Design, synthesis and antitumour evaluation of pyrrolo[1,2-f]-phenanthridine and dibenzo[f,h]pyrrolo[1,2-b]isoquinoline derivatives

Article abstract of DOI:10.1016/j.ejmech.2020.112516

A series of 1,2-bis(hydroxymethyl)pyrrolo[1,2-f]phenanthridine derivatives and their alkyl (ethyl and isopropyl) carbamates and 12,13-bis(hydroxymethyl)-9,14-dihydro-dibenzo[f,h]pyrrolo[1,2-b]isoquinoline derivatives were synthesized for antiproliferative evaluation. The preliminary antitumour studies revealed that these two types of bis(hydroxymethyl) derivatives showed significant antitumour activities and were able to inhibit the growth of various human tumour cell lines in vitro. Several of the derivatives were demonstrated to cause DNA interstrand cross-links by an alkaline agarose gel shifting assay. These conjugates were cytotoxic to a variety of cancer cell lines by inducing DNA damage, delaying cell cycle progression in the G2/M phase and triggering apoptosis. Compound 21a, dissolved in a vehicle suitable for intravenous administration, was selected for antitumour studies in animal models. We demonstrated that at a dose that did not cause body weight loss in mice, compound 21a could significantly suppress the growth of tumour xenografts of human lung cancer H460 and colorectal cancer HCT-116 cells in nude mice. Our present results confirm the antitumour activities of these conjugates.

Full text of DOI:10.1016/j.ejmech.2020.112516

Journal Pre-proof
Design, synthesis and antitumour evaluation of pyrrolo[1,2-f]-phenanthridine and
dibenzo[f,h]pyrrolo[1,2-b]isoquinoline derivatives
Anilkumar S. Patel, Vicky Jain, Vaikar Navakanth Rao, Yi-Wen Lin, Anamik Shah,
Kuo-Chu Lai, Tsann-Long Su, Te-Chang Lee
PII:
S0223-5234(20)30488-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112516
EJMECH 112516
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 February 2020
Revised Date: 18 May 2020
Accepted Date: 29 May 2020
Please cite this article as: A.S. Patel, V. Jain, V.N. Rao, Y.-W. Lin, A. Shah, K.-C. Lai, T.-L. Su,
T.-C. Lee, Design, synthesis and antitumour evaluation of pyrrolo[1,2-f]-phenanthridine and
dibenzo[f,h]pyrrolo[1,2-b]isoquinoline derivatives , European Journal of Medicinal Chemistry, https://
doi.org/10.1016/j.ejmech.2020.112516.
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Design, synthesis and antitumour evaluation of pyrrolo[1,2-f]-phenanthridine and
dibenzo[f,h]pyrrolo[1,2-b]isoquinoline derivatives
a,b,1
a,c,1
a,d
a
e
Anilkumar S. Patel , Vicky Jain , Vaikar Navakanth Rao , Yi-Wen Lin , Anamik Shah ,
f
a*
a*
Kuo-Chu Lai , Tsann-Long Su , Te-Chang Lee
Graphical Abstract

Design, synthesis and antitumour evaluation of pyrrolo[1,2-f]-phenanthridine and
dibenzo[f,h]pyrrolo[1,2-b]isoquinoline derivatives
a,b,1
a,c,1
a,d
a
e
Anilkumar S. Patel , Vicky Jain , Vaikar Navakanth Rao , Yi-Wen Lin , Anamik Shah ,
f
a*
a*
Kuo-Chu Lai , Tsann-Long Su , Te-Chang Lee
a
Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan
b
Department of Chemistry, Atmiya University, Rajkot, Gujarat, India
Department of Chemistry, Marwadi University, Rajkot, Gujarat, India
c
d
PhD Program in Pharmacology and Toxicology, School of Medicine, Tzu Chi University, Hualien
9
70, Taiwan
e
Gujarat Vidyapith (Deemed University), Ahmedabad, Gujarat, India
Department of Pharmacology, Tzu Chi University, Hualien 970, Taiwan
f
*Corresponding authors. E-mail address: bmtcl@ibms.sinica.edu.tw (T.C. Lee),
tlsu@ibms.sinica.edu.tw (T.L. Su). Tel.: +886-2-26253055; fax: +886-2-27829142.
1
These authors contributed equally to this work.
Abstract:
A series of 1,2-bis(hydroxymethyl)pyrrolo[1,2-f]phenanthridine derivatives and their alkyl
(ethyl
and
isopropyl)
carbamates
and
12,13-bis(hydroxymethyl)-9,14-dihydro-dibenzo[f,h]pyrrolo[1,2-b]isoquinoline derivatives were
synthesized for antiproliferative evaluation. The preliminary antitumour studies revealed that these
two types of bis(hydroxymethyl) derivatives showed significant antitumour activities and were able
to inhibit the growth of various human tumour cell lines in vitro. Several of the derivatives were
demonstrated to cause DNA interstrand cross-links by an alkaline agarose gel shifting assay. These
conjugates were cytotoxic to a variety of cancer cell lines by inducing DNA damage, delaying cell
cycle progression in the G2/M phase and triggering apoptosis. Compound 21a, dissolved in a vehicle
suitable for intravenous administration, was selected for antitumour studies in animal models. We
demonstrated that at a dose that did not cause body weight loss in mice, compound 21a could
1

significantly suppress the growth of tumour xenografts of human lung cancer H460 and colorectal
cancer HCT116 cells in nude mice. Our present results confirm the antitumour activities of these
conjugates.
Keywords: antitumour agents, bis(hydroxymethyl)pyrrolo[1,2-f]phenanthridine, cell cycle,
cytotoxicity, DNA cross-linking, apoptosis
2

1. Introduction
Cancer is one of the major leading causes of death worldwide. The design of new antitumour
agents is one of the most challenging tasks in the field of medicinal chemistry. Among anticancer
agents, DNA alkylating agents have attracted attention and have been widely used as potential
therapeutic agents for a long time. Notably, DNA-damaging therapeutic agents are widely used in
combination therapy with targeted therapeutics as well as immunotherapeutics in clinical settings
[
1-4].
Naturally occurring mitomycin C (MMC, 1, Fig. 1) is a clinically useful chemotherapeutic agent
for treating various cancers[5]. Both MMC and synthetic indoloquinone EO9 (2) [6], which possess
two reactive nucleophilic centres on its pyrrole, are capable of inducing DNA cross-linking via
bioreductive activation [7]. Numerous pyrrolizine alkaloids [8-10] and their synthetic analogues
bearing a bis(hydroxymethyl)pyrrolidine moiety, such as IPP (3) [11], are also capable of inducing
DNA interstrand or intrastrand cross-linking (CL), giving them potent antitumour activities [12].
Numerous studies had shown that synthetic bis(hydroxymethyl or alkylcarbamate)pyrroles or
pyrrolizines were able to generate an electrophilic centre on the pyrrole ring, and hence reacted with
DNA to induce DNA interstrand cross-linking (ICL) via an electrophilic reaction (Fig. 2) [13, 14].
Obviously, these agents do not require bioreductive activation to induce DNA CL.
<
<
Insert Figure 1 here >
Insert Figure 2 here >
To explore new bifunctional DNA alkylating agents, we previously synthesized
1
2
3
a-azacyclopenta[a]indene derivatives (4) (wherein R = H or CONH-alkyl; R = alkyl or aryl),
which contain a bis(hydroxymethyl)pyrrole alkylating pharmacophore and was viewed as a
“benzologue” of IPP (3). Among these congeners, compound BO-1012 (5) exhibited significant in
3

vitro cytotoxicity and potent therapeutic efficacy in nude mice bearing cisplatin-resistant lung or
bladder cancer [15]. We further synthesized a series of “bioisosteres” of 3a-azacyclopenta[a]indenes
(4), namely, 5,10-dihydropyrrolo[1,2-b]isoquinolines (6) and benzo[d]pyrrolo[2,1-b]thiazoles (7),
which also displayed potent antitumour activities by inhibiting the growth of a variety of human
leukaemia and solid tumour cell lines in vitro and in vivo [16, 17].
To further broaden the chemical space of bifunctional DNA alkylating agents containing
bis(hydroxymethyl) pyrrole alkylating pharmacophores, we took advantage of a hybrid approach to
synthesize a series of indolizino[6,7-b]indole derivatives (8) [18] and indolizino[8,7-d]indoles (9)
[
19]. These analogues include β-carboline (DNA topo I and II inhibition moiety) and
bis(hydroxymethyl)pyrrole (DNA ICL moiety), as shown in Fig. 1. As expected, these hybrids
exhibited multiple modes of action, including induction of DNA ICLs and inhibition of topo I and II
1
2
[
18]. Of these analogues, BO-1978 (8, wherein R = H, R = Et) significantly suppressed the growth
of EGFR wild-type and mutant non-small-cell lung cancer (NSCLC) cells in xenograft and
orthotopic lung tumour models [18]. Furthermore, the combination of BO-1978 with gefitinib further
suppressed EGFR mutant NSCLC cell growth in vivo [20]. Additionally, we coupled a
bis((hydroxymethyl)pyrrole) pharmacophore with phthalazines (an anti-angiogenic moiety) to
generate new pyrrolo[2,1-a]phthalazine (10) hybrids [21]. We demonstrated that these conjugates
were cytotoxic to a variety of cancer cell lines by inducing DNA ICLs and inhibiting the
phosphorylation of VEGFR in endothelial cells, leading to cancer cell killing as well as the
suppression of vascular formation.
More recently, we synthesized a series of new antitumour bis(hydroxymethyl)pyrrole
derivatives (Fig. 3), namely, 1,2-bis(hydroxymethyl)pyrrolo[1,2-f]phenanthridine derivatives (Class I)
and 12,13-bishydroxymethyl-9,14-dihydrodibenzo[f,h]pyrrolo[1,2-b]isoquinoline derivatives (Class
II), via a hybrid approach for anticancer evaluation. Compounds of Class I contain a phenanthridine
4

moiety, which is commonly found in natural products with anticancer activity [22-25]. For example,
an N-[2-(dimethylamino)ethyl]phenanthridine-4-carboxamide derivative (11, Fig. 4) exhibited
DNA-intercalating activity and displayed moderate in vivo antitumour activity against P388
leukaemia and Lewis lung carcinoma cells [26]. Likewise, the phenanthridinium scaffold was used to
design a number of DNA-intercalating agents with antitumour properties. For instance, ethidium
bromide (12) is a well-known DNA-intercalating agent commonly used as a fluorescent tag (nucleic
acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis [27].
Moreover, phenanthriplatin derivatives (13 and 14) are hybrid molecules of a phenanthridinium
moiety and a platinum(II) diamine. The former was synthesized by directly conjugating cisplatin to
the phenanthridinium cation [28], and the latter was formed by tethering platinum via a
polymethylene chain (n = 3, 5, 8 and 10) to the phenanthridinium cation (14) [29]. The antitumour
activity of phenanthriplatins is substantially greater than that of cisplatin and pyriplatin because of
the hydrophobicity of the phenanthridine ligand.
Compounds of Class II possess a phenanthroindolizine moiety, as phenanthroindolizidine
alkaloids are commonly isolated from plants [30, 31]. Various analogues have been synthesized for
anticancer studies [32-35]. For instance, (R)-antofine (15, Fig. 4) significantly inhibited various
cancer cell lines at nanomolar concentrations and induced cell arrest in the G2/M phase in human
colon Col2 cells [36, 37]. Naturally occurring tylophorine (16) and its synthetic analogues exhibited
significant inhibitory effects against the growth of human hepatocellular carcinoma HepG2 and
human nasopharyngeal carcinoma KB cells and potent tumour growth suppression activity in
xenograft models [38]. Tylophorine inhibited cyclic AMP response elements, activator protein-1
sites, or nuclear factor-κB binding site-mediated downstream transcription in HepG2 cells, indicating
that phenanthroindolizine derivatives have a mode of action different from those of known
antitumour drugs [38].
<
Insert Figure 3 here >
5

<
Insert Figure 4 here >
Based on the above reports, phenanthridine or phenanthroindolizine moieties may play an
important role in enhancing the DNA/drug interaction and thus may increase antitumour activity
with a novel mechanism of action. We have synthesized several Class I and II derivatives (Fig. 3),
including 1,2-bis(hydroxymethyl)pyrrolo[1,2-f]phenanthridine derivatives and their corresponding
1
2
alkylcarbamates (where R = H, Me, Et, aryl; R = CONHEt or CONHi-Pr, Class I) and
1
12,13-bis(hydroxymethyl)-9,14-dihydrodibenzo[f,h]pyrrolo[1,2-b]isoquinoline derivatives (where R
2
=
H, Me, Et, aryl, R = H, Class II). The Class I and II derivatives, bearing various substituents at C3
or C12, respectively, will allow us to study their structure-activity relationships. Additionally, the
heterocyclic nitrogen of the pyrrole moiety fused to the phenanthridine ring (Class I) and the
1,2,3,4-tetrahydro-dibenzo[f,h]isoquinoline (Class II) allowed us to compare the biological activities
of these structurally distinct types of chemical compounds. As mentioned previously, the electronic
properties of the substituent(s) on the pyrrole may influence the ability of the compound to induce
DNA cross-linking and its antitumour activity. We report herein that the newly synthesized
compounds exhibited significant cytotoxicity against various human cancer cell lines and significant
tumour suppression in vivo. Furthermore, these analogues induced DNA ICLs, cell cycle interference
and apoptotic cell death.
2. Results and Discussion
2.1. Chemistry
Synthesis of 1,2-Bis(hydroxymethyl)pyrrolo[1,2-f]phenanthridine Derivatives (Class I)
The syntheses of 1,2-bis(hydroxymethyl)pyrrolo[1,2-f]phenanthridine derivatives (21a-f) and
their bis(alkylcarbamate) derivatives (22a-f and 23a-f) are shown in Scheme 1. Commercially
available phenanthridine (17) was treated with bromoacetic acid in acetonitrile to give the
6

N-(carboxymethyl)phenanthridinium bromide salt (18a), which was then reacted with dimethyl
acetylenedicarboxylate (DMAD) and trimethylamine (TEA) in toluene at reflux to yield diester 20a
1
(
wherein R = H) by using a procedure developed previously [39]. The diester derivatives (20b-f)
1
having R substituents other than H were also prepared starting from phenanthridine (17). The
reaction of 17 with trimethylsilyl cyanide and various acyl chlorides in dichloromethane (DCM) with
a catalytic amount of AlCl afforded phenanthridine-6-carbonitriles 19b-f by following the
3
previously described procedure [21]. Compounds 19b-f were treated with tetrafluoroboric acid in
acetic acid to give the hydrofluoroborate salt intermediates, which were then reacted with DMAD to
give desired diesters 20b-f. The diester functions of 20a-f were reduced to the corresponding
bis(hydroxymethyl) groups (21a-f) with lithium aluminium hydride (LAH) in a mixture of
ether/DCM in an ice bath. Compounds 21a-f were then converted to the corresponding ethyl
carbamates or isopropyl carbamates (22a-f and 23a-f, respectively) in good yields by treatment with
ethyl or isopropyl isocyanates under basic conditions.
<
Insert Scheme 1 here >
Synthesis of 2,13-Bishydroxymethyl-9,14-dihydrodibenzo[f,h]pyrrolo[1,2-b]-isoquinoline
Derivatives (Class II)
The syntheses of Class II compounds 33a-e are shown in Scheme 2. Commercially available
9
-phenanthrene carboxaldehyde (24) was reduced with NaBH to the corresponding known alcohol
4
(
25) [40], which was then treated with PBr to afford 9-(bromomethyl)phenanthrene (26) [41]. The
3
reaction of compound 26 with diphenyl methylene-glycine ethyl ester in the presence of K CO3
2
afforded compound 27, which was further treated with concentrated HCl to yield compound 28. The
Pictet-Spengler cyclization of 28 by treatment with formaldehyde (37%) in a mixture of
DCM/trifluoroacetic acid (TFA) gave dibenzo[f,h]isoquinoline 29. Compound 29 was reacted with
7

various acid chlorides or acid anhydrides in the presence of TEA to produce N-acetyl derivatives
0a-e. Hydrolysis of 30a-e under basic conditions (1 N aqueous sodium hydroxide in ethanol)
yielded corresponding carboxylic acid derivatives 31a-e, which were further converted to diesters
2a-e by treatment with DMAD in Ac O at 100°C. The reaction of diesters 32a-e with LAH in a
3
3
2
mixture of ether/DCM yielded bis(hydroxymethyl) derivatives 33a-e. Attempts to convert 33a-e into
their corresponding bis(alkylcarbamate) derivative congeners failed because of the instability of
3
3a-e under the reaction conditions. A similar result was observed in the synthesis of the
alkylcarbamate of bis(hydroxymethyl)pyrroloindolizino[8,7-d]indoles (9) as previously reported
19].
[
<
Insert Scheme 2 here >
2
2
.2. Biological results
.2.1. In vitro cytotoxicity
We first evaluated the anti-proliferative activities and studied the structure-activity relationships
SARs) of the newly synthesized compounds against human lymphoblastic leukaemia (CCRF-CEM)
(
and various human solid tumour cells in vitro. As shown in Table 1, among the tested
1
bis(hydroxymethyl) derivatives (21a-f), 21a (R = H) was the most cytotoxic against CCRF/CEM
cells, with an IC value of 0.37 µM. The cytotoxicities of the tested compounds clearly decreased as
5
0
the size of the substituent at C3 increased [e.g., H > Me > Et> aryl (Ph, 4’-FPh, and 4’-MeOPh)].
Among the bis(alkylcarbamate)-substituted derivatives (22a-f and 23a-f), their cytotoxicities were
1
influenced by the C3 substituent (R ) and were in the order C3-Me > H > Et > aryl. A similar SAR
was observed among the dibenzo[f,h]pyrrolo[1,2-b]isoquinoline derivatives (33a-e) (Class II); the
size and the electronic properties of the substituent at C11 influenced their cytotoxicity. A Me
1
substituent at C11 (R ) (33a) resulted in the most cytotoxic compound among the Class II series
(33a-e). These results confirmed that the electronegativity of the N atom in the pyrrole affects the
8

DNA ICL and thus the cytotoxicity of the compound, and the electronegativity of the N atom is
decreased when the inductive effect of the substituent at C3 or C11 in Class I or Class II compounds,
respectively, is reduced.
Intriguingly, except for those without a substituent at C3 (H) (21a, 22a and 23a), Class I
compounds with ethyl or isopropyl substituents on the bis(alkylcarbamate) moiety showed
significantly higher cytotoxicities to CCRF/CEM cells relative to those of their Class II counterparts.
These results indicate that the substituents at C3 or C11 in Class I or Class II compounds,
respectively, influence the biological activity of these bis(hydroxymethyl) derivatives. Furthermore,
the C3-4’-MeOPh-substituted compound is more cytotoxic than the corresponding C3-Ph and
C3-4’-F-Ph substituted derivatives.
Drug resistance is one of the main concerns in new drug development [42]. To determine
whether the newly synthesized compounds effectively inhibit multi-drug resistant (MDR) cancer
cells, we compared the in vitro cytotoxic activities of the new derivatives against CCRF-CEM cells
and its drug-resistant sublines resistant to vinblastine, CCRF-CEM/VBL (approximately 278-fold
more resistant than the sensitive parent cell lines). As shown in Table 1, 15 out of the 23 compounds
tested showed a resistance factor (RF) ≤ 1. The RFs of these new compounds were between 0.39 and
1.42, indicating that they are not substrates of p-glycoprotein, which may allow them to overcome
MDR.
<
Insert Table 1 here >
We further evaluated the effects of selected new derivatives on the inhibition of cell growth
against a panel of human solid tumour cell lines in vitro, including colon carcinoma HCT-116, lung
cancer H1650 and H460 and pancreatic cancer PacaS1 cells. The antiproliferative activities of the
tested compounds are summarized in Table 2. Class I compounds with H or Me substituents at C3
displayed the most potent cytotoxicities against the tested tumour cell lines. In Class II, it was also
9

shown that the C11-alkyl (Me and Et) derivatives were generally more cytotoxic than the
corresponding C11-aryl congeners. In general, the values of IC50 were less than 3 folds among the
tumour cell lines tested, except compound 33a. H460 cells were the most sensitive to compound 33a,
while HCT-116 to compound 21a.
<
Insert Table 2 here >
2
.2.3. DNA cross-linking study
Our previous studies revealed that bis(hydroxymethyl)pyrrole analogues [8] are able to induce
DNA ICLs. To determine whether the newly synthesized compounds are also capable of causing
DNA cross-linking, linearized pBR322 DNA was treated with potent Class I and II
bis(hydroxymethyl) derivatives (21a, 21b, 33a, and 33b) at various concentrations (1, 5, 10 and 20
µM) and melphalan (1 and 5 µM) as a positive control. The DNA ICLs were determined by an
alkaline agarose gel shifting assay. The results, shown in Fig. 5, indicated that compounds 33a and
33b (Class II) induced more DNA ICLs than 21a and 21b (Class I), suggesting that the electron
density on the N atom of the pyrrole heterocycle influence potency of the drug-induced DNA ICLs.
As stated previously, the heterocyclic N of the pyrrole moiety in Class I and Class II compounds is
fused to the phenanthridine ring and the dihydroisoquinoline, respectively. These results clearly
show that the lone pair electrons on the N atom in Class II compounds may improve the leaving
group ability of the hydroxymethyl function in 33a and 33b relative to the analogous groups in 21a
and 21b. Therefore, one can expect compounds 33a and 33b to be stronger DNA cross-linkers than
21a and 21b. Although 21a showed lower activity in forming DNA interstrand cross-links than 33a
in vitro, their IC values for all tested cell lines were similar. We can therefore infer that 21a may
5
0
have functions other than DNA cross-linking. However, we cannot rule out other possibilities, such
as that 21a and 33a may have different pharmacokinetics, including differential cellular uptake rates
and different stabilities in the medium.
1
0

<
Insert Figure 5 here >
2.2.4. Cell cycle inhibition
DNA interacting/damaging agents are known to induce cell cycle perturbations and arrest cell
cycle progression predominantly at the G2/M boundary. Therefore, the effect of compound 21a on
cell cycle progression was further investigated in human colorectal cancer HCT-116 and
non-small-cell lung cancer H460 cells. HCT-116 cells were treated with 21a at concentrations of
0.375, 0.75, and 1.5 µM for 12, 24, and 36 h. Similarly, H460 cells were treated with compound 21a
at 0.75, 1.5, and 3 µM. At the end of treatment, cells were harvested by trypsination, fixed with
ethanol, stained with propidium iodide (PI), and subjected to flow cytometric analysis. As shown in
Fig. 6, the cell cycle progression was influenced by compound 21a. Accordingly the cell cycle
progression profiles, we noticed the temporary and dose-dependent increase in G2/M phase at 24 h
after treatment in either HCT-116 and H460 cells. At 36 h after treatment, the jammed G2/M phase
was likely progressing to the next cycle. However, at a higher concentration (i.e., 1.5 µM in
HCT-116 cells and 3.0 µM in H460 cells), we observed the appearance of a large proportion of the
sub-G1 phase at 36 h, indicating that the cells underwent apoptosis. These observations implicate
that compound 21a targets DNA, induces DNA damage, and subsequently triggers apoptotic cell
death. However, we could not exclude other toxic mechanisms.
<
Insert Figure 6 here >
2.2.5. Induction of apoptosis
To confirm that compound 21a triggered apoptotic cell death, we performed an annexin V
binding assay. H460 cells were treated with compound 21a at various concentrations (1.5, 3.0 and
1
1

6.0 µM) for 48 h, stained with annexin V-FITC and PI, and subjected to flow cytometry analysis. As
+
shown in Fig. 7A, there was a significant time-dependent increase in the percentage of annexin V
cells after 48 h of treatment with compound 21a at various concentrations (1.5, 3, and 6 µM). The
+
frequencies of annexin V cells are summarized in Fig. 7B. These results confirm that the cytotoxic
effects of 21a in H460 cells were due to its ability to induce apoptosis.
<
Insert Figure 7 here >
2.2.2. In vivo antitumour activity
Although 33a was as cytotoxic as 21a, inhibiting all the tested tumour cell lines in vitro and
serving as a stronger DNA cross-linker than 21a, this congener was not selected for in vivo
antitumour activity analysis because of its poor solubility. Alternatively, compound 21a has good
solubility in a mixture of ethanol/PEG400/Cremophor-EL/0.9% saline (10:10:10:70; v/v/v/v) that
can be administered via intravenous injection (i.v.). We therefore selected compound 21a for
antitumour activity evaluation using human tumour xenografts in animal models. The therapeutic
efficacy of 21a was analysed in nude mice bearing human lung cancer H460 and human colorectal
cancer HCT116 xenografts by i.v. administration at 30 mg/kg once every day for five days (QD×5),
and this five-day cycle was repeated for twice with a 2-day interval. Oxaliplatin administered at 7.5
mg/kg once a week for 2 weeks (Q7D×2) through i.v. was used as a positive control. As shown in
Fig. 8A and B (left), compound 21a suppressed approximately 66 and 72% of tumour growth
compared to the vehicle control in H460 and HCT116 xenografts, respectively, at day 24. However,
the tumour size in H460 and HCT116 xenografts was reduced by approximately 34 and 40%,
respectively, in mice treated with oxaliplatin at day 24. Based on the average body weight changes
(Fig. 8A and B, right), neither 21a nor oxaliplatin showed significant systematic toxicity in mice at
1
2

the doses used. These results demonstrated that compound 21a was more effective than oxaliplatin in
inhibiting these in vivo models.
<
Insert Scheme 8 here >
3. Conclusion
In the current study, we designed and synthesized a series of pyrrolo[1,2-f]phenanthridine
(Class I) and dibenzo[f,h]pyrrolo[1,2-b]isoquinoline (Class II) derivatives by coupling a DNA
cross-linking bis(hydroxymethyl)pyrrole pharmacophore with phenanthridine or
a
phenanthroindolizine moiety and evaluated their antitumour activities. We demonstrated that
compounds in Classes I and II showed potent cytotoxicities against the growth of lymphoblastic
leukaemia CCRF/CEM and human colon carcinoma HCT-116, lung cancer H1650 and H460, and
pancreatic cancer PacaS1 cells in vitro. The SAR studies showed that compounds having a Me or Et
1
substituent at R in both classes of compounds are generally more cytotoxic than the corresponding
aryl-substituted
compounds.
Interestingly,
bis(hydroxymethyl)dibenzo[f,h]pyrrolo[1,2-b]-
isoquinolines (e.g., 33a and 33b) are more cytotoxic and induce more DNA cross-linking than the
corresponding bis(hydroxymethyl)pyrrolo[1,2-f]phenanthridines (e.g., 21a and 21b). Whether
bis(hydroxymethyl)pyrrolo[1,2-f]phenanthridines have other biological activities warrants further
investigation. The results of the present study further confirmed that the substituents on the pyrrole
affect the degree of electronic perturbation in the participating pyrrole and thus modulate the
properties of the leaving OH or alkylcarbamate group, the ability to induce DNA cross-linking and
the antitumour activity. Among the newly synthesized compounds, we selected compound 21a for
further antitumour activity evaluation in human tumour xenograft models because of its potent in
vitro cytotoxicity and better solubility in the intravenous injection vehicle. We compared the
therapeutic efficacy of 21a with that of oxaliplatin in nude mice bearing H460 and HCT116
xenografts. 21a showed significant tumour growth inhibition in both the human lung and colorectal
1
3

cancer models. Moreover, these compounds are capable of inducing DNA cross-linking, interfering
with cell cycle progression and triggering cell apoptosis.
We previously constructed various hybrid molecules by coupling β-carboline (Topo I/II
inhibitory moiety) or phthalazine (an anti-angiogenic moiety) with bis(hydroxymethyl)pyrrole
pharmacophorer. It was revealed that these hybrids displayed multiple modes of action with
significant antitumor activity in tumour xenograft models. In comparison with that, we applied
phenanthridine or phenanthroindolizine moieties for preparing hybrids in the present studies. It
showed that the main mechanism of action of the new hybrids is DNA ICL. Due to the planar
structure of phenanthridine and phenathriondolizine, we may infer that these moieties may
intercalate into DNA and hence enhance the interaction as well as cleavage activity of these newly
synthesized compounds. As a result, the newly synthesized compounds are less potent than those
previously synthesized. Nevertheless, the current studies suggest that bis(hydroxymethyl)pyrrole is a
valuable scaffold for designing powerful DNA cross-linking agents with potential antitumor activity
for clinical applications. The anticancer activity of conjugation of other functional moiety to
bis(hydroxymethyl)pyrrole warrants our further investigation.
4. Experimental Protocols
4.1. Materials and methods
All commercial chemicals and solvents were reagent grade. Melting points were determined in
open capillaries on a Fargo melting point apparatus and are uncorrected. Thin-layer chromatography
was performed on silica gel G60 F254 plates (Merck, Merck KGaA, Darmstadt, Germany) with
short-wave UV light for visualization. The purity of all the tested compounds was ≥95% based on
analytical HPLC. High-resolution mass spectrometry (HRMS) was conducted on a Waters HDMS
+
1
G1 instrument with ESI , centroid mode, and the samples were dissolved in MeOH. H NMR spectra
1
3
and C NMR spectra were recorded on a Bruker AVANCE 500 DRX and/or a 400 MHz Bruker
Top-Spin spectrometer in the solvents indicated. The proton chemical shifts are reported in parts per
1
4

million (δ ppm) relative to (CH ) Si, coupling constants (J) are reported in Hertz (Hz), and
3
4
multiplicities are given by the following abbreviations: s, singlet; d, doublet; dd, doublet of doublets; t,
1
13
triplet; m, multiplet; and br s, broad singlet. The HPLC chromatograms and H NMR, C NMR and
HRMS spectra of the new compounds are presented in Appendix A.
4
4
.2. Chemistry
.2.1. 5-(Carboxymethyl)phenanthridin-5-ium bromide (18a).
Bromoacetic acid (4.65 g, 33.0 mmol) was added to a stirred solution of phenanthridine (17, 5.0 g,
28.0 mmol) in acetonitrile (50 mL) at rt. The mixture was heated at reflux for 48 h until a solid product
was obtained. After cooling, the product was collected by filtration, washed with acetonitrile and
1
dried to give compound 18a. Yield 5.8 g (65%); mp 260–262°C; H NMR (DMSO-d ) δ 6.15 (s, 2H,
6
CH ), 8.11–8.18 (m, 3H, ArH), 8.45–8.49 (m, 2H, ArH), 8.61–8.62 (m, 1H, ArH), 9.18–9.23 (m, 2H,
2
13
ArH), 10.54 (s, 1H, ArH); C NMR (DMSO-d ) δ 58.11, 119.81, 123.13, 123.40, 124.98, 125.41,
6
+
1
30.43, 130.66, 132.23, 133.12, 133.69, 134.67, 138.81, 157.04, 167.39; HRMS [ESI ] calcd for
+
C H NBrO , 239.0946 [M+H-Br] , found 239.0884.
15
12
2
4.2.2. 5-Acetyl-5,6-dihydrophenanthridine-6-carbonitrile (19b).
Trimethylsilyl cyanide (5.0 mL, 40.0 mmol) and a catalytic amount of AlCl were added to a
3
stirred solution of 17 (3.6 g, 20.0 mmol) in DCM (60 mL) under an argon atmosphere. To this
reaction mixture was dropwise added acetyl chloride (2.2 mL, 30.0 mmol). After stirring for 4 h at rt,
the reaction was poured into cold water, and the organic layer was separated and washed with water,
5% sodium hydroxide aqueous solution and water, dried over sodium sulfate and concentrated to
1
dryness in vacuo to yield 19b. Yield 4.6 g (92%); mp 170–172°C; H NMR (DMSO-d ) δ 2.22 (s,
6
3
H, COCH ), 7.26 (s, 1H, CH), 7.45–7.52 (m, 3H, ArH), 7.58–7.60 (m, 1H, ArH), 7.69–7.71 (m, 1H,
3
1
3
ArH), 7.77–7.79 (m, 1H, ArH), 8.06–8.08 (m, 2H, ArH); C NMR (DMSO-d ) δ 21.94, 117.26,
6
124.30, 124.89, 125.53, 126.87, 127.11, 128.83, 128.90, 129.96, 130.30, 130.41, 134.21, 168.70;
+
+
HRMS [ESI ] calcd for C H N O, 249.1028 [M+H] , found 249.1034.
16
12
2
1
5

By following the same synthetic procedure as that of 19b, the following compounds were
synthesized:
4.2.3. 5-Propionyl-5,6-dihydrophenanthridine-6-carbonitrile (19c).
Compound 19c was prepared from 17 (3.6 g, 20.0 mmol), trimethylsilyl cyanide (5.0 mL, 40.0
1
mmol) and propionyl chloride (2.7 mL, 30.0 mmol). Yield 5.0 g (95%); mp 152–154°C; H NMR
(
(
(
DMSO-d ) δ 0.98 (t, J = 7.3 Hz, 3H, CH ), 2.31–2.32 (m, 1H, CH ), 2.77–2.85 (m, 1H, CH ), 7.27
6 3 2 2
s, 1H, CH), 7.45–7.51 (m, 3H, ArH), 7.56–7.59 (m, 1H, ArH), 7.69–7.70 (m, 1H, ArH), 7.77–7.78
1
3
m, 1H, ArH), 8.05–8.08 (m, 2H, ArH); C NMR (DMSO-d ) δ 9.28, 26.60, 117.30, 124.30, 124.95,
6
125.67, 126.85, 127.14, 127.28, 128.81, 128.84, 130.13, 130.28, 130.45, 133.96, 172.21; HRMS
+
+
[
ESI ]: calcd for C H N O, 263.1184 [M+H] , found 263.1183.
17 14 2
4.2.4. 5-Benzoyl-5,6-dihydrophenanthridine-6-carbonitrile (19d).
Compound 19d was prepared from 17 (3.6 g, 20.0 mmol), trimethylsilyl cyanide (5.0 mL, 40.0
1
mmol) and benzoyl chloride (3.5 mL, 30.0 mmol). Yield 5.2 g (82%); mp 144–146°C; H NMR
(
DMSO-d ) δ 6.76–6.77 (m, 1H, ArH), 7.10–7.12 (m, 2H, 1 × CH and 1 × ArH), 7.29–7.36 (m, 5H,
6
ArH), 7.46–7.53 (m, 2H, ArH), 7.62–7.65 (m, 1H, ArH), 7.83–7.85 (m, 1H, ArH), 8.06–8.07 (m, 1H,
1
3
ArH), 8.13–8.15 (m, 1H, ArH); C NMR (DMSO-d ) δ 45.10, 117.22, 124.27, 124.94, 125.52,
6
126.10, 126.40, 127.13, 128.33, 128.45, 129.03, 129.32, 130.25, 130.42, 131.57, 133.35, 168.19;
+
+
HRMS [ESI ] calcd for C H N O, 311.1184 [M+H] , found 311.1182.
21
14
2
4.2.5. 5-(4-Fluorobenzoyl)-5,6-dihydrophenanthridine-6-carbonitrile (19e).
Compound 19e was prepared from 17 (3.6 g, 20.0 mmol), trimethylsilyl cyanide (5.0 mL, 40.0
1
mmol) and 4-fluorobenzoyl chloride (3.5 mL, 30.0 mmol). Yield 5.6 g (85%); mp 156–158°C; H
NMR (DMSO-d ) δ 6.77–6.79 (m, 1H, ArH), 7.09 (s, 1H, CH), 7.12–7.19 (m, 3H, ArH), 7.29–7.32
6
(m, 1H, ArH), 7.38–7.41 (m, 2H, ArH), 7.49–7.51 (m, 1H, ArH), 7.59–7.63 (m, 1H, ArH), 7.81–7.83
1
3
(
m, 1H, ArH), 8.04–8.06 (m, 1H, ArH), 8.11–8.12 (m, 1H, ArH); C NMR (DMSO-d ) δ 45.17,
6
115.47, 115.64, 117.18, 124.26, 124.98, 125.53, 126.11, 126.47, 127.13, 128.41, 129.03, 129.30,
1
6

+
1
29.79, 129.81, 130.21, 130.40, 131.90, 131.97, 134.73, 162.64, 164.63, 167.16; HRMS [ESI ] calcd
+
for C H FN O, 329.1090 [M+H] , found 329.1089.
2
1
13
2
4.2.6. 5-(4-Methoxybenzoyl)-5,6-dihydrophenanthridine-6-carbonitrile (19f).
Compound 19f was prepared from 17 (3.6 g, 20.0 mmol), trimethylsilyl cyanide (5.0 mL, 40.0
1
mmol) and 4-methoxybenzoyl chloride (4.1 mL, 30.0 mmol). Yield 5.4 g (79%); mp 148–150°C; H
NMR (DMSO-d ) δ 3.75 (s, 3H, OCH ), 6.78–6.79 (m, 1H, ArH), 6.88–6.89 (m, 1H, ArH), 7.04
6
3
(
(
(
1H, s, CH), 7.14–7.17 (m, 1H, ArH), 7.29–7.33 (m, 3H, ArH), 7.49–7.52 (m, 1H, ArH), 7.61–7.64
1
3
m, 1H, ArH), 7.82–7.83 (m, 1H, ArH), 8.05–8.07 (m, 1H, ArH), 8.12–8.13 (m, 1H, ArH); C NMR
DMSO-d ) δ 45.32, 55.36, 113.76, 117.34, 124.19, 124.94, 125.12, 125.25, 125.88, 126.08, 127.10,
6
+
1
28.41, 128.95, 129.40, 130.34, 130.36, 131.39, 135.38, 161.82, 167.80; HRMS [ESI ] calcd for
+
C H N O , 341.1290 [M+H] , found 341.1369.
22
16
2
2
4.2.7. Dimethyl pyrrolo[1,2-f]phenanthridine-1,2-dicarboxylate (20a).
DMAD (9.7 mL, 80.0 mmol) was slowly added to a stirred suspension of 18a (5.0 g, 16.0 mmol)
and TEA (2.6 mL, 19.0 mmol) in toluene (80 mL) at rt. The reaction mixture was stirred at 90°C for 2
h (monitored by TLC). The mixture was cooled to rt, and the solvent was removed in vacuo. The crude
product was purified by silica gel column chromatography (SiO , hexane:ethyl acetate = 80:20 v/v) to
2
1
give 20a. Yield 2.6 g (50%); mp 175–177°C; H NMR (DMSO-d ) δ 3.85 (s, 3H, COOCH ), 3.96 (s,
6
3
3
7
H, COOCH ), 7.55–7.58 (m, 1H, ArH), 7.60–7.62 (m, 2H, ArH), 7.67–7.68 (m, 1H, ArH), 7.95–
3
.97 (m, 1H, ArH), 8.49–8.50 (m, 1H, ArH), 8.56–8.57 (m, 1H, ArH), 8.59–8.61 (m, 1H, ArH), 8.90
1
3
(
s, 1H, ArH); C NMR (DMSO-d ) δ 51.73, 52.73, 111.41, 116.63, 116.74, 118.74, 121.29, 122.99,
6
123.48, 123.54, 124.39, 125.69, 125.83, 126.17, 128.16, 128.95, 129.68, 131.37, 163.27, 167.32;
+
+
HRMS [ESI ] calcd for C H NO , 356.0899 [M+Na] , found 356.0893.
20
15
4
4.2.8. Dimethyl 3-methylpyrrolo[1,2-f]phenanthridine-1,2-dicarboxylate (20b).
1
7

To a solution of 20b (4.0 gm, 16.0 mmol) in hot acetic acid (100 mL) was dropwise added
tetrafluoroboric acid (HBF ) (3.2 mL, 17.6 mmol). The solution was stirred for 30 min at 60–70°C.
4
The mixture was cooled, the white precipitate was collected by filtration, and the filter cake was
washed with ether to give the desired hydrofluoroborate salt. The solid salt was added to a solution
of DMAD (5.0 mL, 40.0 mmol) in dimethylformamide (DMF) (25 mL) and heated at 95-100°C for
14 h. The reaction mixture was concentrated in vacuo, and the residue was crystallized from
1
methanol to give 20b. Yield 3.6 g (64%); mp 145–147°C; H NMR (DMSO-d ) δ 3.11 (s, 3H, CH ),
6
3
3
7
.81 (s, 3H, COOCH ), 3.93 (s, 3H, COOCH ), 7.54–7.58 (m, 3H, ArH), 7.62–7.65 (m, 1H, ArH),
3 3
.92–7.94 (m, 1H, ArH), 8.39 – 8.40 (m, 1H, ArH), 8.51–8.53 (m, 1H, ArH), 8.60–8.61 (m, 1H,
1
3
ArH); C NMR (DMSO-d ) δ 16.13, 51.75, 52.65, 111.29, 115.25, 118.86, 122.73, 123.05, 123.21,
6
123.83, 124.43, 125.41, 125.74, 127.78, 128.59, 128.87, 132.42, 132.90, 164.22, 167.79; HRMS
+
+
[
ESI ] calcd for C H NO , 348.1236 [M+H] , found 348.1247.
21 17 4
By following the same synthetic procedure as that of 20b, the following compounds were
synthesized:
4.2.9. Dimethyl 3-ethylpyrrolo[1,2-f]phenanthridine-1,2-dicarboxylate (20c).
Compound 20c was prepared from 19c (4.0 g, 15 mmol), HBF (3.0 mL, 16.5 mmol) and
4
1
DMAD (4.7 mL, 37.5 mmol). Yield 3.2 g (58%); mp 121–123°C; H NMR (DMSO-d ) δ 1.38 (t, J =
6
7
.3 Hz, 3H, CH ), 3.58 (m, 2H, CH ), 3.82 (s, 3H, COOCH ), 3.94 (s, 3H, COOCH ), 7.55–7.59 (m,
3 2 3 3
3H, ArH), 7.67–7.70 (m, 1H, ArH), 7.91–7.93 (m, 1H, ArH), 8.24–8.26 (m, 1H, ArH), 8.51–8.53 (m,
1
3
1
H, ArH), 8.61 –8.62 (m, 1H, ArH); C NMR (DMSO-d ) δ 13.26, 20.59, 51.80, 52.66, 111.50,
6
115.05, 118.30, 122.73, 123.02, 123.19, 123.87, 124.63, 125.56, 125.62, 125.85, 127.82, 128.89,
+
+
1
29.02, 132.60, 137.71, 164.07, 167.81; HRMS [ESI ] calcd for C H NO , 362.1392 [M+H] ,
22 19 4
found 362.1385.
4.2.10. Dimethyl 3-phenylpyrrolo[1,2-f]phenanthridine-1,2-dicarboxylate (20d).
1
8

Compound 20d was prepared from 19d (5.0 g, 16.0 mmol), HBF (3.2 mL, 17.6 mmol) and
4
1
DMAD (5.0 mL, 40.0 mmol). Yield 3.2 g (48%); mp 132–134°C; H NMR (DMSO-d ) δ 3.59 (s,
6
3
H, COOCH ), 3.95 (s, 3H, COOCH ), 7.04–7.06 (m, 1H, ArH), 7.12–7.16 (m, 1H, ArH), 7.39–7.42
3 3
(m, 1H, ArH), 7.47–7.49 (m, 2H, ArH), 7.54–7.59 (m, 3H, ArH), 7.61–7.63 (m, 2H, ArH), 8.15–8.17
1
3
(
m, 1H, ArH), 8.55–8.58 (m, 2H, ArH); C NMR (DMSO-d ) δ 51.63, 52.68, 111.24, 117.93,
6
1
1
4
18.66, 123.06, 123.28, 123.43, 123.78, 124.69, 125.62, 126.11, 126.88, 127.83, 128.25, 128.89,
+
29.22, 130.18, 132.29, 132.54, 132.60, 163.60, 167.12; HRMS [ESI ] calcd for C H NO ,
2
6
19
4
+
10.1392 [M+H] , found 410.1393.
4.2.11. Dimethyl 3-(4-fluorophenyl)pyrrolo[1,2-f]phenanthridine-1,2-dicarboxylate (20e).
Compound 20e was prepared from 19e (5.3 g, 16.0 mmol), HBF (3.2 mL, 17.6 mmol) and
4
1
DMAD (5.0 mL, 40.0 mmol). Yield 3.1 g, (45%); mp 148–150°C; H NMR (DMSO-d ) δ 3.60 (s,
6
3
H, COOCH ), 3.95 (s, 3H, COOCH ), 7.07–7.09 (m, 1H, ArH), 7.22–7.24 (m, 1H, ArH), 7.37–7.44
3 3
(m, 3H, ArH), 7.54–7.57 (m, 2H, ArH), 7.61–7.64 (m, 2H, ArH), 8.12–8.14 (m, 1H, ArH), 8.55–8.60
1
3
(
m, 2H, ArH); C NMR (DMSO-d ) δ 51.66, 52.71, 111.34, 115.87, 116.04, 118.61, 123.08, 123.28,
6
1
1
4
23.35, 123.74, 124.74, 125.66, 126.11, 126.81, 127.99, 128.26, 128.94, 131.64, 132.27, 132.55,
+
+
32.62, 161.53, 163.49, 167.18; HRMS [ESI ] calcd for C H FNO , 450.1118 [M+Na] , found
2
6
18
4
50.1135.
4.2.12. Dimethyl 3-(4-methoxyphenyl)pyrrolo[1,2-f]phenanthridine-1,2-dicarboxylate (20f).
Compound 20f was prepared from 19f (4.4 g, 13.0 mmol), HBF (2.6 mL, 14.2 mmol) and
4
1
DMAD (4.0 mL, 32.5 mmol). Yield 3.0 g (53%); mp 138–140°C; H NMR (DMSO-d ) δ 3.62 (s,
6
3
2
2
1
1
H, COOCH ), 3.88 (s, 3H, OCH ), 3.95 (s, 3H, COOCH ), 7.09–7.11 (m, 2H, ArH), 7.18–7.20 (m,
3 3 3
H, ArH), 7.38–7.41 (m, 3H, ArH), 7.61–7.63 (m, 2H, ArH), 8.15–8.17 (m, 1H, ArH), 8.54–8.58 (m,
1
3
H, ArH); C NMR (DMSO-d ) δ 51.62, 52.65, 55.22, 111.15, 114.34, 117.95, 118.61, 123.04,
6
23.24, 123.42, 123.85, 124.39, 124.65, 125.54, 126.11, 126.68, 127.91, 128.16, 128.86, 159.75,
+
+
63.70, 167.19; HRMS [ESI ] calcd for C H NO , 462.1317 [M+Na] , found 462.1314.
2
7
21
5
1
9

4.2.13. Pyrrolo[1,2-f]phenanthridine-1,2-diyldimethanol (21a).
To a stirred suspension of LAH (0.48 g, 12.5 mmol) in diethyl ether (25 mL) was portionwise
added 20a (1.7 g, 5.0 mmol) in DCM (50 mL) at 0 to -5°C. The reaction mixture was stirred at this
temperature for 50 min. The excess LAH was decomposed by adding water (1 mL), NH OH (1 mL)
4
and more water (1 mL) at 0°C. The mixture was filtered through a pad of Celite, and the filter cake
was washed several times with DCM. The combined filtrate and washings were sequentially washed
with water and brine, dried over sodium sulfate and concentrated to dryness in vacuo. The residue
1
was crystalized from ether to give 21a. Yield 1.25 g (89%); mp 194–196°C; H NMR (DMSO-d ) δ
6
4
.65 (d, J = 5.1 Hz, 2H, OCH ), 4.82 (d, J = 4.9 Hz, 2H, OCH ), 4.89 (t, J = 5.1 Hz, 1H, OH,
2
2
exchangeable), 4.93 (t, J = 4.9 Hz, 1H, OH, exchangeable), 7.40–7.43 (m, 1H, ArH), 7.46–7.49 (m,
H, ArH), 7.56–7.60 (m, 2H, ArH), 8.08 (s, 1H, ArH), 8.16–8.18 (m, 1H, ArH), 8.41–8.43 (m, 1H,
1
1
3
ArH), 8.46–8.51 (m, 1H, ArH), 8.51–8.53 (m, 1H, ArH); C NMR (DMSO-d ) δ 54.29, 55.40,
6
1
1
2
11.90, 115.29, 117.76, 120.59, 122.72, 124.04, 124.15, 124.45, 124.85, 125.58, 125.85, 126.12,
+
+
28.21, 128.35, 129.15, 132.53; HRMS [ESI ] calcd for C H NO , 260.1075 [M+H-H O] , found
1
8
15
2
2
60.1101.
By following the same synthetic procedure as that of 21a, the following compounds were
synthesized:
4.2.14. (3-Methylpyrrolo[1,2-f]phenanthridine-1,2-diyl)dimethanol (21b).
Compound 21b was prepared from 20b (2.1 g, 6.0 mmol) and LAH (0.6 g, 15 mmol). Yield 1.6
1
g (91%); mp 158–160°C; H NMR (DMSO-d ) δ 2.86 (s, 3H, CH ), 4.60 (d, J = 5.1 Hz, 2H, OCH ),
6
3
2
4
.63 (t, J = 5.0 Hz, 1H, OH, exchangeable), 4.82 (d, J = 5.0 Hz, 2H, OCH ), 4.90 (t, J = 5.1 Hz, 1H,
2
OH, exchangeable), 7.41–7.44 (m, 2H, ArH), 7.52–7.57 (m, 2H, ArH), 8.31–8.33 (m, 1H, ArH),
1
3
8
.42–8.43 (m, 2H, ArH), 8.52–8.54 (m, 1H, ArH); C NMR (DMSO-d ) δ 15.53, 53.31, 54.27,
6
117.43, 117.63, 122.40, 122.43, 123.80, 124.07, 124.34, 124.69, 125.37, 125.43, 125.83, 126.39,
2
0

+
+
1
2
28.11, 128.33, 134.30; HRMS [ESI ] calcd for C H NO , 274.1226 [M+H-H O] , found
19 17 2 2
74.1243.
4.2.15. (3-Ethylpyrrolo[1,2-f]phenanthridine-1,2-diyl)dimethanol (21c).
Compound 21c was prepared from 20c (2.5 g, 7.0 mmol) and LAH (0.65 g, 17.5 mmol). Yield
1
1
.85 g (88%); mp 167–169°C; H NMR (DMSO-d ) δ 1.32 (t, J = 7.3 Hz, 3H, CH ), 3.32 (m, 2H,
6
3
CH ), 4.60 (d, J = 5.0 Hz, 2H, OCH ), 4.65 (t, J = 4.9 Hz, 1H, OH exchangeable), 4.82 (d, J = 4.9
2
2
Hz, 2H, OCH ), 4.92 (t, J = 5.0 Hz, 1H, OH exchangeable), 7.42–7.44 (m, 2H, ArH), 7.53–7.56 (m,
2
1
1
1
H, ArH), 7.58–7.61 (m, 1H, ArH), 8.17–8.19 (m, 1H, ArH), 8.42–8.44 (m, 2H, ArH), 8.53–8.55 (m,
1
3
H, ArH); C NMR (DMSO-d ) δ 14.46, 20.30, 53.20, 54.28, 117.09, 117.73, 123.37, 123.86,
6
24.16, 124.21, 124.66, 125.50, 125.67, 125.77, 126.43, 128.33, 128.46, 130.20, 133.39; HRMS
+
+
[
ESI ] calcd for C H NO , 288.1339 [M+H-H O] , found 288.1387.
20 19 2 2
4.2.16. (3-Phenylpyrrolo[1,2-f]phenanthridine-1,2-diyl)dimethanol (21d).
Compound 21d was prepared from 20d (2.85 g, 7.0 mmol) and LAH (0.65 g, 17.5 mmol). Yield
1
2
.1 g (85%); mp 210–212°C; H NMR (DMSO-d ) δ 4.36 (d, J = 4.1 Hz, 2H, OCH ), 4.77 (t, J = 4.1
6
2
Hz, 1H, OH, exchangeable), 4.93 (d, J = 3.7 Hz, 2H, OCH ), 5.02 (t, J = 4.4 Hz, 1H, OH,
2
exchangeable), 7.11–7.12 (m, 2H, ArH), 7.29–7.30 (m, 1H, ArH), 7.46–7.54 (m, 6H, ArH), 7.59–
1
3
7
.62 (m, 1H, ArH), 8.46–8.51 (m, 3H, ArH); C NMR (DMSO-d ) δ 53.37, 54.34, 118.19, 118.47,
6
122.47, 122.58, 123.83, 124.46, 124.83, 125.39, 126.09, 126.32, 127.15, 127.37, 127.65, 127.94,
+
+
1
28.05, 128.87, 129.90, 133.27, 133.64; HRMS [ESI ] calcd for C H NO , 336.1383 [M+H-H O] ,
24 19 2 2
found 336.1416.
4.2.17. (3-(4-Fluorophenyl)pyrrolo[1,2-f]phenanthridine-1,2-diyl)dimethanol (21e).
Compound 21e was prepared from 20e (2.15 g, 5.0 mmol) and LAH (0.48 g, 12.5 mmol). Yield
1
1
.7 g (91%); mp 190–192°C; H NMR (DMSO-d ) δ 4.34 (d, J = 4.9 Hz, 2H, OCH ), 4.80 (t, J = 4.9
6
2
Hz, 1H, OH, exchangeable), 4.92 (d, J = 4.7 Hz, 2H, OCH ), 5.03 (t, J = 4.7 Hz, 1H, OH,
2
2
1

exchangeable), 7.10–7.12 (m, 1H, ArH), 7.16–7.19 (m, 1H, ArH), 7.29–7.32 (m, 1H, ArH), 7.36–
1
3
7
.40 (m, 2H, ArH), 7.48–7.52 (m, 3H, ArH), 7.59–7.62 (m, 1H, ArH), 8.46–8.52 (m, 3H, ArH); C
NMR (DMSO-d ) δ 53.30, 54.30, 115.89, 115.96, 118.07, 118.40, 122.50, 122.58, 123.89, 124.53,
6
1
1
3
24.83, 125.39, 126.14, 126.26, 126.84, 127.21, 127.52, 127.86, 128.50, 130.04, 130.07, 131.97,
+
+
32.03, 133.20, 160.86, 162.82; HRMS [ESI ] calcd for C H FNO , 354.1294 [M+H-H O] , found
2
4
18
2
2
54.1316.
4.2.18. (3-(4-Methoxyphenyl)pyrrolo[1,2-f]phenanthridine-1,2-diyl)dimethanol (21f).
Compound 21f was prepared from 20f (2.0 g, 4.5 mmol) and LAH (0.43 g, 11.3 mmol). Yield
1
1
.54 g (88%); mp 177–179°C; H NMR (DMSO-d ) δ 3.85 (s, 3H, OCH ), 4.35 (d, J = 5.0 Hz, 2H,
6
3
OCH ), 4.72 (t, J = 5.0 Hz, 1H, OH, exchangeable), 4.92 (d, J = 4.9 Hz, 2H, OCH ), 5.00 (t, J = 4.9
2
2
Hz, 1H, OH, exchangeable), 7.09–7.11 (m, 2H, ArH), 7.13–7.16 (m, 1H, ArH), 7.21–7.23 (m, 1H,
ArH), 7.27–7.30 (m, 1H, ArH), 7.37–7.39 (m, 2H, ArH), 7.46–7.49 (m, 1H, ArH), 7.58–7.61 (m, 1H,
1
3
ArH), 8.44–8.50 (m, 3H, ArH); C NMR (DMSO-d ) δ 53.46, 54.41, 55.18, 114.32, 117.99, 118.33,
6
122.45, 122.54, 123.75, 124.41, 124.76, 125.31, 125.81, 125.95, 126.41, 126.75, 127.43, 127.46,
+
+
1
27.90, 128.45, 131.23, 133.50, 159.00; HRMS [ESI ] calcd for C H NO , 366.1494 [M+H-H O] ,
2
5
21
3
2
found 366.1516.
4
.2.19. General procedures for the preparation of bis(alkylcarbamate) derivatives (22a-f and 23a-f).
Alkyl isocyanate (4.0 equivalents) and TEA (4.0 equivalents) were added to a solution of
bis(hydroxymethyl) derivative (21a-e, 1.0 equivalent) in anhydrous THF or DMF. The reaction
mixture was stirred at ambient temperature for 24–48 h under an argon atmosphere. After completion
of the reaction, the reaction mixture was concentrated to dryness in vacuo. The desired product was
obtained by crystallization.
4.2.19.1. Pyrrolo[1,2-f]phenanthridine-1,2-diylbis(methylene)bis(ethylcarbamate (22a).
2
2

Compound 22a was prepared from 21a (0.42 g, 1.5 mmol), TEA (0.84 mL, 6.0 mmol) and ethyl
1
isocyanate (0.48 mL, 6.0 mmol). Yield 0.38 g (60%); mp 145–147°C; H NMR (DMSO-d ) δ 1.01 (t,
6
J = 7.3 Hz, 6H, 2 × CH ), 3.01–3.04 (m, 4H, 2 × CH ), 5.20 (s, 2H, OCH ), 5.40 (s, 2H, OCH ),
3
2
2
2
7.09–7.13 (br s, 2H, 2 × NH, exchangeable), 7.47–7.62 (m, 4H, ArH), 8.17–8.26 (m, 3H, ArH),
1
3
8
.54–8.56 (m, 2H, ArH); C NMR (DMSO-d ) δ 15.02, 35.05, 57.19, 57.50, 112.99, 114.13, 115.49,
6
120.67, 123.09, 123.46, 123.59, 124.24, 124.68, 125.20, 125.38, 126.32, 126.53, 128.60, 129.33,
+
+
1
32.06, 155.99, 156.18; HRMS [ESI ] calcd for C H N O , 244.1125 [M+H-2(OCONHC H )] ,
24 25 3 4 2 5
found 244.1144.
4.2.19.2. (3-Methylpyrrolo[1,2-f]phenanthridine-1,2-diyl)bis(methylene)bis(ethylcarbamate) (22b).
Compound 22b was prepared from 21b (0.3 g, 1.0 mmol), TEA (0.55 mL, 4.0 mmol) and ethyl
1
isocyanate (0.33 mL, 4.0 mmol). Yield 0.24 g (52%); mp 172–174°C; H NMR (DMSO-d ) δ 0.98
6
(
t, J = 7.3 Hz, 3H, CH ), 1.01 (t, J = 7.3 Hz, 3H, CH ), 2.87 (s, 3H, CH ), 2.97–3.04 (m, 4H, 2 ×
3 3 3
CH ), 5.22 (s, 2H, OCH ), 5.40 (s, 2H, OCH ), 7.05 (br s, 1H, NH, exchangeable), 7.12 (br s, 1H,
2
2
2
NH, exchangeable), 7.45–7.50 (m, 2H, ArH), 7.55–7.60 (m, 2H, ArH), 8.13–8.15 (m, 1H, ArH),
1
3
8
1
1
.33–8.34 (m, 1H, ArH), 8.47–8.49 (m, 1H, ArH), 8.55–8.57 (m, 1H, ArH); C NMR (DMSO-d ) δ
6
5.00, 15.50, 35.06, 56.14, 57.37, 112.73, 117.78, 121.58, 122.46, 122.77, 123.18, 124.15, 124.37,
+
25.11, 125.66, 126.12, 126.31, 128.29, 128.57, 133.80, 156.04, 156.16; HRMS [ESI ] calcd for
+
C H N O , 258.1282 [M+H-2(OCONHC H )] , found 258.1289.
25
27
3
4
2
5
4.2.19.3. ((3-Ethylpyrrolo[1,2-f]phenanthridine-1,2-diyl)bis(methylene)bis(ethylcarbamate)) (22c).
Compound 22c was prepared from 21c (0.32 g, 1.0 mmol), TEA (0.55 mL, 4.0 mmol) and ethyl
1
isocyanate (0.33 mL, 4.0 mmol). Yield 0.3 g, (64%); mp 180–182°C; H NMR (DMSO-d ) δ 0.99 (t,
6
J = 7.1 Hz, 3H, CH ), 1.02 (t, J = 7.1 Hz, 3H, CH ), 1.30 (t, J = 6.0 Hz, 3H, CH ), 2.99–3.05 (m, 4H,
3
3
3
2
× CH ), 3.34 (m, 2H, CH ), 5.22 (s, 2H, OCH ), 5.40 (s, 2H, OCH ), 7.05 (br s, 1H, NH,
2 2 2 2
exchangeable), 7.13 (br s, 1H, NH, exchangeable), 7.48–7.50 (m, 2H, ArH), 7.55–7.58 (m, 1H, ArH),
.61–7.64 (m, 1H, ArH), 8.13–8.14 (m, 1H, ArH), 8.19–8.23 (m, 1H, ArH), 8.48–8.50 (m, 1H, ArH),
7
2
3

1
3
8
1
1
.57–8.58 (m, 1H, ArH); C NMR (DMSO-d ) δ 14.20, 15.00, 20.31, 35.02, 35.05, 57.98, 57.39,
6
12.80, 117.39, 121.50, 22.38, 122.76, 123.22, 124.30, 124.46, 125.08, 125.69, 126.20, 126.62,
+
28.58, 128.67, 132.04, 133.47, 155.97, 156.15; HRMS [ESI ] calcd for C H N O , 272.1438
2
6
29
3
4
+
[
M+H-2(OCONHC H )] , found 272.1464.
2 5
4.2.19.4. (3-Phenylpyrrolo[1,2-f]phenanthridine-1,2-diyl)bis(methylene)bis(ethylcarbamate) (22d).
Compound 22d was prepared from 21d (0.7 g, 2.0 mmol), TEA (1.1 mL, 8.0 mmol) and ethyl
1
isocyanate (0.64 mL, 8.0 mmol). Yield 0.53 g (54%); mp 200–202°C; H NMR (DMSO-d ) δ 1.02
6
(
t, J = 4.9 Hz, 6H, 2 × CH ), 2.99–3.05 (m, 4H, 2 × CH ), 4.92 (s, 2H, OCH ), 5.47 (s, 2H, OCH ),
3 2 2 2
7.05–7.12 (m, 4H, 2 × NH, exchangeable, and 2 × ArH), 7.33–7.34 (m, 1H, ArH), 7.43–7.44 (m, 2H,
1
3
ArH), 7.55–7.63 (m, 5H, ArH), 8.20–8.21 (m, 1H, ArH), 8.52–8.53 (m, 2H, ArH); C NMR
(
DMSO-d ) δ 14.99, 35.00, 35.08, 56.32, 56.38, 113.21, 118.26, 122.48, 122.95, 123.08, 123.76,
6
1
1
24.41, 124.52, 125.58, 125.69, 126.72, 127.57, 127.67, 128.63, 128.74, 129.10, 129.66, 129.99,
+
32.81, 133.01, 155.77, 156.14; HRMS [ESI ]: calcd for C H N O , 320.1438
3
0
29
3
4
+
[
M+H-2(OCONHC H )] , found 320.1451.
2 5
4.2.19.5.
(3-(4-Fluorophenyl)pyrrolo[1,2-f]phenanthridine-1,2-diyl)bis(methylene)bis(ethylcarbamate) (22e).
Compound 22e was prepared from 21e (0.37 g, 1.0 mmol), TEA (0.55 mL, 4.0 mmol) and ethyl
1
isocyanate (0.33 mL, 4.0 mmol). Yield 0.28 g (55%); mp 175–177°C; H NMR (DMSO-d ) δ 0.99
6
(
t, J = 6.9 Hz, 3H, CH ), 1.02 (t, J = 7.1 Hz, 3H, CH ), 2.94–3.06 (m, 4H, 2 × CH ), 4.91 (s, 2H,
3 3 2
OCH ), 5.46 (s, 2H, OCH ), 7.04 (br s, 1H, NH, exchangeable), 7.10–7.11 (m, 1H, ArH), 7.15 (br s,
2
2
1
7
H, NH, exchangeable), 7.20–7.22 (m, 1H, ArH), 7.34–7.42 (m, 3H, ArH), 7.47–7.50 (m, 2H, ArH),
.54–7.57 (m, 1H, ArH), 7.61–7.64 (m, 1H, ArH), 8.20–8.21 (m, 1H, ArH), 8.53–8.55 (m, 2H, ArH);
1
3
C NMR (DMSO-d ) δ 15.01, 35.01, 35.09, 56.23, 57.36, 113.20, 116.08, 116.25, 118.18, 122.52,
6
122.98, 123.37, 123.78, 124.49, 124.61, 125.53, 125.71, 126.79, 127.76, 128.51, 128.78, 129.44,
2
4

+
1
3
32.21, 132.28, 132.77, 155.75, 156.14, 161.16, 163.12; HRMS [ESI ]: calcd for C H FN O ,
30 28 3 4
+
38.1344 [M+H-2(OCONHC H )] , found 338.1376.
2
5
4.2.19.6.
(3-(4-Methoxyphenyl)pyrrolo[1,2-f]phenanthridine-1,2-diyl)bis(methylene)bis(ethyl-carbamate)
(22f).
Compound 22f was prepared from 21f (0.4 g, 1.0 mmol), TEA (0.55 mL, 4.0 mmol) and ethyl
1
isocyanate (0.33 mL, 4.0 mmol). Yield 0.35 g (64%); mp 155–157°C; H NMR (DMSO-d ) δ 0.99
6
(
t, J = 7.1 Hz, 3H, CH ), 1.02 (t, J = 7.2 Hz, 3H, CH ), 2.97–3.06 (m, 4H, 2 × CH ), 3.86 (s, 3H,
3 3 2
OCH ), 4.90 (s, 2H, OCH ), 5.45 (s, 2H, OCH ), 7.04 (br s, 1H, NH, exchangeable), 7.10–7.22 (m,
3
2
2
5H, 1 × NH, exchangeable, and ArH), 7.32–7.35 (m, 3H, ArH), 7.52–7.55 (m, 1H, ArH), 7.60–7.63
1
3
(
m, 1H, ArH), 8.18–8.19 (m, 1H, ArH), 8.51–8.53 (m, 2H, ArH); C NMR (DMSO-d ) δ 15.01,
6
3
1
1
5.00, 35.08, 55.19, 56.45, 57.43, 113.06, 114.54, 118.08, 122.46, 122.94, 123.69, 124.35, 124.49,
25.02, 125.63, 125.67, 126.62, 127.35, 127.67, 128.71, 129.64, 131.34, 133.04, 155.82, 156.16,
+
+
59.34; HRMS [ESI ] calcd for C H N O , 350.1544 [M+H-2(OCONHC H )] , found 350.1565.
3
1
31
3
5
2
5
4.2.19.7. Pyrrolo[1,2-f]phenanthridine-1,2-diylbis(methylene)bis(isopropylcarbamate) (23a).
Compound 23a was prepared from 21a (0.42 g, 1.5 mmol), TEA (0.84 mL, 6.0 mmol) and
1
isopropyl isocyanate (0.6 mL, 6.0 mmol). Yield 0.36 g (53%); mp 166–168°C; H NMR (DMSO-d ) δ
6
1
7
7
.04–1.06 (m, 12H, 4 × CH ), 3.60–3.64 (m, 2H, 2 × CH), 5.19 (s, 2H, OCH ), 5.39 (s, 2H, OCH ),
3
2
2
.02–7.06 (br s, 2H, 2 × NH, exchangeable), 7.45–7.48 (m, 1H, ArH), 7.52–7.55 (m, 1H, ArH),
1
3
.58–7.64 (m, 2H, ArH), 8.16–8.21 (m, 2H, ArH), 8.28 (s, 1H, ArH), 8.52–8.57 (m, 2H, ArH); C
NMR (DMSO-d ) δ 22.54, 12.31, 57.03, 57.37, 113.04, 114.13, 115.49, 120.67, 123.10, 123.45,
6
123.60, 124.26, 124.69, 125.20, 125.39, 126.29, 126.53, 128.60, 129.35, 132.07, 155.30, 155.52;
+
+
HRMS [ESI ] calcd for C H N O , 244.1126 [M+H-2(OCONHC H )] , found 244.1156.
26
29
3
4
3
7
2
5

4
.2.19.8. (3-Methylpyrrolo[1,2-f]phenanthridine-1,2-diyl)bis(methylene)bis(isopropylcarbamate)
23b).
Compound 23b was prepared from 21b (0.3 g, 1.0 mmol), TEA (0.55 mL, 4.0 mmol) and
(
1
isopropyl isocyanate (0.4 mL, 4.0 mmol). Yield 0.3 g (63%); mp 186–188°C; H NMR (DMSO-d ) δ
6
1
5
.02–1.05 (m, 12H, 4 × CH ), 2.88 (s, 3H, CH ), 3.59–3.65 (m, 2H, 2 × CH), 5.22 (s, 2H, OCH ),
3 3 2
.40 (s, 2H, OCH ), 6.96–6.97 (br s, 1H, NH, exchangeable), 7.02–7.04 (br s, 1H, NH,
2
exchangeable), 7.46–7.50 (m, 2H, ArH), 7.54–7.60 (m, 2H, ArH), 8.14–8.15 (m, 1H, ArH), 8.33–
1
3
8
1
1
.35 (m, 1H, ArH), 8.48–8.50 (m, 1H, ArH), 8.56–8.58 (m, 1H, ArH); C NMR (DMSO-d ) δ
6
5.51, 22.52, 42.24, 42.31, 55.96, 57.20, 112.80, 117.80, 121.61, 122.46, 122.78, 123.21, 124.17,
+
24.39, 125.11, 125.68, 126.13, 126.30, 128.32, 128.55, 133.81, 155.35, 155.50; HRMS [ESI ] calcd
+
for C H N O , 258.1283 [M+H-2(OCONHC H )] , found 258.1302.
2
7
31
3
4
3
7
4
.2.19.9. (3-Ethylpyrrolo[1,2-f]phenanthridine-1,2-diyl)bis(methylene)bis(isopropylcarbamate)
23c).
Compound 23c was prepared from 21c (0.31 g, 1.0 mmol), TEA (0.55 mL, 4.0 mmol) and
(
1
isopropyl isocyanate (0.4 mL, 4.0 mmol). Yield 0.28 g (58%); mp 196–198°C; H NMR (DMSO-d )
6
δ 0.99–1.06 (m, 12H, 4 × CH ), 1.29 (t, J = 6.4 Hz, 3H, CH ), 3.31–3.35 (m, 2H, CH ), 3.59–3.65 (m,
3
3
2
2
7
7
8
1
1
H, 2 × CH), 5.21 (2H, s, OCH ), 5.40 (s, 2H, OCH ), 6.96–6.97 (br s, 1H, NH, exchangeable),
2 2
.05–7.06 (br s, 1H, NH, exchangeable), 7.49–7.51 (m, 2H, ArH), 7.54–7.57 (m, 1H, ArH), 7.62–
.65 (m, 1H, ArH), 8.13–8.14 (m, 1H, ArH), 8.22–8.24 (m, 1H, ArH), 8.49–8.50 (m, 1H, ArH),
1
3
.57–8.59 (m, 1H, ArH); C NMR (DMSO-d ) δ 14.18, 20.33, 22.53, 23.26, 42.30, 55.80, 57.22,
6
12.88, 117.40, 121.55, 122.40, 122.78, 123.27, 124.33, 124.49, 125.10, 125.72, 126.22, 126.63,
+
28.57, 128.70, 132.04, 132.49, 155.32, 155.50; HRMS [ESI ] calcd for C H N O , 272.1439
2
8
33
3
4
+
[
M+H-2(OCONHC H )] , found 272.1461.
3 7
4.2.19.10. (3-Phenylpyrrolo[1,2-f]phenanthridine-1,2-diyl)bis(methylene)bis(isopropylcarbamate)
(23d).
2
6

Compound 23d was prepared from 21d (0.7 g, 2.0 mmol), TEA (1.1 mL, 8.0 mmol) and
1
isopropyl isocyanate (0.8 mL, 8.0 mmol). Yield 0.57 g (55%); mp 212–214°C; H NMR (DMSO-d )
6
δ 1.05 (d, J = 5.5 Hz, 12H, 4 × CH ), 3.57–3.65 (m, 2H, 2 × CH), 4.91 (s, 2H, OCH ), 5.47 (s, 2H,
3
2
OCH ), 6.98 (br s, 1H, NH, exchangeable), 7.10–7.14 (m, 3H, 1 × NH, exchangeable, and ArH),
2
7
8
1
1
3
.33–7.34 (m, 1H, ArH), 7.42–7.45 (m, 2H, ArH), 7.55–7.64 (m, 5H, ArH), 8.19–8.20 (m, 1H, ArH),
1
3
.52–8.54 (m, 2H, ArH); C NMR (DMSO-d ) δ 22.54, 42.34, 56.12, 57.20, 113.29, 118.28, 122.51,
6
22.98, 123.18, 123.78, 124.44, 124.55, 125.61, 125.70, 126.73, 127.60, 127.65, 128.65, 128.74,
+
29.12, 129.66, 130.00, 132.81, 133.02, 155.10, 155.50; HRMS [ESI ] calcd for C H N O ,
3
2
33
3
4
+
20.1409 [M+H-2(OCONHC H )] , found 320.1449.
3
7
4.2.19.11.
(3-(4-Fluorophenyl)pyrrolo[1,2-f]phenanthridine-1,2-diyl)bis(methylene)bis(isopropylcarbamate)
(23e).
Compound 23e was prepared from 21e (0.37 g, 1.0 mmol), TEA (0.55 mL, 4.0 mmol) and ethyl
1
isocyanate (0.4 mL, 4.0 mmol). Yield 0.16 g (55%); mp 184–186°C; H NMR (DMSO-d ) δ 1.02–
6
1
.06 (m, 12H, 4 × CH ), 3.56–3.66 (m, 2H, 2 × CH), 4.91 (s, 2H, OCH ), 5.47 (s, 2H, OCH ), 6.97
3
2
2
(br s, 1H, NH, exchangeable), 7.08–7.11 (m, 2H, 1 × NH, exchangeable, and ArH), 7.19–7.22 (m,
1
1
5
1
1
H, ArH), 7.34–7.41 (m, 3H, ArH), 7.48–7.49 (m, 2H, ArH), 7.54–7.57 (m, 1H, ArH), 7.60–7.63 (m,
1
3
H, ArH), 8.19–8.20 (m, 1H, ArH), 8.54–8.55 (m, 2H, ArH); C NMR (DMSO-d ) δ 22.53, 22.35,
6
6.03, 57.17, 116.07, 116.25, 118.19, 122.53, 122.98, 123.79, 124.49, 124.61, 125.55, 125.71,
26.78, 127.69, 127.76, 128.49, 128.74, 129.46, 132.20, 132.26, 132.77, 155.05, 155.49, 161.16,
+
+
63.12; HRMS [ESI ] calcd for C H FN O , 338.1345 [M+H-2(OCONHC H )] , found 338.1394.
3
2
32
3
4
3
7
4.2.19.12.
(3-(4-Methoxyphenyl)pyrrolo[1,2-f]phenanthridine-1,2-diyl)bis(methylene)bis(iso-propylcarbamate)
(23f).
2
7

Compound 23f was prepared from 21f (0.4 g, 1.0 mmol), TEA (0.55 mL, 4.0 mmol) and
1
isopropyl isocyanate (0.4 mL, 4.0 mmol). Yield 0.38 g (66%); mp 170–172°C; H NMR (DMSO-d )
6
δ 1.03–1.07 (m, 12H, 4 × CH ), 3.57–3.66 (m, 2H, 2 × CH), 3.87 (s, 3H, OCH ), 4.90 (s, 2H, OCH ),
3
3
2
5
.45 (s, 2H, OCH ), 6.97 (br s, 1H, NH, exchangeable), 7.06–7.12 (m, 3H, 1 × NH, exchangeable,
2
and ArH), 7.16–7.22 (m, 2H, ArH), 7.34–7.35 (m, 3H, ArH), 7.50–7.55 (m, 1H, ArH), 7.59–7.62 (m,
1
3
1
4
1
H, ArH), 8.17–8.19 (m, 1H, ArH), 8.51–8.53 (m, 2H, ArH); C NMR (DMSO-d ) δ 22.53, 42.24,
6
2.33, 55.20, 56.25, 57.29, 113.14, 114.55, 118.10, 122.47, 122.95, 123.71, 124.35, 124.51, 125.05,
25.63, 125.68, 126.61, 127.32, 127.67, 128.69, 129.63, 131.35, 133.05, 155.14, 155.52, 159.35;
+
+
HRMS [ESI ] calcd for C H N O , 350.1545 [M+H-2(OCONHC H )] , found 350.1563.
33
35
3
5
3
7
4.2.20. Phenanthren-9-ylmethanol (25).
Sodium borohydride (5.7 g, 150.0 mmol) was suspended in a solution of 9-phenanthrene
carboxaldehyde (24, 10.3 g, 50.0 mmol) in dry THF (200 mL). Isopropanol (100 mL) was slowly
added at rt. The mixture was allowed to stir for 1 h until the reaction was complete. The solvents
were evaporated under reduced pressure to afford a dry residue, and the crude product was triturated
with water (1000 mL), filtered and washed with water to give 25. Yield 10.0 g (96%); mp 146–
1
1
48°C; H NMR (DMSO-d ) δ 5.02 (d, J = 4.1 Hz, 2H, OCH ), 5.42 (t, J = 4.7 Hz, 1H, OH
6
2
exchangeable), 7.61–7.72 (m, 4H, ArH), 7.88 (s, 1H, ArH), 7.96–7.98 (m, 1H, ArH), 8.12–8.14 (m,
1
3
1
H, ArH), 8.79–8.80 (m, 1H, ArH), 8.85–8.86 (m, 1H, ArH); C NMR (DMSO-d ) δ 61.40, 122.71,
6
+
1
23.24, 126.49, 126.73, 126.85, 128.35, 129.43, 129.84, 129.89, 131.20, 136.02; HRMS [ESI ] calcd
+
for C H O, 191.0860 [M+H-H O] , found 191.0861.
1
5
12
2
4.2.21. 9-(Bromomethyl)phenanthrene (26).
Phosphorus tribromide (4.6 mL, 24.0 mmol) was slowly added to a solution of 25 (10.0 g, 48.0
mmol) in DCM (100 mL) at 0°C. The reaction mixture was stirred at 0°C for 2 h. The reaction
mixture was sequentially washed with 5 % NaHCO aqueous solution (100 mL), 10% sodium
3
thiosulfate solution (100 mL) and water (100 mL). The separated organic layer was dried over
2
8