general procedure described above, the reaction of 9
(209 mg, 0.47 mmol) and 1 N NaOH provided 128 mg of 8 as a
white solid in 76% yield. TLC Rf 0.36 (chloroform/methanol 15:1);
mp 233.6 °C; 1H NMR (DMSO-d6) d 6.26 (br s, 2H, NH2, exch),
7.09–7.13 (m, 1H, Ar), 7.15–7.20 (m, 2H, Ar), 7.23–7.25 (m, 1H,
Ar), 7.28–7.30 (m, 1H, Ar), 7.94–7.96 (m, 1H, Ar), 8.01–8.02 (m,
1H, Ar), 8.08–8.09 (m, 1H, Ar), 8.44 (s, 1H, 4-NH, exch), 11.32 (s,
1H, 9-NH, exch). Anal. Calcd for C16H12BrN5: C, 54.25; H, 3.41; N,
19.77; Br, 22.55. Found: C, 54.38; H, 3.48; N, 19.56; Br, 22.29.
Using the general procedure described above, the reaction of 21
(180 mg, 0.59 mmol) and 3-methoxy-N-methylaniline 26 (612 mg,
4.46 mmol) was run for 120 h, to provide 88 mg of 11 as a brown
solid in 37% yield. TLC Rf 0.57 (chloroform/methanol 15:1); mp
288.5–289.4 °C; 1H NMR (DMSO-d6) d 1.27 (s, 9H, C(CH3)3,) 3.62
(s, 3H, OCH3), 3.71 (s, 3H, NCH3), 5.89–5.91 (m, 1H, Ar), 6.68–
6.81 (m, 2H, Ar), 6.96–7.34 (m, 5H, Ar), 9.60 (s, 1H, 2-NH, exch),
11.89 (s, 1H, 9-NH, exch). HRMS (ESI) Calcd for C23H25N5O2
(M+H)+: 404.2087. Found: 404.2080.
5.17. N4-(3-Methoxyphenyl)-N4-methyl-9H-pyrimido[4,5-
b]indole-2,4-diamine (10)
5.12. General procedure for the synthesis of 2, 4, 6, 8, and 10
Compounds 3, 5, 7, 9, and 11 were dissolved individually in iso-
propanol. About 4 mL of 1 N NaOH was added to this solution, and
the resulting mixture was stirred and heated to reflux for 14 h. For
Using the general procedure described above, the reaction of 11
(100 mg, 0.24 mmol) and 1 N NaOH provided 58 mg of 10 as a white

A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 891–902
901
solid in 53% yield. TLC Rf 0.34 (chloroform-methanol 15:1); mp
233.6 °C; 1H NMR (DMSO-d6) d 3.55 (s, 3H, OCH3), 3.58 (s, 3H,
NCH3), 5.87–5.89 (m, 1H, Ar), 6.17–6.18 (m, 1H, Ar), 6.27 (br s, 2H,
NH2, exch), 6.55–6.59 (m, 1H, Ar), 6.67–6.74 (m, 2H, Ar), 6.96–7.03
(m, 1H, Ar), 7.14–7.22 (m, 1H, Ar), 11.27 (s, 1H, 9-NH, exch). HRMS
(ESI) Calcd for C18H17N5O (M+H)+: 320.1503. Found: 320.1511.
CH2), 5.66–5.68 (d, 1H, Ar), 6.15 (s, 2H, 2-NH2, exch), 6.50–6.54 (t,
1H, Ar), 6.90–6.97 (m, 3H, Ar), 7.12–7.16 (m, 3H, Ar), 11.20 (s, 1H,
9-NH, exch). Anal. Calcd for C19H19N5Oꢀ0.90 CH3OH: C, 65.98; H,
6.29; N, 19.33. Found: C, 65.81; H, 6.15; N, 19.31.
5.23. N4-(4-Ethoxyphenyl)-N4-methyl-9H-pyrimido[4,5-
b]indole-2,4-diamine (15)
5.18. 4-((4-Methoxyphenyl)(methyl)amino)-9H-pyrimido[4,5-
b]indol-2-aminium chloride (6ꢀHCl)
In a round bottom flask, compound 21 (80 mg, 0.26 mmol), 4-
ethoxy-N-methylaniline 30 (120 mg, 0.78 mmol) and 1 drop of
concd HCl were heated to reflux in 30 mL isopropanol for 120 h.
After cooling to rt, silica gel (500 mg) was added, and isopropanol
was removed under reduced pressure. Purification was performed
by column chromatography using 0.5% methanol in chloroform to
provide 63 mg of 15 as a white solid in 71% yield. TLC Rf 0.43 (chlo-
roform/methanol 15:1); mp 238.8–239.6 °C; 1H NMR (DMSO-d6) d
1.29–1.32 (t, 3H, J = 6.9 Hz, CH3), 3.50 (s, 3H, N4-CH3), 3.97–4.02 (q,
2H, J = 6.9 Hz, CH2), 5.76–5.78 (d, 1H, Ar), 6.22 (s, 2H, 2-NH2, exch),
6.53–6.57 (t, 1H, Ar), 6.88–6.90 (m, 2H, Ar), 6.95–6.98 (t, 1H, Ar),
7.13–7.15 (m, 3H, Ar), 11.24 (s, 1H, 9-NH, exch). Anal. Calcd for
Compound 6 (55 mg) was dissolved in chloroform (0.5 mL), ethyl
acetate (2 mL) and anhydrous ether (20 mL), and then HCl gas was
bubbled into the solution till precipitation ceased. The white solid
was collected by filtration and dried over P2O5 to afford 6ꢀHCl in
70% yield. TLC Rf 0.27 (chloroform/methanol 15:1 and 4 drops of
NH4OH); mp >250 °C; 1H NMR (DMSO-d6) d 3.66 (s, 3H, OCH3),
3.79 (s, 3H, NCH3), 5.50–5.52 (m, 1H, Ar), 6.65–6.69 (m, 1H, Ar),
7.00–7.02 (m, 2H, Ar), 7.10–7.13 (m, 1H, Ar), 7.33–7.35 (m, 1H,
Ar), 7.41–7.43 (m, 2H, Ar), 7.74 (br s, 3H, NH3+, exch), 12.36 (s, 1H,
9-NH, exch). Anal. Calcd for C18H18ClN5O: C, 60.76; H, 5.10; N,
19.68; Cl, 9.96. Found: C, 60.55; H, 5.14; N, 19.41; Cl, 9.75.
C19H19N5O: C, 68.45; H, 5.74; N, 21.01. Found: C, 68.40; H, 5.61;
N, 21.01.
5.19. General procedure for the synthesis of 12–14
5.24. N-Ethyl-4-methoxyaniline (29)
To a round bottom flask were added 21, the appropriate substi-
tuted aniline, 1–3 drops of concentrated HCl and 50 mL of n-buta-
nol. The reaction mixture was heated under reflux. After cooling to
rt, n-butanol was removed under reduced pressure, and purifica-
tion was performed by column chromatography using 1% metha-
nol in chloroform as the eluant.
4-Methoxyaniline 31 (246.3 mg, 2.0 mmol), triethylamine
(303.57 mg, 0.42 mL, 3 mmol), 10% Pd/C (32 mg, 0.03 mmol,
1.5 mol % relative to 31), and dry toluene (2.5 mL) were added to
an argon-filled 2–5 mL biotage microwave vial. The vial was sealed,
and the reaction was run in a microwave at 175 °C for 1.5 h. The
reaction vessel was cooled to room temperature, and the palladium
catalyst was removed by filtration through celite and washed with
ethyl acetate. Silica gel (1 g) was added, and the solvent was re-
moved under reduced pressure. Purification was performed by col-
umn chromatography with hexane/ethyl acetate (95/5) to afford N-
ethyl-4-methoxyaniline 29 as a pale yellow liquid (211.69 mg,
70%). The analytical values agree well with the literature.19 TLC
Rf 0.61 (hexane/ethyl acetate 1:1); 1H NMR (CDCl3) d 1.26–1.29
(t, 3H, J = 7.1 Hz, CH3), 3.12–3.17 (q, 2H, J = 7.1 Hz, N-CH2), 3.79
(s, 3H, 4-OCH3), 6.62–6.64 (m, 2H, Ar), 6.82–6.84 (m, 2H, Ar).
5.20. N4-(3,4,5-Trimethoxyphenyl)-9H-pyrimido[4,5-b]indole-
2,4-diamine (12)
Using the general procedure described above, the reaction of 21
(100 mg, 0.33 mmol) with 3,4,5-trimethoxyaniline 27 (302 mg,
1.65 mmol) and 3 drops of concd HCl for 6 h afforded 12 in 79%
yield. TLC Rf 0.13 (chloroform/methanol 15:1 with 2 drops concen-
trated NH4OH); mp 248–248.7 °C; 1H NMR (DMSO-d6) d 3.64 (s,
3H, 4-OCH3), 3.80 (s, 6H, 3-OCH3, 5-OCH3), 6.23 (br s, 2H, NH2,
exch), 7.11–7.21 (m, 2H, Ar), 7.28–7.31 (m, 2H, Ar), 8.08–8.10 (m,
1H, Ar), 8.15 (s, 1H, 4-NH, exch), 11.29 (s, 1H, 9-NH, exch). HRMS
(ESI) Calcd for C19H19N5O3 (M+H)+: 365.1561. Found: 365.1543.
5.25. 4-Ethoxy-N-methylaniline (30)
To a solution of sodium methoxide (540.2 mg, 10 mmol) in 5 mL
of methanol in a 25 mL round bottom flask was added 4-ethoxyan-
iline 32 (274.36 mg, 2 mmol). The resultant solution was then
added to a 100 mL round bottom flask containing paraformalde-
hyde (84.08 mg, 2.8 mmol) in 5 mL of methanol. The reaction mix-
ture was stirred at room temperature under an argon atmosphere.
After 5 h, sodium borohydride (121.02 mg, 2 mmol) was added,
and the reaction mixture was heated to reflux for 2 h. After cooling
to rt, silica gel (1 g) was added, and the solvent was removed in va-
cuo. The resultant plug was purified using column chromatography
with hexane/ethyl acetate (95/5) to afford 4-ethoxy-N-methylani-
line 30 as a light brown liquid (227.6 mg, 75%). The analytical val-
ues agree well with the literature.20 TLC Rf 0.6 (hexane/ethyl
acetate 1:1); 1H NMR (CDCl3) d 1.43–1.46 (t, 3H, J = 7.0 Hz, CH3),
2.82 (s, 3H, N-CH3), 3.56 (s, 1H, NH), 3.99–4.04 (q, 2H, J = 7.0 Hz,
O-CH2), 6.61–6.63 (m, 2H, Ar), 6.86–6.88 (m, 2H, Ar).
5.21. N4-Methyl-N4-(4-methylphenyl)-9H-pyrimido[4,5-
b]indole-2,4-diamine (13)
Using the general procedure described above, the reaction of 21
(100 mg, 0.33 mmol) with N,4-dimethylaniline 28 (300 mg,
2.47 mmol) and 3 drops of concd HCl for 18 h afforded 13 in 71%
yield. TLC Rf 0.12 (chloroform/methanol 15:1 with 2 drops concen-
trated NH4OH); mp 252.7–253.5 °C; 1H NMR (DMSO-d6) d 2.27 (s,
3H, CH3), 3.53 (s, 3H, NCH3), 5.79–5.81 (m, 1H, Ar), 6.25 (br s,
2H, NH2, exch), 6.52–6.56 (m, 1H, Ar), 6.95–6.99 (m, 1H, Ar),
7.07–7.17 (m, 5H, Ar), 11.26 (s, 1H, 9-NH, exch). HRMS (ESI) Calcd
for C18H17N5 (M+H)+: 304.1557. Found: 304.1539.
5.22. N4-Ethyl-N4-(4-methoxyphenyl)-9H-pyrimido[4,5-
b]indole-2,4-diamine (14)
6. Cellular studies
6.1. Cell culture
Using the general procedure described above, the reaction of 21
(80 mg, 0.26 mmol) with N-ethyl-4-methoxyaniline 29 (120 mg,
0.78 mmol) and 1 drop of concd HCl for 72 h afforded 30 mg of
14 as a white solid in 31% yield. TLC Rf 0.50 (chloroform/methanol
15:1); mp 223.9–224.6 °C; 1H NMR (DMSO-d6) d 1.15–1.19 (t, 3 H,
J = 6.9 Hz, CH3), 3.74 (s, 3H, O-CH3), 4.09–4.13 (q, 2 H, J = 6.9 Hz, N4-
A-10, SK-OV-3 and HeLa cells were purchased directly from the
American Type Culture Collection (Manassas, VA). A-10 cells are rat

902
A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 891–902
aortic smooth muscle cells, SK-OV-3 human ovarian carcinoma
cells and HeLa, human cervical carcinoma cells. MDA-MB-435 hu-
man melanoma cells were obtained from the Lombardi Cancer
Center, Georgetown University. The SK-OV-3 MDR-1-6/6 and HeLa
WTbIII cell lines have been previously described.23
S.L.M.) and the Duquesne University Adrian Van Kaam Chair in
Scholarly Excellence (A.G.). We acknowledge support from the
CTRC Cancer Center Support Grant, CCSG (CA054174) (S.L.M.).
Supplementary data
6.2. Evaluation of cellular microtubule effects
Supplementary data (results from elemental analysis and High
Resolution Mass Spectra (HRMS)) associated with this article can
be found, in the online version, at http://dx.doi.org/10.1016/
j.bmc.2012.12.010.
The effects of the compounds on microtubules were evaluated
in A-10 cells using a b-tubulin antibody and indirect immunofluo-
rescence techniques. The EC50, the concentration required to cause
50% loss of interphase microtubules was calculated from at least 3
independent experiments.32 The EC50 for each experiment was cal-
culated and the numbers represent the mean SD.
References and notes
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6.3. Antiproliferative effects
The sulforhodamine B (SRB) assay21,22 was used to measure the
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pipeline/zybrestat_in_oncology/; Combretastatin A4 phosphate in treating
patients with advanced solid tumors, http://clinicaltrials.gov/ct2/show/
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cell lung cancer (FALCON), http://clinicaltrials.gov/ct2/show/
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8. Clinical trials: A phase I clinical trial of OXi4503 for relapsed and refractory
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CA1P&rank=1.
Inhibition of tubulin polymerization was measured turbidimet-
rically by absorbance at 340 nm. The assays utilized 2.5 mg/mL
purified porcine brain tubulin incubated with tubulin assembly
buffer (80 mM Na-Pipes, pH 6.9, 1 mM EGTA, 1 mM MgCl2, 1 mM
GTP and 10% glycerol) with vehicle or compounds in a final volume
of 100 lL. Tubulin polymerization was monitored at 37 °C in a
Spectromax Plus 96 well plate spectrophotometer (Molecular
Devices).
A safety and
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as propidium iodide intensity versus the number of events.
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6.6. Reagents
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Paclitaxel was purchased from Sigma–Aldrich (St. Louis, MO)
and CA-4 used in the cellular studies was synthesized by Dr. Doug
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20. Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D. J.
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6.7. Quantitative tubulin studies
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24. We thank the NCI for providing the tumor cell growth inhibitory activity for 6
in the NCI 60 cell line panel.
25. Hamel, E.; Lin, C. M. Biochemistry 1984, 23, 4173.
The purification of electrophoretically homogeneous tubulin
from bovine brains was described previously.25 Inhibition of
assembly was performed as described in detail previously,26 using
10 lM (1.0 mg/mL) tubulin and various compound concentrations.
The GTP (0.4 mM) was added to reaction mixtures following a
15 min preincubation at 30 °C. The parameter measured to obtain
IC50 values was the extent of assembly after 20 min at 30 °C. Mea-
surement of inhibition of colchicine binding was measured as de-
scribed in detail previously.27 Reaction mixtures contained
26. Hamel, E. Cell Biochem. Biophys. 2003, 38, 1.
27. Verdier-Pinard, P.; Lai, J.-Y.; Yoo, H.-D.; Yu, J.; Marquez, B.; Nagle, D. G.; Nambu,
M.; White, J. D.; Falck, J. R.; Gerwick, W. H.; Day, B. W.; Hamel, E. Mol.
Pharmacol. 1998, 53, 62.
28. Ravelli, R. B. G.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar, S.; Sobel, A.;
Knossow, M. Nature 2004, 428, 198.
29. Molecular Operating Environment (MOE 2008.10); Chemical Computing
Group, Inc.: Montreal, Quebec, Canada, 2008; www.chemcomp.com.
30. Nguyen, T. L.; McGrath, C.; Hermone, A. R.; Burnett, J. C.; Zaharevitz, D. W.; Day,
B. W.; Wipf, P.; Hamel, E.; Gussio, R. J. Med. Chem. 2005, 48, 6107.
31. Taylor, E. C.; Cocuzza, A. J. J. Org. Chem. 1979, 44, 1125–1128.
32. Lee, L.; Robb, L. M.; Lee, M.; Davis, R.; Mackay, H.; Chavda, S.; Babu, B.; O’Brien,
E. L.; Risinger, A. L.; Mooberry, S. L.; Lee, M. J. Med. Chem. 2010, 53, 325.
33. Krishan, A. J. Cell Biol. 1975, 66, 188–193.
1.0
lM tubulin, 5.0
l
M [3H]colchicine, obtained from Perkin–El-
mer, and 5.0
lM inhibitor, with incubation for 10 min at 37 °C.
CA-4 was generously provided by Dr. G. A. Pettit of Arizona State
University, Tempe, AZ.
Acknowledgments
This work was supported, in part, by the National Institutes of
Health and National Cancer Institute Grant CA142868 (A.G. and