Discovery and preclinical evaluation of 7-benzyl-N-(substituted)-pyrrolo[3,2-d]pyrimidin-4-amines as single agents with microtubule targeting effects along with triple-acting angiokinase inhibition as antitumor agents

Article abstract of DOI:10.1016/j.bmc.2016.11.026

The utility of cytostatic antiangiogenic agents (AA) in cancer chemotherapy lies in their combination with cytotoxic chemotherapeutic agents. Clinical combinations of AA with microtubule targeting agents (MTAs) have been particularly successful. The discovery, synthesis and biological evaluations of a series of 7-benzyl-N-substituted-pyrrolo[3,2-d]pyrimidin-4-amines are reported. Novel compounds which inhibit proangiogenic receptor tyrosine kinases (RTKs) including vascular endothelial growth factor receptor-2 (VEGFR-2), platelet-derived growth factor receptor-β (PDGFR-β) and epidermal growth factor receptor (EGFR), along with microtubule targeting in single molecules are described. These compounds also inhibited blood vessel formation in the chicken chorioallantoic membrane (CAM) assay, and some potently inhibited tubulin assembly (with activity comparable to that of combretastatin A-4 (CA)). In addition, some of the analogs circumvent the most clinically relevant tumor resistance mechanisms (P-glycoprotein and β-III tubulin expression) to microtubule targeting agents (MTA). These MTAs bind at the colchicine site on tubulin. Two analogs displayed two to three digit nanomolar GI₅₀values across the entire NCI 60 tumor cell panel and one of these, compound 7, freely water soluble as its HCl salt, afforded excellent in vivo antitumor activity against an orthotopic triple negative 4T1 breast cancer model and was superior to doxorubicin.

Full text of DOI:10.1016/j.bmc.2016.11.026

Accepted Manuscript
Discovery and Preclinical Evaluation of 7-benzyl-N-(substituted)-pyrrolo[3,2-
d]pyrimidin-4-amines as Single Agents with Microtubule Targeting Effects
along with Triple-acting Angiokinase Inhibition as Antitumor Agents
Roheeth Kumar Pavana, Shruti Choudhary, Anja Bastian, Michael A. Ihnat,
Raouli Bai, Ernest Hamel, Aleem Gangjee
PII:
S0968-0896(16)30958-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.026
BMC 13393
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 October 2016
8 November 2016
11 November 2016
Please cite this article as: Kumar Pavana, R., Choudhary, S., Bastian, A., Ihnat, M.A., Bai, R., Hamel, E., Gangjee,
A., Discovery and Preclinical Evaluation of 7-benzyl-N-(substituted)-pyrrolo[3,2-d]pyrimidin-4-amines as Single
Agents with Microtubule Targeting Effects along with Triple-acting Angiokinase Inhibition as Antitumor Agents,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.026
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Discovery and Preclinical Evaluation of 7-benzyl-N-(substituted)-pyrrolo[3,2-d]pyrimidin-
4-amines as Single Agents with Microtubule Targeting Effects along with Triple-acting
Angiokinase Inhibition as Antitumor Agents
Roheeth Kumar Pavana,a Shruti Choudhary, a Anja Bastian, b Michael A. Ihnat,c Raouli Bai,d
Ernest Hamel,d Aleem Gangjee a,*
aDivision of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne
University, Pittsburgh, PA 15282, gangjee@duq.edu, Fax: 412-396-5593
bDepartment of Physiology, University of Oklahoma College of Medicine, Oklahoma City, OK
73104
cDepartment of Pharmaceutical Sciences, University of Oklahoma College of Pharmacy,
Oklahoma City, OK 73117
dScreening Technologies Branch, Developmental Therapeutics Program, Division of Cancer
Treatment and Diagnosis, Frederick National Laboratory for Cancer Research, National Cancer
Institute, Frederick, MD 21702
Abbreviations: AA, antiangiogenic agents; MTA, microtubule targeting agents; VEGFR-2,
vascular endothelial growth factor receptor-2; PDGFR-β, platelet-derived growth factor receptor-
β; CAM, chorioallantoic membrane; CA, combretastatin A-4; Pgp, P-glycoprotein; EGFR,
epidermal growth factor receptor; TBS, Tris-buffered saline; HRP, horseradish peroxidase; PY,
phosphotyrosine
1

Abstract
The utility of cytostatic antiangiogenic agents (AA) in cancer chemotherapy lies in their
combination with cytotoxic chemotherapeutic agents. Clinical combinations of AA with
microtubule targeting agents (MTAs) have been particularly successful. The discovery, synthesis
and biological evaluations of a series of 7-benzyl-N-substituted-pyrrolo[3,2-d]pyrimidin-4-
amines are reported. Novel compounds which inhibit proangiogenic receptor tyrosine kinases
(RTKs) including vascular endothelial growth factor receptor-2 (VEGFR-2), platelet-derived
growth factor receptor-β (PDGFR-β) and epidermal growth factor receptor (EGFR), along with
microtubule targeting in single molecules are described. These compounds also inhibited blood
vessel formation in the chicken chorioallantoic membrane (CAM) assay, and some potently
inhibited tubulin assembly (with activity comparable to that of combretastatin A-4 (CA)). In
addition, some of the analogs circumvent the most clinically relevant tumor resistance
mechanisms (P-glycoprotein and β-III tubulin expression) to microtubule targeting agents
(MTA). These MTAs bind at the colchicine site on tubulin. Two analogs displayed two to three
digit nanomolar GI50 values across the entire NCI 60 tumor cell panel and one of these,
compound 7, freely water soluble as its HCl salt, afforded excellent in vivo antitumor activity
against an orthotopic triple negative 4T1 breast cancer model and was superior to doxorubicin.
Keywords
antiangiogenic, combination chemotherapy, receptor tyrosine kinase inhibitors, multitargeted
agents, microtubule targeting agents
2

1. Introduction
Angiogenesis is the process of formation of new blood vessels from pre-existing
vasculature. When a tumor grows beyond 2 mm3 it requires nutrients and oxygen for
maintenance and growth and initiates the process of acquiring additional blood supply.1 The
activation of receptor tyrosine kinases (RTKs) regulates signal transduction from the
extracellular domain of endothelial cells to the nucleus2 and represents the most important factor
for triggering angiogenesis. Under hypoxic conditions, the tumor secretes several proangiogenic
factors.1 The principal mediator of angiogenesis is the proangiogenic vascular endothelial growth
factor (VEGF) and its RTK, VEGFR-2.3 Rapid regrowth of tumor vasculature after the removal
of VEGFR-2 inhibitors from the treatment regimen attests to their cytostatic mechanism of
action and also promotes metastases.4, 5 Thus, VEGFR-2 inhibitors are known to cause the
regression of impaired tumor blood vessels but transiently normalize the surviving vessels,
which are less leaky and are more like normal blood vessels. VEGFR-2 inhibitors transiently
increase blood flow in the surviving vasculature.6 Administration of a cytotoxic agent during this
transient phase of tumor vasculature "normalization" provides improved delivery of the drug to
the tumor via the surviving vasculature. 6, 7 In combination chemotherapy, dosing separate
antiangiogenic and cytotoxic agents may miss the timing window of the transient normalization.8
Single molecular entities with both antiangiogenic and cytotoxic attributes allow the cytotoxicity
to be manifested as soon as the antiangiogenic effect and vasculature normalization occur. Such
single agents could circumvent pharmacokinetic problems of multiple agents, avoid drug-drug
interactions, and could be used at lower doses to minimize toxicities and tumor cell resistance
than two or more agents dosed separately in combination chemotherapy.
3

Microtubule targeting agents (MTAs) are among the most successful anticancer agents in
clinical use. These compounds produce tumor cytotoxic effects by interfering with microtubule
dynamics or by stabilizing or destabilizing tubulin polymerization.9 Several clinical trials are
ongoing for a variety of cancers including ovarian, breast, and lung, among others, and involve
FDA approved receptor tyrosine kinase inhibitors (RTKIs), as well as those in development, and
cytotoxic MTAs. These combinations include paclitaxel, docetaxel, or vinorelbine with RTKIs
bevacizumab, vandetanib, gefitinib, sorafenib, dasatinib or famitinib among others.10
It has been our interest to design single entities with RTKI attributes and cytotoxic
mechanisms of action.11-17 Our choice of MTAs as the mechanism for the cytotoxic component
was predicated, in part, on the finding that half of all human tumors have mutations in the p53
gene and p53 status effects vulnerability of tumor cells for cell cycle arrest. The most effective
drugs in cell lines with p53 gene mutations are MTAs.18, 19Additionally, there is clinical evidence
for the success of RTKIs in combination with MTAs.20-22
Development of resistance to chemotherapeutic agents is one of the major hallmarks of
cancer.23 Clinically significant mechanisms of resistance to MTAs, especially to paclitaxel, are
overexpression of the multidrug resistance protein P-glycoprotein (Pgp) and the β-tubulin
isoform βIII.24 With RTKIs, resistance to VEGFR-2 inhibition is associated with increased
platelet-derived growth factor receptor-β (PDGFR-β) expression in tumor endothelial cells,
increased recruitment of pericytes to tumor vasculature, and increases in other proangiogenic
factors.25 Similarly, epidermal growth factor receptor (EGFR) inhibition can lead to tumor
VEGFR-2 up-regulation, and this subsequently promotes tumor growth signaling independent of
EGFR and thus contributes to tumor resistance of EGFR inhibitors.26, 27 The effect of EGFR
inhibition can also be partially overcome by activation of PDGFR-β.26, 28 Due to the complexity
4

of angiogenic pathways, disrupting a single pathway of angiogenesis may not result in significant
clinical success. Multiple RTKs are co-activated in tumors and redundant inputs drive and
maintain downstream signaling, thereby limiting the efficacy of therapies targeting single
RTKs.26- 28 Moreover, high intratumoral heterogeneity has been reported with different
subpopulations producing distinct growth factors. 29-31 Thus, targeting a single RTK could be less
effective due to subpopulations of cells that are either not affected by the drug mechanism and
possess or acquire a greater drug resistance.32 Hence, targeting multiple angiokinases such as
VEGFR-2, PDGFR-β and/or EGFR maximizes the proportion of angiogenic signaling that is
effectively targeted. If such a multiple angiokinase inhibitor, which targets the principal
angiogenic growth factor receptors, could be structurally designed to include cytotoxic activity
by incorporating the effects of a MTA in single molecular entities, multi-agent combination
chemotherapy potential in single molecules could be realized. Such agents would be expected to
thwart RTK resistance mechanisms involving redundant angiogenic pathways and also provide a
cytotoxic MTA to effectively kill tumor cells.
We recently reported the preclinical evaluation of 7-benzyl-N-substituted-pyrrolo[3,2-
d]pyrimidin-4-amines as MTAs with VEGFR-2 inhibition.15, 33 In this study, we extend our
original reports to the design, discovery and preclinical evaluation of 7-benzyl-N-substituted-
pyrrolo[3,2-d]pyrimidin-4-amines with MTA and triple angiokinase (VEGFR-2, PDGFR-β,
EGFR) inhibitory activities in single molecules.
5

2. Rationale
Figure 1. Lead compounds
We previously reported that compound 1 (Fig. 1) containing the pyrrolo[3,2-d]pyrimidine
scaffold afforded potent MTAs.34 The on-going clinical trials of antiangiogenic agents in
combination with tumor-cytotoxic MTAs prompted the design and development of single agents
with both VEGFR-2 and tubulin inhibitory activities. As an initial study we appended the 7-
benzyl group, which dictates RTK inhibitory activity in substituted 7-benzyl pyrrolo[2,3-
d]pyrimidines of general structure 235 (Fig. 1), to the pyrrolo[3,2-d]pyrimidine scaffold of 1. Our
hybrid design to structurally engineer VEGFR-2 inhibitory activity into our existing MTA 1
afforded 3, a single agent with potent activities against both VEGFR-2 and tubulin assembly.15
Compound 3 reduced tumor size and vascularity in two flank xenograft models [the BLBC
MDA-MB-435 and U251 glioma models] and in a 4T1 triple negative breast orthotopic allograft
model, without overt toxicity to animals.15 A structure-activity study suggested that the methyl
group attached to the nitrogen bridge in the 4-position and the 4'-methoxy group in 3 were
crucial for inhibition of tubulin and removal of either of these moieties resulted in a complete
loss of the ability of the compound to inhibit tubulin assembly and also decreased VEGFR-2
inhibition in cells.15
6

Figure 2. Structures of compounds 4-10
In this study we explore the effect of conformational changes in compound 3 on the RTK
and tubulin inhibitory properties to extend our previous studies of agents which inhibit multiple
RTKs including VEGFR-2, PDGFR-β, and EGFR.11, 36, 37 Thus, it was of interest to explore the
effect of structural variations in 1 on activity against the RTKs VEGFR-2, PDGFR-β and EGFR,
in addition to cytotoxic MT effects with the goal of identifying single molecules as MTAs with
multiple angiokinase inhibitory potential. The clinical successes of the triple angiokinase
inhibitor nintedanib in combination with docetaxel38-41 makes a compelling argument for triple
angiokinase inhibitors with MTAs in single molecular entities. Compounds 4 - 8 (Fig. 2) were
designed as conformationally restricted analogs of 3. Compounds 4 and 5 incorporate the
bicyclic 6-methoxy-tetrahydroquinoline or the bicyclic 5-methoxy-dihydroindole respectively
onto the 4-position of the 2-methyl pyrrolo[3,2-d]pyrimidine. Compared to the N-methylaniline,
these bicyclics incorporated on the 4-position restrict the rotation of the phenyl ring, thus
affording more rigid structures than 3 while maintaining the phenyl and alkyl substitutions on the
N4 as in 3. Compounds 6 -8 were designed to determine whether the addition of a 5-methyl
group is conducive to microtubule targeting and RTKI activities. The 5-methyl group could
allow additional hydrophobic interactions with the targets and further restrict rotation around the
N-phenyl bond, thus providing further conformational rigidity which could increase potency.
7

Compounds 9 and 10 were designed to study the effect of a 2-amino group on multiple
RTK inhibition. We42 previously reported that a 2-amino group on fused pyrimidine ring systems
improves hinge region binding in the ATP-binding site of RTKs and provides multiple-RTK
inhibition. Compounds 9 and 10 replace the 2-methyl group in the dual acting, VEGFR-2
inhibiting, MTAs 3 and 5, respectively, with a 2-amino group that could potentially increase
binding to the hinge region of EGFR and/or PDGFR-β via hydrogen bonds, without loss of MT
and VEGFR-2 inhibitory activities.
To further support our structural modifications, we carried out molecular modeling
studies using MOE 2015.10, in which the proposed compounds 3-10 were docked in the x-ray
crystal structures of tubulin, 43 VEGFR-244 and EGFR45 and a homology model of PDGFR-β. 11
A
B
8

Figure 3. A) Superimposition of the docked pose of 3 (magenta) and colchicine (pink) in tubulin
(PDB ID: 4O2B).43 B) Superimposition of docked pose of 8 (green) and colchicine (pink) in
tubulin (PDB ID: 4O2B).43
Compounds 3‒10 were docked in the x-ray crystal structure of the colchicine site in tubulin
(PDB: 4O2B, 2.3 Å),43 using the parameters specified in the experimental section. Multiple low
energy conformations were obtained on docking. As representative examples, figure 3A shows
the docked conformation of 3 (magenta), superimposed on the co-crystallized ligand, colchicine
(pink). The pyrrolo[3,2-d]pyrimidine scaffold of 3 forms hydrophobic interactions with Alaα180,
Valα181, Leuβ248, Asnβ258 and Lysβ352 and partially occupies the region where the C-ring of
colchicine binds. 43 The benzylic group lies in the pocket lined by residues Serα178, Glnβ247,
Leuβ248 and Lysβ352. The N4-methyl interacts with Leuβ255, Asnβ258, Metβ259 and
Alaβ316. The phenyl group of 3 superimposes on the A ring of colchicine and interacts with
Cysβ241, Leuβ248, Alaβ250, and Leuβ255 and the 4'-methoxy interacts with Cysβ241, Leuβ242
and Leuβ255. In addition, the oxygen atom of the 4'-methoxy group lies within H-bonding
distance with a water molecule (3.16 Å for 3 and 3.11 Å for 8, figure 3B) present in the crystal
structure in the vicinity of Cysβ241. The best docked pose of 3 had a score of -8.82 kcal/mol.
Figure 3B shows the docked pose of 8 (green), superimposed on the crystallized ligand
colchicine (pink). It’s mode of binding in tubulin was similar to that of 3 and had a better docked
score of -9.50 kcal/mol compared to 3. Compounds 4-10, when similarly docked in the
colchicine site of tubulin, had scores that ranged between -8.82 and -9.50 kcal/mol and exhibited
similar modes of binding. These docking scores suggest that the designed analogs 4-10 should
have attributes of MTAs comparable to or better than those of 3.
9

A
B
Figure 4. A) Docked pose of 3 (magenta) in the binding site of axitinib (pink) in VEGFR-2
(PDB ID: 4AG8).44 B) Docked pose of 9 (green) in the binding site of axitinib (pink) in VEGFR-
2 (PDB ID: 4AG8).44 H-bond highlighted in red circle.
Since the design objective was to provide multiple RTKIs as well as MTAs in single molecules,
it was also of interest to dock compounds 4-10 in the RTKs VEGFR-2, EGFR and the homology
model of PDGFR-β with 3 as the lead compound. Figure 4A shows the docked conformation of
3 (magenta) superimposed on the co-crystallized ligand axitinib (pink) in the crystal structure of
VEGFR-2. 44 The pyrrolo[3,2-d]pyrimidine scaffold of 3 form hydrophobic interactions with
Val848, Lys868, Val916, Cys1045 and Phe1047. The N5-H of pyrrole ring undergoes H-bonding
interaction with backbone carbonyl of Asp1046. The 7-benzyl group interacts with Leu840,
Val848, Ala866, Leu1035 and Phe1047 and lies in the region occupied by the indazole group of
axitinib. The N4-methyl of 3 is oriented towards Leu889 and Val914. The N-phenyl group of 3
10

interacts with residues Leu889, Val899, Cys1045 and Asp1046. The best docked pose of 3 had a
score of -6.98 kcal/mol. Figure 4B shows the docked pose of 9 (green), as a representative
example, superimposed on the co-crystallized ligand axitinib (pink). The mode of binding of 9,
in VEGFR-2 was similar to that of 3, in addition the 2-amino of 9 formed H-bond interactions
with the backbone carbonyl of Ala866 and had a docked score of -7.77 kcal/mol, somewhat
better than the value obtained with 3. Compounds 4-10, when similarly docked into the crystal
structure of VEGFR-2, had scores ranging between -5.33 to -7.77 kcal/mol and exhibited similar
modes of binding as 3 and 9. These results indicate that, like 3, analogs 4-10 should inhibit
VEGFR-2.
A
B
11

Figure 5. A) Docked pose of 3 (magenta) in the binding site of gefitinib (pink) in EGFR (PDB
ID: 4WKQ).45 B) Docked pose of 9 (green) in the binding site of gefitinib (pink) in EGFR (PDB
ID: 4WKQ).45 H-bond highlighted in red circle.
Figure 5A and 5B shows the docked conformations of 3 (magenta) and 9 (green), respectively,
superimposed on the co-crystallized ligand gefitinib (pink) in the crystal structure of EGFR. 45
The pyrrolo[3,2-d]pyrimidine scaffold of 3 and 9 form hydrophobic interactions with Leu718,
Val726, Ala743, Thr790, Met793 and Leu844. The N3 atom in both 3 and 9 undergo a H-
bonding interaction with HOH1320. As predicted in our rationale, the 2-amino group in 9 makes
an additional H-bonding interaction in the hinge region with the carbonyl backbone of Gln791.
This additional bonding is anticipated to increase the potency of 9 as compared to 3. The
benzylic group of 3 and 9 lies in the region occupied by the morpholinopropoxy side chain of
gefitinib and interacts with Leu718, Gly796 and Leu844. The N4-methyl group of 3 and 9
interacts with Lys745 and Thr790, whereas the 4'-methoxyanilino group undergoes hydrophobic
interactions with the side chain carbons of Lys745, Thr854 and Asp855. The best docked pose of
3 had a score of -6.97 kcal/mol and, for 9, the score was -7.08 kcal/mol. Compounds 4-10 when
docked into EGFR had scores ranging between -6.68 to -7.30 kcal/mol and exhibited modes of
binding similar to those of 3 and 9 indicating that these analogs are expected to possess EGFR
inhibitory properties.
12

B
A
Figure 6. A) Docked pose of 3 (magenta) in the homology model of PDGFR-β.11 B) Docked
pose of 9 (green) in the homology model of PDGFR-β. H-bond highlighted in red circle.
In the absence of an x-ray crystal structure, docking of compounds 3 and 4-10 was carried out in
our validated homology model of PDGFR-β.11 The docked conformations of 3 (magenta) and 9
(green) in the homology model of PDGFR-β are shown in figure 6A and 6B, respectively, as
representative examples. The pyrrolo[3,2-d]pyrimidine scaffold of 3 and 9 form hydrophobic
interactions with Val614, Tyr683 and Gly687. The benzylic group interacts with residues
Leu606, Gly607, Val614 and Ala848. The N4-methyl group lies in a pocket lined by residues
Val665, Cys684 and Leu833 and the 4'-methoxyaniline moiety undergoes hydrophobic
interactions with Val614, Lys634, Ala632, Leu833, Phe845 and Ala848. The 2-amino group in 9
makes an additional H-bonding interaction in the hinge region with the carbonyl backbone of
Cys684 as compared with 3, as predicted.
3. Chemistry
13

Scheme 1. Reagents and conditions: (a) 5-methoxyindoline or 6-methoxy-1,2,3,4-
tetrahydroquinoline, isopropanol, reflux, 8-12 h (76-79%); (b) NaH, CH3Br, 3 h (84%); (c)
appropriately substituted aromatic amine, isopropanol, reflux, 4-24 h (74-79%)
The key intermediate 7-benzyl-4-chloro-2-methyl-5H-pyrrolo[3,2-d]pyrimidine 1115
(Scheme 1) was synthesized from benzaldehyde as previously described.15 Nucleophilic aromatic
substitution reactions of 11 with appropriately substituted amines in isopropanol at reflux
afforded compounds 4 and 5 in 76 and 79% yields, respectively. Compound 11 was deprotonated
with sodium hydride and methylated with methyl bromide to afford the N5-methylated
pyrrolo[3,2-d]pyrimidine 12, which, on treatment with appropriately substituted anilines in
isopropanol at reflux afforded compounds 6 and 7 in 74 and 79% yields, respectively. Treatment
of 12 with 6-methoxy-1,2,3,4-tetrahydroquinoline gave 8 in 76% yield. Displacement reactions
with the bulky nucleophile 6-methoxy-1,2,3,4-tetrahydroquinoline required longer reaction times
as compared to other amines for complete disappearance of starting material, as estimated from
TLC.
14

Figure 7. Comparison of 1H NMRs of 3, 6, 7 and 13
Comparison of 1H NMRs (in DMSO-d6) of the 4-N-CH3 compounds 3 and 7 with their
corresponding 4-NH analogs 1315 and 6, respectively, suggests a conformational preference of
the 4-anilino moiety. As shown in figure 7, the chemical shift of the N5-H proton in 3, compared
with that in 13, indicates that this proton in 3 is in the shielding zone of the 4-methoxyphenyl
ring and adopts the conformation shown in figure 7 in DMSO-d6, with the phenyl ring positioned
over the N5-H proton. Similarly, comparing the chemical shift of the N5-CH3 protons in 7 with
that in 6 suggests a deshielding and a similar conformational preference for 7 in DMSO-d6, as
shown in figure 7 with the phenyl ring positioned above the N5-CH3. These conformational
preferences from solution 1H-NMR in DMSO-d6 corroborate the molecular modeling
conformations in the docked poses of 3 and 7 in tubulin (Fig. 3) and 3 and 9 in VEGFR-2, EGFR
and PDGFR-β (Fig. 4, Fig. 5 and Fig. 6, respectively), suggesting that not only are these
conformations the most stable in solution but are also the preferred bound conformations in
tubulin as well as RTKs.
15

Scheme 2. Reagents and conditions: (a) trimethylacetic anhydride, 100 °C, 2 h (92%); (b) POCl3,
110 °C, 3 h; (c) 4-methoxy-N-methylaniline or 6-methoxy-1,2,3,4-tetrahydroquinoline,
isopropanol, reflux, 4-16 h; (d)1 N NaOH, CH3OH, reflux, 10 h (66-68%).
Chlorination of the 2-amino-4-oxo-pyrrolo[3,2-d]pyrimidine 1446 (Scheme 2) gave poor
yields (<20%). The subsequent aniline displacement reactions also did not proceed to
completion, and a separation of the product from the starting materials was tedious and required
extensive column chromatography. Hence, compound 14 (Scheme 2) was first protected with
trimethylacetic anhydride to provide amide 15 in 92% yield. The pivaloyl protected 4-oxo
pyrrolo[3,2-d] pyrimidine 15, with significantly improved solubility in organic solvents
compared to 14, was chlorinated with phosphorus oxychloride to generate the 4-chloro-
pyrrolo[3,2-d]pyrimidine 16, which was subjected to nucleophilic aromatic substitution reactions
using the appropriately substituted amines, followed by base catalyzed amide deprotection to
afford 9 and 10 in 66 and 68% yields, respectively.
16

4. Biological evaluations and discussion
4.1 Tubulin Assembly and RTK- inhibitory effects
Figure 8. Standards used in biological assays: A) RTKIs B) MTAs
Table 1: Inhibition of tubulin assembly, [3H]colchicine binding and cellular VEGFR-2, EGFR
and PDGFR-β RTKs
17

[3H]colchicine binding
% Inhibition ± SD
tubulin
assembly
A431
VEGFR-2
EGFR
PDGFR-β
COMPOUND
Cytotoxicity
IC50 (nM) ± SD
IC50 (nM) ± SD IC50 (nM) ± SD IC50 (nM) ± SD
IC50 (µM) ± SD
5 µM inhibitor 1 µM inhibitor
3∙HCl15
4∙HCl
21 ± 1
> 20
ND
ND
ND
ND
21.3 ± 3.2
>200
23.6 ± 5.8
167.1 ± 27.6
33.6 ± 5.6
2.6 ± 0.42
2.3 ± 0.30
4.9 ± 0.6
1.1 ± 0.2
47.1 ±5.0
ND
>200
>200
9.2 ± 0.8
ND
5∙HCl
3.3 ± 0.3
> 20
43 ± 3
ND
ND
38.7 ± 7.1
26.7 ± 4.6
33.0 ± 5.0
32.9 ± 4.9
27.3 ± 5.1
84.3 ± 3.9
ND
90.3 ± 18.3
7.2 ± 0.9
10.3 ± 1.7
30.2 ± 7.2
8.1 ± 0.91
>200
40.1 ± 5.6
2.3 ± 0.3
1.2 ± 0.07
8.1 ± 0.8
0.7 ± 0.09
ND
6∙HCl
ND
7∙HCl
0.48 ± 0.008
0.91 ± 0.03
> 20
92 ± 0.2
72 ± 2
ND
73 ±3
ND
8∙HCl
ND
9
13 ± 1
1.2 ± 0.01
ND
ND
ND
10
CA
99 ± 0.3
ND
90 ± 3
ND
ND
ND
semaxinib
sunitinib
erlotinib
doxorubucin
12.9
ND
ND
ND
ND
ND
ND
18.9 ± 2.7
ND
ND
83.1 ± 10.1
ND
ND
ND
ND
ND
1.2 ± 0.2
ND
ND
ND
ND
ND
ND
ND
1.35 ± 0.03
18

ND: not determined
19

Inhibition of tubulin assembly: Quantitative studies were conducted to evaluate the effects of
compounds 4 - 10 on the polymerization of purified bovine brain tubulin (Table 1). As
inhibitors of tubulin assembly, these compounds were compared with combretastatin A-4 (CA,
figure 8), the active metabolite of the water soluble prodrug combretastatin A-4 phosphate
(Zybrestat, Fosbretabulin), which has orphan drug status for ovarian cancer.47 Compounds 5, 7
and 8 were effective and potent inhibitors of bovine tubulin assembly, with activity comparable
to that of CA. Compound 5, the conformationally restricted tetrahydroquinoline analog of 3,
was almost 7-fold more potent than 3. In contrast with 5, conformational restriction using a 5-
membered ring (compound 4) was not favorable to inhibition of tubulin assembly. Compound 7,
the N5-methyl analog of 3, was the most potent inhibitor of tubulin assembly in this series. It
was almost 44-fold more inhibitory than 3 and almost 7-fold more inhibitory than 5, indicating
that N5-methylation is highly conducive for a strong interaction with tubulin. Clearly N5-
methylation of 3 to 7 affords further conformational restriction for the N-phenyl ring in addition
to providing hydrophobic interactions (Leuβ248 and Alaβ354) with the N5-methyl moiety
instead of a hydrogen donor at the pyrrole N5 in 3. In contrast, compound 8, the N5-methyl
analog of 5, wherein the N-phenyl bond is completely restricted via a tertrahydroquinoline ring,
was only 3.6-fold more potent against tubulin assembly than 5. However, compound 8 with
added conformational restriction via a N5-methyl over 5 was highly potent and only 2-fold less
potent than the most potent analog 7. As with previously described pyrimidine fused bicyclic
and tricyclic compounds, 15, 34, 48-50 the methyl group attached to the 4-nitrogen bridge is crucial
for inhibition of tubulin assembly. Removal of this N-methyl group (6 compared with 7)
resulted in significant loss of tubulin inhibitory activity. The 2-aminopyrrolo[3,2-d]pyrimidine
20

analogs 9 and 10 (as free bases) were less potent in the tubulin assembly inhibitory assay as
compared to the corresponding 2-methylpyrrolo[3,2-d]pyrimidine analogs 3 and 5, respectively,
indicating that the 2-amino was less important than the 2-methyl moiety for an interaction with
tubulin. This, along with the results for 9, indicates the importance of conformational restriction
via the N5-methylation in both 7 and 8 as being important for a strong interaction with tubulin.
However, further increase in conformational restriction, as in 4 with the 5-
methoxydihydroindole, abolished the interaction with tubulin. Thus for these analogs, limited
conformational restriction is an important facet for potent interactions with tubulin.
B
A
Figure 9. A) Superimposition of docked pose of 3 (cyan) and 7 (orange) in the colchicine site of
tubulin (PDB ID: 4O2B).43 B) Space fill view of selected residues (N5-methyl of compound 7,
Leuβ248 and Alaβ354) in the colchicine site of tubulin (PDB ID: 4O2B).43 The green surface
indicates hydrophobic regions and the pink surface indicates hydrophilic region in the pocket.
In order to rationalize the 40‒fold improvement in the inhibition of tubulin assembly when
comparing the activities of 3 and 7, their docked poses in the colchicine site of tubulin were
compared. Figure 9A and 9B shows the superimposed docked conformations of 3 and 7,
respectively, in the colchicine site. In figure 9, compound 7 retains all the other interactions at
21

the binding site as discussed above, and, in addition, the N5-methyl group of 7 undergoes
additional hydrophobic interactions with Leuβ248 and Alaβ354. The best docked pose of 7 had a
score of -9.21 kcal/mol. An additional reason for the overall improved binding of 7 over 3 can be
attributed to the further conformational restriction imposed on the rotation of the C4-N bond due
to the presence of the N5-methyl moiety in 7.
A conformational search carried out by a 1°rotation about C4-N bond using Sybyl-X
2.1.151 to compare the number of low energy conformations generated within 5 kcal/mol for 3
afforded 111 conformers, whereas for 7 only 53 conformers were possible. Similar improvement
in binding affinity by introducing methyl groups has been recently reviewed in the literature and
has been attributed to a combination of conformational, hydrophobic and desolvation effects.52 It
was also observed that 6 generated 112 conformers, whereas 7 generated 53 conformers. Thus,
the increased rotational flexibility about the C4-N bond might be one of the contributing factors
responsible for the loss of inhibitory activity against tubulin assembly for compound 6.
Compounds 5, 7 and 8, which showed potent tubulin inhibitory activities, were evaluated
for their ability to inhibit the binding of radiolabelled colchicine to tubulin (Table 1). These
compounds inhibited [3H]colchicine binding by percentages proportional to their tubulin
assembly IC50s, indicating that they are probably colchicine site MTAs, as is CA. 53
Inhibition of RTKs: Compounds 4 - 10 were evaluated for their activity against RTKs that are
overexpressed by tumor and endothelial cells (Table 1). Compounds 5 - 9 have potencies
comparable with standards sunitinib (clinically used) and semaxinib against VEGFR-2,
although these compounds are less potent than 3 (Table 1). The EGFR cellular potency of 5, the
conformationally restricted tetrahydroquinoline analog of 3, is comparable to 3. The N5-methyl
22

analogs 6 - 8, have significantly improved EGFR potencies compared to 3 and are also
comparable with clinically used erlotinib against EGFR. Compound 9, the 2-amino analog of 3,
is the most potent analog against EGFR and indicates the important contribution of the 2-amino
moiety to EGFR inhibitory potency. Although compound 5 was not significantly potent against
PDGFR-β, the N5-methyl analogs 6 - 8, along with 9, have potencies better than the clinically
used standard sunitinib against PDGFR-β. Replacing the 2-methyl group of 3 with a 2 amino
group (compound 9) resulted in better activity against both EGFR and PDGFR-β. These
activities were comparable to those of standard erlotinib and better than sunitinib, respectively,
perhaps due to improved binding to the hinge region of the ATP-pocket of these RTKs, as
predicted by our molecular modeling studies. Compounds 6 - 9 showed potent inhibition of
A431 tumor cells comparable to that obtained with doxorubicin.
23

4.2 Effect on Pgp overexpression and βIII-tubulin mediated resistance to paclitaxel
Table 2: Inhibition of growth of β-III and Pgp overexpressing cells.
β-III
Pgp
WT
HeLa
Parental
OVCAR-8
overexpressing
HeLa
overexpressing
NCI/ADR-RES
IC50 (µM) ± SD
COMPOUND
IC50 (µM) ± SD
IC50 (µM) ± SD
IC50 (µM) ± SD
5∙HCl
7∙HCl
8∙HCl
CA
250 ± 0
14 ± 2
250 ± 0
14 ± 1
120 ± 4
70 ± 10
60 ± 0
58 ± 2
1.8 ± 0.4
2.5 ± 0.7
1.3 ± 0.6
5.0 ± 1
1.3 ± 0.6
2,500 ± 0
Paclitaxel
Docetaxel
5.3 ± 2
4.0 ± 2
16 ± 1
13 ± 4
The ability of MTAs 5, 7 and 8 to overcome βIII-tubulin drug resistance was evaluated
by using an isogenic HeLa cell line pair (Table 2). While paclitaxel was 3-fold less potent in the
βIII-tubulin overexpressing cell line than in the wild type HeLa cells, compounds 5, 7 and 8
inhibited both cell lines with equal potency without regard to their expression of βIII-tubulin.
The ability of 5 to circumvent Pgp-mediated drug resistance was evaluated by using an
ovarian cancer cell line pair (Table 3). In this cell line pair, paclitaxel, which is a well-known
Pgp substrate, was 500-fold less potent in the Pgp overexpressing cell line than in the wild type
OVCAR-8 cells. Surprisingly, compound 5 is about 1.4-fold more potent in the Pgp
overexpressing cell line. Hence, compound 5 could have advantages over some clinically useful
24

microtubule targeting drugs, such as paclitaxel, docetaxel, vinblastine and vinorelbine, since 5 is
a poor substrate for transport by Pgp.
25

4.3 Antiproliferative effects in NCI 60 tumor panel
Table 3. Tumor cell inhibitory activity (NCI) GI50 (10-8 M) for 5 and 7
GI50 (10-8 M)
Compound
GI50 (10-8 M)
Compound
GI50 (10-8 M)
Compound
GI50 (10-8 M)
Compound
Panel/ Cell
line
Panel/ Cell line
Panel/ Cell line
Panel/ Cell line
5∙HCl 7∙HCl
5∙HCl 7∙HCl
5∙HCl 7∙HCl
5∙HCl 7∙HCl
Leukemia
CCRF-CEM
HL-60(TB)
K-562
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
Melanoma
LOX IMVI
MALME-3M
M14
Renal Cancer
786 - 0
22.6
22.2
11.1
34.5
26.6
92.2
3.35
2.68
3.37
5.08
3.69
3.38
23.2
31.8
36.8
36.6
2.63
7.11
3.50
3.81
3.51
4.45
3.84
52.1
33.0
25.2
34.1
5.88
1.77
2.95
2.14
2.86
4.87
5.42
7.82
47.5
93.4
19.7
83.0
34.1
21.2
61.8
12.3
39.0
9.32
2.02
17.2
5.66
2.54
8.87
A498
ACHN
MOLT-4
RPMI-8226
SR
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian
CAKI-1
RXF 393
SN12C
HT29
KM12
36.6
37.0
36.8
32.5
10.8
65.9
SW-620
TK10
NSCLC
A549/ATCC
EKVX
40.6
46.3
4.40
4.78
UO-31
20.9
CNS Cancer
SF-268
72.3
19.2
35.0
3.04
Prostate Cancer
HOP-62
37.9
SF-295
PC-3
4.96
4.44
cancer
HOP-92
18.9
13.6
32.0
4.71
30.4
4.58
SF-539
SNB-19
SNB-75
26.0
54.9
18.2
2.54
5.47
2.78
IGROVI
44.0
28.5
46.8
9.58
3.52
DU-145
38.3
NCI-H226
NCI-H23
OVCAR-3
OVCAR-4
Breast Cancer
MCF7
24.8
64.1
3.33
6.37
MDA-MB-
231/ATCC
HS 578T
NCI-H322M
36.2
7.36
U251
31.6
4.43
OVCAR-5
54.3
26.9
NCI-H460
NCI-H522
33.7
31.6
3.87
2.39
OVCAR-8
48.7
26.5
42.3
4.91
3.28
4.54
58.2
75.5
12.8
15.3
6.23
4.82
NCI/ADR-RES
SK-OV-3
BT-549
T-47D
MDA-MB-468
3.01
Compounds 5 (a tubulin and VEGFR-2 inhibitor) and 7 (a tubulin, VEGFR-2, EGFR and
PDGFR-β inhibitor) were selected for tumor cytotoxicity evaluation in the NCI 60 tumor cell
line panel (Table 3). Both compounds show 2- to 3-digit nanomolar GI50 values across all tumor
26

types, with 7 more active than 5. Antiangiogenic activities of these compounds are not pertinent
in these cell culture assays since there is no angiogenesis involved in these proliferation studies.
4.4 Antiangiogenic effects in CAM Assay
Table 4. Chorioallantoic membrane assay
IC50 [µM] ± SD
6∙HCl
7∙HCl
8∙HCl
9
8.7 ± 0.9
2.2 ± 0.4
26.1 ± 4.3
2.3 ± 0.3
1.3 ± 0.07
29.1 ± 1.9
sunitinib
erlotinib
Compounds 6 - 9 were evaluated for their effects on blood vessel formation in the chicken
chorioallantoic membrane (CAM) antiangiogenic activity assay against standards sunitinib and
erlotinib (Table 4). Compounds 6, 7 and 9, which inhibit VEGFR-2, EGFR and PDGFR-β,
were found to have strong antiangiogenic activities, comparable with those obtained with the
standards.
27

4.5 Effect of 7∙HCl on primary tumor growth and lung metastasis in the 4T1 orthotopic
breast tumor model
A.
B.
C.
4T1 lung metastases
animal weight
60
60
40
20
0
40
20
0
*
*
untreated 7•HCl
dox
untreated 7•HCl
Dox
Figure 10: Treatment with 7∙HCl decreased primary tumor growth in the 4T1 orthotopic
breast model.
[Legend]
(A) 4T1-luc2-GFP tumor bearing BALB/cJ mice were treated with 7∙HCl (IP 50 mg/kg, three
times weekly), or doxorubicin (dox) (IP 1 mg/kg, weekly) and tumor volumes were determined.
Statistical analysis was performed with two-way ANOVA repeated measures post test. (B)
Animal weights were graphed as percent weight change at day 40 over the starting weight.
Statistical analysis was performed with one-way ANOVA. (C) At the end of the experiment,
animals were euthanized and lungs removed and number of metastases per lung counted and
graphically represented. n =9 - 10, *P<0.05, **P<0.01 versus untreated control group.
Compound 7∙HCl was selected for in vivo studies, since it is a unique compound with cytotoxic
MTA effects and triple angiokinase (PDGFR-β, VEGFR-2 and EGFR) inhibitory mechanisms of
action in a single entity. The ability of 7∙HCl in reducing tumor growth and metastasis was
evaluated in a relevant triple negative breast cancer model in which metastasis can also be
assessed. The 4T1 triple negative orthotopic allograft model was used because it mimics human
28

metastatic progression and does not require the use of immunodeficient mice or Matrigel.
Treatment with 7∙HCl significantly reduced primary tumor growth as compared to untreated
control. In addition, 7∙HCl significantly reduced tumor growth compared to doxorubicin
treatment, which did not have a significant effect on tumor volume (Fig 7A). Compound 7∙HCl-
treated mice had similar weight gain as the untreated control group, indicating that 7∙HCl lacks
systemic toxicity. In contrast, doxorubicin-treated mice showed significantly less weight gain
compared to untreated control animals (Fig 7B). Next, the effect of 7∙HCl on lung metastasis was
evaluated by counting the number of GFP positive cell clusters. Compound 7∙HCl reduced the
average number of lung metastases better than did doxorubicin (Fig 7C), which is an important
finding since most cancer patients succumb to metastatic disease rather than the primary tumor.54
Compound 7 serves as a lead analog for further preclinical development, analog design and
synthesis for potential use in triple negative breast cancer.
29