Design and Synthesis of 1,2-Bis(hydroxymethyl)pyrrolo[2,1- a]phthalazine Hybrids as Potent Anticancer Agents that Inhibit Angiogenesis and Induce DNA Interstrand Cross-links

Article abstract of DOI:10.1021/acs.jmedchem.8b01689

Hybrid molecules are composed of two pharmacophores with different biological activities. Here, we conjugated phthalazine moieties (antiangiogenetic pharmacophore) and bis(hydroxymethyl)pyrrole moieties (DNA cross-linking agent) to form a series of bis(hydroxymethyl)pyrrolo[2,1-a]phthalazine hybrids. These conjugates were cytotoxic to a variety of cancer cell lines by inducing DNA damage, arresting cell cycle progression at the G2/M phase, triggering apoptosis, and inhibiting vascular endothelial growth factor receptor 2 (VEGFR-2) in endothelial cells. Among them, compound 29d encapsulated in a liposomal formulation (e.g., 29dL) significantly suppressed the growth of small-cell lung cancer cell (H526) xenografts in mice. Based on immunohistochemical staining, the tumor xenografts in mice treated with 29dL showed time-dependent decreases in the intensity of CD31, a marker of blood vessels, whereas the intensity of γ-H2AX, a marker of DNA damage, increased. The present data revealed that the conjugation of antiangiogenic and DNA-damaging agents can generate potential hybrid agents for cancer treatment.

Full text of DOI:10.1021/acs.jmedchem.8b01689

Subscriber access provided by Macquarie University
Article
Design and Synthesis of 1,2-Bis(hydroxymethyl)pyrrolo[2,1-a]phthalazine
Hybrids as Potent Anticancer Agents that Inhibit
Angiogenesis and Induce DNA Interstrand Crosslinks
Sue-Ming Chang, Vicky Jain, Tai-Lin Chen, Anilkumar Patel, Hima Bindu Pidugu, Yi-Wen Lin, Ming-
Hsi Wu, Jiao-Ren Huang, Han-Chung Wu, Anamik K. Shah, Tsann-Long Su, and Te-Chang Lee
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01689 • Publication Date (Web): 18 Feb 2019
Downloaded from http://pubs.acs.org on February 19, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Design and Synthesis of 1,2-Bis(hydroxymethyl)pyrrolo[2,1-
a]phthalazine Hybrids as Potent Anticancer Agents that Inhibit
Angiogenesis and Induce DNA Interstrand Crosslinks
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Sue-Ming Chang,†,# Vicky Jain,†,#, ¶ Tai-Lin Chen, Anilkumar S. Patel, Hima Bindu Pidugu,
†,#
†
†
†
†,¶
§
§
‡
Yi-Wen Lin, Ming-Hsi Wu, Jiao-Ren Huang, Han-Chung Wu, Anamik Shah, Tsann-Long
†
†
Su, * and Te-Chang Lee *
†Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
§Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan
‡Center of Excellence in Drug Discovery, Saurashtra University, Rajkot 360005, India
KEYWORDS: Pyrrolophthalazine hybrids, anti-angiogenesis, DNA crosslinking, VEGFR
inhibition, anticancer
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract:
Hybrid molecules are composed of two pharmacophores with different biological activities.
Here, we conjugated phthalazine moieties (anti-angiogenetic pharmacophore) and
bis(hydroxymethyl)pyrrole moieties (DNA crosslinking agent) to form a series of
bis(hydroxymethyl)pyrrolo[2,1-a]phthalazine hybrids. These conjugates were cytotoxic to a
variety of cancer cell lines by inducing DNA damage, arresting cell cycle progression at the G2/M
phase, triggering apoptosis and inhibiting vascular endothelial growth factor receptor 2 (VEGFR-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
) in endothelial cells. Among them, compound 29d encapsulated in a liposomal formulation (e.g.,
29dL) significantly suppressed the growth of small cell lung cancer cell (H526) xenografts in mice.
Based on immunohistochemical staining, the tumor xenografts in mice treated with 29dL showed
time-dependent decreases in the intensity of CD31, a marker of blood vessels, whereas the
intensity of -H2AX, a marker of DNA damage, increased. The present data revealed that the
conjugation of anti-angiogenic and DNA-damaging agents can generate potential hybrid agents
for cancer treatment.
2
ACS Paragon Plus Environment

Page 3 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Introduction
Anti-angiogenic therapy (AAT) is a strategy for treating cancer involving starving cancer cells
1
to death. Bevacizumab, a monoclonal antibody that neutralizes vascular endothelial growth factor
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1
A (VEGF-A), is an FDA-approved drug used for the treatment of metastatic solid tumors. Several
receptor tyrosine kinase inhibitors, which inhibit vascular endothelial growth factor receptors
1
(VEGFRs), are also approved for cancer treatment as AAT agents. Unfortunately, the rapid
appearance of resistance to AAT agents causes treatment failure and even promotes
aggressiveness.3, 4 However, AAT is still a relevant and promising strategy for fighting highly
5
aggressive cancers. Therefore, the development of novel AAT agents is desirable. Recently, a
large number of small molecule VEGFR inhibitors with diverse chemical scaffolds have been
6
synthesized and evaluated for their therapeutic potential in oncology. Some of these compounds,
such as sunitinib and sorafenib, were approved for the treatment of selected cancers.7, 8
Several phthalazine derivatives exhibited potent angiogenesis inhibition and anticancer
activities.9-13 Vatalanib (1) (Figure 1), an anilinophthalazine derivative,9, 14, 15 can inhibit all
VEGFRs, but it shows especially high selectivity for VEGFR-2, which is the principle endothelial
1
6
VEGF signaling receptor and primary mediator for tumor angiogenesis. This agent has been
included in several clinical trials on the treatment of metastatic colorectal cancer, pancreatic
adenocarcinoma and relapsed lymphoma.17-19 Substitution of the bioactive pharmacophores at
positions 1 and 4 of the phthalazine core, such as with compounds 2 and 3 (Figure 1), provided
derivatives with improved inhibitory activity toward VEGFR-2 and cancer cells.10-12, 20, 21 The
conjugation of phthalazine and a triazole ring was an alternative approach for generating
phthalazine-based VEGFR-2 inhibitors (4) with enhanced anticancer activity (Figure 1).13
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Moreover, phthalazine-based compound exerted diverse pharmacological activities, such as
AMG900 (5), which acts as a potent inhibitor of aurora kinase, and phthalazino[1,2-
b]quinazolinones (6), which is a p53 activator (Figure 1).22-24 These studies revealed important
information for phthalazine-based drug development.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
<
Fig. 1>
On the other hand, DNA crosslinking agents are clinically used for cancer chemotherapy,
2
5
particularly for late-stage and drug-resistant tumors. In general, designed DNA crosslinking
agents comprising two reactive electrophilic centers that attack DNA to form intrastrand or
interstrand crosslinks have potent cytotoxic activity as they can interfere with replication and
2
6
transcription, leading to cell death. Mitomycin C (7) (Figure 2), a DNA crosslinking therapeutic
frequently used in the clinic, possesses two reactive nucleophilic centers that are converted into
DNA interactive groups via bioreductive activation,27-30 and several naturally occurring
pyrrolizidine alkaloids, such as retrorsine and its metabolite, dihydropyrrolizine alcohol (8 and 9)
(
Figure 2), are able to induce DNA crosslinking without enzymatic activation.31-33 In addition, the
pyrrolizine core, as in 1,2-bis(hydroxymethyl)pyrrolizines (10) (Figure 2), is a privileged motif in
3
4
designing bifunctional DNA crosslinking agents with broad structural diversity. We designed
and synthesized several series of compounds containing pyrrolizine units that can form DNA
interstrand crosslinks (ICLs) (11-12) (Figure 2).35, 36
<
Fig. 2>
4
ACS Paragon Plus Environment

Page 5 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The design of hybrid molecules is a potential strategy for developing new drugs. Hybrid
molecules comprise two distinct biologically active domains that attack different targets, i.e., one
3
7
molecule that displays two modes of action. Thus, hybrid molecules may mimic the combination
therapies but with enhanced efficacy and reduced adverse side effects. We recently conjugated β-
carbolines and bis(hydroxymethyl)pyrroles (13) to form indolizino[6,7-b]indole (14) and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
indolizino[8,7-b]indole hybrids (15) and their bis(alkylcarbamates) for antitumor studies (Figure
40
2
).38, 39 The β-carboline component intercalates into DNA and inhibits topoisomerases, while the
bis(hydroxymethyl)pyrrole component induces DNA crosslink formation.41 Of these hybrid
1
2
3
molecules, BO-1978 (14, wherein R = Me, R = H, and R = Et) potently inhibits topoisomerase
I and II and induces DNA interstrand crosslinking (Figure 2),38 but it also exhibits potent
therapeutic efficacy against the growth of human non-small cell lung cancer (NSCLC)
4
2
xenografts. These studies support that conjugation of bis(hydroxymethyl)pyrrole with another
active pharmacophore is an effective strategy for identifying potent anticancer agents with dual
mechanisms.41
While angiogenesis as a hallmark of tumor development and metastasis is now a validated
4
3
44,
target for cancer treatment, the outcomes of AAT are still far from the desired overall benefits.
4
5
In the clinic, anti-angiogenic drugs are most effective when they are used in combination with
additional therapies, usually chemotherapy or immunotherapy.46-49 On the other hand, the adverse
5
0
effects of DNA crosslinking agents also limit their clinical application. The development of novel
cytotoxic drugs with increased efficacy but lowered incidence of adverse events is of great interest.
Therefore, we propose to generate novel hybrid anticancer agents, which may display both anti-
angiogenesis and DNA crosslinking properties and provide increased anticancer efficacy and
reduced toxicity.
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In the present work, we coupled a phthalazine base (16) bis(hydroxymethyl)pyrrol (13) to
form 1,2-bis(hydroxymethyl)pyrrolo[2,1-a]phthalazines (17) (Figure 3). To better understand the
structure-activity relationship (SAR), a series of new hybrids were synthesized with various
substituents, such as Me, Et, or a substituted aryl group at C3 and secondary amine side chains at
C6 (Figure 3). The systematic chemical modifications of the phthalazine derivatives allowed us to
determine the SAR through cytotoxicity analysis. In addition, these diols were converted to the
corresponding alkylcarbamates to understand whether the alkylcarbamate group could serve as a
better leaving group and favor the interaction with DNA over what was achieved with the OH
group. Experiments were also conducted to demonstrate that these novel hybrids not only
possessed significant anti-angiogenic activity in endothelial cells but also displayed potent DNA
crosslinking activity in cancer cells. Furthermore, we confirmed the in vivo anti-small cell lung
cancer (SCLC) activity of one of the derivatives using liposomes as a delivery system.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
<
Fig. 3>
Results and Discussion
Chemistry. As shown in Scheme 1, commercially available 1-phthalazinone (18) was treated
51
with phosphorus oxychloride to give 1-chlorophthalazine (19) by the reported procedure. The
reaction of 18 with morpholine in ethanol in the presence of TEA gave compound 20, which was
then reacted with trimethylsilyl cyanide (Me SiCN) and various alkyl or aryl acid chlorides in
3
dichloromethane (DCM) to afford compounds 21a-c. Treatment of 21a-c with tetrafluoroboric
acid (HBF ) in ether yielded the intermediate hydrofluoroborate salts, which were immediately
4
6
ACS Paragon Plus Environment

Page 7 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
reacted with dimethyl acetylenedicarboxylate (DMAD) to afford diester derivatives 22a-c52.
Diesters 22a-c were reduced to generate the corresponding 6-morpholinopyrrolo[2,1-
a]phthalazine dimethanol derivatives 23a-c by treatment with lithium aluminum hydride (LAH)
in a mixture of ether/DCM in an ice bath. Compounds 23a-c were further converted into their
corresponding bis(alkylcarbamate) congeners 24a-c (R2 = Et) or 25a-c (R = i-Pr) in good yields
by treatment with ethyl isocyanate or isopropyl isocyanate, respectively, in the presence of
trimethylamine (TEA). These derivatives (23a-c, 24a-c, and 25a-c) with alkyl (such as Me or Et)
and aryl (4’-MeO-Ph) substituents at C3 would allow us to study the effect of the substituent at C3
on the cytotoxicity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
<
Scheme 1>
Our preliminary antitumor screening showed that C3-Me derivatives (e.g., 23a, 24a, and 25a)
were the more cytotoxic than the corresponding C3-Et or C3-phenyl substituted compounds (e.g.,
2
3b-c, 24b-c, and 25b-c). We therefore focused on preparing C3-Me-pyrrolo[2,1-a]phthalazines
bearing various secondary amines at C6 for antitumor evaluation. As shown in Scheme 2,
compound 19 was first reacted with various secondary amines in ethanol in the presence of TEA
to obtain compounds 26a-d by the previously described method.53 As described above, the
reactions of compounds 26a-d with Me SiCN and acetyl chloride in the presence of a catalytic
3
amount of AlCl yielded compounds 27a-d, which were converted into the corresponding
3
hydrofluoroborate salts (tetrafluoroboric acid/ether) and then reacted with DMAD to give diesters
2
8a-d. The reductions of diesters 28a-d by treatment with LAH/ether/DCM afforded the
corresponding bis(hydroxymethyl) derivatives 29a-d. Compounds 29a-d were further converted
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
into their corresponding bis(alkylcarbamate) congeners (30a-d or 31a-d) in good yields by
treatment with ethyl isocyanate or isopropyl isocyanate, respectively, in the presence of
trimethylamine (TEA). These derivatives (29a-d, 30a-d, and 31a-d) with Me at the C3 position
and different secondary amine side chains at C6 would allow us to study the effect of the
substituent at C6 on the cytotoxicity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
<
Scheme 2>
Biological data. For structure-activity relationship (SAR) analysis, the in vitro cytotoxicity
of the 1,2-bis(hydroxymethyl)pyrrolo[2,1- a]phthalazines (23a-c, 24a-c, and 25a-c) with
1
morpholine substituents at C6 (R = morpholine) was first examined in human lymphoblastic
leukemia CCRF-CEM (Table 1). In these derivatives, different sized functional groups at C3 (e.g.,
2
R = Me, Et, or 4’-MeO-Ph) were introduced. The SAR analysis clearly showed that increasing
the size of the substituent at the C3 position generally attenuated the cytotoxicity of the
bis(hydroxymethyl)pyrrolo[2,1-a]phthalazine derivative toward CCRF-CEM cells (i.e., 4’-MeO-
Ph < Et < Me), except that the C3-4’-MeO-Ph-substituted compound (25c) was slightly more
cytotoxic than the C3-Et-substituted derivative (25b). Although good leaving groups were
3
introduced to C1 and C2 (i.e., R = ethyl or iso-pro carbamate) (24a-c or 25a-c), their cytotoxicity
toward leukemia cells was similar to those of the corresponding compounds with a bis-diol moiety
at C1 and C2 (23a-c).
8
ACS Paragon Plus Environment

Page 9 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Since C3-Me-substituted bis(hydroxymethyl)pyrrolo[2,1-a]phthalazine derivatives (23a, 24a,
and 25a) were generally more cytotoxic than those with ethyl or 4’-MeO-Ph substituents at the C3
position, C3-Me-substituted bis(hydroxymethyl)pyrrolo[2,1-a]phthalazine derivatives with
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
various secondary aliphatic and heterocyclic amines at C6 (e.g., R = dimethylamine, pyrrolidine,
piperidine, and 1,4’-bipiperidine) (29a-d, 30a-d, and 31a-d) were synthesized. Together with
morpholine substituents at C6, compounds with different C6 substituents, i.e., those in different
series of derivatives, such as bis-diol (23a and 29a-d), bis-ethylcarbamate (24a and 30a-d), and
bis-iso-pro carbamate (25a and 31a-d), did not exhibit consistent cell killing potency against
CCRF-CEM cells (Table 1). However, among those with various secondary aliphatic and
heterocyclic amines at C6, the derivatives with morpholine substituents at C6 were generally the
most cytotoxic toward leukemia cells. Intriguingly, a bipiperidine substituent at C6 of a bis-diol
derivative (compound 29d) afforded the most potent compound in this study; its IC against
5
0
CCRF-CEM cells was 0.23  0.03 M. Compound 29d was thus selected for mechanistic and
animal studies.
Furthermore, as shown in Table 1, these newly synthesized compounds were 2.5- to 72-fold
more cytotoxic to CCRF-CEM cells than cisplatin, and more than 90% of them had IC values
5
0
less than 5 M. In addition, the CCRF-CEM/VBL cell line, an acquired multidrug resistant (MDR)
cell line featuring P-glycoprotein overexpression, was used to determine whether these newly
synthesized compounds displayed cross-resistance to MDR reagents. Compared to the parental
CCRF-CEM cells, CCRF-CEM/VBL cells were first confirmed as remarkably resistant cells to
vinblastine (Table 1). However, CCRF-CEM/VBL cells showed no obvious cross-resistance to
any of the newly synthesized molecules. Some of the new compounds were even more cytotoxic
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
to CCRF-CEM/VBL than they were to parental CCRF-CEM. The range of the resistant factor (RF)
values was 0.43–1.48. Among all compounds examined, compound 29d, with RF = 1.09, was the
most cytotoxic toward drug-resistant CCRF-CEM/VBL cells.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
<
Table 1 >
In addition to leukemia cells, several compounds were selected for further evaluation of their
antiproliferative activity in a number of human solid tumor cell lines, including colorectal cancer
HCT-116 cells, pancreatic cancer PaCa-S1 cells, NSCLC H460 cells, and SCLC H526 cells (Table
2). Among 23a-c, compound 23a displayed the most activity toward these four solid tumor cell
lines, consistent with the previous observation that smaller substituents at C3 result in stronger
cytotoxicity. Intriguingly, SCLC H526 cells were the most susceptible to the tested compounds;
the IC values ranged from nanomolar to submicromolar levels. Of the compounds examined,
5
0
compound 29d exhibited highly potent antitumor activity against a broad spectrum of tumor cell
lines.
<
Table 2 >
The pyrrolo[2,1-a]phthalazine hybrid molecules were designed by assembling a DNA
crosslinking moiety and an anti-angiogenic moiety. This DNA crosslinking moiety,
bis(hydroxymethyl)pyrrole, has been widely used as a scaffold for the development of a variety of
therapeutic agents.34, 54 The two adjacent hydroxymethyl groups serve as leaving groups and lead
to the formation of electrophilic centers that attack DNA to form crosslinks. The alkaline gel
10
ACS Paragon Plus Environment

Page 11 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
electrophoresis was performed to demonstrate the DNA ICL-inducing activity of the pyrrolo[2,1-
a]phthalazine derivatives. As shown in Figure 4A, compound 29d dose-dependently induces a
significant amount of DNA ICLs in the concentration range of 0.1 to 1 M, while compound 23b
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
required higher concentrations (> 10 M) to achieve the same effect. These results showed the
consistency between the cytotoxicity toward cancer cell lines and in vitro DNA crosslinking
activity of the pyrrolo[2,1-a]phthalazine derivatives, confirming that the induction of DNA ICLs
is one of the mechanisms causing cell death.
While cell cycle interference occurs in cells treated with DNA crosslinking agents, the effects
of compound 29d were further investigated on cell cycle progression in H460 cells. As shown in
Figure 4B and Supporting Information Figure S1, cell cycle progression was obviously influenced
by compound 29d. By increasing the concentration of compound 29d, the G2/M phase
accumulation was observed at low concentrations (0.125 and 0.25 M), the S phase at a moderate
concentration (0.5 M), and the G1 phase at high concentrations (1 and 2 M). Accordingly, the
pattern of cell cycle interference induced by compound 29d was typically attributed to the
formation of DNA crosslinks.38, 39 In addition, we noticed time- and dose-dependent accumulation
of the Sub-G1 population, which is an indicator of apoptotic cells. Furthermore, it was confirmed
that compound 29d induced apoptotic cell death by using an Annexin V staining assay (Supporting
+
Information, Figure S2). As summarized in Figure 4C, Annexin V cells (the first and fourth
quadrants) were significantly and dose-dependently increased in H460 cells treated with various
concentrations of compound 29d. Together, these results indicate that compound 29d induces
DNA damage and hence interferes with cell cycle progression, triggering apoptotic cell death.
<
Fig. 4>
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
There are several critical steps involved in angiogenesis and blood capillary formation, such
as endothelial cell survival, proliferation, migration, organization, and remodeling into a capillary-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
55
like structure. Tumor-induced angiogenesis is facilitated by the production of a number of
angiogenic factors.56 Approximately 30 endogenous pro-angiogenic factors are known to
participate in angiogenesis. These hybrids were designed to challenge traditionally combinational
therapies using anti-angiogenic and DNA interstrand crosslinking agents. To demonstrate the
antiangiogenic properties of these hybrids, the cytotoxicity of compounds 29d and 23b and
vatalanib were first evaluated toward human endothelial EA.hy926 cells. As shown in Supporting
Information Figure S3, compound 29d was more cytotoxic to EA.hy926 cells (IC = 1.41  0.31
5
0

M) than were compound 23b (IC = 8.84  3.89 M) and vatalanib (IC = 19.98  7.54 M).
5
0
50
However, compared with the IC values of these compounds against the four human solid tumor
5
0
cell lines analyzed, compounds 29d and 23b were approximately 5- to 100-fold less toxic to
EA.hy926 cells.
5
6
VEGF, the most potent mediator of angiogenesis, stimulates endothelial cell survival,
proliferation, migration, and invasion via activation of specific tyrosine kinase receptors,
5
7
VEGFRs, through autophosphorylation. The in vivo angiogenic response to VEGF in tumors
5
8
is mainly mediated via the activation of VEGFR-2. Phthalazine is the core structure of vatalanib,
9
and it is the moiety that binds VEGFR-2 and inhibits its activation. We therefore performed
western blotting analysis to examine the levels of VEGFR-2 and phosphorylated VEGFR-2 (p-
5
9
VEGFR-2) in endothelial EA.hy926 cells treated with 29d, 23b, and vatalanib for 12 h. As shown
in Figure 5, these compounds at noncytotoxic doses did not significantly change the protein
levels of VEGFR-2 but dose-dependently suppressed p-VEGFR-2 protein levels, indicating that
12
ACS Paragon Plus Environment

Page 13 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
9d and 23b could inhibit VEGFR-2 activation to a similar extent as achieved with vatalanib.
However, compound 29d was more potent than 23b and vatalanib in decreasing p-VEGFR-2.
Since one of the main effects of the VEGFR pathway is to enhance the migration of endothelial
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
cells, it was observed that compound 29d inhibited the migration of EA.hy926 cells at the same
dose range in which VEGFR-2 activation was inhibited using the transwell migration assay
(Supporting Information, Figure S4). These results support that our hybrid designs retain the ability
to inhibit VEGFR-2.
< Fig. 5>
Angiogenesis is the process of generating new blood vessels as extensions of the existing
vasculature. Evaluating the vascular behavior and monitoring tube formation are generally used to
6
0
experimentally determine whether a compound is capable of interrupting angiogenesis. A tube
formation assay using human endothelial EA.hy926 cells was conducted to compare the
antiangiogenic activity of newly synthesized hybrids 29d with vatalanib. The results (Figure 6A)
show that robust tubular structures were formed in the control culture, whereas preincubation with
2
9d or vatalanib dose-dependently decreased tube formation. However, compound 29d was
approximately 50-fold more potent than vatalanib in suppressing tube formation (Figure 6B),
indicating that hybrid compound 29d obviously retained antiangiogenic activity by the inhibition
of VEGFR-2 activation.
<
Fig. 6>
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Based on in vitro data, the therapeutic efficacy of compound 29d was subsequently evaluated
in nude mice bearing human SCLC H526 xenografts. To overcome the poor solubility of 29d, a
liposomal formulation was successfully developed to encapsulate compound 29d (29dL).
Liposomal 29dL was prepared by mixing 29d with DSPC, SPC, cholesterol, and PEG-2000. For
the antitumor experiments, mice bearing H526 tumors were intravenously (i.v.) administered
various doses of liposomal 29dL via the tail vein every other day 6 times (Q2D6). As shown in
Figure 7A, the average volume of the H526 tumors was dose-dependently decreased by treatment
with 29dL. At the maximal achievable dose (10 mg/kg), liposomal 29dL suppressed tumor growth
by approximately 55% on D30. In addition, no body weight change was observed in mice treated
with 29dL, indicating that mice could tolerate 29dL at the doses used. Then, the efficacy of 29dL
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
10 mg/kg, QD9 via i.v. injection) was compared with that of vatalanib (100 mg/kg, QD9 given
orally) and cisplatin (4 mg/kg, Q4D3 via i.v. injection). The results of Figure 7B revealed that
9dL was more potent than vatalanib and almost as efficacious as cisplatin on D33, i.e., it
2
suppressed H526 tumors by approximately 60%. Notably, liposomal 29dL at the tested dosages
did not cause body weight loss, that further supports its low toxicity, whereas approximately 25%
body weight loss was observed in cisplatin-treated mice (Figure 7B). These results suggest that
liposomal 29dL potently suppresses H526 xenografts but is less systematically toxic.
< Fig. 7>
In a separate experiment, tumors at days 3, 6, and 9 were excised after drug treatment for
histopathological examination. Tumor xenograft tissue sections were immunohistochemically
(IHC) stained with primary antibodies (anti-rabbit CD31 antibody and anti-mouse γ-H2AX
antibody) followed by HRP-conjugated secondary antibody. The intensities of the anti-CD31 and
14
ACS Paragon Plus Environment

Page 15 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
anti-γ-H2AX staining indicated the blood vessel density and the DNA damage, respectively. As
shown in Figure 8A, significantly reduced blood vessel formation was observed in tumors treated
with 29dL, vatalanib, or cisplatin compared to that in the vehicle control. The CD31 intensity
increased in a time-dependent manner in untreated tumors, whereas on day 9, the intensity was
reduced to 4% in tumors treated with 29dL or vatalanib (Figure 8A). Since cisplatin at the tested
dose also significantly suppressed tumor growth, decreased CD31 expression in cisplatin-treated
tumors was also observed but to a lesser extent than what was seen in those treated with 29dL or
vatalanib. These results indicated that 29dL was almost as potent as vatalanib in inhibiting
angiogenesis in an in vivo system.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Since compound 29d is also a DNA-damaging agent, remarkably increased signals of γ-H2AX
were observed in tumors treated with cisplatin or 29dL, but the signals increase by much less in
tumors treated with vatalanib (Figure 8B). The quantitative analysis data are shown in Figure 8B.
Signals for γ-H2AX were almost absent in the control tumor. However, it was observed that time-
dependent increases in the γ-H2AX signals in tumors treated with cisplatin and 29dL. In tumors
treated with vatalanib, a moderate signal from γ-H2AX was observed, which may be indirectly
caused by the inhibition of blood vessel formation by vatalanib. Taken together, compound 29dL
displays unique activity, e.g., this hybrid compound may kill cancer cells via DNA damage and
suppress blood vessel formation via the inhibition of VEGFR-2.
< Fig. 8>
Conclusions
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Antiangiogenic therapy for patients with various types of cancers initially shows clinical
benefits, but those benefits rapidly disappear because most patients became refractory or acquire
6
1
resistance to angiogenic inhibitors. In animal studies, insufficient doses of VEGF blockers were
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
62
apt to result in vascular regrowth and limit the tumor-suppression effects. Furthermore, several
studies have shown that VEGF blockades suppressed not only tumor vessel formation but also
healthy vessel formation, resulting in life-threatening problems such as hemorrhagic and
thrombotic events.63, 64 The identification of novel anti-angiogenic therapies specific for tumors
that do not impact the signaling pathways essential for the maintenance of healthy vessels is of
utmost importance.
Combination therapy has been frequently used to treat patients with cancer. Since each drug
has its own ADME characteristics, it remains to be a great challenge to overcome the
pharmacology and toxicology of various drugs given separately. It may also limit the designs of
hybrid molecules. However, we have successfully synthesized a series of new compounds with
potential anticancer effects. They may simultaneously target cancer cells and tumor
microenvironment. Of these novel 1,2-bis(hydroxymethyl)pyrrolo[2,1-a]phthalazine hybrids, we
found that compound 29d was able to inhibit the phosphorylation of VEGFR, leading to the
suppression of vascular formation, and induce DNA crosslinking, resulting in cell cycle arrest
followed by apoptosis. Furthermore, compound 29d exhibited significant antitumor and
antivascular properties in tumor xenograft models. Compound 29d is composed of two different
biological pharmacophores allowing it to target cancer cells by inducing DNA damage and
endothelial cells by suppressing VEGFR activation. Thus, compound 29d is a promising new
compound with potential as an antitumor and antivascular agent that could be superior to common
combinational anticancer therapies.
16
ACS Paragon Plus Environment

Page 17 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Experimental Section
General. All commercial chemicals and solvents were reagent grade and were used without
further purification unless otherwise specified. Melting points were determined in open capillaries
on a Fargo MP-2D melting point apparatus. Thin-layer chromatography (TLC) analyses were
performed on silica gel 60 F254 (Merck KGaA, Darmstadt, Germany) with shortwavelength
ultraviolet (UV) light for visualization. High-performance liquid chromatography (HPLC) was
performed on an Elite Lachrom instrument (HITACHI) with a Mightysil RP-18 (250×4.6 mm)
column. Compounds were detected by UV light at 260 nm. The mobile phase was acetonitrile/THF
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(80:20 v/v) at a flow rate of 1 mL/min. The purities of all tested compounds were ≥95% based on
1
13
analytical HPLC. H NMR spectra and C NMR spectra were taken on Bruker AVANCE Top-
Spin spectrometers (400 or 500 MHz) in the solvents indicated. Proton chemical shifts (δ) are
reported in parts per million (ppm) relative to (CH ) Si (TMS), and coupling constants (J) are
3
4
reported in Hertz (Hz). NMR peak splittings are given by the following abbreviations: s, singlet;
d, doublet; t, triplet; m, multiplet; and br, broad. Most of the 1,2-bis(hydroxymethyl)pyrrolo[2,1-
a]phthalazine analogs were characterized by high-resolution mass spectrometry (HRMS) on a
Waters HDMS G1 instrument with ESI+ in centroid mode, and the samples were dissolved in
MeOH. Notably, some of the carbamate analogs were decomposed by the ESI+ ion source.
However, these carbamate phthalazine analogs were characterized using a Thermo Scientific
Orbitrap Elite Mass Spectrometer with a low-power ESI+ source. The HPLC chromatograms and
1
13
H NMR, C NMR and HRMS spectra of the new compounds are presented in Supporting
Information Figure S5.
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
65
1
-Chlorophthalazine (19). A mixture of phthalazin-1(2H)-one (18, 5.0 g, 34.0 mmol) and
phosphorus oxychloride (POCl ) (25 mL) was heated with stirring at 100°C for 2 h. After cooling
3
to rt, the excess POCl was completely removed by distillation under reduced pressure. The residue
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was triturated with toluene (2  25 mL) followed by THF (100 mL). The solid product was
collected by filtration and washed with THF. The solid product was then dissolved in DCM,
washed with saturated aqueous NaHCO solution, dried over sodium sulfate, and concentrated
3
65
1
under reduced pressure to give 19. Yield 4.6 g (82%); mp 119–121°C (lit. mp 132–134°C). H
1
3
NMR (DMSO-d ) δ 8.18–8.22 (2H, m, ArH), 8.31–8.35 (2H, m, ArH), 9.73 (1H, s, ArH). C
6
+
NMR (DMSO-d )  124.8, 126.1, 128.4, 128.7, 135.0, 151.5, 155.3. HRMS [ESI ]: calculated for
6
+
C H ClN , 165.0220 [M+H] , found 165.0212.
8
5
2
4
-(Phthalazin-1-yl)morpholine (20).66 Morpholine (1.89 mL, 22.0 mmol) was added
dropwise to a solution of 19 (3.64 g, 20.0 mmol) in ethanol (120 mL) containing triethylamine
6.96 mL, 50.0 mmol). The reaction mixture was heated at reflux for 18 h, and the solvent was
(
removed under reduced pressure to give a dry residue. The residue was cooled to rt. The obtained
crude material was diluted with water and extracted twice with DCM. The separated organic layer
was dried over sodium sulfate and concentrated in vacuo to give (20). Brown solid; yield 3.8 g
1
(
80%); mp 125–127°C H NMR (DMSO-d ) δ 3.40 (4H, t, J = 4.8 Hz, 2 × CH ), 3.88 (4H, t, J =
6
2
1
3
4
.8 Hz, 2 × CH ), 7.94–7.97 (2H, m, ArH), 8.09–8.14 (2H, m, ArH), 9.31 (1H, s, ArH). C NMR
2
(
DMSO-d )  51.4, 51.7, 66.5, 120.7, 124.4, 124.5, 127.5, 128.5, 132.4, 132.5, 148.4,
6
+
+
159.7. HRMS [ESI ]: calculated for C H N O, 216.1137 [M+H] , found 216.1094.
1
2
13
3
2-Acetyl-4-morpholino-1,2-dihydrophthalazine-1-carbonitrile (21a). To a solution of 20
(
2.0 g, 9.3 mmol) in DCM (30 mL) containing a catalytic amount of AlCl was added dropwise
3
18
ACS Paragon Plus Environment

Page 19 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Me SiCN (2.32 mL, 18.6 mmol). Acetyl chloride (1.0 mL, 14.0 mmol) was then added dropwise
3
to the above mixture, and the reaction was stirred for 4 h at rt. The reaction mixture was poured
into ice water, and the organic layer was washed sequentially with water, 5% NaOH solution and
water. The solution was dried over sodium sulfate and concentrated under vacuum to give 21a.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Yield 2.36 g (89%); mp 140–142°C. H NMR (CDCl ) δ 2.32 (3H, s, COCH ), 3.13–3.18 (2H, m,
3
3
CH ), 3.44–3.49 (2H, m, CH ), 3.81–3.86 (2H, m, CH ), 3.94–3.98 (2H, m, CH ), 6.70 (1H, s,
2
2
2
2
13
CH), 7.42–7.44 (1H, m, ArH), 7.53–7.59 (3H, m, ArH). C NMR (CDCl )  16.9, 49.8, 55.8,
3
+
6
6.7, 116.4, 121.5, 124.0, 123.7, 129.2, 130.3, 132.8,155.5, 169.6. HRMS [ESI ]: calculated for
+
C H N O , 285.1352 [M+H] , found 285.1341.
1
5
16
4
2
Compounds 21b and 21c were prepared following the synthetic procedure used to prepare 21a.
-Morpholino-2-propionyl-1,2-dihydrophthalazine-1-carbonitrile (21b). Compound 21b
4
was prepared from 20 (2.0 g, 9.3 mmol), Me SiCN (2.3 mL, 18.6 mmol) and propionyl chloride
3
1
(1.2 mL, 14.0 mmol). Yield 2.46 g (89%); mp 146–148°C. H NMR (DMSO-d ) δ 1.05 (3H, t, J
6
=
5.6 Hz, CH ), 2.53–2.58 (1H, m, CH), 2.71–2.75 (1H, m, CH), 3.03 (2H, m, CH ), 3.35 (2H, m,
3 2
CH ), 3.70 (2H, m, CH ), 3.87 (2H, m, CH ), 7.04 (1H, s, CH), 7.64 (3H, m, ArH), 7.80 (1H, m,
2
2
2
ArH). 13C NMR (DMSO-d )  49.8, 51.8, 65.7, 116.4, 121.5, 124.0, 123.7, 129.2, 130.3,
6
+
+
1
32.8,155.5, 169.6. HRMS [ESI ]: calculated for C H N O , 299.1508 [M+H] , found 299.1519.
16 18 4 2
2
-(4-Methoxybenzoyl)-4-morpholino-1,2-dihydrophthalazine-1-carbonitrile
(21c).
Compound 21c was prepared from 20 (3.0 g, 14.0 mmol), Me SiCN (3.5 mL, 28.0 mmol) and p-
3
1
anisoyl chloride (2.26 mL, 16.0 mmol). Yield 4.2 g (87%); mp 162–164°C; H NMR (DMSO-d )
6
δ 2.92–2.96 (2H, m, CH ), 3.23–3.26 (2H, m, CH ), 3.69–3.71 (2H, m, CH ), 3.80–3.83 (2H, m,
2
2
2
CH ), 3.83 (3H, s, OCH ), 6.99–7.01 (2H, m, ArH), 7.05 (1H, s, CH), 7.70–7.73 (5H, m, ArH),
2
3
13
7
.87–7.89 (1H, m, ArH). C NMR ((DMSO-d )  42.1, 49.8, 55.8, 66.0, 113.6,116.8, 121.2, 124.8,
6
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
1
26.7, 127.7, 129.6, 130.8, 132.8, 132.9, 155.0, 162.4, 169.0; HRMS [ESI ]: calculated for
+
C H N O , 377.1614 [M+H] , found 377.1623.
2
1
20
4
3
Dimethyl-3-methyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-dicarboxylate (22a). To
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a solution of 21a (2.0 g, 7.0 mmol) in warm acetic acid (50 mL) was added dropwise HBF (1.55
4
mL). The mixture was allowed to stir at 50–60°C for 30 min. After cooling to rt, the yellow solid
salt was collected by filtration, and the filter cake was washed with dry ether. The solid salt was
dissolved in DMF (20 mL), and DMAD (1.6 mL, 13 mmol) was slowly added to this solution. The
reaction mixture was heated at 90–100°C for 16 h. The solvent was removed by evaporation in
vacuo. The residue was crystallized from MeOH to give 22a. Yield 1.5 g (56%); mp 182–183°C.
1
H NMR (DMSO-d ) δ 2.64 (3H, s, CH ), 3.30–3.31 (4H, m, 2 × CH ), 3.80 (3H, s, COOCH ),
6
3
2
3
3
.88 (3H, s, COOCH ), 3.87–3.88 (4H, m, 2 × CH ), 7.62–7.65 (1H, m, ArH), 7.79–7.82 (1H, m,
3 2
1
3
ArH), 8.05–8.07 (1H, m, ArH), 8.24–8.26 (1H, m, ArH). C NMR (DMSO-d )  13.9, 52.4, 52.7,
6
55.6, 66.2, 106.7, 115.8, 117.7, 121.0, 124.6, 126.7, 128.9, 130.3, 133.0, 156.2, 159.7, 165.6,
+
+
1
65.9; HRMS [ESI ]: calculated for C H N O , 406.1379 [M+Na] , found 406.1385.
20 21 3 5
Compounds 22b and 22c were prepared following the synthetic procedure used to prepare 22a.
Dimethyl 3-ethyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-dicarboxylate (22b).
Compound 22b was prepared from 21b (2.0 g, 6.7 mmol), HBF (1.30 mL) and DMAD (1.5 mL,
4
1
1
2.0 mmol). Yield 1.6 g (60%); mp 182–183°C. H NMR (DMSO-d ) δ 1.21 (3H, t, CH ), 3.14–
6
3
3.15 (2H, m, CH ), 3.30 (4H, m, 2 × CH ), 3.80 (3H, s, COOCH ), 3.87 (7H, m, 2 × CH and
2
2
3
2
COOCH ), 7.63 (1H, m, ArH), 7.80 (1H, m, ArH), 8.05–8.06 (1H, m, ArH), 8.26–8.27 (1H, m,
3
1
3
ArH). C NMR (DMSO-d )  13.9, 23.0, 52.4, 52.7, 55.6, 66.2, 106.7, 115.8, 117.7, 121.0, 124.6,
6
+
126.7, 128.9, 130.3, 133.0, 156.2, 159.7, 165.6, 165.9. HRMS [ESI ]: calculated for C H N O ,
2
1
23
3
5
+
4
20.1535 [M+Na] , found 420.1552.
20
ACS Paragon Plus Environment

Page 21 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Dimethyl
3-(4-methoxyphenyl)-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-
dicarboxylate (22c). Compound 22c was prepared from 21c (2.0 g, 5.3 mmol), HBF (1.02 mL)
4
1
and DMAD (1.7 mL, 13.0 mmol). Yield 1.9 g (77%); mp 192–194°C. H NMR (DMSO-d ) δ 3.17
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
4H, t, J = 4.5 Hz, 2 × CH ), 3.72 (3H, s, OCH ), 3.81 (4H, t, J = 4.5 Hz, 2 × CH ), 3.83 (3H, s,
2 3 2
COOCH ), 3.88 (3H, s, COOCH ), 7.04 (2H, d, J = 9.0 Hz), 7.57 (2H, d, J = 9.0 Hz, ArH), 7.66–
3
3
7
.69 (1H, m, ArH), 7.82–7.85 (1H, m, ArH), 8.06–8.07 (1H, m, ArH), 8.73–8.75 (1H, m, ArH).
13
C NMR (DMSO-d )  51.5, 52.4, 52.7, 55.6, 66.2, 106.7, 113.7, 115.8, 117.7, 121.0, 124.6,
6
+
1
26.7, 128.3, 128.9, 130.2, 133.0, 156.2, 159.7, 165.6, 165.9. HRMS [ESI ]: calculated for
+
C H N O , 498.1641 [M+Na] , found 498.1637.
2
6
25
3
6
(
3-Methyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-diyl)dimethanol (23a). A solution
of 22a (1.5 g, 3.9 mmol) in DCM (50 mL) was added dropwise to a stirred suspension of LAH
0.37 g, 9.7 mmol) in diethyl ether (20 mL) at 0–5°C. After completion of the reaction (2 h), the
(
excess LAH was decomposed by the addition of water (2 mL) and NH OH (2 mL). The reaction
4
mixture was filtered through a pad of Celite and washed well with DCM. The combined filtrate
and washings were concentrated to dryness in vacuo. The residue was crystallized from ethanol to
1
give 23a. Yield 1.0 g, (80%), mp 184–186°C. H NMR (DMSO-d ) δ 2.46 (3H, s, CH ), 3.23 (4H,
6
3
m, 2 × CH ), 3.88 (4H, m, 2 × CH ), 4.58 (3H, br s, OCH and OH, exchangeable), 4.78 (1H, d, J
2
2
2
=
4.5 Hz, OH, exchangeable), 4.82–4.83 (2H, m, OCH ), 7.44–7.47 (1H, m, ArH), 7.72–7.75 (1H,
2
1
3
m, ArH), 7.96–7.98 (1H, m, ArH), 8.28–8.30 (1H, m, ArH). C NMR (DMSO-d )  9.3, 51.9,
6
5
3.8, 54.4, 66.5, 114.4, 115.7, 118.3, 121.6, 123.2, 123.6, 125.3, 126.0, 130.2, 132.3, 154.1. HRMS
+
+
[
ESI ]: calculated for C H N O , 310.1556 [M+H-H O] , found 310.1569.
18 21 3 3 2
Compounds 23b and 23c were prepared following the synthetic procedure used to prepare 20a.
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
3-Ethyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-diyl)dimethanol (23b). Compound
2
3b was prepared from 22b (1.4 g, 3.5 mmol) and LAH (0.43 g, 10.5 mmol). Yield 1.2 g (90%);
1
mp 180–182°C. H NMR (DMSO-d ) δ 1.21 (3H, t, J = 7.5 Hz, CH ), 2.96 (2H, q, J = 7.5 Hz,
6
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CH ), 3.21 (4H, t, J = 4.5 Hz, 2 × CH ), 3.86 (4H, t, J = 4.5 Hz, 2 × CH ), 4.57 (3H, br s, OCH
2
2
2
2
and OH, exchangeable), 4.76 (1H, t, J = 5.1 Hz, OH, exchangeable), 4.82 (2H, d, J = 5.1 Hz,
OCH ), 7.42–7.45 (1H, m, ArH), 7.70–7.74 (1H, m, ArH), 7.95–7.96 (1H, m, ArH), 8.28–8.30
2
13
(
1H, m, ArH). C NMR (DMSO-d )  13.5, 16.5, 51.3, 53.1, 53.8, 65.8, 113.7, 115.2, 117.5,
6
+
1
20.4, 123.0, 124.7, 125.4, 128.3, 129.7, 131.7, 153.4. HRMS [ESI ]: calculated for C H N O ,
1
9
23
3
3
+
342.1818 [M+H-H O] , found 324.1712.
2
(3-(4-Methoxyphenyl)-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-diyl)dimethanol
(
23c). Compound 23c was prepared from 22c (1.2 g, 2.86 mmol) and LAH (0.30 g, 8.86 mmol).
1
Yield 0.9 g (83%); mp 190–192°C. H NMR (DMSO-d ) δ 3.14 (4H, t, J = 4.0 Hz, 2 × CH ), 3.83
6
2
(7H, m, 2 × CH and OCH ), 4.52 (2H, d, J = 5.0 Hz, OCH ), 4.85 (1H, t, J = 5.0 Hz, OH,
2
3
2
exchangeable), 4.86–4.90 (3H, m, OCH and OH, exchangeable), 7.05 (2H, d, J = 8.8 Hz, ArH),
2
7
.47–7.50 (1H, m, ArH), 7.73 (2H, d, J = 8.8 Hz, ArH), 7.75–7.78 (1H, m, ArH), 7.97–7.98 (1H,
1
3
m, ArH), 8.37–8.39 (1H, m, ArH). C NMR (DMSO-d )  51.8, 54.0, 54.4, 55.5, 66.4, 113.8,
6
1
15.7, 116.5, 119.1, 122.3, 123.1, 124.2, 125.9, 126.0, 127.1, 130.2, 131.5, 132.5, 154.2, 158.8.
+
+
+
HRMS [ESI ]: calculated for C H N O , 419.1923, [M+H-H O] , found 402.1818, [M+Na] ,
24 25
3
4
2
found 442.1743.
General procedure for the preparation of bis(alkylcarbamate) derivatives (24a-c and
2
6a-c). The alkyl isocyanate (4.0 equiv) was added to a solution of bis(hydroxymethyl) derivative
(23a-c, 1.0 equiv) and TEA (4.0 equiv) in dry DMF or THF. The reaction mixture was stirred for
4-48 h at rt under argon. After completion of the reaction, the reaction mixture was concentrated
2
22
ACS Paragon Plus Environment

Page 23 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
to dryness in vacuo. The residue was triturated with ether, and the desired product was collected
by filtration.
(3-Methyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-diyl)bis(methylene)
bis(ethylcarbamate) (24a). Compound 24a was prepared from 23a (0.15 g, 0.5 mmol), TEA (0.25
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mL, 2.0 mmol) and ethyl isocyanate (0.15 mL, 2.0 mmol). Yield 0.16 g (75%); mp 171–173°C.
1
H NMR (DMSO-d ) δ 0.97 (6H, t, J = 6.5 Hz, 2 × CH ), 2.46 (3H, s, CH ), 2.97–2.98 (4H, m, 2
6
3
3
×
CH ), 3.23 (4H, m 2 × CH ), 3.86 (4H, m, 2 × CH ), 5.17 (2H, s, OCH ), 5.38 (2H, s, OCH ),
2 2 2 2 2
7
8
5
.01 (2H, br s, 2 × NH, exchangeable), 7.48–7.51 (1H, m, ArH), 7.75–7.77 (1H, m, ArH), 7.99–
1
3
.01 (1H, m, ArH), 8.10–8.11 (1H, m, ArH), C NMR (DMSO-d )  9.35, 15.5, 35.4, 51.9, 56.5,
6
7.3, 66.4, 109.5, 116.1, 117.6, 119.2, 122.9, 125.2, 126.2, 129.6, 132.7, 154.6, 156.5, 156.6.
+
+
HRMS [ESI ]: calculated for C H N O , 470.2403 [M+Na] , found 492.2222.
2
4
31
5
5
(3-Ethyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-diyl)bis(methylene)
bis(ethylcarbamate) (24b). Compound 24b was prepared from 23b (0.14 g, 0.44 mmol), TEA
(0.2 mL, 1.7 mmol) and ethyl isocyanate (0.14 mL, 1.7 mmol). Yellow solid; Yield 0.16 g (75%);
1
mp 165–167°C. H NMR (DMSO-d ) δ 0.97 (6H, t, J = 7.0 Hz, 2 × CH ), 1.19 (3H, t, J = 6.5 Hz,
6
3
CH ), 2.98–3.00 (6H, m, 3 × CH ), 3.20 (4H, m, 2 × CH ), 3.85–3.87 (4H, m, CH ), 5.17 (2H, s,
3
2
2
2
OCH ), 5.38 (2H, s, OCH ), 7.00–7.05 (2H, br s, 2 × NH, exchangeable), 7.49–7.52 (1H, m, ArH),
2
2
1
3
7
.75–7.78 (1H, m, ArH), 7.99–8.01 (1H, m, ArH), 8.09–8.10 (1H, m, ArH). C NMR (DMSO-
d6)  13.9, 15.5, 15.6, 17.2, 35.5, 51.9, 66.4, 109.4, 116.1, 116.9, 119.1, 122.9, 126.2, 126.4, 129.7,
+
+
1
30.7, 132.7, 154.5, 156.4, 156.6. HRMS [ESI ]: calculated for C H N O , 484.2560 [M+Na] ,
25 33 5 5
found 506.2379.
(4-Methoxyphenyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-diyl)
bis(methylene)-
bis(ethylcarbamate) (24c). Compound 24c was prepared from 23c (0.2 g, 0.47 mmol), TEA (0.25
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
mL, 1.7 mmol) and ethyl isocyanate (0.15 mL, 1.7 mmol). Yield 0.16 g (64%); mp 168–170°C.
1
H NMR (DMSO-d) δ 0.98 (3H, t, J = 7.0 Hz, CH ), 1.00 (3H, t, J = 7.0 Hz, CH ), 2.99–3.02 (4H,
3
3
m, 2 × CH ), 3.13 (4H, m, 2 × CH ), 3.81 (4H, m, 2 × CH ), 3.82 (3H, s, OCH ), 5.10 (2H, s,
2
2
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OCH ), 5.45 (2H, s, OCH ), 7.05–7.06 (2H, br s, 2 × NH, exchangeable), 7.05 (2H, d, J = 8.5 Hz,
2
2
ArH), 7.53–7.56 (1H, m, ArH), 7.60 (2H, d, J = 8.5 Hz, ArH), 7.78–7.81 (1H, m, ArH), 7.97–8.02
1
3
(1H, m, ArH), 8.16–8.18 (1H, m, ArH). C NMR (DMSO-d )  14.9, 34.9, 51.1, 54.9, 56.6, 56.7,
6
6
1
5.8, 110.0, 113.2, 113.4, 116.2, 117.3, 119.3, 121.7, 122.8, 125.8, 126.1, 127.6, 129.0, 131.0,
+
31.1, 132.3, 154.1, 155.7, 156.0, 158.6. HRMS [ESI ]: calculated for C H N O , 562.2665
3
0
35
5
6
+
[M+Na] , found 584.2484.
3-Methyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-diyl)bis(methylene)
bis(isopropylcarbamate)) (25a). Compound 25a was prepared from 23a (0.15 g, 0.5 mmol), TEA
(
(0.25 mL, 2.0 mmol) and isopropyl isocyanate (0.18 mL, 2.0 mmol). Yield 0.18 g (79%); mp 159–
1
161°C. H NMR (DMSO-d ) δ 1.01 (12H, d, J = 6.0 Hz, 4 × CH ), 2.48 (3H, s, CH ), 3.24 (4H,
6
3
3
m, 2 × CH ), 3.59 (2H, d, J = 6.0 Hz, 2 × CH), 3.87 (4H, m, 2 × CH ), 5.19 (2H, s, OCH ), 5.40
2
2
2
(
2H, s, OCH ), 6.95 (2H, br s, 2 × NH, exchangeable), 7.49–7.52 (1H, m, ArH), 7.75–7.78 (1H,
2
1
3
m, ArH), 8.00–8.02 (1H, m, ArH), 8.10–8.12 (1H, m, ArH). C NMR (DMSO-d )  9.36, 23.0,
6
2
3.7, 42.7, 51.9, 56.3, 57.1, 66.4, 109.5, 116.1, 117.6, 119.2, 122.9, 125.2, 126.1, 126.4, 129.6,
+
+
1
32.7, 154.6, 155.8, 156.0. HRMS [ESI ]: calculated for C H N O , 498.2716 [M+Na] , found
26 35 5 5
520.2535.
(3-Ethyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-diyl)bis(methylene)
bis(isopropylcarbamate) (25b). Compound 25b was prepared from 23b (0.15 g, 0.44 mmol),
TEA (0.22 mL, 1.7 mmol) and isopropyl isocyanate (0.17 mL, 1.7 mmol). Yield 0.18 g (81%); mp
1
1
47–149°C; H NMR (DMSO-d ) δ 1.00 (12H, d, J = 6.5 Hz, 4 × CH ), 1.19 (3H, t, J = 7.5 Hz,
6
3
24
ACS Paragon Plus Environment

Page 25 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
CH ), 2.96 (2H, q, J = 7.5 Hz, CH ), 3.23 (4H, m, 2 × CH ), 3.56–3.62 (2H, m, CH), 3.86 (4H, m,
3
2
2
2
× CH ), 5.17 (2H, s, OCH ), 5.38 (2H, s, OCH ), 6.91–6.97 (2H, br s, 2  NH, exchangeable),
2
2
2
7.49–7.52 (1H, m, ArH), 7.74–7.77 (1H, m, ArH), 7.99–8.01 (1H, m, ArH), 8.08–8.10 (1H, m,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
ArH). C NMR (DMSO-d )  13.9, 17.2, 23.0, 51.9, 66.4, 109.5, 116.1, 122.9, 126.2, 126.4,
6
+
1
29.7, 130.7, 132.7, 154.5, 155.7, 156.0. HRMS [ESI ]: calculated for C H N O , 512.2873
27 37
5
5
+
[
M+Na] , found 534.2692.
(3-(4-Methoxyphenyl)-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-diyl) bis(methylene)
bis(isopropylcarbamate) (25c). Compound 25c was prepared from 23c (0.2 g, 0.47 mmol), TEA
(0.25 mL, 1.8 mmol) and isopropyl isocyanate (0.2 mL, 1.8 mmol). Yield 0.18 g (64%); mp 152–
1
1
54°C. H NMR (DMSO-d ) δ 0.99–1.06 (12H, m, 4 × CH ), 3.14 (4H, m, 2 × CH ), 3.61–3.63
6
3
2
(
2H, m, 2 × CH), 3.80 (4H, m, 2 × CH ), 3.84 (3H, s, OCH ), 5.11 (2H, s, OCH ), 5.46 (2H, s,
2 3 2
OCH ), 6.97–7.02 (2H, br s, 2 × NH, exchangeable), 7.06 (2H, d, J = 8.5 Hz, ArH), 7.54–7.57
2
(1H, m, ArH), 7.62 (2H, d, J = 8.5 Hz, ArH), 7.79–7.82 (1H, m, ArH), 8.02–8.03 (1H, m, ArH),
1
3
8
.17–8.18 (1H, m, ArH). C NMR (DMSO-d )  22.4, 23.1, 46.2, 51.1, 55.0, 65.8, 110.0, 113.3,
6
+
114.8, 121.7, 122.8, 125.8, 128.8, 131.2, 133.5, 147.4, 154.1, 158.6. HRMS [ESI ]: calculated for
+
C H N O , 590.2979 [M+Na] , found 612.2797.
3
2
39
5
6
N,N-dimethylphthalazin-1-amine (26a). Dimethylamine (90.0 mL, 240.0 mmol) was added
slowly to a solution of 1-chlorophthalazine (19) (10.0 g, 60.0 mmol) and TEA (28.0 mL, 200.0
mmol) in ethanol (200 mL). The reaction mixture was stirred at rt for 48 h. After completion of
the reaction, the solvent was evaporated, and the residue was diluted with water and then extracted
with DCM (2  200 mL). The organic layer was dried over sodium sulfate and concentrated in
44
1
vacuo to give desired product 26a. Yield 8.0 g (80%); mp 69–71°C (lit. mp 67°C). H NMR
DMSO-d ) δ 3.12 (6H, s, N(CH ) ), 7.90–7.93 (2H, m, ArH), 8.03–8.05 (1H, m, ArH), 8.14–8.15
(
6
3 2
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
(
1H, m, ArH), 9.18 (1H, s, ArH). C NMR (DMSO-d ) δ 42.8, 120.3, 125.0, 127.1, 128.6, 131.9,
6
1
32.1, 146.8, 159.8.
Compounds 26b-d were prepared following the synthetic procedure used to prepare 26a.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
-(Pyrrolidin-1-yl)phthalazine (26b). Compound 26b was prepared from 19 (10.0 g, 60.0
mmol), pyrrolidine (20.0 mL, 240.0 mmol) and TEA (30.0 mL, 200.0 mmol). Yield 10.0 g (83%);
4
5
1
1
mp 90–91°C (lit. mp 88–89°C). H NMR (DMSO-d )  H NMR (DMSO-d ) δ 1.95–1.98 (4H,
6
6
m, 2 × CH ), 3.80–3.83 (4H, m, 2 × CH ), 7.80–7.88 (2H, m, ArH), 7.94–7.95 (1H, m, ArH), 8.27–
2
2
1
3
8
.29 (1H, m, ArH), 8.97 (1H, s, ArH). C NMR (DMSO-d ) δ 25.8, 51.1, 119.2, 125.2, 126.5,
6
+
128.7, 130.9, 131.6, 144.1, 155.7. HRMS [ESI+]: calculated for C H N , 200.1188 [M+H] ,
1
2
13
3
found 200.1140.
-(Piperidin-1-yl)phthalazine (26c). Compound 26c was prepared from 19 (10.0 g, 60.0
1
mmol), piperidine (24.0 mL, 240.0 mmol) and TEA (34.0 mL, 240.0 mmol). Yield 10.0 g, (78%),
1
mp 130–132°C. H NMR (DMSO-d )  1.64–1.67 (2H, m, CH ), 1.75–1.79 (4H, m, 2 × CH ),
6
2
2
3
.35–3.37 (4H, m, 2 × CH ), 7.91–7.94 (2H, m, ArH), 8.01–8.07 (2H, m, ArH), 9.25 (1H, s, ArH).
2
1
3
C NMR (DMSO-d )  24.6, 26.0, 52.4, 121.0, 124.4, 127.3, 128.5, 132.2, 132.4, 147.8, 160.4.
6
+
+
HRMS [ESI ]: calculated for C H N , 214.1344 [M+H] , found 214.1364.
1
3
15
3
1
-([1,4'-Bipiperidin]-1'-yl)phthalazine (26d). Compound 26d was prepared from 19 (10.0
g, 60.0 mmol), 1,4'-bipiperidine (20.0 g, 120.0 mmol) and TEA (34 mL, 240.0 mmol). Yield 10.2
1
g (57%); mp 140–142°C. H NMR (DMSO-d ) δ 1.37–1.42 (2H, m, CH ), 1.48–1.53 (4H, m,
6
2
CH ), 1.75–1.81 (2H, m, CH ), 1.88–1.91 (2H, m, CH ), 2.43 (1H, m, CH), 2.57 (4H, m, 2 × CH ),
2
2
2
2
2
.94–3.00 (2H, m, CH ), 3.87–3.90 (2H, m, CH ), 7.92–7.94 (2H, m, ArH), 8.04–8.06 (2H, m,
2 2
+
ArH), 9.25 (1H, s, ArH). HRMS [ESI+]: calculated for C H N , 297.2079 [M+H] , found
1
8
24
4
2
97.2092.
26
ACS Paragon Plus Environment

Page 27 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Compounds 27a-d were prepared following the synthetic procedure used to prepare 21a.
2
-Acetyl-4-(dimethylamino)-1,2-dihydrophthalazine-1-carbonitrile (27a). Compound
27a was prepared from 26a (2.5 g, 14.4 mmol), Me SiCN (3.6 mL, 28.8 mmol) and acetyl chloride
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
(
1.5 mL, 20.0 mmol). Yield 2.78 g (80%); mp 118–120°C. H NMR (DMSO-d ) δ 2.25 (3H, s,
6
COCH ), 2.90 (6H, s, (NCH ) ), 7.08 (1H, s, CH), 7.61–7.68 (3H, m, ArH), 7.82–7.83 (1H, m,
3
3 2
1
3
ArH). C NMR (DMSO-d ) δ 20.9, 116.7, 121.7, 127.0, 127.5, 129.6, 130.6, 132.6, 155.9, 171.2.
6
+
HRMS [ESI+]: calculated for C H N O, 243.1246 [M+H] , found 243.1241.
1
3
14
4
2
-Acetyl-4-(pyrrolidin-1-yl)-1,2-dihydrophthalazine-1-carbonitrile (27b). Compound
2
7b was prepared from 26b (5.0 g, 25.0 mmol), Me SiCN (6.3 mL, 50.0 mmol) and acetyl chloride
3
1
(
2.7 mL, 37.5 mmol). Yield 5.7 g (85%); mp 123–125°C. H NMR (DMSO-d )  1.81–1.85 (2H,
6
m, CH ) 2.00 (2H, m, CH ), 2.21 (3H, s, COCH ), 3.28–3.34 (2H, m, CH ), 3.70–3.75 (2H, m,
2
2
3
2
1
3
CH ), 7.06 (1H, s, CH), 7.59–7.66 (2H, m, ArH), 7.81–7.82 (2H, m, ArH). C NMR (DMSO-d )
2
6
20.8, 25.3,49.8, 116.9, 122.7, 126.7, 127.3, 129.7, 130.4, 132.3, 153.5, 170.8. HRMS [ESI+]:

+
calculated for C H N O, 269.1402 [M+H] , found 269.1416.
1
5
16
4
2
-Acetyl-4-(piperidin-1-yl)-1,2-dihydrophthalazine-1-carbonitrile (27c). Compound 27c
was prepared from 26c (5.0 g, 23.5 mmol), Me SiCN (6.0 mL, 47.0 mmol) and acetyl chloride
3
1
(
2.5 mL, 35.0 mmol). Yield 5.7 g (87%); mp 135–137°C. H NMR (DMSO-d )  1.59–1.64 (4H,
6
m, 2 × CH ), 1.78–1.80 (2H, m, CH ), 2.24 (3H, s, COCH ), 3.08–3.12 (2H, m, CH ), 3.30–3.34
2
2
3
2
13
(
2H, m, CH ), 7.07 (1H, s, CH), 7.58–7.67 (3H, m, ArH), 7.80–7.82 (1H, m, ArH). C NMR
2
(
DMSO-d )  21.0, 24.5, 25.3, 50.3, 116.7, 121.8, 126.6, 127.5, 129.5, 130.7, 132.7, 155.6, 171.3.
6
+
+
HRMS [ESI ]: calculated for C H N O, 283.1559 [M+H] , found 283.1536.
1
6
18
4
4
-([1,4'-Bipiperidin]-1'-yl)-2-acetyl-1,2-dihydrophthalazine-1-carbonitrile
(27d).
Compound 27d was prepared from 26d (2.0 g, 6.7 mmol), Me SiCN (1.69 mL, 13.5 mmol) and
3
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
acetyl chloride (0.88 mL, 10.0 mmol). Yield 1.45 g, (60%); mp 160–162°C. H NMR (DMSO-d )
6
δ 1.42–1.45 (1H, m, CH ), 1.70–1.82 (6H, m, 3 × CH ), 2.10–2.26 (6H, m, 3 × CH ), 2.65 (3H, s,
2
2
2
COCH ), 2.66–2.73 (1H, m, CH), 2.95–3.00 (3H, m, CH ), 3.75–3.78 (1H, m, CH ), 3.86–3.89
3
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
(
1H, m, CH ), 7.00 (1H, s, CH), 7.61–7.69 (3H, m, ArH), 7.79–7.81 (1H, m, ArH). HRMS [ESI ]:
2
+
calculated for C H N O, 366.2294 [M+H] , found 366.2308.
21 27
5
Compounds 28a-d were prepared following the synthetic procedure used to prepare 22a.
Dimethyl 6-(dimethylamino)-3-methylpyrrolo[2,1-a]phthalazine-1,2-dicarboxylate
(
28a). Compound 28a was prepared from 27a (2.7 g, 11.0 mmol), HBF (2.15 mL) and DMAD
4
1
(2.5 mL, 22.0 mmol) in DMF (30 mL). Yield 1.5 g (45%); mp 164–166°C. H NMR (DMSO-d )
6
δ 2.63 (3H, s, CH ), 2.98 (6H, s, N(CH ) ), 3.80 (3H, s, COOCH ), 3.88 (3H, s, COOCH ), 7.60–
3
3 2
3
3
7
.63 (1H, m, ArH), 7.76–7.79 (1H, m, ArH), 8.03–8.05 (1H, m, ArH), 8.23–8.24 (1H, m, ArH).
1
3
C NMR (DMSO-d ) δ 10.5, 52.0, 52.8, 107.0, 112.2, 117.5, 120.5, 123.2, 127.3, 128.1, 128.2,
6
+
+
1
30.2, 132.8, 156.9, 164.9, 167.1. HRMS [ESI ]: calculated for C H N O , 364.1273 [M+Na] ,
18 19 3 4
found 364.1308.
Dimethyl-3-methyl-6-(pyrrolidin-1-yl)pyrrolo[2,1-a]phthalazine-1,2-dicarboxylate
(
28b). Compound 28b was prepared from 27b (5.0 g, 18.6 mmol), HBF (3.6 mL) and DMAD
4
1
(
4.5 mL, 37.0 mmol). Yield 2.5 g (48%); mp 176–178°C. H NMR (DMSO-d ) 1.92 (4H, m, 2 ×
6
CH ), 2.57 (3H, s, CH ), 3.65 (4H, m, 2 × CH ), 3.78 (3H, s, COOCH ), 3.86 (3H, s, COOCH ),
2
3
2
3
3
7.53–7.57 (1H, m, ArH), 7.72–7.75 (1H, m, ArH), 8.13–8.15 (1H, m, ArH), 8.20–8.21 (1H, m,
1
3
ArH). C NMR (DMSO-d )  10.5, 25.7, 51.3, 51.9, 52.7, 106.7, 111.7, 117.8, 120.1, 123.0,
6
+
1
27.4, 127.7, 128.1, 129.4, 132.4, 153.7, 165.0, 167.3. HRMS [ESI ]: calculated for C H N O ,
20 21
3
4
+
368.1610 [M+H] , found 368.1581.
28
ACS Paragon Plus Environment

Page 29 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Dimethyl-3-methyl-6-(piperidin-1-yl)pyrrolo[2,1-a]phthalazine-1,2-dicarboxylate (28c).
Compound (28c) was prepared from (27c) (5.0 g, 17.7 mmol), HBF (3.4 mL) and DMAD (3.7
4
1
mL, 30.0 mmol). Yield 3.0 g (54%); mp 182–183°C. H NMR (DMSO-d )  1.67 (2H, m, CH ),
6
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
.79 (4H, m, 2 × CH ), 2.65 (3H, s, CH ), 3.28 (4H, m, 2 × CH ), 3.82 (3H, s, COOCH ), 3.90
2 3 2 3
(
3H, s, COOCH ), 7.64–7.67 (1H, m, ArH), 7.79–7.82 (1H, m, ArH), 7.99–8.00 (1H, m, ArH),
3
1
3
8.25–8.26 (1H, m, ArH). C NMR (DMSO-d )  10.5, 24.5, 25.7, 52.1, 52.4, 52.8, 107.0, 112.4,
6
1
17.6, 120.6, 123.2, 126.9, 128.1, 128.3, 130.4, 133.0, 157.0, 164.9, 167.1. HRMS [ESI+]:
+
calculated for C H N O , 382.1767 [M+H] , found 382.1796.
21 23
3
4
Dimethyl-6-([1,4'-bipiperidin]-1'-yl)-3-methylpyrrolo[2,1-a]phthalazine-1,2-
dicarboxylate (28d). Compound 28d was prepared from 27d (1.3 g, 3.5 mmol), HBF (0.7 mL)
4
1
and DMAD (0.8 mL, 6.5 mmol). Yield 1.0 g (70%); mp 189–191°C. H NMR (DMSO-d ) δ 1.43–
6
1.46 (3H, m, CH ), 1.68–1.72 (2H, m, CH ), 1.88–1.91 (2H, m, CH ), 2.02–2.04 (2H, m, CH ),
2
2
2
2
2
.14–2.16 (2H, m, CH ), 2.65 (3H, s, CH ), 3.01 (4H, m, 2 × CH ), 3.17 (1H, m, CH), 3. 49–3.51
2 3 2
(
3H, m, CH ), 3.81 (3H, s, COOCH ), 3.86–3.88 (2H, m, CH ), 3.89 (3H, s, COOCH ), 7.64–7.68
2 3 2 3
1
3
(
1H, m, ArH), 7.81–7.85 (1H, m, ArH), 7.99–8.01 (1H, m, ArH), 8.26–8.28 (1H, m, ArH), . C
NMR (DMSO-d )  12.6, 24.5, 26.2, 28.1, 50.4, 51.5, 58.2, 58.9, 67.5, 70.4, 111.6, 118.1, 123.4,
6
+
1
27.6, 128.5, 129.8, 132.8, 133.5, 147.4, 165.9, 175.7. HRMS [ESI ]: calculated for C H N O ,
2
6
32
4
4
+
465.2502 [M+H] , found 465.2501.
Compounds 29a-d were prepared following the synthetic procedure used to prepare 23a.
6-(Dimethylamino)-3-methylpyrrolo[2,1-a]phthalazine-1,2-diyl)dimethanol (29a).
(
Compound 29a was prepared from 28a (1.5 g, 4.3 mmol) and LAH (0.4 g, 10.9 mmol). Yield 1.0
1
g (80%), mp 164–166°C. H NMR (DMSO-d ) δ 2.45 (3H, s, CH ), 2.92 (6H, s, N(CH ) ), 4.56
6
3
3 2
(
3H, s, OCH and OH, exchangeable), 4.75 (1H, t, J = 5.3 Hz, OH, exchangeable), 4.80 (2H, d, J
2
29
ACS Paragon Plus Environment