Single agents with designed combination chemotherapy potential: Synthesis and evaluation of substituted pyrimido[4,5-b]indoles as receptor tyrosine kinase and thymidylate synthase inhibitors and as antitumor agents

Article abstract of DOI:10.1021/jm9011142

Combinations of antiangiogenic agents (AAs) with cytotoxic agents have shown significant promise in cancer treatment, and several such clinical trials are currently underway. We have designed, synthesized, and evaluated two compounds that each inhibit vascular endothelial growth, factor receptor-2 (VEGFR-2) and platelet-derived growth, factor receptor-β (PDGFR-β) for antiangiogenic effects and also inhibit human thymidylate synthase (hTS) for cytotoxic effects in single agents. The synthesis of these compounds involved, the nucleophilic displacement of the common intermediate 5-chloro-9H-pyrimido[4, 5-b]indole-2,4-diamine with appropriate benzenethiols. The inhibitory potency of both these single agents against VEGFR-2, PDGFR-β, and hTS is better than or close to standards. In a. COLO-205 xenograft mouse model, one of the analogs significantly decreased tumor growth (tumor growth inhibition (TGI) = 76% at 35 mg/kg), liver metastases, and tumor blood vessels compared with a standard drug and with control and thus demonstrated potent tumor growth inhibition, inhibition of metastasis, and antiangiogenic effects in vivo. These compounds afford combination chemotherapeutic potential in single agents.

Full text of DOI:10.1021/jm9011142

J. Med. Chem. 2010, 53, 1563–1578 1563
DOI: 10.1021/jm9011142
Single Agents with Designed Combination Chemotherapy Potential: Synthesis and Evaluation
of Substituted Pyrimido[4,5-b]indoles as Receptor Tyrosine Kinase and Thymidylate Synthase
Inhibitors and as Antitumor Agents
Aleem Gangjee,*,† Nilesh Zaware,† Sudhir Raghavan,† Michael Ihnat,‡ Satyendra Shenoy,‡ and Roy L. Kisliuk§
†Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh,
Pennsylvania 15282, ‡Department of Cell Biology, The University of Oklahoma Health Science Center, Oklahoma City, Oklahoma
73104, and §Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111
Received July 27, 2009
Combinations of antiangiogenic agents (AAs) with cytotoxic agents have shown significant promise in
cancer treatment, and several such clinical trials are currently underway. We have designed, synthesized,
and evaluated two compounds that each inhibit vascular endothelial growth factor receptor-2
(VEGFR-2) and platelet-derived growth factor receptor-β (PDGFR-β) for antiangiogenic effects and
also inhibit human thymidylate synthase (hTS) for cytotoxic effects in single agents. The synthesis of
these compounds involved the nucleophilic displacement of the common intermediate 5-chloro-9H-
pyrimido[4,5-b]indole-2,4-diamine with appropriate benzenethiols. The inhibitory potency of both
these single agents against VEGFR-2, PDGFR-β, and hTS is better than or close to standards. In a
COLO-205 xenograft mouse model, one of the analogs significantly decreased tumor growth (tumor
growth inhibition (TGI) = 76% at 35 mg/kg), liver metastases, and tumor blood vessels compared with
a standard drug and with control and thus demonstrated potent tumor growth inhibition, inhibition of
metastasis, and antiangiogenic effects in vivo. These compounds afford combination chemotherapeutic
potential in single agents.
Antiangiogenic agents (AA)a have established a new para-
digm in cancer chemotherapy and have allowed significant
progress toward the control and treatment of various can-
cers.1 Normal cell angiogenesis is initiated under conditions of
injured or hypoxic tissues, occurs in wound healing, menstrual
cycle and pregnancy, and is promoted by vascular endothelial
growth factor (VEGF) and similar growth factors. Except for
these conditions, angiogenesis is mostly absent in normal
tissues.2 Heterogeneous solid growing tumors however are
in a state of angiogenesis and continually overexpress growth
factors.
receptor (EGFR), platelet-derived growth factor receptor
(PDGFR), and vascular endothelial growth factor receptor
(VEGFR) families of receptors are all RTKs and, despite their
diverse biological roles, share similarities in structure and
domain arrangement.
Clinically useful RTK inhibitors have resulted from small
molecule ATP competitive inhibitors (Figure 1) that target the
kinase domain of RTKs. While RTK inhibitors have afforded
a new mechanism for the treatment of a variety of cancers, it
was quickly realized that single RTK inhibitors allowed the
development of resistance by point mutations in the ATP
binding site. In addition, the molecular pathways responsible
for tumor growth, survival, and metastasis are redundant and
adaptable between individual patients and within tumors in
the same patient. Thus treatment targeting a single RTK
would be highly unlikely to provide long-term tumor control
in most patients. Proangiogenic growth factors are redun-
dantly expressed by both tumor and stromal cells.5 Thus there
is now a paradigm shift in the utility of RTK inhibitors for
cancer treatment in that rather than single RTK inhibition it is
now desirable to use multitargeted RTK inhibitors to over-
come possible resistance and thwart the escape mechanism of
alternative pathways for tumor growth. Recent FDA ap-
provals of just such multitargeted RTK inhibitors sorafenib
(VEGFR-2, PDGFR-β, FMS-like tyrosine kinase 3 (Flt-3),
raf kinase, and stem cell factor receptor (c-kit)) and sunitinib
(VEGFR-1, -2, and -3, PDGFR-β and -R, Flt-3, and colony
stimulating factor 1 receptor (CSF-1R)) attest to the impor-
tance of this new paradigm in cancer chemotherapy.3-12
Receptor tyrosine kinases (RTKs) are a subclass of
cell surface growth factor receptors with intrinsic, ligand-
controlledtyrosinekinase activity.3,4 Epidermalgrowthfactor
*To whom correspondence should be addressed. Telephone: 412-396-
6070. Fax: 412-396-5593. E-mail address: gangjee@duq.edu.
aAbbreviations: AA, antiangiogenic agents; VEGF, vascular endo-
thelial growth factor; RTK, receptor tyrosine kinases; EGFR, epidermal
growth factor receptor; PDGFR, platelet-derived growth factor recep-
tor; abl, abelson proto-oncogene; c-kit, stem cell factor receptor; CSF-
1R, colony stimulating factor 1 receptor; Flt-3, FMS-like tyrosine kinase
3; ErbB-2, human epidermal growth factor receptor 2; hTS, human
thymidylate synthase; hDHFR, human dihydrofolate reductase;
GARFT, glycinamide-ribonucleotide formyl transferase; AICARFT,
aminoimidazole-4-carboxamide-ribonucleotide formyl transferase; 5-FU,
5-fluorouracil; PMX, pemetrexed; RTX, raltitrexed; MTX, methotrexate;
TMQ, trimetrexate; PDB, protein data bank; DMSO2, methyl sulfone;
DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; POCl3,
phosphorus oxychloride; NMP, N-methyl-2-pyrrolidone; PI3K, phospha-
tidylinositol 3-kinase; CAM, chorioallantoic membrane; TGI, tumor
growth inhibition; MTD, maximum tolerated dose; ADR, adverse drug
reaction.
r
2010 American Chemical Society
Published on Web 01/21/2010
pubs.acs.org/jmc

1564 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Gangjee et al.
Figure 1. Single and multitargeted RTK inhibitors.
In addition, vandetanib is a VEGFR-2 inhibitor that com-
bines EGFR (rather than PDGFR-β) inhibitory activity, and
lapatinib also combines EGFR and human epidermal growth
factor receptor-2 (ErbB-2) inhibitory activity. All of these
multitargeting RTK inhibitors have some monotherapy po-
tential but are mostly cytostatic (some cytotoxicity has been
observed via apoptosis in a limited number of tumor types);
thus for optimal benefit they must be combined with cyto-
toxic, conventional chemotherapeutic agents, radiation ther-
apy, or both.13 The rationale and mechanisms for the success
of these combinations continues to provide discussion in the
literature.13,14 Hundreds of such ongoing clinical trials
(clinicaltrials.gov) attest to the importance of the concept
and the individual drugs, both cytostatic and cytotoxic, in
the combinations.
Combination therapy with VEGFR-2 (endothelial cell
inhibition) along with PDGFR-β inhibition (pericytes in-
hibition) increases the antiangiogenic effect even in the often
intractable late state of solid tumors.15,16 Thus targeting both
VEGFR-2 and PDGFR-β simultaneously is a desirable goal
for AAs that have cytostatic and perhaps cytotoxic activity.
The clinical success of sunitinib9,11 and sorafenib11 that target
both VEGFR and PDGFR attests to the viability of this
combination in single agent multi-RTK inhibitors. Preclinical
and clinical evidence indicates that combinations of AAs with
conventional cytotoxic agents, radiation therapy, or both
results in additive or even synergistic antitumor effects15-17
and that monotherapy is usually unsuccessful with antiangio-
genic agents. Rapid vascular regrowth in tumors after the
removal of AAs attests to their cytostatic mechanism.18,19
Combination cancer chemotherapy is not a new idea.
Recent studies indicate that the combination of antiangio-
genic agents with cytotoxic agents is more effective in cancer
treatment.20 However, what would be novel is if a single agent
could be found that had both antiangiogenic activity by
multitargeting RTKs and also possessed cytotoxic activity
to afford combination chemotherapy in a single agent. We21
synthesized such agents with VEGFR-2, PDGFR-β, and
dihydrofolatereductase (DHFR) inhibitionwithgood results.
However, the potency of the cytotoxic component in these
agents was extremely low.21 Thus we elected to structurally
engineer multi-RTK inhibitory attributes along with the
deliberate design of cytotoxicity in single agents as a second
iteration of our previous report. Such single agents would
circumvent the pharmacokinetic problems of multiple agents,
would avoid drug-drug interactions, could be used at lower
doses to alleviate toxicity, could be devoid of overlapping
toxicities, and could delay or prevent tumor cell resistance.
Most importantly providing the cytotoxic agent, by structural
design, in the same molecule allows the cytotoxicity to be
mainfested as soon as the antiangiogenic effects are operable.
A separately dosed cytotoxic agent may miss the timing
window and hence preclude the intent of the combination.
Such multitargeted agents could exert their cytotoxic action as
soon as or even during transient tumor vasculature normal-
ization13,22 due to the antiangiogenic effects. Thus such agents
might not need to be as potent as conventional separately
dosed cytotoxic agents. Dosing of such an antiangiogenic
multitargeted RTK inhibitor with a built-in cytotoxic me-
chanism would be tantamount to providing a combination of
multitargeted RTK inhibitors along with a metronomic dos-
ing of a cytotoxic agent. Thus these single agents would be in
keeping with the two important mechanisms that explain the
rationale for the success of separate antiangiogenic and
cytotoxic chemotherapeutic agents in combination for cancer
chemotherapy.13,22-25 Other advantages of such single agents
are in the reduced cost and increased patient compliance,
which are sometimes as significant contributors to chemother-
apy failure as resistance, toxicity, and lack of efficacy.
The antiangiogenic component is usually targeted to tumor
cells and is, under most circumstances, not targeted to normal
cells. However, the cytotoxic component is targeted to the
tumor cells but not exclusively. Thus the challenge in design-
ing single agents with a cytotoxic component is that the
cytotoxic component should be potent enough to kill tumor
cells that have been compromised via the antiangiogenic effect
but not potent enough to cause serious toxicity to normal cells
not affected by the antiangiogenic effect. Clearly one of the
problems with conventional cytotoxic chemotherapeutic
agents is dose-limiting toxicities. These single agents should
avoid these toxicities since they do not need to be as potent as
conventional chemotherapeutic agents.

Article
We have designed and synthesized compounds 1 and 2
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1565
TS inhibitor antimetabolites (Figure 3) that are clinically used
alone or in combination in a variety of cancers include PMX37
and in Europe raltitrexed (RTX) a derivative of PDDF. In
addition, 3 (Figure 3), also a TS inhibitor, and its derivatives
are in various stages of clinical development alone and in
combinations against a variety of cancers.38
(Figure 2), which each target VEGFR-2 and PDGFR-β and
provide antiangiogenic effects in vivo and also have human
thymidylate synthase (hTS) inhibitory activity to afford the
cytotoxic component in vivo in single agents. The choice of
VEGFR-226 and PDGFR-β27 inhibition for antiangiogenic
activity was obvious since these are the principal direct and
indirect mediators of angiogenesis. The choice of inhibition of
human dihydrofolate reductase (hDHFR) or hTS as the
cytotoxic component was based in part on our long-standing
interest in inhibitors of folate metabolizing enzymes and on
structural, architectural, and molecular modeling considera-
tions in the design of the molecules. In addition, the successful
clinical and preclinical combinations of capecitibine,28,29
5-FU30 (TS inhibitors) and pemetrexed (PMX)31,32 (TS and
DHFR inhibitor) (Figure 3) withAAs in combinationtherapy
with and without radiation was also an important factor in
selecting TS or DHFR as the possible cytotoxic targets in the
design of 1 and 2.
DHFR carries out the reduction of dihydrofolate to tetra-
hydrofolate and maintains the pool of reduced folate cofac-
tors that function in one carbon transfers in a variety of
metabolic transformations crucial for cell survival.33 Thus
inhibitors of DHFR such as methotrexate (MTX) and PMX
(Figure 3) have found utility as antitumor agents. TS carries
out the sole de novo biosynthesis of TMP from dUMP. It
utilizes 5,10-methylenetetrahydrofolate as a cofactor to trans-
fer the methyl group to dUMP.34 Because of its pivotal role in
DNA synthesis and cell growth, it is a viable target for several
clinically used cancer chemotherapeutic agents.35 The fluoro-
pyrimidine 5-FU and its derivatives, in particular, capecita-
bine (Figure 3), have found extensive utility in ovarian, breast,
colon, and several other cancers alone and in combinations
and are a mainstay in cancer chemotherapy.36 Folate-based
Tricyclic scaffolds reported in the literature with appro-
priate 4-anilino substitutions have shown excellent RTK
inhibition.39,40 An example of such an analog is presented in
Figure 4A.39 In a general RTK model, it was shown41-44 that
the 2-NH2, the N3, and the 4-anilino nitrogen of the pyrimi-
dine ring of RTK inhibitors H-bond with the hinge region as
depicted in Figure 4A. In addition, the phenyl ring of the
4-anilino moiety lies in the hydrophobic region 1, and the
tricyclic scaffold mimics the purine ring of ATP.39,40 We
reasoned that transposing the phenyl ring from the 4-position
(Figure 4A) to the 5-position of the tricyclic scaffold as shown
in Figure 4B maintains access to hydrophobic region 1 and
allows the H-bonds with the hinge region. Such compounds
with phenyl substitutions in the 5-position should maintain
RTK inhibitory activity. However, moving the phenyl ring
from the 4- to the 5-position unveils a 2,4-diaminopyrimidine
motif on the tricyclic scaffold (Figure 4B) that is highly
conducive for DHFR or TS inhibition or both.45,46 MTX
and trimetrexate (TMQ) (Figure 3) are well-known 2,4-dia-
mino fused pyrimidines withhDHFR inhibition andforMTX
hTS inhibitory activity as well.47 Usually DHFR and TS
inhibitors have a 2,4-diamino and a 2-amino-4-oxo substitu-
tion on the pyrimidine ring, respectively. There are however
reports where analogs with a 2,4-diamino substitution have
TS and DHFR inhibitory activity45,46 while a 2-amino-4-oxo
substitution provides DHFR and TS inhibitory activity.48,49
Thus we designed 2,4-diamino-5-thio phenyl substituted
pyrimido[4,5-b]indoles as potential RTK inhibitors with
DHFR or TS inhibitory attributes or both.
Molecular modeling with hDHFR confirmed that the third
ring of the tricyclic scaffold could afford additional interac-
tion with Ile60 of hDHFR50 over the previously synthesized
bicyclic analogs.21 Modeling (Figure 5) using SYBYL7.351
and superimposition of one of the energy-minimized confor-
mations of 1 with the pyrimidine ring of 1 on the X-ray crystal
structure of the pyrimidine ring of the pyrido[2,3-d]pyrimidine
inhibitor (not shown) (1PDB)50 in hDHFR shows that
the 2,4-diamino motif is maintained for hDHFR inhibitory
Figure 2. Target compounds 1 and 2.
Figure 3. Dihydrofolate reductase and thymidylate synthase inhibitors.

1566 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Gangjee et al.
Figure 4. Rationale for the synthesis of target molecules.
Figure 5. Stereoview (SYBYL7.3) of the pyrimidine ring of 1 superimposed on the pyrimidine ring of the DHFR inhibitor in the X-ray crystal
structure of hDHFR (1PDB).50
activity as is the S-Ph, which mimics the phenyl ring of
MTX52,53 and TMQ.50 The 2NH2 and N1 make a salt bridge
with Glu30 in hDHFR and the S-Ph ring makes hydrophobic
contact with Phe31 and Val115 much like TMQ and MTX and
the third ring of the tricyclic scaffold contacts Ile60. The
conformation of the phenyl ring of 1 in Figure 5 is not the usual
conformation of this ring in other reported hDHFR inhibitors.
With this exception all other interactions of the scaffold of 1
were similar to the pteridine of MTX and the quinazoline of
TMQ. Thus we anticipated DHFR inhibitory activity for 1.
For TS inhibitory activity, modeling 1 by superimposing
the pyrimidine ring of the energy minimized 1 (yellow) onto
the pyrimidine ring of the X-ray crystal structure of PMX
(green) in hTS (PDB 1JU6),54 Figure 6 (SYBYL 7.3), showed
that the superimposition is perfect for the two scaffolds. In the
molecular modeling, the hydrophobic interactions of the
tricyclic C-ring of 1 with Trp109 (bottom right) are somewhat
better than those of the bicyclic B-ring of PMX. In addi-
tion, the S-phenyl ring of 1 and its close proximity to the
benzoyl ring of PMX (green) is evident, as are the hydro-
phobic interactions with Ile108, Leu221, and Phe225 (top
center) (and Met311 not shown). Also evident is the stacking
interaction of 1 and dUMP (directly below 1). Thus on the
basis of molecular modeling, we anticipated compound 1 to
have DHFR or TS inhibitory activity or both.
In Figure 7, energy-minimized 1 was superimposed
(SYBYL7.3) onto a furo[2,3-d]pyrimidine55 inhibitor (not
shown) of VEGFR-2 in the X-ray crystal structure of
VEGFR-2 (PDB 1YWN)55 that indicated the hinge region
binding of the 4NH2 group of 1 with Glu915 (CdO); N3 with
Cys917 (N-H); 2NH2 with Cys917 (CdO). The 5-thiophenyl
ring lies in hydrophobic region 1 and interacts with Val897,
Leu1033, and Cys1043. Thus molecular modeling supports
the inhibition of VEGFR-2 by 1.
There is currently no crystal structure of PDGFR-β bound
to a ligand. Using the structure of colony-stimulating factor-1
receptor (cFMS) (PDB 1PKG, chain A)56 as the template, a
homology model of inactive PDGFR-β was generated using
the Molecular Operating Environment (MOE 2007.09)57 suite
and methods indicated in the literature.58-60 A conforma-
tional database was built for 1 and minimized and docked into
the homology-modeled active site shown in Figure 8. The
docked poses were then ranked. The 2- and 4-NH2 groups of 1
form hydrogen bonds with the backbone residues of the hinge

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1567
Figure 6. Stereoview (SYBYL7.3) of 1 superimposed on the X-ray crystal structure of pemetrexed (green) in hTS. (PDB ID 1JU6).54
Figure 7. Stereoview (SYBYL7.3) of 1 superimposed on furo[2,3-d]pyrimidine X-ray crystal structure (not shown) in VEGFR2. Hinge region,
4NH2-Glu915 (CdO), N3-Cys917 (N-H), 2NH2-Cys917 (CdO); hydrophobic region 1, 5-thiophenyl-(Leu1033, Cys1043) (PDB ID
1YWN).55
region (Tyr683, Cys684). Additionally, the 5-S-Ph is involved
in a cation-π interaction (10-15 kcal/mol stabilization) with
the protonated Arg604. Figure 8 shows only one of the low-
energy binding poses of 1 with PDGFR-β and provides a
working model for binding to PDGFR-β.
Thus we anticipated that the synthesis and evaluation of 1
and 2 would afford VEGFR-2 or PDGFR-β inhibition or
both and that these compounds would also possess DHFR or
TS inhibitory activity or both, thus providing combination
chemotherapy potential in single molecules.
with the potassium salt of ethyl cyanoacetate anion provi-
ded adduct 2-(2-chloro-6-nitrophenyl)-2-cyanoacetate 5. Zinc
dust reduction of 5, utilizing literature conditions,39 furnished
ethyl 2-amino-4-chloro-1H-indole-3-carboxylate 6. Cyclo-
condensation of 6 with carbamimidic chloride hydrochlor-
ide61 afforded 2-amino-5-chloro-3H-pyrimido[4,5-b]indol-
4(9H)-one 7. Protection of the 2-amino group of 7 using
2,2-dimethyl propanoic anhydride provided 8. The 4-chloro
derivative 9 was prepared by treating 8 with phosphorus
oxychloride at reflux. Displacement of the 4-chloro group
with ammonia and concomitant deprotection of the 2-amino
group of 9 was achieved by means of a sealed vessel reaction;
thus the 2,4-diamino compound 10 was obtained as the
common intermediate. Treatment of 10 with the appropriate
Chemistry
The synthesis of the target compounds is outlined in
Scheme 1. Condensation of 1,2-dichloro-3-nitro-benzene 4

1568 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Gangjee et al.
Figure 8. Stereoview of a docked pose of 1 in the putative PDGFR-β active site model.
Scheme 1a
a Reagents and conditions: (a) EtO2CCH2CN, t-BuOK, THF, reflux, 48 h; (b) Zn, HOAc, 50-55 °C, 2 h, 30 min; (c) methyl sulfone, 110 °C, 30 min;
(d) 2,2-dimethylpropanoic anhydride, DMAP, NEt3, DMF, 60 °C, 48 h; (e) POCl3, reflux, 4 h; (f) NH3/CH3OH, sealed vessel, 130 °C, 48 h; (g) K2CO3,
NMP, microwave, 250 °C, 30 min.
substituted benzenethiols in a microwave apparatus (from
Biotage) provided target compounds 1 and 2.
of the assay dictate the IC50 values. Variations in assay
conditions afford different IC50 values. Thus we have elected
to evaluate the RTK inhibitory activity of our compounds
using human tumor cells known to express high levels of the
appropriate RTK.
We believe that tumor cell inhibitory assays are the most
meaningful assays of the activity of the analogs evaluated and
allow the most appropriate extrapolation regarding candi-
date selection and chances of success for in vivo evaluations.
Biological Evaluation and Discussion
Discrepancies in IC50 values obtained from isolated RTK
inhibitory assays compared with IC50 values obtained against
whole tumor cell assays are sometimes quite large (as much as
1000-fold). There are several reasons for this, and the condi-
tions (e.g., ATP concentrations for ATP competitive agents)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1569
Figure 9. Standard drugs and control agents.
Table 1. IC50 Values (μM) of Kinase Inhibition and in the A431 Cytotoxicity Assay and EC50 (μM) against P13Ka
EGFR
compd no. kinase inhibition
VEGFR-2 (Flk-1) VEGFR-1 (Flt-1)
kinase inhibition
PDGFR-β
kinase inhibition A431 cytotoxicity PI3K activity Flt-3 activity
kinase inhibition
1
15.07 ( 3.1
10.41 ( 1.2
0.23 ( 0.05
22.6 ( 4.5
56.3 ( 7.1
118.1 ( 19.4
160.1 ( 28.9
2.8 ( 0.42
40.3 ( 5.1
49.2 ( 4.7
14.1 ( 2.0
21.6
12.2
41.2 ( 5.7
39.6 ( 4.1
2
13
semaxinib
14
12.9 ( 2.9
14.1 ( 2.8
DMBI
cisplatin
15
3.75 ( 0.31
10.6 ( 3.5
1.5
16
2.9 ( 0.031
a In-cell kinase activity was assessed by a “cytoblot” developed in our laboratory using A431 cells, which overexpress EGFR, SF539 cells for
PDGFRβ, U251 cells for VEGFR2, A498 cells for VEGFR1, MV 3:11 cells for Flt-3, and HeLa cells for PI3K as described in the Detailed Methods
section.
In addition to this, we recognize that the IC50’s of standard
compounds also vary under different assay conditions, thus
we use a standard (control) compound in each of our assays.
These standard compounds were evaluated side by side with
our analogs and afford direct comparison of IC50’s of our
analogs with the standard compounds.
much less potent. Since neither compound is a potent EGFR
inhibitor compared with standard 13, the marginal inhibitory
activityagainst A431 tumorcells of 1 and 2 was not surprising.
The involvement of Flt-311 inhibition for both sorafenib and
sunitinibinadditiontootherRTKsprompted the modelingof
1 in the X-ray crystal structure of Flt-3.62 This afforded an
excellent docked conformation, and hence we initiated the
whole cell assay for Flt-3 kinase for 1 and 2 (Table 1).
Compounds 1 and 2 were about 13-fold less active against
Flt-3 compared with standard 16. Due to the importance of
PI3K in tumor growth,63 we also evaluated 1 and 2 in the
PI3K assay using 15 as the standard (Table 1); compounds 1
and 2 are 8-fold and 11-fold less active, respectively, than the
standard 15 against PI3K.
The compounds were also evaluated against isolated
DHFR and TS for the cytotoxic component. The results are
listed in Table 2 and are compared with standards for DHFR
(MTX, trimethoprim (TMP)) and TS (PMX, RTX). The
compounds were potent inhibitors of hTS at submicromolar
levels that were 54-fold (1) and 76-fold (2) more active than
clinically used PMX as its monoglutamate and comparable
to RTX. The better hTS inhibitory activity of both 1 and 2
compared with PMX could be due to a better C-ring interac-
tion with Trp109.
Compounds 1 and 2 were evaluated as inhibitors of EGFR,
VEGFR-1, VEGFR-2, PDGFR-β, phosphatidylinositol 3-ki-
nase (PI3K), and Flt-3 and for antiproliferative activity against
the A431 tumor cell line that overexpresses EGFR. For the
RTK and PI3K assays, cells overexpresssing each kinase were
exposed to compound followed by the ligand for each RTK as
described in the Detailed Methods section. This was followed
by a “cytoblot”, or in-cell ELISA, developed by our labora-
tory for the evaluation of kinase activity.42 The compounds
were compared with standards N-(3-bromophenyl)-6,7-
dimethoxyquinazolin-4-amine (13) for EGFR (Figure 9),
semaxinib for VEGFR-2, N-(4-chloro-2-fluorophenyl)-6,7-
dimethoxyquinazolin-4-amine (14) for VEGFR-1, (Z)-3-((4-di-
methylamino)benzylidene)indolin-2-one (DMBI) for PDGFR-β,
cisplatin (CDDP) for A431, 2-morpholino-8-phenyl-4H-chro-
men-4-one (15) for P13K, and 2-(3,4-dimethoxybenzamido)-
4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (16) for
Flt-3 (Figure 9). The results are listed in Table 1. The
compounds are active against VEGFR-2 and within 2-4-fold
of the standard semaxinib with 1 more potent than 2. Against
PDGFR-β, 1 is somewhat more potent than the standard
DMBI and 2 is about 11-fold less potent. Against the growth
of A431 tumor cells in culture 2 is similar to cisplatin while 1 is
However 1 and 2 were not appreciably potent against
hDHFR as well as other DHFR. Why was 1 poorly active
against hDHFR? Molecular modeling had suggested that
superimposition of 1 onto DHFR inhibitors in X-ray crystal
structures (Figure 5) should afford inhibition of hDHFR.

1570 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Gangjee et al.
Table 2. Inhibitory Concentrations (IC50, μM) against DHFR and TS
TS inhibitory activity,a IC50 (μM)
DHFR inhibitory activity,a IC50 (μM)
compd
humanc
Escherichia colic
Toxoplasma gondiid
humane
Escherichia colif
Toxoplasma gondiid
1
2
0.54
0.39
29.0
0.29
>27
>26
15
0.11
>26
14
>33 (17)b
>31 (7)
>33 (35)
>31 (27)
33
>31 (22)
pemetrexedg
raltitrexedh
MTX
2.3
0.48
0.022
680
0.0066
0.02
0.019
2.9
trimethoprim
a The percent inhibition was determined at a minimum of four inhibitor concentrations within 20% of the 50% point. The standard deviations for
determination of 50% points were within (10% of the value given. b Numbers in parentheses indicate the percent inhibition at the stated concentration.
c Kindly provided by Dr. Frank Maley, New York State Department of Health. d Kindly provided by Dr. Karen Anderson, Yale University, New
Haven, CT. e Kindly provided by Dr. Andre Rosowsky, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA. f Kindly provided by
Dr. R. L. Blakley, St. Jude Children’s Hospital, Memphis, TN. g Kindly provided by Dr. Chuan Shih, Eli Lilly and Co. h Kindly provided by
Dr. Ann Jackman, CRC Centre for Cancer Therapeutics, Surrey SM2 5NG, England.
Figure 10. Cell viability assay after 48 h treatment.
The modeling provides the 5-S-phenyl of 1 in a binding mode
that is at 90° to other hDHFR inhibitor (such as MTX52,53
and TMQ50) sidechainphenylrings. Onthe basisofmolecular
modeling, other binding modes of 1 afford clashes of the
C-ring with side chains and backbone of hDHFR. This could
perhaps explain, in part, the poor activity against hDHFR.
Thus 1 and 2 were good to moderate inhibitors of VEGFR-
2 and PDGFR-β, comparable or better than the standards
(except 2 for PDGFR-β) for RTK inhibition and potential
antiangiogenic activity, and also had hTS inhibitory activity
(IC50) in the submicromolar range to provide the cytotoxic
effects needed for combination chemotherapy potential in
single agents. The hTS IC50 values were not in the single digit
nanomolar range and hence should not cause severe toxicity
to normal cells not compromised by the antiangiogenic effects
of 1 and 2. The usual next step is to determine the inhibitory
activity against tumor cells in culture. The NCI preclinical
60 tumor panel was the obvious choice; however since angio-
genesis is not part of these tumors in culture, only the
cytotoxic effects were expected to play a role in the inhibition
of these cells in culture. Compound 1 was evaluated against
the growth of the NCI 60 tumor cell lines, and the results
were as anticipated; 1 inhibited the tumors at GI50 values of
1-10 μM. This was about 10-fold less than the isolated hTS
inhibitory activity (Table 2) and indicates a relatively low
potency completely in keeping with the absence of the anti-
angiogenic component and perhaps an indication of low
toxicity to normal cells.
Pharmacodynamic (PD) Data in MDA-MB-435 Cells.
Compounds 1 and 2 were evaluated for cell viability
(IC50 of cell kill) in several cell types (Figure 10). In addi-
tion, MDA MB 435 metastatic dermal breast cancer cells
were exposed to 20 μM of 1 and 2 for cell cycle analysis
(Figure 11), and cell signaling analysis (Figure 12) was
carried out. First, cell viability was evaluated on cells
serum-starved overnight before the addition of 1% FCS to
the cancer cells, 200 ng/mL hrVEGF-165 to the endothelial
cells, and 100 ng/mL hrPDGF-BB to the SMC variants
together with drug for 24 h followed by 24 h incubation in
serum-containing media and a cell viability assay carried out
as described in the Experimental Section. It was found that 1
had varied effects (Figure 10) on three cancer cell lines
(MDA-MB-435, A431, Capan-1) and fairly consistent
effects on two endothelial cell types (HUVEC, HMEC-1).
Human retinal pericytes (Huperi-3)64 (not human aortic
SMCs) were very sensitive to 1 cell kill (Figure 10, left panel).
Compound 2 had a different profile of sensitivity than did 1
with tumor cells being the most sensitive, endothelial cells
falling someplace in the middle, and SMC variants being the
least sensitive to 2 (Figure 10, right panel). For cell cycle
analysis, it was found that after 24 h exposure, 1 resulted in a
buildup in G2/M (Figure 11, right panel) while 2 resulted in
an increase in S phase (Figure 11, left panel). Next, a cell
signaling experiment was performed on the MDA-MB-435
breast cells exposed for either 1 or 4 h to 20 μM 1 or 2. The
same cells exposed to 30 J/m2 UV-B for 4 h were used as a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1571
Figure 11. Cell cycle analysis.
perhaps decreasing cell proliferation. The data from com-
pound 2 are more complex; it appears that this compound
acts both on the tumor and on cells of the vasculature to
decrease RTK and MAPK signaling. The increase in S phase
content observed in response to 2 could indicate that cells
enter into DNA synthesis but cannot exit into G2, a phenom-
enon consistent with TS inhibition.
The logical next step was to demonstrate antitumor acti-
vity of 1 via an in vivo animal model. Compound 1 was the
first choice over 2 mainly because of its better PDGFR-β
inhibitory activity. To determine the antitumor effect of 1 in
vivo, athymic mice were first implanted with COLO-
205 metastatic colon cancer cells, shown to overexpress
PDGFR-β.65 At day 9 after implantation, treatment was
initiated with the PDGFR-β inhibitor DMBI66 at the MTD
of 15 mg/kg three times weekly and with 1 at two doses, 25
and 35 mg/kg three times weekly. Tumor growth was mea-
sured using calipers. It was found that DMBI and 1 resulted
in significant decreases in primary tumor growth rate (Figure
14A,B) and that 1 resulted in a further significant decrease in
tumor growth rate compared with the control compound,
DMBI. At the end of the experiment, tumors were taken and
stained for blood vessels. It was found that 1 at 35 mg/kg
three times weekly resulted in a significant decrease in
primary tumor vascularity compared with control treated
animals (Figure 14C) suggesting an antiangiogenic effect in
vivo. Finally, livers were stained for the presence of meta-
stases, and it was found that 1 at both 25 and 35 mg/kg, but
not DMBI, resulted in significantly reduced numbers of
COLO-205 metastases to the liver (Figure 14D). TGI for 1
is 76% at 35 mg/kg in this study. No MTD was determined
for 1; doses up to 75 mg/kg were well tolerated without any
visual ADRs or loss in weight. Thus in vivo compound 1 has
considerable antitumor activity, antiangiogenic activity, and
antimetastatic activity. Further in vitro and in vivo studies
are currently underway to afford further evidence of the
triple mechanism(s) of action of 1 and 2 with a triple
combination of a VEGFR2, a PDGFR-β, and a hTS in-
hibitor as standard as well as a combination of two of these
standards together to determine the mechanism of action, in
vivo, of 1 and 2 responsible for the in vivo antitumor,
antiangiogenic, and antimetastatic activities of 1.
Figure 12. Cell signaling.
positive control. The first downstream target of RTKs was
activated phospho-p42/44 MAPkinase (ERK 1/2), a MAP
kinase member involved in cell proliferation in many can-
cers. It was found that both 1 and 2 reduced levels of
activated ERK 1/2, but not to the same extent as UV-B
exposure (Figure 12; top gel). In contrast, exposure to neither
agent resulted in increased levels of activated phospho-Akt, a
cell survival pathway also downstream of RTKs with levels
of (nonphospho) Akt used as a loading control (Figure 12,
middle two gels). Finally, neither 20 μM 1 nor 20 μM 2
resulted in increased levels of cleaved caspase 3, the terminal
step in apoptosis (Figure 12; bottom most gel).
MDA-MB-435 Chorioallantoic Membrane (CAM) Xeno-
graft. We have developed an intermediate systemic in vivo
xenograft system using the chicken embryo CAM. This
method allows for in vivo testing of more compounds in a
given time and cost than the mouse tumor xenograft studies.
For the CAM xenograft model, MDA-MB-435 cells
(250 000) were implanted under the vascularized CAM of
10 days of incubation (DI) chicken embryos, and then the
embryos were treated systemically with 25 mg/kg 1 on days
one and two, and CAMs were fixed, excised, and imaged
with representative images shown from five to six CAMs
(Figure 13). It was found that 1 reduced both the size and the
vascularity of resulting tumors, at least to the same extent as the
standard VEGFR2 kinase inhibitor semaxanib at 10 mg/kg
(Figure 13). These results indicate the antitumor and anti-
angiogenic activity of 1. Taken together the preliminary in
vitro and the in vivo results (see below) suggest that com-
pound 1 has profound antiangiogenic activity both in vitro
and in vivo. Further, these data indicate that 1 may act more
on the vasculature to decrease RTK growth factor signaling
We designed, synthesized, and discovered two com-
pounds, 1 and 2, that inhibit VEGFR-2 and PDGFR-β
in whole cells at IC50 values comparable to standard com-
pounds. In addition, 1 and 2 also inhibit isolated hTS
better than the monoglutamate of clinically used PMX. Thus

1572 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Gangjee et al.
Figure 13. MDA-MB-435 CAM 72 h xenograft.
Figure 14. Tumor growth: (A) primary tumor volume, (B) primary tumor growth rate, (C) primary tumor vascularity, and (D) liver metastasis
of COLO-205 tumor xenografts after various treatments. COLO-205 cells were implanted into male athymic mice as in the Detailed Methods
section. Compounds were administered at the listed doses three times weekly, and tumor growth was measured using calipers. At the
experiment end, tumors were stained for vascularity and livers were stained for metastases as in the Detailed Methods section. Data represent
the average ( SD of 7-11 animals: /, P > 0.05; //, P < 0.01; ///, P < 0.001 by one-way ANOVA and Neuman-Keuls post-test. The average
tumor growth rate for control treated animals (B) is 58.9 mm3/day, average number of tumor vessels in control-treated animals (C) is 11.2/field,
and average metastases in control treated animals (D) are 0.65/lobe.
remarkably 1 and 2 possess both multi-RTK inhibitory
activity (VEGFR-2 and PDGFR-β) for antiangiogenic
effects and hTS inhibitory activity (cytotoxic effects) com-
parable to or better than clinically used single agents.
Compound 1 in vivo in a COLO-205 metastatic colon cancer
xenograft mouse model, at both 25 and 35 mg/kg, showed
significantly decreased tumor growth, significantly de-
creased liver metastasis, and significantly decreased primary
tumor blood vessels (angiogenesis), remarkably without any
toxicity, compared with untreated control and a standard
PDGFR-β inhibitor DMBI. The combination chemothera-
peutic attributes of 1 and 2 of VEGFR-2, PDGFR-β, and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1573
hTS inhibitory activity makes them unique and distinct from
all other known agents in clinical or investigational use. The
relevance of this combination of activities in a single agent is
that it mimics the clinically relevant combinations of RTK
inhibitors with TS inhibitors. In addition, the in vivo anti-
tumor, antiangiogenic, and antimetastatic activity of 1 sup-
ports the optimization and development of this agent and
design of analogs as potential clinically useful antitumor
agents that afford combination chemotherapy potential in
single agents. Such single agents are designed to be used as
monotherapy but could also be used with other antitumor
agents or radiation as part of the therapy for cancers.
in CHCl3. The fractions containing the pure product (TLC)
were pooled and evaporated to give a pink solid. The overall
yield from 4 to 6 was 63% (7.7 g of 6 was obtained). TLC Rf 0.18
(hexane/CHCl3, 1:1); mp 140-142 °C; 1H NMR (DMSO-d6) δ
1.26-1.30 (t, 3 H, CH3), 4.15-4.23 (q, 2 H, CH2), 6.82-6.87 (t,
J = 7.8 Hz, 1 H, C6-CH), 6.92-6.96 (dd, J = 7.8 Hz, 1 H, Ar),
6.96 (bs, 2 H, 2-NH2, exch), 7.05-7.08 (dd, J = 7.8 Hz, 1 H, Ar),
11.35 (bs, 1 H, 9-NH, exch). Anal. (C11H11ClN2O2) C, H, N.
2-Amino-5-chloro-3,9-dihydro-4H-pyrimido[4,5-b]indol-4-one (7).
A mixture of ethyl 2-amino-4-chloro-1H-indole-3-carboxylate
(6) (200 mg, 0.837 mmol), carbamimidic chloride hydrochlor-
ide61 (106.22 mg, 1.37 mmol), and methyl sulfone (1 g) was
stirred and heated at 110-120 °C for 30 min. About 10 mL of
water was added to quench the reaction. Ammonia-water was
added to neutralize the reaction mixture. The precipitated solid
was filtered, dissolved in chloroform and methanol, and dried
(Na2SO4), the solvent was evaporated, and the solid was recrys-
tallized from chloroform and methanol (1:1) in overall yield of
78%. TLC Rf 0.33 (CHCl3/MeOH, 1:1); mp >250 °C; 1H NMR
(DMSO-d6) δ 6.58 (bs, 2 H, 2-NH2, exch), 7.04-7.06 (t, J = 3.3
Hz, 1 H, C7-CH), 7.17-7.20 (m, 2 H, Ar), 10.42 (s, 1 H, 3-NH,
exch), 11.65 (s, 1 H, 9-NH, exch). HRMS calcd for C10H7ClN4O
234.0309, found 234.0308.
Experimental Section
Analytical samples were dried in vacuo (0.2 mmHg) in a
CHEM-DRY drying apparatus over P2O5 at 80 °C. Melting
points were determined on a 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 a Bruker WH-300 (300 MHz) spectro-
meter. 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 spots were visualized under 254 and 366 nm
illumination. Proportions of solvents used for TLC are by
N-(5-Chloro-4-oxo-4,9-dihydro-3H-pyrimido[4,5-b]indol-2-
yl)-2,2-dimethyl Propanamide (8). To a round bottomed flask
were added 7 (300 mg, 1.27 mmol), 2,2-dimethylpropanoic
anhydride (713.32 mg, 3.83 mmol), dimethylaminopyridine
(7 mg, 0.06 mmol), and triethylamine (514.05 mg, 5.08 mmol),
along with 30 mL of DMF. The mixture was stirred at 60 °C for
48 h. The DMF was removed using an oil pump to afford a
residue, which was purified by column chromatography, eluting
sequentially with 0%, 1%, and 5% CH3OH in CHCl3. Fractions
containing the product 8 (TLC) were pooled and evaporated to
give solid compound. The yield was 40% (163 mg). TLC Rf 0.45
(CHCl3/MeOH, 10:1); mp 185.8-190.1 °C; 1H NMR (DMSO-
d6) δ 1.27 (s, 9 H, C(CH3)3), 7.18-7,21 (t, J = 2.4 Hz, 1 H, C7-
CH), 7.24-7.26 (dd, J = 7.5 Hz, 1 H, Ar), 7.37-7.40 (dd, J =
6.3 Hz, 1 H, Ar), 11.15 (s, 1 H, 3-NH, exch), 11.93 (s, 1 H, 2-NH,
exch), 12.11 (s, 1 H, 9-NH,exch). HRMS calcd for C15H15-
ClN4O2 [M þ Na]þ 341.0757, found 341.0781.
volume. Column chromatography was performed on
a
230-400 mesh silica gel (Fisher, Somerville, NJ) column. Ele-
mental analyses were performed by Atlantic Microlab, Inc.,
Norcross, GA. Element compositions are within (0.4% of the
calculated values. Fractional moles of water or organic solvents
frequently found in some analytical samples of antifolates could
not be prevented despite 24-48 h of drying in vacuo and were
confirmed where possible by their presence in the 1H NMR
spectra. Elemental analysis was used to determine the purity of
the final compounds 1 and 2. All solvents and chemicals were
purchased from Aldrich Chemical Co. or Fisher Scientific and
were used as received. Purity of the final compounds 1 and 2 were
>95% and were determined by elemental (C, H, N, S) analysis.
Ethyl 2-Amino-4-chloro-1H-indole-3-carboxylate (6). To an
ice-cold solution of ethyl cyanoacetate (10.9 mL, 102.4 mmol) in
anhydrous THF (170 mL) under N2 was added potassium tert-
butoxide (12.07 g, 107.5 mmol). The formed white suspension
was stirred for 15 min then treated with 1,2-dichloro-3-nitro-
benzene 4 (9.83 g, 51.2 mmol). The suspension was heated at
reflux for 48 h. The resulting reddish brown solution was poured
into H2O, and the aqueous mixture was acidified to pH 2 with
concentrated HCl. The mixture was extracted with ether (3ꢀ),
and then the combined organic phases were dried using sodium
sulfate and concentrated to give ethyl (2-chloro-2-nitrophenyl)-
(cyano)acetate 5 as a dark oil. The oil was purified by column
chromatography eluting with 10:1 hexane/EtOAc. TLC Rf 0.23
(hexane/EtOAc, 3:1). 1H NMR (DMSO-d6) δ 1.17-1.22 (t, 3 H,
CH3), 4.18-4.25 (q, 2 H, CH2), 6.36 (bs, 2 H, 2-NH2, exch),
7.75-7.81 (t, J = 8.1 Hz, 1 H, C4-CH), 8.04-8.06 (dd, J = 6.9,
1.2 Hz, 1 H, Ar), 8.13-8.16 (dd, J = 6.9, 1.2 Hz, 1 H, Ar). The
material was used directly for the next step.
N-(4,5-Dichloro-9H-pyrimido[4,5-b]indol-2-yl)-2,2-dimethyl
Propanamide (9). Compound 8 (2 g, 6.274 mmol) was added to a
round bottomed flask and dissolved in 30 mL of POCl3. The
reaction mixture was refluxed at 110-120 °C for 4 h. After
evaporation of the POCl3, ice-cold water was added. The
reaction mixture was neutralized with NH3 H2O and extracted
3
with CHCl3. The organic phase was dried with Na2SO4. The
crude mixture was then purified by column chromatography,
eluting sequentially with 0%, 1%, and 5% methanol in chloro-
form. Fractions containing the product 9 (TLC) were pooled
and evaporated to give a solid. The yield was 70% (1.48 g). TLC
Rf 0.86 (CHCl3/MeOH, 5:1); mp 245.6-246.1 °C; 1H NMR
(DMSO-d6) δ 1.25 (s, 9 H, C(CH3)3), 7.36-7.39 (t, J = 4.5 Hz,
1 H, C7-CH), 7.49-7.51 (m, 2 H, Ar), 10.30 (s, 1 H, 9-NH,
exch), 12.96 (s, 1 H, 2-NH, exch). HRMS calcd for C15H14Cl2-
N4O2 [M þ Na]þ 359.0428, found 359.0442.
5-Chloro-9H-pyrimido[4,5-b]indole-2,4-diamine Propanamide
(10). Compound 9 (200 mg, 0.6 mmol) was added to 5 mL of
methanol saturated with ammonia. The solution was stirred at
130 °C for 2 days in a sealed vessel. The methanol was evapo-
rated to give a solid that was purified by column chromatogra-
phy, eluting sequentially with 0% and 1% methanol in
chloroform. Fractions containing the product 10 (TLC) were
pooled and evaporated to give a solid. The yield was 39%
(54 mg). TLC Rf 0.43 (CHCl3/MeOH, 5:1); mp 245.2-
246.3 °C; 1H NMR (DMSO-d6) δ 6.15 (bs, 2 H, 4-NH2, exch),
6.85 (bs, 2 H, 2-NH2, exch), 7.10-7.15 (t, J = 7.2 Hz, 1 H,
C7-CH), 7.15-7.18 (dd, J = 9 Hz, 1 H, Ar), 7.22-7.24 (dd,
J = 6.9 Hz, 1 H, Ar), 11.54 (bs, 1 H, 9-NH, exch). Anal. (C10H8-
ClN5) C, H, N, Cl.
A solution of impure 5 (18 g, 67 mmol) in glacial AcOH (185
mL) was treated with a single charge of Zn dust (12.1 g, 185
mmol). The mixture was heated at 55 °C for 45 min, then treated
with more Zn dust (6 g). After heating for another 105 min, the
mixture was filtered through a pad of Celite. The pad was
washed well with AcOH, and the filtrate was concentrated to
a residue that was distributed between CHCl3 and H2O. The
organic phase was washed with 5% aq NaHCO3 and concen-
trated to a residue that was purified by column chromato-
graphy, eluting sequentially with 0%, 5%, and 10% EtOAc

1574 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Gangjee et al.
General Procedure for the Synthesis of 5-Substituted
(Phenylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines
The docking was restricted to the active site pocket residues
using the Alpha triangle placement method. Refinement of the
docked poses was carried out using the Forcefield refinement
scheme and scored using the Affinity dG scoring system.
Around 30 poses were returned for each compound at the end
of each docking run. The docked poses were manually examined
in the binding pocket to ensure quality of docking and to
confirm absence of steric clashes with the amino acid residues
of the binding pocket.
Detailed Methods. Specific Assays. CYQUANT Cell Proli-
feration Marker Assay. As a measure of cell proliferation, the
CYQUANT cell counting/proliferation assay was used as pre-
viously described.69 We have found that this assay is comparable
in sensitivity to a clonogenic (colony-forming) assay70 and lacks
many of the problems associated with metabolic-based assays.
For the CYQUANT assay, cells were serum-starved overnight;
1% FCS was then added along with drug to the media of cancer
cells (MDA-MB-435, Capan-1, J82, A431, U251), 200 ng/mL
hrVEGF-165 was added with drug to the media of endothelial
cells (HUVEC, HMEC-1), and 100 ng/mL hrPDGF-BB was
added to the media of SMCs (HASMCs, HuPeri-3) for 24 h;
then media were replaced with fresh (drug-free) completed
media for an additional 24 h. The cells were then lysed, and
the CYQUANT dye, which intercalates into the DNA of cells,
was added, and after 5 min, the fluorescence of each well was
measured using a plate reader. A positive control used for
cytotoxicity was cisplatin. Data are graphed as a percent of cells
receiving no compound, and IC50 values were estimated from
two to three separate experiments (n = 6-15) using sigmoidal
dose-response analysis with Prism 3.0 (GraphPad software,
San Diego).
1 and 2.
Compound 10 (50 mg, 0.2 mmol), the appropriate thiol (0.9
mmol), and potassium carbonate (120 mg, 0.9 mmol) were added
to a 2-5 mL biotage microwave vial. Three milliliters of NMP
was added as solvent, and the tube was sealed. The reaction was
run in a microwave for 30 min at 250 °C. After cooling to room
temperature, the reaction mixture was transferred on top of a
silica gel column and eluted with 0% and 4% methanol in
chloroform. Fractionscontainingthe product (TLC) werepooled
and evaporated to afford the products 1 and 2.
5-(Phenylthio)-9H-pyrimido[4,5-b]indole-2,4-diamine (1). Using
the general procedure described above, the reaction of 10 with
benzene thiol 11 afforded 1 as an off-white solid: yield 97% (64
1
mg). TLC Rf 0.23 (CHCl3/MeOH 10:1); mp 251 °C; H NMR
(DMSO-d6) δ 6.04 (bs, 2 H, 4-NH2, exch), 7.03-7.04 (d, J = 4.5
Hz, 2 H, Ar), 7.13-7.16 (t, J = 4.2 Hz, 1 H, C7-CH), 7.21-7.27
(m, 2 H, Ar), 7.24 (bs, 2 H, 2-NH2, exch), 7.32-7.33 (dd, J = 4.2
Hz, 1 H, Ar), 7.40-7.42 (dd, J = 4.5 Hz, 1 H, Ar), 11.49 (bs, 1 H,
9-NH, exch). Anal. (C16H13N5S) C, H, N, S.
5-[(4-Methylphenyl)thio]-9H-pyrimido[4,5-b]indole-2,4-diamine
(2). Using the general procedure described above, the reaction of
10 with 4-methyl benzene thiol 12 afforded 2 as an off-white
solid: yield 87% (60 mg). TLC Rf 0.54 (CHCl3/MeOH 5:1); mp
>250 °C; 1H NMR (DMSO-d6) δ 2.19 (s, 3 H, CH3), 6.02 (bs,
2 H, 4-NH2, exch); 7.19 (bs, 2 H, 2-NH2, exch), 6.95-6.97 (d,
J = 6 Hz, 2 H, Ar), 7.05-7.07 (d, J = 6 Hz, 2 H, Ar), 7.18-7.22
(t, J = 6 Hz, 1 H, C7-CH), 7.29-7.31 (dd, J = 5.7 Hz, 1 H, Ar),
7.38-7.40 (dd, J = 5.7 Hz, 1 H, Ar), 11.46 (bs, 1 H, 9-NH, exch).
Anal. (C17H15N5S 0.4H2O) C, H, N, S.
3
Molecular Modeling. There is currently no known crystal
structure of PDGFR-β bound to a ligand. A homology model
was hence built for evaluating the binding of 1 in PDGFR-β.
The amino acid sequence of the PDGFR-β kinase domain was
obtained from the SWISS-PROT database (ID PGFRB_HU-
MAN [P09619]). As has been reported earlier in the literature,67
a DISOPRED2.068 analysis of the amino acid sequence was
performed to predict the ordered and disordered regions. The
results from this analysis predicted amino acids 700-792 were
disordered. A homology model was then built using MOE
2007.09 using the structure of c-KIT kinase complex (PDB
1PKG, chain A) as the template. A BLASTP58 search indicated
that chain A of the c-KIT kinase complex shows high sequence
similarity with PDGFR-β (E-value: 1e-58). Sequence alignment
was performed using MOE_Align using the “actual secondary
structure” option in MOE. Using the “actual secondary struc-
ture” option in MOE was necessary to correctly identify the
disordered kinase insertion domain (amino acids 700-792). The
final homology model returned by the program was subjected to
further energy minimization using Amber99 as the forcefield
and a 0.5 rms gradient. A Ramachandran plot of the model
showed the presence of six outlying residues (Tyr562, Glu563,
Asp590, Ser623, Ser642, Glu911). Since these residues were not
in the proximity of the ATP binding site, the model was used
without further refinement. Docking studies were performed
using the docking suite of MOE 2007.09. After addition of
hydrogen atoms, the protein was then “prepared” using the
LigX function in MOE. LigX is a graphical interface and
collection of procedures for conducting interactive ligand mod-
ification and energy minimization in the active site of a flexible
receptor. In LigX calculations, the receptor atoms far from the
ligand are constrained and not allowed to move while receptor
atoms in the active site of the protein are allowed to move but are
subject to tether restraints that discourage gross movement. The
procedure was performed with the default settings.
Phosphotyrosine (PY) Cell-Based ELISA. Because of the need
for screening many compounds in multiple replicates, a higher
throughput 96-well phosphotyrosine (PY) ELISA was devel-
oped.71,72 Briefly, cells at 60-75% confluence were placed in
serum-free medium for 18 h to reduce the background of
phosphorylation. Cells used for these experiments have been
shown to overexpress particular RTKs; specifically A431 for
EGFR, SF539 for PDGFRβ, U251 for VEGFR2, A498 for
VEGFR1, and MV 3:11 cells for Flt-3 (but not VEGFR1/2).
Cells were then pretreated for 60 min with 10, 3.33, 1.11, 0.37,
and 0.12 μM compound followed by an optimized dose and time
of purified growth factor (EGF, PDGF-β, VEGF). The reaction
was stopped, and cells were permeabilized by quickly removing
the media from the cells and adding ice-cold Tris-buffered saline
(TBS) containing 0.05% triton X-100, protease inhibitor cock-
tail, and tyrosine phosphatase inhibitor cocktail (both from
Sigma Chemical). The TBS solution was then removed, and cells
were fixed to the plate by heat and further incubation in 70%
ethanol. Cells were further exposed to block (TBS with 1%
BSA) and washed, and then a horseradish peroxidase (HRP)-
conjugated phosphotyrosine antibody was added. The antibody
was removed; cells were washed again and exposed to an
enhanced luminol ELISA substrate, and light emission was
measured using a plate reader. The known RTK-specific kinase
inhibitors (PD153035, SU5416, CB67645, DMBI, and com-
pound 16) discussed above were used as positive controls for
kinase inhibition. Data were graphed as a percent of cells
receiving growth factor alone, and EC50 values were estimated
from two to three separate experiments (n = 8-24) using
sigmoidal dose-response relations in Prism 3.0 software
(GraphPad). In every case, the activity of a positive control
inhibitor did not deviate more than 10% from the EC50 values
historically collected in this assay.21,42,73
PI3 Kinase Assay. The PI3 kinase assay is a cytoblot for the
phosphorylated (active) form of the p85 subunit of PI3K. HeLa
cells have been shown to possess large PI3K activity without
any stimulus,74 so for these studies, no inducer is necessary.
HeLa cells grown in log phase for one day after passaging
in serum-containing media were exposed as above to various
Ligands were built using the molecule builder function in
MOE and were energy-minimized to their local minima using
the MMF94X forcefield to a constant of 0.05 kcal/mol.
Ligands were docked into the active site of the prepared
protein using the docking suite as implemented in MOE.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1575
concentrations of unknown compounds or the known PI3K
inhibitor, LY294002 for 1 h at 37 °C. As above, cells were
permeabilized with Triton, fixed by heat and ethanol and
blocked, and a phospho-(Tyr) p85 PI3K binding motif antibody
(Cell Signaling) was added at a 1:100 dilution overnight at 4 °C.
The antibody was then removed, and an anti-rabbit HRP-
conjugated secondary antibody was then added (Pierce
Chemical) for 30 min at RT, the antibody was removed, cells
were washed again and exposed to an enhanced luminol ELISA
substrate, and light emission was measured using a chemilumi-
nescent plate reader.
1 were given at doses of 2.5, 5, 10, 15, 25, and 35 mg/kg embryo
body weight81 and at 3 days after injection embryo lethality was
evaluated. It was found that 10 mg/kg of gemcitabine and 25 mg/
kg of compound 1 were the highest doses resulting in no lethality
or overt toxicity in embryos; thus these doses were used in
subsequent experiments. Three days after tumor implantation,
CAMs were fixed in situ by pipetting on a solution of 0.1%
Triton X-100 in 4% paraformaldehyde for 1 min. The solution
was removed and CAM/chorion were excised in an area sur-
rounding the tumor implantation site. The CAM/chorion were
placed into 4% paraformaldehyde and allowed to fix an addi-
tion 2 min, and the chorion was separated from the CAM.
CAMs were removed to 6-well plates containing 4% parafor-
maldehyde. CAMs were spread gently to remove wrinkles, and
images were captured at 6.25ꢀ magnification using a brightfield
dissecting microcope (Wild M400 photomakroscop, Bern,
Switzerland) connected to a cooled camera (QImaging, Burna-
by, BC, Canada). Images were then converted to gray scale, the
number of vessels surrounding the tumor was manually
counted, and the size of the tumor was determined using NIH
Image J densitometry software.
Animal Numbers. Consulting with the Biostatistics Program
at OUHSC and using StatMate software (www.graphpad.com)
and a power analysis, we have found that a sample size of 10
animals is optimal for 80% power, a significance level of 0.05 in
a (two-tailed) unpaired t test, and an expected SD of 24 (from
previous experiments). Four animals per treatment group are
used for short-term toxicity studies, and five animals per group
are used for the longer term toxicity studies.
Whole Animal Toxicity Assay. To first determine the maximal
tolerated dose (MTD) of a drug, male NCr nu/nu mice at 8
weeks of age without tumors were injected with 5, 10, 12.5, 15,
17.5, and 20 mg/kg compound 1 or the PDGFR-β kinase
inhibitor DMBI three times weekly, on Monday morning,
Wednesday noon, and Friday afternoon. Weights were taken,
and animals were observed for acute distress during the first 96 h
after injection and in a 3 and 6 week period. Significance in
weights was calculated after each weighing using one-way
ANOVA and Neuman-Keuls post-test with the null hypothesis
rejected if P < 0.05. Animals with significant (>7%) weight loss
were humanely euthanized and inspected for any overt toxicity,
and organs (heart, lung, kidney, liver, colon) were removed for
inspection by a veterinarian pathologist at OUHSC.
COLO-205 Human Metastatic Colon Mouse Xenograft. It has
been shown that the natural killer (NK) T cell activity in athymic
mice affects the metastasis of human tumor xenografts.82,83
Thus for these experiments, NIH-III nude mice, deficient in
NK cells, were used. One million COLO-205 (liver colonizing;
ATCC) human metastatic colon cancer cells were injected
subcutaneously (SQ) in the lateral flank of athymic NIH-III
male mice, 8 weeks in age. Animals are monitored every other
day for the presence of tumors. At the time in which most tumors
are measurable by calipers (day 9 after implantation), animals
with tumors were randomly sorted into treatment groups.
DMSO stocks (30 mM) of drugs were further dissolved into
sterile water for injection, and the optimal dose (from toxicity
studies) was injected intraperitoneally (IP). The length (long
side), width (short side), and depth of the tumors were measured
using digital Vernier calipers each Monday, Wednesday, and
Friday. Tumor volume was calculated using the formula length ꢀ
width ꢀ depth. Tumor growth rate was then calculated using
a linear regression analysis algorithm with the software Graph-
Pad Prism 4.0.c. At the experiment’s end, animals were humanly
euthanized, tumors and liver were excised, fixed in 20% neutral
buffered formalin for 8-10 h, and embedded into paraffin, and
hematoxylin-eosin (H&E) stain of three separate tissue sections
was completed to span the tumor and liver. Together with the
OUHSC Department of Pathology core, metastases per liver
lobe were counted using the H&E stained sections. The meta-
stases can be seen as purple clusters of disorganized cells on the
Dihydrofolate Reductase (DHFR) Assay.75 All enzymes were
assayed spectrophotometrically in a solution containing 50 μM
dihydrofolate, 80 μM NADPH, 50 mM Tris-HCl, 0.001 M
2-mercaptoethanol, and 0.001 M EDTA at pH 7.4 at 30 °C.
The reaction was initiated with an amount of enzymes yielding
a change in optical density at 340 nm of 0.015 units/min.
Thymidylate Synthase (TS) Assay. TS was assayed spectro-
photometrically at 30 °C and pH 7.4 in a mixture containing 0.1
M 2-mercaptoethanol, 0.0003 M (6R,S)-tetrahydrofolate, 0.012
M formaldehyde, 0.02 M MgCl2, 0.001 M dUMP, 0.04 M Tris-
HCl, and 0.000 75 M NaEDTA. This was the assay described by
Wahba and Friedkin76 except that the dUMP concentration was
increased 25-fold according to the method of Davisson et al.77
The reaction was initiated by the addition of an amount of
enzyme yielding a change in absorbance at 340 nm of 0.016
units/min in the absence of inhibitor.
In Vitro Pharmacodynamic Studies. Cell Cycle Analysis.
After exposure to drug, cells were fixed in 50% ethanol over-
night, permeabilized, and labeled with PBS containing 0.1%
Triton X-100, 100 μg/mL Rnase A, and 25 μg/mL propidium
iodide for 45 min to 1 h at 37 °C before being subjected to flow
cytometry as previously described.70 Cell cycle analysis of mean
fluorescence data was performed using MODFIT software to
generate percent S phase, G1/G0 phase, and G2/M phase
(Verity Software, Topsham, ME). Aphidicolin (250 ng/mL),
an agent that inhibits DNA polymerase, was used as a control
for G1 arrest,78 and 50 ng/mL nocodazole, a reversible mitotic
spindle poison, was used as a control for G2/M arrest (data not
shown).79
Cell Signaling Analysis. Cells were treated with compounds at
around the IC50 dose for 1 and 4 h, and whole cell lysates were
prepared using M-PER reagent (Pierce Chemical, Rockford,
IL) with 300 mM NaCl to lyse the nucleus. Western blots against
phosphorylated (activated) (ERK1/2 (Cell Signaling cat. no.
4370)) and against phospho (Cell Signaling cat. no. 4060) and
nonphosphorylated Akt (Cell Signaling cat. no. 2920), two
major signaling pathways downstream of RTKs, were done
per manufacturer’s instructions. Additionaly, cleaved caspase 3
(Cell Signaling cat. no. 9661) was assessed as a marker of
apoptosis.
Chorioallantoic Membrane (CAM) Xenograft Assay. The
procedure used was a modification of a previously published
procedure80 Fertile Leghorn chicken eggs (CBT Farms, Ches-
tertown, MD) were incubated until 10 DI, eggs were candled for
viability, then a small window was made in the superior shell to
expose the chorion and CAM. The CAM was then candled to
visualize the vasculature, and a small mark was made on the
chorion in a location away from major vessels. Human MDA-
MB-435 cancer cells (American Type Culture Collection, Man-
assas, VA), 250 000 in 50 μL of inert extracellular matrix
(Humatrix, Care-Tech Laboratories, St Louis, MO), were im-
planted just under the CAM using a 1 mL syringe with a 25G
needle. Neomycin-polymyxin ointment was then applied to the
injection site, and the shell hole was covered with Micropore
surgical tape (3M, St. Paul, MN). Four hours and 48 h after
tumor implantation, the tape was partially removed and drug or
solvent (DMSO in SWFI) was placed onto the chorion using a
micropipettor, yielding a systemic dose to the embryo. In an
initial dose-finding experiment, either semaxanib or compound

1576 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Gangjee et al.
highly organized largely pink liver. Together with the OUHSC
Department of Pathology core, blood vessels per unit area on
primary tumors were also counted in five fields at 100ꢀ magni-
fication and averaged. Tumor growth rate per day, tumor
vascularity, and liver metastases were calculated, and signifi-
cance was determined from the growth curves using two-way
ANOVA with a repeated measures post-test, and the null
hypothesis was rejected if P < 0.05.
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Acknowledgment. This work was supported, in part, by a
grant from the National Institutes of Health, National Cancer
Institute (Grant CA136944 (A.G.)).
Supporting Information Available: Results from elemental
analysis and high-resolution mass spectrometry. This material is
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