Discovery of potent indenoisoquinoline topoisomerase i poisons lacking the 3-nitro toxicophore

Article

pubs.acs.org/jmc

Discovery of Potent Indenoisoquinoline Topoisomerase I Poisons

Lacking the 3‑Nitro Toxicophore

Daniel E. Beck,^†Monica Abdelmalak,^‡Wei Lv,^†P. V. Narasimha Reddy,^†Gabrielle S. Tender,^‡

Elizaveta O’Neill,^†Keli Agama,^‡Christophe Marchand,^‡Yves Pommier,^‡and Mark Cushman*^,†

^†Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, and the Purdue Center for Cancer

Research, Purdue University, 575 Stadium Mall Drive, West Lafayette, Indiana 47907, United States

^‡Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NCI-Frederick, Frederick, Maryland

21702, United States

S

* Supporting Information

ABSTRACT: 3-Nitroindenoisoquinoline human topoisomer-

ase IB (Top1) poisons have potent antiproliferative eﬀects on

cancer cells. The undesirable nitro toxicophore could hypo-

thetically be replaced by other functional groups that would

retain the desired biological activities and minimize potential

safety risks. Eleven series of indenoisoquinolines bearing 3-

nitro bioisosteres were synthesized. The molecules were

evaluated in the Top1-mediated DNA cleavage assay and in the National Cancer Institute’s 60 cell line cytotoxicity assay.

The data reveal that ﬂuorine and chlorine may substitute for the 3-nitro group with minimal loss of Top1 poisoning activity. The

new information gained from these eﬀorts can be used to design novel indenoisoquinolines with improved safety.

lines overcome the inherent chemical instability of the

camptothecins, and blood plasma protein binding has not

been a signiﬁcant problem in their development. Additional

advantages of the indenoisoquinolines over camptothecins

include the fact that some are not substrates for drug eﬄux

pumps, their ability to overcome Top1 mutations that confer

resistance to camptothecins, and their longer residence times in

the binding site.^1,10,11

Camptothecins and indenoisoquinolines stabilize the cata-

lytic intermediate in the Top-1-mediated DNA relaxation

process known as the cleavage complex, wherein Top1 and its

DNA substrate are covalently bound. Under normal conditions,

this intermediate exists only transiently as the enzyme removes

excessive supercoiling from DNA. Top1 poisons intercalate

between the DNA base pairs at the cleavage site and inhibit

reversal of the complex. Collision of advancing DNA replication

forks with Top1 cleavage sites produces double-strand breaks in

the DNA which, if not properly dealt with, can lead the cell to

enter apoptosis.^1,12,13

Numerous 3-nitroindenoisoquinolines have been identiﬁed

that display Top1 poisoning activities that are greater than the

camptothecins, along with mean graph midpoint (MGM) GI₅₀

values in the double-digit nanomolar range.¹⁴⁻¹⁸3-Position

nitration also contributes to inhibitory activity against tyrosyl

DNA phosphodiesterase 1 (TDP1), an enzyme involved in the

repair of DNA damage caused by Top1 poisons.¹⁹Although an

aromatic nitro group may be acceptable if the drug is selectively

delivered to cancer cells, systemic therapy with a nontargeted

INTRODUCTION

■

The camptothecin (1) derivatives topotecan (2) and irinotecan

(3) are cancer chemotherapeutic drugs that inhibit DNA

religation in the ternary drug−DNA−topoisomerase IB (Top1)

cleavage complex, and they are therefore referred to as Top1

“poisons”, as opposed to Top1 “suppressors”, which inhibit the

initial enzyme-catalyzed DNA cleavage reaction. Both topo-

tecan and irinotecan were approved by the FDA for the

treatment of solid tumors in 1996. Although these drugs are

able to limit the growth of solid tumors, neither is curative and

both have signiﬁcant drawbacks to their use which include

dose-limiting bone marrow suppression and the requirement of

prolonged infusion times. The latter drawback is caused by the

rapid diﬀusion of camptothecins out of their target site. In

addition, the camptothecin core structure is susceptible to

lactone hydrolysis in human blood plasma, and its ring-opened

carboxylate form readily binds to blood plasma proteins.¹New

camptothecins are currently in clinical development, and several

PEGylated and liposomal camptothecins have been granted

orphan drug status by the FDA.²Another strategy for

overcoming the challenges associated with camptothecins is

to develop structurally dissimilar Top1 poisons.

Top1 poisons based on the indenoisoquinoline core scaﬀold

could oﬀer solutions to the problems posed by the current

Top1 poison arsenal. Our group designed and synthesized two

indenoisoquinolines, indotecan (4, LMP400)³and indimitecan

(5, LMP776),³which are being studied in phase I clinical trials

at the National Institutes of Health.^4,5These agents are well

tolerated, and preliminary evidence of eﬃcacy has been found.⁶

Their side chain analogue MJ-III-65 (6, LMP744)⁷is under

consideration for human clinical study.^8,9The indenoisoquino-

Received: February 21, 2015

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deazaﬂavins display submicromolar IC₅₀values against TDP2

(e.g., 8), the series is plagued by cell membrane permeability

issues.³⁰

The pharmacological data for our compounds were

rationalized using molecular mechanics-based molecular

modeling and quantum mechanics calculations. Molecular

mechanics calculations indicate hydrogen bonding and van

der Waals forces stabilize the bioisosteric compounds in the

Top1−DNA cleavage complex. Quantum mechanics calcula-

tions provide support for the hypothesis that electron-

withdrawing groups in the 3-position favor stabilization of the

drugs between DNA base pairs in the binding site.¹⁵

CHEMISTRY

■

Benzaldehyde 9 was reduced with NaBH₄to provide the

bromobenzylic alcohol 10 (Scheme 1). Rosenmund−von

a

Scheme 1

a

Reagents and conditions: (a) NaBH₄, MeOH, 0 °C to room temp;

(b) (i) CuCN, DMF, reﬂux, (ii) H₂O, reﬂux; (c) NBS, AIBN, CCl₄,

reﬂux; (d) KOH, H₂O, reﬂux; (e) (i) NaOMe, MeOH, EtOAc, reﬂux,

(ii) Ac₂O, reﬂux.

molecule would expose healthy cells to the nitro toxicophore.²⁰

Metabolic reduction of aryl nitro groups converts them to

potentially toxic aryl nitroso and hydroxylamine species that

can covalently modify the DNA and proteins of healthy cells.²¹

The NCI-60 screening service recently instituted a policy

wherein submission of molecules containing “problematic”

functionalities, including nitro groups, is discouraged.²²For

these reasons, indenoisoquinolines were sought that lack this

potential metabolic and toxicological liability but maintain

similar pharmacological activities.

Braun reaction with CuCN and in situ hydrolysis and

lactonization of the intermediate yielded compound 11.

Phthalide 11 was subjected to radical bromination, and the

obtained 3-bromophthalide intermediate 12 was hydrolyzed to

produce 3-hydroxyphthalide 13. Compound 13 was condensed

with phthalide (14) in reﬂuxing methanol−EtOAc with

NaOMe and then dehydratively cyclized in reﬂuxing Ac₂O to

aﬀord 3-ﬂuoroindenobenzopyran 15.

This report details the design, synthesis, and biological

evaluation of bioisosteric indenoisoquinolines that display

potent Top1 poisoning and growth inhibitory activities. In

nearly all cases, the analogues were outperformed by their 3-

nitro counterparts in one or both of these regards. One of the

new Top1 poisons exhibits inhibitory activity against the DNA

repair enzymes TDP1 and tyrosyl DNA phosphodiesterase 2

(TDP2, an enzyme that participates in the repair of DNA

damage caused by topoisomerase II poisons).²³These triple

enzyme inhibitory activities increase the attractiveness of

indenoisoquinolines for continued development as cancer

chemotherapeutic agents. At present, there are few reported

TDP1 and TDP2 inhibitors.^19,24−33We discovered bis-

(indenoisoquinoline) 7, which is a potent dual Top1 poison

and TDP1 inhibitor.³²A separate group recently disclosed a

series of deazaﬂavin TDP2 inhibitors.³⁰Although some of the

Indenobenzopyrans 30−33 were synthesized by following

nearly identical synthetic routes, whose only point of diﬀerence

was in step c (Scheme 2). 6-Aminophthalide (17) was prepared

in two steps from phthalide (14) following known

procedures.^34,35Sandmeyer chemistry was employed to

introduce halogens or a nitrile group. Radical bromination

and hydrolysis of each 3-bromophthalide 18−21 gave 3-

hydroxyphthalides 26−29, which were used to make

indenobenzopyrans 30, 31,³⁶32, and 33.¹⁹

A fortuitous byproduct of the synthetic route to

indenobenzopyran 43 permitted a comparison of the eﬀect of

substituting either the 2- or 3-positions with a triﬂuoromethyl

group (Scheme 3). Phthalic acid 34 was converted to its

anhydride 35 in reﬂuxing AcCl. Anhydride 35 was reduced with

NaBH₄in THF to give a 1:1 mixture of 5- and 6-

(triﬂuoromethyl)phthalides 36 and 37, which were readily

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a

condensation of 3-hydroxyphthalide 40 or 41 with phthalide

(14), and dehydrative cyclization of the unisolated intermediate

was carried out as before in reﬂuxing Ac₂O.

Scheme 2

3-Hydroxyphthalide 13 underwent nucleophilic aromatic

substitution with NaSMe to deliver product 44 (Scheme 4). 3-

a

Scheme 4

a

Reagents and conditions: (a) NaSMe, DMF, 120 °C; (b) (i) NaOMe,

MeOH, EtOAc, reﬂux, (ii) Ac₂O, reﬂux; (c) m-CPBA, CHCl₃, room

temp.

Hydroxyphthalide 44 was condensed with phthalide (14) and

dehydratively cyclized as described before to provide lactone

45. Oxidation of lactone 45 with m-CPBA provided

indenobenzopyran 46.

(S)-3-Amino-1,2-propanediol (48) was condensed with

indenobenzopyrans 15, 30, 31,³⁶32, 33,¹⁹42, 43, 45, 46,

and 47³⁶to produce indenoisoquinolines 49−58 (Scheme 5).

1-(3-Aminopropyl)imidazole (59) was condensed with inden-

obenzopyrans 15, 30, 32, 42, 43, 45, and 46 to yield

indenoisoquinolines 60−66.

3-Morpholinopropylamine (67) was condensed with inden-

obenzopyrans 15, 30, and 32 to produce indenoisoquinolines

68−70 (Scheme 6).

a

Reagents and conditions: (a) KNO₃, H₂SO₄, 0 °C to room temp; (b)

H₂, Pd/C, EtOAc; (c) (i) NaNO₂, 37% HCl, 0 °C, (ii) CuCl, 37%

HCl, 0 °C to reﬂux (18), or (i) NaNO₂, 48% HBr, 0 °C, (ii) CuBr,

48% HBr, 0−80 °C (19), or (i) NaNO₂, 37% HCl, 0 °C, (ii) KI, 0 °C

to room temp (20), or (i) NaNO₂, 37% HCl, 0 °C, (ii) NaCN, CuCN,

0 °C to room temp (21); (d) NBS, AIBN, CCl₄, reﬂux; (e) KOH,

H₂O, reﬂux; (f) (i) NaOMe, MeOH, EtOAc, reﬂux, (ii) Ac₂O, reﬂux.

a

Scheme 3

Anhydride 71 was reduced with NaBH₄in PhMe-DMF, and

the reduction product was cyclized in reﬂuxing 5 M HCl to

provide phthalide 72 (Scheme 7). Phthalide 72 was subjected

to radical bromination, and hydrolysis of the product 73

produced 3-hydroxyphthalide 74. 3-Hydroxyphthalide 74 and

phthalide (14) were condensed under basic conditions, and the

intermediate was cyclized in situ in reﬂuxing Ac₂O to yield

lactone 75.

In similar fashion to its carbocyclic analogues, lactone 75 was

condensed with primary amines 67, N,N-dimethyl-1,3-diami-

nopropane (77), or 59 to yield indenoisoquinolines 76, 78, and

79, respectively (Scheme 8).

RATIONALE, BIOLOGICAL RESULTS, AND

DISCUSSION

■

The objective of this study was to discover a suitable

bioisosteric replacement for the 3-nitro group on the

indenoisoquinoline system that would maintain or improve

Top1 poisoning activity and growth inhibitory potency.

Accordingly, the newly synthesized compounds were subjected

to the Top1-mediated DNA cleavage assay to assess Top1

poisoning activity and the NCI-60 human tumor cell line screen

to evaluate growth inhibitory activity. The ﬁrst assay scores the

performances of Top1 poisons according to a rubric that is

based on the activity of 1 μM camptothecin. Test agents are

incubated at 0.1, 1, 10, and 100 μM concentrations with a 3′-

[³²P]-labeled double-stranded DNA fragment and Top1

a

Reagents and conditions: (a) AcCl, reﬂux; (b) (i) NaBH₄, THF, 0

°C, (ii) HCl; (c) NBS, AIBN, CCl₄, reﬂux; (d) KOH, H₂O, reﬂux; (e)

(i) NaOMe, MeOH, EtOAc, reﬂux, (ii) Ac₂O, reﬂux.

separated by silica gel column chromatography. The identity of

1

each isomer was conﬁrmed by comparing H NMR spectral

information to that reported in the literature.³⁷Phthalides 36

and 37 were separately subjected to radical bromination and

subsequent hydrolysis to provide 3-hydroxyphthalides 40 and

41. Indenobenzopyrans 42 and 43 were obtained by the

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a

Scheme 5

a

Reagents and conditions: (a) MeOH−CHCl₃, reﬂux; (b) CHCl₃, reﬂux.

a

Scheme 6

Scheme 7

a

Reagents and conditions: (a) (i) NaBH₄, PhMe, DMF, −20 to 35 °C,

(ii) 5 M HCl, reﬂux; (b) NBS, AIBN, CCl₄, reﬂux; (c) H₂O, reﬂux;

(d) (i) NaOMe, MeOH, EtOAc, reﬂux, (ii) Ac₂O, reﬂux.

intensities of the DNA cleavage bands in test agent lanes do

not increase in a simple dose-dependent manner. The

intensities appear to subside at high drug concentration after

increasing steadily at lower concentrations (e.g., compound 69,

bands at positions 92 and 119). One possible cause is that high

drug concentrations produce a signiﬁcant population of DNA

with intercalated test agent, which is a poorer enzyme substrate

than nonintercalated DNA. Intercalation of drug molecules into

DNA suppresses Top1 nonspeciﬁcally, and this mechanism is

distinct from Top1 poisoning.³⁸Second, the DNA cleavage

patterns induced by test agents indicate preferences for

diﬀerent ﬂanking base pairs at the binding site, or cleavage

site speciﬁcities, versus the control compounds. For example,

the dark band at position 62 for compound 6, which has a

unique hydroxyethylaminopropyl side chain, is not observed for

1 or the test agents. Diﬀerent cleavage site speciﬁcities may

translate into unique antitumor activity proﬁles for Top1

poisons.^8,10

a

Reagents and conditions: (a) CHCl₃, reﬂux.

enzyme. Top1 poisons bind to and trap Top1−DNA cleavage

complexes. Separation of the resulting cleaved DNA via

denaturing gel electrophoresis reveals a DNA cleavage pattern.

The number and intensity of the bands observed in test agent

lanes is visually compared to that seen with 1 μM CPT. The

relative activity is estimated and is used to assign a

semiquantitative score which ranges from 0 (no activity) to

++++ (activity equal to that of 1 μM CPT; see Table 2 caption

for a complete description of the scoring rubric). A

representative gel is shown in Figure 1.

The representative Top1 assay gel reveals additional

information beyond Top1 poisoning activity. First, the

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a

Scheme 8

a

Reagents and conditions: (a) CHCl₃, reﬂux.

Figure 1. Top1-mediated DNA cleavage assay gel: lane 1, DNA alone; lane 2, DNA and Top1; lane 3, DNA, Top1, and 1 μM 1; lane 4, DNA, Top1,

and 1 μM 6; remaining lanes, DNA, Top1, and indicated concentration (μM) of test compound. The numbers and arrows at the left indicate

cleavage site positions (see Experimental Section). Gel-based assays are performed twice for each active compound, and they are always run with

positive controls, including camptothecin (1) and the indenoisoquinoline 6.

Recent investigations have focused on mapping out the

structural features of indenoisoquinolines that contribute to

potent TDP1 inhibition.^19,31−33TDP1 is a DNA-repair enzyme

that is speciﬁcally involved in the recognition and repair of

DNA lesions induced by Top1 poisons. Co-treatment with a

TDP1 inhibitor and Top1 poison may achieve a selective

cytotoxic eﬀect on cancer cells versus normal cells because

3. Similar to the idea with dual Top1 and TDP1 activities, dual

Top2 and TDP2 activities may produce a synergistic eﬀect that

selectively targets cancer cells. TDP1 and TDP2 inhibitors

would be useful not just for DNA-damaging cancer chemo-

therapy but also in radiation and antiviral therapies, where

DNA breakage is induced to create a therapeutic eﬀect.²³Only

one compound prepared in the present investigation, 78,

showed activity against either TDP1 or TDP2. The activity of

78 was scored as “+” (IC₅₀37−111 μM) against both TDP1

and TDP2. Its activity was not surprising, given that sterically

undemanding aminopropyl lactam side chains are usually

necessary for indenoisoquinolines to inhibit TDP1.¹⁹

cancer cells often have a reduced capacity for DNA repair.³⁹

A

representative TDP1 inhibition assay gel is shown in Figure 2.

TDP2 is a distinct DNA repair enzyme that is involved in the

removal of stalled topoisomerase II (Top2) cleavage complexes.

A representative TDP2 inhibition assay gel is shown in Figure

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Figure 2. TDP1 inhibition assay gel. The concentrations of test compounds were 12.3, 37, and 111 μM (left to right). N14Y is 5′-end labeled DNA

oligonucleotide with a 3′-phosphate group, and N14P is 5′-end labeled DNA oligonucleotide (see Experimental Section). Gel-based assays are

commonly acquired twice for each compound. The positive control used for the TDP1 assay was 7. The “+”-based scoring system rubric is as

follows: 0, IC₅₀> 111 μM; +, IC₅₀37−111 μM; ++, IC₅₀12−37 μM; +++, IC₅₀> 1−12 μM; ++++, IC₅₀≤ 1 μM. The image was cropped to remove

irrelevant compounds that also appeared on this gel.

Figure 3. TDP2 inhibition assay gel. The concentrations of test compounds were 12.3, 37, and 111 μM (left to right). The TDP2 substrate Y19

corresponds to a 3′-end labeled DNA oligonucleotide with a 5′ phosphotyrosyl, and the TDP2 product p19 corresponds to a 3′-end labeled DNA

oligonucleotide with a 5′ phosphate group (see Experimental Section). Gel-based assays are commonly acquired twice for each compound. The

positive control used for the TDP2 assay was 8. The “+”-based scoring system rubric is the same as that for the TDP1 assay (see Figure 2 caption).

The image was cropped to remove irrelevant compounds that also appeared on this gel.

The NCI-60 human tumor cell line screen⁴⁰provides

information on the cytotoxic eﬀect of a test agent across 60

cell lines originating from human breast, colon, central nervous

system (CNS), melanoma, and other tissues. Compounds are

ﬁrst screened at 10 μM, and an averaged growth percent across

all of the tested cell lines is reported as a mean growth percent.

If the mean growth percent is suﬃciently low (normally below

ca. 60%), the compound is promoted to a ﬁve-dose assay. The

ﬁve testing concentrations range from 10⁻⁸to 10⁻⁴μM, and the

resulting dose−response curves are used to calculate the

concentration required for 50% growth inhibition relative to

control, or GI₅₀, for each cell line. In cases where a GI₅₀is

calculated to fall outside of the testing range, it is replaced with

either the minimum (10⁻⁸μM) or maximum (10⁻⁴μM)

concentration. The mean GI₅₀calculated this way for all cell

lines tested is called a mean graph midpoint (MGM) GI₅₀.

Molecular modeling studies performed by Morrell et al.

suggest two explanations for the marked enhancement in

biological activity observed with nitrated indenoisoquinolines.¹⁵

The ﬁrst possibility is that a 3-nitro is appropriately positioned

to engage the side chain carboxamide of the nearby residue

Asn722 in a direct hydrogen bond. Replacement substituents

CN, SO₂Me, CO₂Me, and 3-aza were investigated here because

they too could function as hydrogen bond acceptors. A second

possibility is that the electronegative 3-nitro group creates

favorable polar interactions between the ligand and surrounding

DNA bases. All of the atoms and groups selected for study exert

an electron-withdrawing eﬀect. A comparison of the electro-

negativities of several of these chemical groups, obtained from

ab initio calculations performed by de Proft et al.,⁴¹is displayed

in Table 1.

F

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+). MGM GI₅₀values were calculated to be 0.692 μM for 60,

0.229 μM for 61, and 2.75 μM for 68. Compound 86 was not

advanced to ﬁve-dose testing for determination of MGM GI₅₀

because it failed to signiﬁcantly reduce the mean growth of the

same cancer cell lines at 10 μM. Compounds 60, 61, 68, and 86

are substituted with the halogens F, Cl, and Br. It was not

immediately clear why “smaller” halogens were better replace-

ments for nitro than other atoms and functional groups. For

this reason, several model systems were studied using molecular

mechanics and quantum mechanics calculations. It is not

unprecedented that F should act as a bioisostere of nitro.⁴³In

the course of optimizing adrenoceptor antagonists, a p-

nitrophenyl ring was replaced by a 3,4-diﬂuorophenyl one

with retention or improvement of K_i. This exchange provided

additional value by side-stepping toxicity concerns about the

nitro group.⁴⁴

Table 1. Electronegativities of Bioisosteres from the Present

Study⁴¹

a

group

electronegativity (eV)

NO₂

F

7.84

10.01

7.65

8.63

6.30

5.48

4.99

Cl

CN

CF₃

CO₂Me

SMe

a

Calculated at the CISD level.

Biological testing data are organized in Table 2. Compounds

80−88 were previously studied^17,19,36,42and are included in the

table for easy referencing. Among the bioisosteric compounds,

60, 61, 68, and 86 display the best Top1 poisoning activities.

The Top1-mediated DNA cleavage induced by these four

compounds is between 75 and 95% that of 1 μM CPT (i.e., ++

Indenoisoquinoline lactam side chains are known to exert a

signiﬁcant inﬂuence on Top1 poisoning and growth inhibitory

activities.^45,46Unexpectedly, dihydroxy side chain analogues 49

Table 2. Antiproliferative and Top1 Poisoning Activities of Indenoisoquinolines Substituted with 3-Nitro Bioisosteres

cytotoxicity (GI₅₀, μM)

Top1

compd cleavage

mean growth

lung

colon

CNS

melanoma

ovarian

renal

prostate

breast

MCF7

a

b

c

percent

HOP-62 HCT-116 SF-539 UACC-62 OVCAR-3 SN12C DU-145

MGM

2

++++

+++

++

+++

++

+++

++

+++

++

+

ND

0.01

0.089

0.19

0.03

0.01

0.22

0.02

0.01

0.016

2.17

0.0405 0.0187

0.156 0.061

0.370 0.28

3-NO₂

3-F

80

81

82

49

60

68

50

61

69

51

83

86

52

62

70

53

84

87

54

63

55

64

56

65

57

66

58

85

88

76

78

79

0.025

0.274

0.038

0.157

0.016

0.095

0.098

0.012

0.380

0.309

0.864

0.241

0.015

0.309

0.040

0.017

0.085

ND

0.021

0.632 0.029

d

61.14

−8.15

30.31

14.53

0.06

NS

0.490

2.09

0.309

1.29

0.417

2.95

0.245

1.38

1.29

4.27

2.24

0.407

6.03

0.437

2.34

0.575

2.95

0.135

0.347

0.257

0.031

0.372

0.692

2.75

1.12

0.229

4.17

NS

3-Cl

3-Br

3-I

0.692

0.186

2.14

0.437

0.107

1.62

0.417

0.204

2.40

0.398

0.074

3.31

0.977

0.110

5.01

2.09

0.309

4.07

44.73

46.84

27.33

84.03

39.73

43.54

96.00

42.65

41.11

101.69

82.67

49.34

52.24

6.57

0.513

0.251

0.676

0.427

3.31

0.468

0.676

0.191

1.26

NS

e

NT

0.631

0.200

3.02

2.14

2.29

5.27

3.89

4.27

3.02

0.331

0.074

3.16

1.62

NS

0.437

0.550

0.708

0.603

3-CN

+(+)

++

+

NS

0.537

0.112

0.501

0.407

4.68

0.575

0.562

0.033

0.944 0.015

NS

2-CF₃

NS

0/+

+

NS

3-CF₃

NS

0/+

+

0.407

1.82

0.646

0.741

1.29

2.34

0.537

1.38

1.51

4.37

1.78

1.95

1.10

2.51

0.257

0.427

1.07

2.51

NS

3-SMe

39.68

42.65

96.74

59.38

+

3-SO₂Me

3-CO₂Me

++

+

NS

f

NA

++

+

62.13

87.47

NA

0.589

0.871

0.398

0.380

0.490

0.708

100

0.525

0.955

0.692

0.977

0.316

0.331

5.37

NS

3-aza

++

+

NA

−9.25

47.53

1.23

2.00

0.977

NS

a

Compound-induced DNA cleavage due to Top1 poisoning, with scores given according to the following system based on the activity of 1 μM

camptothecin: 0, no activity; +, between 20 and 50% activity; ++, between 50 and 75% activity; +++, between 75 and 95% activity; ++++, equal

b

c

activity. Growth percent of cultured cells of ∼60 cancer cell lines treated with 10 μM compound relative to vehicle-treated control. Mean graph

d

e

f

midpoint of growth inhibition from 5 dose assay, ranging from 10⁻⁸−10⁻⁴M. NS = not selected for 5 dose assay. NT = cell line not tested. NA =

not accepted by the National Cancer Institute for screening in the NCI-60.

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Figure 4. Energy-minimized hypothetical binding pose of 60 (green) within the X-ray crystal structure of a stalled Top1−DNA cleavage complex

cocrystallized with indenoisoquinoline 89 (PDB code 1SC7). Potential interactions are indicated by dashed lines. The distances between heavy

atoms appear next to the dashed lines. The stereoview is programmed for wall-eyed (relaxed) viewing.

Figure 5. Energy-minimized hypothetical binding pose of 64 (blue) within the X-ray crystal structure of a stalled Top1−DNA cleavage complex

cocrystallized with indenoisoquinoline 89 (PDB code 1SC7). Potential interactions are indicated by dashed lines. For clarity, only one potential

interaction is shown. The distance between heavy atoms appears next to the dashed line. The stereoview is programmed for wall-eyed (relaxed)

viewing.

and 50 displayed worse Top1 poisoning activity and

cytotoxicity than imidazolylpropyl side chain analogues 60

and 61. This trend held generally true for all of the other

potential bioisosteres. The (S)-2,3-dihydroxypropyl lactam side

chain was recently reported to impart equivalent or greater

Top1 poisoning activity, and improved MGM GI₅₀values,

compared to imidazolylpropyl and morpholinopropyl lactam

side chains.³¹This trend is applicable to 2,3-dimethoxy-8,9-

methylenedioxy- and 3-nitro-substituted indenoisoquinolines

but clearly not for all aromatic ring substitution patterns.

There are several reports in the literature of successful nitro

aryl replacement with a pyridine ring.⁴³It might therefore be

expected that the 3-azaindenoisoquinolines would display Top1

poisoning and growth inhibitory activities comparable to 3-

nitroindenoisoquinolines 80−82. Two possibilities to explain

why this “swap” has turned out to be successful in other studies

include (1) the pyridine nitrogen, like nitro group oxygens, can

function as a hydrogen bond acceptor, and (2) the electro-

negative nitrogen within the pyridine ring mimics the electron-

withdrawing eﬀect of nitro. On the contrary, the 3-

azaindenoisoquinolines’ Top1 poisoning scores were mediocre

to poor (++ or +). A single compound, 78, progressed to the

ﬁve-dose growth inhibition assay, and its MGM GI₅₀was 0.977

The other potential bioisosteres (i.e., I, CN, CF₃, SMe,

μM. None of the other 3-aza compounds displayed

submicromolar MGM GI₅₀values or Top1 poisoning scores

above ++.

SO₂Me, and CO₂Me) did not display Top1-mediated DNA

cleavage assay scores above ++. Although all of the new

compounds had some activity, the majority were clustered with

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“+” and “++” Top1 assay scores. Only four of these

compounds, 52, 62, 63, and 84, were advanced to the ﬁve-

dose assay for MGM GI₅₀determination. However, none of

these four displayed MGM GI₅₀values better than 0.944 μM.

In an eﬀort to rationalize the observed biological activities on

the basis of molecular mechanics, several ligands were docked

into the PDB code 1SC7 X-ray crystal structure of the

indenoisoquinoline 89−Top1−DNA ternary complex.⁴⁷The

ligands, Top1−DNA cleavage complex, and resulting ligand−

Top1−DNA ternary complexes were energy minimized as

described in the Experimental Section. The graphics in Figures

4 and 5 were prepared in PyMOL.⁴⁸

CF₃compounds exhibit very poor Top1 poisoning activities

(0/+ or +).

Previous quantum mechanics studies suggest that the

interactions between an indenoisoquinoline and its ﬂanking

base pairs within the cleavage complex are predictive of ligand

binding orientation.⁵²Electrostatic potential surfaces calculated

using quantum mechanics showed that the electron with-

drawing eﬀect of 3-nitro pulls electron density away from the

lactam carbonyl. This polar eﬀect serves to minimize poor

charge complementarity at the lactam carbonyl and to

maximize the charge complementarity just outside of the A-

ring 3-position with the ﬂanking base pairs.⁵²The theoretical

binding energy, electron correlation, and dispersion forces that

stabilize azaindenoisoquinolines in their binding site are

sensitive to both incorporation and position of the nitrogen

atom in the D-ring.⁵³In the present study, it was predicted that

the quantum mechanics-calculated binding energies of

bioisosterically modiﬁed indenoisoquinolines would be similar

to those for nitrated indenoisoquinolines.

The binding mode of 60 may provide insight into its

relatively potent Top1 poisoning activity (Figure 4). The 3-

ﬂuoro substituent is calculated to be within hydrogen bonding

distance (2.98 Å) of the carboxamide nitrogen of Asn722. The

angle deﬁned by the ﬂuorine of 60 and the carboxamide

hydrogen and nitrogen of Asn722 would ideally be >150° for

hydrogen bonding,⁴⁹but the measured angle in the docking

pose is 116°. The ﬂuorine atom occupies a pocket deﬁned by

two deoxyribose sugars and Top1 residues Asn722 and Thr718

(not shown). Its close contacts (≤3.5 Å) with heavy atoms

from each of these four structures favorably impact the van der

Waals contribution to the ligand’s binding energy.

Compound 61 was also studied by molecular mechanics

molecular modeling. The binding mode of 61 (not shown),

which is similar to that obtained for 60, does not suggest a

halogen bonding interaction between the chlorine and Asn722.

In halogen bonding, chlorine, bromine, or iodine interacts as a

Lewis acid with a Lewis basic atom such as oxygen or

nitrogen.⁵⁰The electron density surrounding the halogen atom

is unevenly distributed, such that a small region of electro-

positivity exists at one face.⁴⁹This type of bonding seems

unlikely for 61, given that the distance between the chlorine

and the carboxamide oxygen of Asn722 is calculated to be 4.2

Å. The carbonyl does not align with the C−Cl bond on the

ligand. Furthermore, its 3-bromo and 3-iodo analogues do not

show the marked enhancement in activity relative to 3-ﬂuoro

observed with some halogen-bonding ligands.^49,51Another

possibility is that the 3-chloro substituent has appropriate shape

complementarity for its surroundings. Signiﬁcantly larger

monatomic bioisosteres (i.e., bromine and iodine) and

multiatom groups (e.g., CO₂Me and SO₂Me) were unable to

confer similar Top1 poisoning and growth inhibitory activities.

Triﬂuoromethylated indenoisoquinoline 64 exhibits poor

Top1 poisoning activity. In its molecular model (Figure 5), the

CF₃group packs closely with the Asn722 residue. The same

attractive intermolecular interactions that could stabilize the

binding of 60 and 61 seem plausible. As can be observed from

close visual inspection of the ligands in Figures 4 and 5, 64 is

rotated relative to 60 so as to maximize its interaction with

Asn722, an eﬀect which may consequently weaken other

important interactions for ligand binding. The triﬂuoromethyl

group is unusual among the bioisosteres selected for this study

because of its tetrahedral geometry (the only other being

SO₂Me). Its geometry may help to explain why the 2- and 3-

Figure 6. Two- and three-dimensional depictions of the model

complex used for quantum mechanics calculations. The three-

dimensional depiction is shown in stereoview and is programmed

for wall-eyed (relaxed) viewing.

The binding energies of ﬁve model indenoisoquinolines 90−

94 in a simpliﬁed version of a Top1−DNA cleavage complex

(Figure 6) were calculated by quantum mechanics at the MP2/

6-31G* level of theory. The quantum mechanics calculations

were performed with a model Top1−DNA cleavage complex

containing only the heterocyclic bases shown in Figure 6 and

simpliﬁed indenoisoquinolines in which the lactam side chains

were replaced by a methyl group. These approximations were

made because (1) previous theoretical studies have demon-

strated that the experimentally observed indenoisoquinoline

binding orientation can be predicted using this simpliﬁed

binding site model⁵²and (2) the lactam side chain of the

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bonding, van der Waals forces, and interactions between the

ﬂanking base pairs and the indenoisoquinoline could stabilize

bioisosterically modiﬁed indenoisoquinolines within their

binding site in the Top1−DNA cleavage complex.

Table 3. Calculated Binding Energies of Hypothetical

Indenoisoquinolines A−E in the Model Binding Site

The ﬁndings from this study are valuable because they can

aid the design of novel Top1 poisons. 3,9-Diﬂuorinated

indolocarbazole glycosides have demonstrated eﬃcacy in

preclinical models and advanced to human clinical trials.^56,57

The 3-position substituents on the indenoisoquinoline and

indolocarbazole scaﬀolds project toward Asn722.⁴⁷This

observation provides support for a universal structure−activity

relationship for the Top1 poisons, and may therefore aid in the

structure-based design and optimization of new and diﬀerent

Top1 poisons.⁵⁸⁻⁶¹

a

b

ligand

R

E_inter(kcal/mol)

electronegativity of R (eV)

7.84

90

91

92

93

94

NO₂

H

−21.34

−15.87

−17.36

−18.60

−16.09

F

10.01

7.65

6.30

Cl

CF₃

EXPERIMENTAL SECTION

a

■

Binding energies were calculated at the MP2/6-31G* level and

include the basis set superposition error. Calculated at CISD level by

b

Reactions were monitored by silica gel analytical thin-layer

chromatography, and 254 nm UV light was used for visualization.

Melting points were determined using capillary tubes and are

uncorrected. ¹H Nuclear magnetic resonance spectroscopy was

performed using a 300 MHz spectrometer. Infrared spectra were

obtained using an FTIR spectrometer. High-resolution mass spectra

were recorded on a double-focusing sector mass spectrometer with

magnetic and electrostatic mass analyzers. Compound purities were

estimated by reversed phase C18 HPLC, with UV detection at 254

nm. The major peak area of each tested compound was ≥95% of the

combined total peak area.

de Proft et al.⁴¹

indenoisoquinolines projects out of the simpliﬁed binding site

model and into the DNA major groove.⁴⁷

The quantum mechanics-derived binding energies (Table 3)

display a correlation with Top1 assay scores. The two nitro

bioisosteres that result in +++ Top1 assay scores, F and Cl (i.e.,

compounds 60 and 61), also have the most favorable

theoretical binding energies, at −17.36 and −18.60 kcal/mol,

respectively. In line with Top1 assay scores, neither ﬂuorinated

92 nor chlorinated 93 were calculated to bind as favorably as

90, the nitrated analogue. The negative control ligand 91 is

substituted with 3-H, and it has the least favorable calculated

binding energy. Ligand 94 is substituted with a CF₃group in

the 3-position. Its calculated binding energy was slightly better

than 91’s, although considerably worse than that of 90, 92, or

93.

The calculated binding energies of the ligands also show a

correlation with group electronegativity. The substituents on

ligands 90 and 93, NO₂and Cl respectively, have similar

electronegativities, and their theoretical binding energies are the

most favorable of all ﬁve systems studied. With its substantially

greater electronegativity, ﬂuorinated analogue 92 could be

anticipated to have a more favorable theoretical binding energy

than 90 and 93, but it does not. The CF₃group present on

ligand 94 has the lowest electronegativity among the nitro

bioisosteres, and this may factor into its unfavorable calculated

binding energy and low Top1 assay scores. It is possible that

both, or either, direct substituent-substituent interactions and

polarized aromatic system interactions dominate the calculated

theoretical binding energies.^54,55

(2-Bromo-4-ﬂuorophenyl)methanol (10). Aldehyde 9 (5.014 g,

24.70 mmol) was dissolved in MeOH (50 mL), and the solution was

cooled to 0−5 °C. NaBH₄(1.873 g, 49.51 mmol) was added all at

once, and the mixture was stirred at 0−5 °C for 1 h, and then at room

temperature for 24 h. Saturated NaHCO₃solution (50 mL) was added

to quench the reaction, and the mixture was concentrated in vacuo to

remove MeOH. The resulting suspension was extracted with EtOAc (3

× 75 mL). The combined organic layers were washed with brine (1 ×

50 mL), dried over Na₂SO₄, and concentrated in vacuo. The obtained

oil was puriﬁed by silica gel column chromatography, eluting with

CHCl₃, to yield 10 (4.240 g, 84%) as a white solid: mp 63−66 °C. ¹H

NMR (300 MHz, CDCl₃) δ 7.46 (dd, J = 8.6 Hz, J = 6.1 Hz, 1 H),

7.31 (dd, J = 8.2 Hz, J = 2.6 Hz, 1 H), 7.06 (td, J = 8.3 Hz, J = 2.6 Hz,

1 H), 4.72 (d, J = 6.4 Hz, 2 H), 2.02 (t, J = 6.3 Hz, 1 H).

6-Fluorophthalide (11). (2-Bromo-4-ﬂuorophenyl)methanol (10,

6.720 g, 32.78 mmol) and CuCN (5.891 g, 65.78 mmol) were diluted

in DMF (35 mL). The system was ﬂushed with argon and then heated

to reﬂux with stirring for 2 h 45 min. The mixture was cooled to 100

°C, H₂O (3.5 mL) was added, and the mixture was stirred with heating

to reﬂux for 21 h. EtOAc (75 mL) was added to the reaction mixture

once it cooled to room temperature, and the mixture was ﬁltered

through a plug of Celite, washing with EtOAc (25 mL). Brine (150

mL) was added, the mixture partitioned between the two phases, and

the organic layer was separated. The aqueous layer was extracted with

EtOAc (2 × 70 mL). The combined organic layers were washed with

brine (2 × 70 mL) and saturated LiCl solution (70 mL), dried over

Na₂SO₄, and concentrated in vacuo. The residue was puriﬁed by silica

gel column chromatography, eluting with CHCl₃, to provide 11 (2.945

g, 59%) as an oﬀ-white solid: mp 100−105 °C (lit.³⁵mp 100−102

CONCLUSIONS

■

Twenty-three new 3-substituted indenoisoquinolines were

synthesized in order to investigate the eﬀect of bioisosteric

replacements of the nitro group in the 3-nitroindenoisoquino-

line Top1 poison family. Although many 3-nitroindenoisoqui-

nolines display potent activity in the Top1-mediated DNA

cleavage assay and growth inhibitory eﬀect in the NCI-60

antiproliferative assay, it would be advantageous to identify

bioisosteres that mitigate potential toxicity risks associated with

aryl nitro groups. Fluorine and chlorine were identiﬁed as

bioisosteres on the basis of Top1 poisoning activities and

growth inhibitory potencies. Molecular mechanics modeling

and quantum mechanics calculations indicate that hydrogen-

1

°C). H NMR (300 MHz, CDCl₃) δ 7.58 (dd, J = 7.2, 2.3 Hz, 1 H),

7.53−7.37 (m, 2 H), 5.32 (s, 2 H). CIMS m/z (rel intensity) 153

(MH⁺, 100).

3-Bromo-6-ﬂuorophthalide (12). 6-Fluorophthalide (11, 1.001

g, 6.580 mmol), NBS (1.204 g, 6.764 mmol), and AIBN (39 mg) were

diluted in CCl₄(25 mL) and heated to reﬂux with stirring under argon

for 1 h 30 min. The mixture was cooled to room temperature, ﬁltered,

and the residue was washed with CCl₄(10 mL). The ﬁltrate was

concentrated in vacuo to provide a yellow oil, which was puriﬁed by

silica gel column chromatography, eluting with 80:20 hexanes−EtOAc,

1

to provide 12 (1.230 g, 81%) as a yellow oil. H NMR (300 MHz,

CDCl₃) δ 7.64 (ddd, J = 8.4, 4.2, 0.7 Hz, 1 H), 7.56−7.44 (m, 2 H),

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7.40 (s, 1 H). CIMS m/z (rel intensity) 231/233 (MH⁺, 6/6); 153

(MH⁺− Br, 100).

combined organic layers were washed with saturated aq Na₂S₂O₃(2 ×

100 mL) and brine (50 mL), dried over Na₂SO₄, and concentrated in

vacuo. The residue was puriﬁed by silica gel column chromatography,

eluting with CHCl₃, to provide 20 (5.973 g, 63%) as an oﬀ-white solid:

mp 87−90 °C. ¹H NMR (300 MHz, CDCl₃) δ 8.26 (dd, J = 1.5 Hz, J

= 0.7 Hz, 1 H), 7.99 (dd, J = 8.0 Hz, J = 1.5 Hz, 1 H), 7.29 (d, J = 0.8

Hz, 1 H), 5.28 (s, 2 H). CIMS m/z (rel intensity) 261 (MH⁺, 100).

6-Cyanophthalide (21). 3-Aminophthalide (17, 1.100 g, 7.375

mmol) was suspended in 37% HCl (2 mL) and H₂O (12 mL) at 0 °C.

An ice-cold solution of NaNO₂(691 mg, 10.0 mmol) in H₂O (2 mL)

was added dropwise, and the resulting mixture was added dropwise to

a prepared solution of CuCN (804 mg, 9.00 mmol) and NaCN (1.240

g, 25.30 mmol) in H₂O (10 mL) stirring at 0 °C. The mixture was

stirred with warming to room temperature for 3.5 h and then ﬁltered

through a plug of Celite (2 cm height), which was then washed with

EtOAc (50 mL). The aqueous layer of the ﬁltrate was extracted with

EtOAc (50 mL), and the combined organic layers were washed with

brine (25 mL), dried over Na₂SO₄, and concentrated in vacuo. The

residue was puriﬁed by silica gel column chromatography, eluting with

a 50:50 EtOAc−hexanes →80:20 EtOAc−hexanes gradient, to provide

21 (498 mg, 42%) as a yellow solid: mp 185−190 °C. ¹H NMR (300

MHz, CDCl₃) δ 8.23 (s, 1 H), 7.97 (dd, J = 7.9, 1.5 Hz, 1 H), 7.67

(dq, J = 7.9, 0.9 Hz, 1 H), 5.41 (s, 2 H).

6-Chloro-3-hydroxyphthalide (26). 6-Chlorophthalide (18,

2.786 g, 16.58 mmol), NBS (2.962 g, 16.64 mmol), and benzoyl

peroxide (33 mg) were suspended in CCl₄(22 mL). The mixture was

heated to reﬂux with stirring under an argon atmosphere for 1 h,

cooled to room temperature, and ﬁltered. The residue was washed

with CCl₄(2 × 20 mL), and the ﬁltrate was concentrated in vacuo.

H₂O (25 mL) and KOH (1.88 g, 33.5 mmol) were added to the

obtained yellow oil, and the mixture was heated to reﬂux with stirring

for 1 h. After cooling to room temperature, the mixture was acidiﬁed

with NaHSO₄(1.9 g) and extracted with EtOAc (3 × 50 mL). The

combined organic layers were dried over Na₂SO₄and concentrated in

vacuo to provide 26 (2.60 g, 85%) as a white solid: mp 111−116 °C.

¹H NMR (300 MHz, DMSO-d₆) δ 8.29 (d, J = 7.4 Hz, 1 H), 7.98−

7.77 (m, 2 H), 7.77−7.59 (m, 1 H), 6.67 (d, J = 8.2 Hz, 1 H). CIMS

m/z (rel intensity) 185/187 (MH⁺, 15/4), 167/169 (MH⁺− H₂O,

100/47).

6-Fluoro-3-hydroxyphthalide (13). Potassium hydroxide (0.542

g, 9.66 mmol) and H₂O (15 mL) were added to 3-bromo-6-

ﬂuorophthalide (12, 1.077 g, 4.662 mmol). The mixture was heated to

reﬂux with stirring for 1 h, cooled to room temperature, and EtOAc

(20 mL) and NaHSO₄(1.8 g) were added. The organic layer was

removed, and the aqueous layer was extracted twice more with EtOAc

(2 × 20 mL). The combined organic layers were washed with brine

(20 mL), dried over Na₂SO₄, and concentrated in vacuo to yield 13

1

(0.617 g, 79%) as a white solid: mp 95−97 °C. H NMR (300 MHz,

DMSO-d₆) δ 8.26 (s, 1 H), 7.85−7.55 (m, 3 H), 6.66 (s, 1 H). CIMS

m/z (rel intensity) 169 (MH⁺, 13), 151 (MH⁺− H₂O, 100).

3-Fluoroindeno[1,2-c]isochromene-5,11-dione (15). 6-Fluo-

ro-3-hydroxyphthalide (13, 0.582 g, 3.46 mmol) and phthalide (14,

0.479 g, 3.57 mmol) were suspended in EtOAc (10 mL), and a freshly

prepared solution of NaOMe (0.349 g, 15.2 mmol of Na) in MeOH

(10 mL) was added. The resulting solution was heated to reﬂux with

stirring for 18 h. The resulting mixture was cooled to room

temperature, acidiﬁed to pH 1 with 37% HCl (3 mL), concentrated

in vacuo, and azeotroped with PhMe (10 mL). Ac₂O (20 mL) was

added to the residue, and the mixture was heated to reﬂux with stirring

for 1 h. The mixture was cooled to room temperature, and hexanes (20

mL) were added. The mixture was ﬁltered, and the residue was washed

with hexanes (20 mL) and H₂O (10 mL) to provide 15 (196 mg,

1

31%) as an orange−red solid: mp 215−218 °C. H NMR (300 MHz,

CDCl₃) δ 8.39 (dd, J = 8.8, 5.2 Hz, 1 H), 7.95 (dd, J = 8.6, 2.7 Hz, 1

H), 7.65−7.37 (m, 5 H). EIMS m/z (rel intensity) 266 (M⁺, 100).

HREIMS calcd for C₁₆H₈FO₃(M⁺) 266.0374, found 266.0380.

6-Chlorophthalide (18). 3-Aminophthalide (17, 3.500 g, 23.49

mmol) was suspended in 37% HCl (25 mL) 0 °C. An ice-cold solution

of NaNO₂(1.713 g, 24.83 mmol) in H₂O (10 mL) was added

dropwise, and the resulting solution was stirred at 0−5 °C for 10 min.

An ice-cold suspension of CuCl (2.91 g, 29.4 mmol) in 37% HCl (10

mL) was added dropwise. The mixture was stirred at room

temperature for 10 min, then at reﬂux for 3 h, and room temperature

for 16 h. The suspension was extracted with EtOAc (3 × 80 mL), and

the combined organic layers were washed with saturated NaHCO₃

solution (1 × 50 mL) and H₂O (1 × 50 mL). The organic solution

was dried over Na₂SO₄and concentrated in vacuo. The residue was

puriﬁed by silica gel column chromatography, eluting with CHCl₃, to

yield 18 (2.852 g, 72%) as a white solid: mp 106−107 °C (lit.⁶²mp

6-Bromo-3-hydroxyphthalide (27). 6-Bromophthalide (19, 2.69

g, 12.6 mmol), NBS (2.25 g, 12.6 mmol), and benzoyl peroxide (30

mg) were suspended in CCl₄(15 mL). The mixture was heated to

reﬂux with stirring under an argon atmosphere for 50 min, cooled to

room temperature, and ﬁltered. The residue was washed with CCl₄(2

× 10 mL), and the ﬁltrate was concentrated in vacuo. H₂O (25 mL),

THF (10 mL), and KOH (1.409 g, 25.11 mmol) were added to the

obtained yellow oil, and the mixture was heated to reﬂux with stirring

for 1 h. After cooling to room temperature, the mixture was acidiﬁed

with NaHSO₄(1.9 g) and extracted with EtOAc (3 × 50 mL). The

combined organic layers were dried over Na₂SO₄and concentrated in

vacuo to provide 27 (2.78 g, 96%) as a white solid: mp 118−120 °C.

¹H NMR (300 MHz, DMSO-d₆) δ 8.28 (d, J = 7.0 Hz, 1 H), 8.06−

7.92 (m, 2 H), 7.64 (dd, J = 7.9 Hz, J = 3.9 Hz, 1 H), 6.65 (d, J = 7.6

Hz, 1 H).

1

107−108 °C). H NMR (300 MHz, CDCl₃) δ 7.89 (d, J = 1.9 Hz, 1

H), 7.66 (dd, J = 8.2 Hz, J = 1.9 Hz, 1 H), 7.45 (dd, J = 8.2 Hz, J = 0.8

Hz, 1 H), 5.31 (s, 2 H). CIMS m/z (rel intensity) 169/171 (MH⁺,

100/32).

6-Bromophthalide (19). 3-Aminophthalide (17, 2.98 g, 20.0

mmol) was suspended in 48% HBr (15 mL) and H₂O (15 mL) at 0

°C. A room temperature solution of NaNO₂(1.45 g, 21.0 mmol) in

H₂O (7 mL) was added dropwise, and the resulting solution was

stirred at 0−5 °C for 20 min. A room temperature suspension of CuBr

(3.58 g, 25.0 mmol) in 48% HBr (20 mL) was added dropwise. The

mixture was stirred at room temperature for 20 min, at 80 °C for 2 h,

and room temperature for 16 h. EtOAc (70 mL) was added, the

suspension shaken vigorously, the aqueous layer removed, and the

organic layer washed with 1 N HCl (2 × 25 mL), saturated NaHCO₃

solution (1 × 25 mL), and H₂O (2 × 25 mL), and concentrated. The

residue was puriﬁed by silica gel column chromatography, eluting with

CHCl₃, to yield 19 (2.71 g, 64%) as a white solid: mp 96−98 °C (lit.⁶²

mp 114−115 °C). ¹H NMR (300 MHz, CDCl₃) δ 8.06 (d, J = 1.7 Hz,

1 H), 7.80 (dd, J = 8.1 Hz, J = 1.8 Hz, 1 H), 7.39 (dd, J = 8.1 Hz, J =

0.7 Hz, 1 H), 5.29 (s, 2 H). CIMS m/z (rel intensity) 213/215 (MH⁺,

100/100).

6-Iodophthalide (20). 3-Aminophthalide (17, 5.414 g, 36.34

mmol) was suspended in 37% HCl (25 mL) at 0 °C. An ice-cold

solution of NaNO₂(3.027 g, 43.87 mmol) in H₂O (8 mL) was added

dropwise, and the resulting solution was stirred at 0 °C for 25 min. A

solution of KI (30.5 g, 184 mmol) in H₂O (30 mL) was added

dropwise. The mixture was stirred at room temperature for 18 h. The

aqueous suspension was extracted with EtOAc (3 × 100 mL), and the

3-Hydroxy-6-iodophthalide (28). 6-Iodophthalide (20, 0.806 g,

3.10 mmol), NBS (0.553 g, 3.11 mmol), and benzoyl peroxide (23

mg) were suspended in CCl₄(15 mL). The mixture was heated to

reﬂux with stirring under an argon atmosphere for 2.5 h, cooled to

room temperature, and ﬁltered. The residue was washed with CCl₄(2

× 20 mL), and the ﬁltrate was concentrated in vacuo. H₂O (25 mL)

and KOH (0.34 g, 6.1 mmol) were added to the obtained yellow oil,

and the mixture was heated to reﬂux with stirring for 3.5 h. After

cooling to room temperature, the mixture was acidiﬁed with NaHSO₄

(2.0 g) and extracted with EtOAc (3 × 50 mL). The combined organic

layers were dried over Na₂SO₄and concentrated in vacuo to provide

1

28 (0.954 g, 96%) as a white solid: mp 127−130 °C. H NMR (300

MHz, DMSO-d₆) δ 8.26 (d, J = 7.0 Hz, 1 H), 8.19−8.09 (m, 2 H),

7.49 (d, J = 7.8 Hz, 1 H), 6.63 (d, J = 7.6 Hz, 1 H). CIMS m/z (rel

intensity) 277 (MH⁺, 8), 259 (MH⁺− H₂O), 100.

K

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6-Cyano-3-hydroxyphthalide (29). 6-Cyanophthalide (21, 802

mg, 5.04 mmol), NBS (990 mg, 5.56 mmol), and AIBN (57 mg) were

suspended in CCl₄(50 mL). The mixture was heated to reﬂux with

stirring under an argon atmosphere for 2 h, cooled to room

temperature, and concentrated in vacuo. The residue was puriﬁed by

ﬂash silica gel column chromatography (10 cm × 3 cm, 80:20

hexanes−EtOAc 50:50 hexanes−EtOAc, 10 mL fractions). The

product-containing fractions were pooled and concentrated in vacuo

to yield a solid residue. H₂O (15 mL) was added to the solid, and the

mixture was heated to reﬂux with stirring for 18 h. The product 29

(408 mg, 46%) precipitated from solution upon cooling to room

temperature as a yellowish-white solid: mp 167−169 °C (lit.¹⁹mp

H), 8.24 (dd, J = 8.3 Hz, J = 1.9 Hz, 1 H), 7.97 (d, J = 8.3 Hz, 1 H),

7.64−7.48 (m, 4 H). CIMS m/z (rel intensity) 375 (MH⁺, 100).

HRCIMS calcd for C₁₆H₈IO₃(MH⁺) 374.9518, found 374.9512.

3-Cyanoindeno[1,2-c]isochromene-5,11-dione (33). 6-Cyano-

3-hydroxyphthalide (29, 404 mg, 2.31 mmol) and phthalide (14, 318

mg, 2.37 mmol) were suspended in EtOAc (10 mL), and a freshly

prepared solution of NaOMe (0.20 g, 8.7 mmol of Na) in MeOH (20

mL) was added. The resulting solution was heated to reﬂux with

stirring for 17.5 h. The resulting mixture was cooled to room

temperature, acidiﬁed to pH 1 with 37% HCl (1 mL), concentrated in

vacuo, and azeotroped with PhMe (2 × 10 mL). Ac₂O (20 mL) was

added to the residue, and the mixture was heated to reﬂux with stirring

for 18.5 h. The resulting mixture was azeotroped with PhMe (4 × 10

mL) to remove the solvents. The residue was puriﬁed by silica gel

column chromatography, eluting with CHCl₃, to provide an oil, which

was triturated with hexanes (10 mL) and ﬁltered to collect a solid. 33

(225 mg, 36%) was obtained as a yellow-orange solid: mp 215−218

°C (lit.¹⁹mp 225−227 °C). ¹H NMR (300 MHz, CDCl₃) δ 8.57 (dd,

J = 1.7, 0.6 Hz, 1 H), 8.49 (dd, J = 8.3, 0.7 Hz, 1 H), 8.00 (dd, J = 8.3,

1.8 Hz, 1 H), 7.66 (dt, J = 6.4, 1.2 Hz, 1 H), 7.61−7.45 (m, 3 H).

CIMS m/z (rel intensity) 274 (MH⁺, 100).

5-(Triﬂuoromethyl)isobenzofuran-1,3-dione (35). Diacid 34

(2.519 g, 10.759 mmol) was suspended in AcCl (15 mL), and the

mixture was heated to reﬂux with stirring for 17 h. The mixture was

concentrated in vacuo, and the product was puriﬁed by silica gel

column chromatography, eluting with 4:1 EtOAc−hexanes, to yield a

yellow oil. The yellow oil was dried under high vacuum to provide 35

(1.683 g, 72%) as a faint-yellow solid: mp 54−58 °C (lit.⁶³mp 65−69

°C). ¹H NMR (300 MHz, DMSO-d₆) δ 8.47 (dt, J = 1.5, 0.7 Hz, 1 H),

8.41−8.23 (m, 2 H).

1

138−140 °C). H NMR (300 MHz, DMSO-d₆) δ 8.43 (s, 1 H), 8.39

(s, 1 H), 8.24 (dd, J = 7.9, 1.4 Hz, 1 H), 7.89 (d, J = 7.9 Hz, 1 H), 6.77

(s, 1 H).

3-Chloroindeno[1,2-c]isochromene-5,11-dione (30). 6-

Chloro-3-hydroxyphthalide (26, 2.437 g, 13.24 mmol) and phthalide

(14, 1.779 g, 13.28 mmol) were suspended in EtOAc (20 mL), and a

freshly prepared solution of NaOMe (1.16 g, 50.4 mmol of Na) in

MeOH (40 mL) was added. The resulting solution was heated to

reﬂux with stirring under an argon atmosphere for 23 h. The resulting

mixture was cooled to room temperature, acidiﬁed to pH 1 with 37%

HCl (4.5 mL), concentrated in vacuo, and azeotroped with PhMe (1 ×

25 mL). Ac₂O (75 mL) was added to the residue, and the mixture was

heated to reﬂux with stirring under an argon atmosphere for 21 h.

After cooling to room temperature, the mixture was concentrated in

vacuo, the residue taken up in CHCl₃(100 mL), and the organic

solution washed with H₂O (3 × 30 mL). The organic solution was

dried over Na₂SO₄and concentrated in vacuo. The residue was

washed with 70:30 hexanes−EtOAc (3 × 30 mL) to provide 30 (2.009

1

g, 54%) as an orange solid: mp 244−246 °C. H NMR (300 MHz,

5-(Triﬂuoromethyl)phthalide (36) and 6-(Triﬂuoromethyl)-

phthalide (37). Anhydride 35 (2.476 g, 11.46 mmol) was dissolved in

THF (20 mL) with stirring, and the mixture was cooled to 0 °C.

NaBH₄(436 mg, 11.5 mmol) was added all at once, and the mixture

was stirred at 0 °C for 1 h 25 min. The mixture was concentrated in

vacuo, and a solution of H₂O (5 mL) and aqueous HCl (37%, 1 mL)

was added to the residue. The aqueous solution was extracted with

CHCl₃(3 × 25 mL), and the combined organic layers were washed

with H₂O (10 mL) and brine (10 mL), dried over Na₂SO₄, and

concentrated in vacuo. The products were ﬁrst puriﬁed on a silica gel

column, eluting with 50:50 hexanes−EtOAc, to obtain a mixture of 36

and 37, and then the two regioisomers were separated on a second

silica gel column, eluting with 80:20 hexanes−EtOAc. Products 36

(405 mg, 17%) and 37 (408 mg, 18%) were each obtained as white

solids: 36 mp 65−68 °C (lit.³⁷mp 65−67 °C); 37 mp 88−89 °C

(lit.³⁷mp 88−90 °C). 36 ¹H NMR (300 MHz, CDCl₃) δ 8.06 (d, J =

DMSO-d₆) δ 8.24−8.14 (m, 2 H), 7.98 (dd, J = 8.5 Hz, J = 2.3 Hz, 1

H), 7.70−7.48 (m, 4 H). CIMS m/z (rel intensity) 283/285 (MH⁺,

100/36). HRCIMS calcd for C₁₆H₈ClO₃(MH⁺) 283.0162, found

283.0153.

3-Bromoindeno[1,2-c]isochromene-5,11-dione (31). 6-

Bromo-3-hydroxyphthalide (27, 2.52 g, 11.0 mmol) and phthalide

(14, 1.47 g, 11.0 mmol) were suspended in EtOAc (20 mL), and a

freshly prepared solution of NaOMe (0.91 g, 40 mmol of Na) in

MeOH (40 mL) was added. The resulting solution was heated to

reﬂux with stirring under an argon atmosphere for 72 h. The resulting

mixture was cooled to room temperature, acidiﬁed to pH 1 with 37%

HCl (4 mL), concentrated in vacuo, and azeotroped with PhMe (1 ×

25 mL). Ac₂O (50 mL) was added to the residue, and the mixture was

heated to reﬂux with stirring under an argon atmosphere for 17 h.

After cooling to room temperature, H₂O (30 mL) and CHCl₃(50

mL) were added and the mixture was ﬁltered to obtain some product.

The ﬁltrate was washed with H₂O (2 × 20 mL) and brine (1 × 20

mL), concentrated in vacuo, and azeotroped with PhMe (2 × 10 mL)

to provide additional product. The combined crude products were

washed with CHCl₃(2 × 30 mL) and EtOAc (1 × 30 mL) to provide

31 (1.920 g, 53%) as an orange solid: mp 236−238 °C (lit.³⁶mp 230−

232 °C). ¹H NMR (300 MHz, DMSO-d₆) δ 8.26 (d, J = 1.6 Hz, 1 H),

8.13−8.08 (m, 2 H), 7.64−7.48 (m, 4 H). CIMS m/z (rel intensity)

327/329 (MH⁺, 100/96), 248 (MH⁺− Br, 77).

3-Iodoindeno[1,2-c]isochromene-5,11-dione (32). 3-Hydroxy-

6-iodophthalide (28, 0.465 g, 1.68 mmol) and phthalide (14, 0.232 g,

1.73 mmol) were suspended in EtOAc (10 mL), and a freshly

prepared solution of NaOMe (0.19 g, 8.3 mmol of Na) in MeOH (10

mL) was added. The resulting solution was heated to reﬂux with

stirring for 27 h. The resulting mixture was cooled to room

temperature, acidiﬁed to pH 1 with 37% HCl (1.5 mL), concentrated

in vacuo, and azeotroped with PhMe (15 mL). Ac₂O (20 mL) was

added to the residue, and the mixture was heated to reﬂux with stirring

for 4 h. The mixture was concentrated in vacuo, the residue taken up

in CHCl₃(30 mL), and the organic layer washed with H₂O (2 × 15

mL). The organic solution was dried over Na₂SO₄and concentrated in

vacuo. The crude product was washed with 80:20 hexanes−EtOAc (2

× 10 mL) to provide 32 (196 mg, 31%) as an orange−red solid: mp

199−201 °C. ¹H NMR (300 MHz, DMSO-d₆) δ 8.43 (d, J = 1.8 Hz, 1

1

7.9 Hz, 1 H), 7.86−7.78 (m, 2 H), 5.41 (s, 2 H). 37 H NMR (300

MHz, CDCl₃) δ 8.20 (s, 1 H), 7.95 (dd, J = 8.1 Hz, J = 0.8 Hz, 1 H),

7.67 (d, J = 8.0 Hz, 1 H), 5.41 (s, 2 H).

3-Hydroxy-5-(triﬂuoromethyl)phthalide (40). Phthalide 36

(1.101 g, 5.447 mmol), NBS (982 mg, 5.52 mmol), and AIBN (39

mg) were suspended in CCl₄(25 mL) with stirring, and the mixture

was heated to reﬂux for 2 h. The product mixture was then cooled and

ﬁltered to remove succinimide. The residue was washed with CCl₄(10

mL), and the combined ﬁltrate was concentrated in vacuo to provide

an oil that was puriﬁed by silica gel column chromatography eluting

with 80:20 hexanes−EtOAc. The pure product-containing fractions

were concentrated in vacuo to provide a red oil. H₂O (20 mL) and

KOH (630 mg, 11.2 mmol) were added to the oil, and the mixture was

stirred and heated to reﬂux for 2 h 30 min. NaHSO₄(1.1 g) was added

to the obtained solution, and the product was extracted with EtOAc (3

× 20 mL). The combined organic layers were washed with brine (20

mL), dried over Na₂SO₄, and concentrated in vacuo to provide 40

1

(480 mg, 40%) as a yellow solid: mp 110−112 °C. H NMR (300

MHz, CDCl₃) δ 8.17 (s, 1 H), 8.00 (dd, J = 8.1, 0.8 Hz, 1 H), 7.82 (d,

J = 8.0 Hz, 1 H), 6.75 (s, 1 H), 3.81 (s, 1 H). CIMS m/z (rel intensity)

201 [(M + H⁺− H₂O)⁺, 100], 219 (MH⁺, 92).

3-Hydroxy-6-(triﬂuoromethyl)phthalide (41). Phthalide 37

(873 mg, 4.32 mmol), NBS (784 mg, 4.40 mmol), and AIBN (32

mg) were suspended in CCl₄(25 mL) with stirring, and the mixture

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was heated to reﬂux for 2 h 10 min. The product mixture was then

cooled and ﬁltered to remove succinimide. The residue was washed

with CCl₄(10 mL), and the combined ﬁltrate was concentrated in

vacuo to provide an oil which was puriﬁed by silica gel column

chromatography, eluting with 80:20 hexanes−EtOAc. The pure

product-containing fractions were concentrated in vacuo to provide

a colorless oil. H₂O (20 mL) and KOH (538 mg, 9.59 mmol) were

added to the oil, and the mixture was stirred and heated to reﬂux for 3

h 10 min. NaHSO₄(1.1 g) was added to the obtained solution, and the

product was extracted with EtOAc (3 × 20 mL). The combined

organic layers were washed with brine (20 mL), dried over Na₂SO₄,

and concentrated in vacuo to provide 41 (595 mg, 63%) as a white

solid: mp 107−110 °C. ¹H NMR (300 MHz, CDCl₃) δ 8.03 (d, J = 8.0

Hz, 1 H), 7.95 (s, 1 H), 7.90 (d, J = 8.2 Hz, 1 H), 6.86 (s, 1 H), 5.08

(s, 1 H). CIMS m/z (rel intensity) 219 (MH⁺, 100).

with stirring under an argon atmosphere for 24.5 h. The mixture was

cooled to room temperature, acidiﬁed to pH 1 with 37% HCl (2 mL),

concentrated in vacuo, and azeotroped with PhMe (2 × 10 mL). Ac₂O

(15 mL) was added to the residue, and the mixture was heated to

reﬂux with stirring under an argon atmosphere for 1 h 10 min. The

obtained suspension was azeotroped with PhMe (3 × 20 mL) to

provide a red solid residue, which was suspended in CHCl₃(125 mL)

and washed with H₂O (2 × 25 mL). The organic solution was dried

over Na₂SO₄and concentrated in vacuo. The residue was washed with

CHCl₃−hexanes (50:50, 50 mL) and ﬁltered. The ﬁltrate was

concentrated in vacuo until 10 mL of solvent remained, and the

suspension was ﬁltered. The collected solids were combined, yielding

45 (844 mg, 56%) as an orange solid: mp 220−222 °C. ¹H NMR (300

MHz, CDCl₃) δ 8.26 (dd, J = 8.4 Hz, J = 0.5 Hz, 1 H), 8.05 (d, J = 2.1

Hz, 1 H), 7.65 (dd, J = 8.4 Hz, J = 2.1 Hz, 1 H), 7.58 (td, J = 6.8 Hz, J

= 0.9 Hz, 1 H), 7.51−7.36 (m, 3 H), 2.59 (s, 3 H). CIMS m/z (rel

intensity) 295 (MH⁺, 100). HRCIMS calcd for C₁₇H₁₁O₃S (MH⁺)

295.0423, found 295.0429.

2-(Triﬂuoromethyl)indeno[1,2-c]isochromene-5,11-dione

(42). 3-Hydroxy-5-(triﬂuoromethyl)phthalide (40, 480 mg, 2.20

mmol) and phthalide (14, 310 mg, 2.31 mmol) were suspended in

EtOAc (5 mL), and a freshly prepared solution of NaOMe (0.21 g, 9.0

mmol of Na) in MeOH (10 mL) was added. The solution was heated

to reﬂux with stirring for 20.5 h. The mixture was cooled to room

temperature, acidiﬁed to pH 1 with 37% HCl (1 mL), concentrated in

vacuo, and azeotroped with PhMe (10 mL). Ac₂O (10 mL) was added

to the residue, and the mixture was heated to reﬂux with stirring for 5

h. After cooling to room temperature, the mixture was azeotroped with

PhMe (2 × 20 mL). The orange solid residue was taken up in CHCl₃

(50 mL) and washed with H₂O (2 × 25 mL). The organic solution

was dried over Na₂SO₄and concentrated in vacuo. The residue was

washed with 50:50 CHCl₃−hexanes (15 mL) to yield 42 (220 mg,

3-(Methylsulfonyl)indeno[1,2-c]isochromene-5,11-dione

(46). Lactone 45 (224 mg, 0.761 mmol) was dissolved with stirring in

CHCl₃(35 mL) at room temperature, and m-CPBA (labeled 57−86%,

assumed 57%, 477 mg, 1.58 mmol) was added. The color of the

solution changed from orange to yellow−orange almost instanta-

neously upon addition of m-CPBA. After stirring for 48.5 h, the

reaction mixture color had changed back to orange. The suspension

was ﬁltered to yield 46 (131 mg, 53%) as an orange solid: mp 305 °C

1

(dec). H NMR (300 MHz, DMSO-d₆) δ 8.60 (t, J = 1.2 Hz, 1 H),

8.40 (d, J = 1.3 Hz, 2 H), 7.70−7.54 (m, 4 H), 3.34 (s, 3 H). CIMS m/

z (rel intensity) 327 (MH⁺, 100). HRESIMS calcd for C₁₇H₁₀O₅SNa

(MNa⁺) 349.0147, found 349.0130.

1

32%) as an orange solid: mp 224−225 °C. H NMR (300 MHz,

CDCl₃) δ 8.56 (s, 1 H), 8.51 (d, J = 8.4 Hz, 1 H), 8.01 (dd, J = 8.5 Hz,

J = 1.9 Hz, 1 H), 7.65 (dt, J = 6.6 Hz, J = 1.1 Hz, 1 H), 7.57−7.45 (m,

3 H). CIMS m/z (rel intensity) 317 (MH⁺, 100). HRCIMS calcd for

C₁₇H₈F₃O₃(MH⁺) 317.0420, found 317.0412.

(S)-3-Fluoro-5,6-dihydro-6-(2,3-dihydroxypropyl)-5,11-

dioxo-11H-indeno[1,2-c]isoquinoline (49). Lactone 15 (86 mg,

0.32 mmol) was suspended in MeOH (15 mL) and CHCl₃(10 mL),

and (S)-3-amino-1,2-propanediol (48, 75 mg, 0.82 mmol) was added

with stirring. The mixture was heated to reﬂux for 15 h, cooled to

room temperature, and ﬁltered to provide a red−orange solid. The

ﬁltrate was concentrated in vacuo, suspended in CHCl₃(5 mL), and

ﬁltered to provide additional product. The products were combined

and washed with CHCl₃(5 mL) and hexanes (10 mL) to yield 49 (88

mg, 81%) as a red-orange solid: mp 183−185 °C. IR (ﬁlm) 3369,

1692, 1647, 1554, 1509 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆) δ 8.64

(dd, J = 9.0, 5.4 Hz, 1 H), 8.04 (d, J = 7.5 Hz, 1 H), 7.88 (dd, J = 9.5,

2.8 Hz, 1 H), 7.74 (td, J = 8.8, 2.9 Hz, 1 H), 7.65−7.41 (m, 3 H), 5.14

(d, J = 5.1 Hz, 1 H), 4.99 (t, J = 5.6 Hz, 1 H), 4.65−4.41 (m, 2 H),

4.06−3.94 (m, 1 H), 3.59 (t, J = 5.2 Hz, 2 H). CIMS m/z (rel

intensity) 340 (MH⁺, 84). HRCIMS calcd for C₁₉H₁₅FNO₄(MH⁺)

340.0980, found 340.0977. HPLC purity, 95.01% (MeOH−H₂O,

90:10).

(S)-3-Chloro-5,6-dihydro-6-(2,3-dihydroxypropyl)-5,11-

dioxo-11H-indeno[1,2-c]isoquinoline (50). Lactone 30 (88 mg,

0.31 mmol) was suspended in MeOH (15 mL) and CHCl₃(10 mL),

and (S)-3-amino-1,2-propanediol (48, 64 mg, 0.70 mmol) was added

with stirring. The mixture was heated to reﬂux for 15 h, cooled to

room temperature, and ﬁltered to provide a red−orange solid. The

product was washed with CHCl₃(5 mL) and hexanes (10 mL) to yield

50 (62 mg, 56%) as a red−orange solid: mp 240−242 °C. IR (ﬁlm)

3435, 1690, 1647, 1540, 1500 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆)

δ 8.59 (d, J = 8.7 Hz, 1 H), 8.14 (d, J = 2.3 Hz, 1 H), 8.06 (d, J = 7.4

Hz, 1 H), 7.87 (dd, J = 8.7, 2.4 Hz, 1 H), 7.63−7.43 (m, 3 H), 5.13 (d,

J = 5.0 Hz, 1 H), 4.98 (t, J = 5.6 Hz, 1 H), 4.62−4.46 (m, 2 H), 3.99

(s, 1 H), 3.58 (t, J = 5.3 Hz, 2 H). ESIMS m/z (rel intensity) 378/380

(MNa⁺, 100/42). HRCIMS calcd for C₁₉H₁₄ClNO₄Na (MNa⁺)

378.0509, found 378.0504. HPLC purity, 95.09% (MeOH−H₂O,

85:15).

3-(Triﬂuoromethyl)indeno[1,2-c]isochromene-5,11-dione

(43). 3-Hydroxy-6-(triﬂuoromethyl)phthalide (41, 595 mg, 2.73

mmol) and phthalide (14, 377 mg, 2.81 mmol) were suspended in

EtOAc (5 mL), and a freshly prepared solution of NaOMe (0.25 g, 11

mmol of Na) in MeOH (10 mL) was added. The solution was heated

to reﬂux with stirring for 20.5 h. The mixture was cooled to room

temperature, acidiﬁed to pH 1 with 37% HCl (1 mL), concentrated in

vacuo, and azeotroped with PhMe (10 mL). Ac₂O (10 mL) was added

to the residue, and the mixture was heated to reﬂux with stirring for 5

h. After cooling to room temperature, the mixture was azeotroped with

PhMe (2 × 10 mL). The orange solid residue was taken up in CHCl₃

(50 mL) and washed with H₂O (2 × 25 mL). The organic solution

was dried over Na₂SO₄and concentrated in vacuo. The residue was

washed with 50:50 CHCl₃−hexanes (15 mL) to yield 43 (287 mg,

1

33%) as an orange solid: mp 200−202 °C. H NMR (300 MHz,

CDCl₃) δ 8.64 (s, 1 H), 8.42 (d, J = 8.4 Hz, 1 H), 7.73 (dd, J = 8.4 Hz,

J = 1.4 Hz, 1 H), 7.65 (dt, J = 6.7 Hz, J = 1.1 Hz, 1 H), 7.58−7.42 (m,

3 H). CIMS m/z (rel intensity) 317 (MH⁺, 100). HRCIMS calcd for

C₁₇H₈F₃O₃(MH⁺) 317.0420, found 317.0433.

3-Hydroxy-6-(methylthio)phthalide (44). 6-Fluoro-3-hydrox-

yphthalide 12 (1.604 g, 9.541 mmol) was dissolved with stirring in

DMF (11 mL), and solid NaSMe (1.358 mg, 19.38 mmol) was added.

The mixture was heated between 115−120 °C for 2 h 30 min. The

reaction mixture was cooled, and H₂O (20 mL) and 37% HCl (3 mL)

were added. The product was extracted with Et₂O (6 × 30 mL). The

combined Et₂O layers were washed with brine (2 × 30 mL), dried over

Na₂SO₄, and concentrated in vacuo to provide a yellow−orange oil.

The oil was puriﬁed by silica gel column chromatography, eluting with

4:1 EtOAc−hexanes, to yield 44 (1.299 g, 69%) as a peach solid: mp

95−100 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.72 (s, 1 H), 7.65−7.47

(m, 2 H), 2.55 (s, 3 H). CIMS m/z (rel intensity) 197 (MH⁺, 100).

3-(Methylthio)indeno[1,2-c]isochromene-5,11-dione (45). 3-

Hydroxy-6-(methylthio)phthalide (44, 1.000 g, 5.096 mmol) and

phthalide (14, 692 mg, 5.159 mmol) were suspended in EtOAc (15

mL), and a freshly prepared solution of NaOMe (0.50 g, 22 mmol of

Na) in MeOH (30 mL) was added. The solution was heated to reﬂux

(S)-3-Bromo-5,6-dihydro-6-(2,3-dihydroxypropyl)-5,11-

dioxo-11H-indeno[1,2-c]isoquinoline (51). Lactone 31 (84 mg,

0.26 mmol) was suspended in MeOH (15 mL) and CHCl₃(10 mL),

and (S)-3-amino-1,2-propanediol (48, 55 mg, 0.60 mmol) was added

with stirring. The mixture was heated to reﬂux for 15 h, cooled to

room temperature, and ﬁltered to provide a red−orange solid. The

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product was washed with CHCl₃(5 mL) and hexanes (10 mL) to yield

(MH⁺, 100). HRCIMS calcd for C₂₀H₁₅F₃NO₄(MH⁺) 390.0948,

found 390.0952. HPLC purity, 96.12% (MeOH−H₂O, 90:10).

(S)-5,6-Dihydro-6-(2,3-dihydroxypropyl)-3-(methylthio)-

5,11-dioxo-11H-indeno[1,2-c]isoquinoline (56). Lactone 45 (57

mg, 0.19 mmol) was suspended in MeOH (10 mL) and CHCl₃(15

mL), and (S)-3-amino-1,2-propanediol (48, 39 mg, 0.43 mmol) was

added as a solution in MeOH (1 mL) with stirring. The mixture was

heated to reﬂux with stirring under an argon atmosphere for 14.5 h,

cooled to room temperature, and concentrated to a volume of ∼1 mL.

Hexanes (0.5 mL) were added, the mixture was vigorously stirred, and

the mixture was ﬁltered. The solid residue that collected was washed

with hexanes (1 mL) to yield 56 (51 mg, 72%) as a red−orange solid:

51 (44 mg, 42%) as a red−orange solid: mp 224−226 °C. IR (ﬁlm)

1

3436, 1664, 1643, 1540, 1315 cm⁻¹. H NMR (300 MHz, DMSO-d₆)

δ 8.49 (d, J = 8.7 Hz, 1 H), 8.26 (d, J = 2.2 Hz, 1 H), 8.14−7.89 (m, 2

H), 7.65−7.37 (m, 3 H), 5.13 (d, J = 5.0 Hz, 1 H), 4.99 (t, J = 5.5 Hz,

1 H), 4.68−4.39 (m, 2 H), 3.98 (d, J = 9.8 Hz, 1 H), 3.57 (t, J = 5.2

Hz, 2 H). CIMS m/z (rel intensity) 400/402 (MH⁺, 94/100).

HRCIMS calcd for C₁₉H₁₅BrNO₄(MH⁺) 400.0184/402.0166, found

400.0192/402.0173. HPLC purity, 95.18% (MeOH−H₂O, 85:15).

(S)-5,6-Dihydro-6-(2,3-dihydroxypropyl)-3-iodo-5,11-dioxo-

11H-indeno[1,2-c]isoquinoline (52). Lactone 32 (52 mg, 0.14

mmol) was dissolved in MeOH/CHCl₃(15:10 mL), and (S)-3-amino-

1,2-propanediol (48, 32 mg, 0.35 mmol) was added. The mixture was

heated at reﬂux under Ar overnight, cooled, and concentrated. The

residue was suspended in CHCl₃−hexanes (10:5 mL), and the

suspension was ﬁltered to provide 52 (58 mg, 93%) as orange solid:

mp 186−193 °C (dec). IR (ﬁlm) 3488, 1706, 1624, 1533, 1304 cm⁻¹.

¹H NMR (300 MHz, DMSO-d₆) δ 8.49 (d, J = 1.9 Hz, 1 H), 8.36 (d, J

= 8.5 Hz, 1 H), 8.13 (dd, J = 8.5, 1.9 Hz, 1 H), 8.07 (d, J = 7.6 Hz, 1

H), 7.62−7.44 (m, 3 H), 5.14 (d, J = 5.1 Hz, 1 H), 4.99 (t, J = 5.5 Hz,

1 H), 4.63−4.45 (m, 2 H), 4.06−3.93 (m, 1 H), 3.59 (t, J = 5.3 Hz, 2

H). ESIMS m/z (rel intensity) 470 (MNa⁺, 52). HRESIMS calcd for

C₁₉H₁₄INO₄Na (MNa⁺) 469.9865, found 469.9855. HPLC purity,

96.81% (MeOH−H₂O, 90:10).

(S)-3-Cyano-5,6-dihydro-6-(2,3-dihydroxypropyl)-5,11-

dioxo-11H-indeno[1,2-c]isoquinoline (53). Lactone 33 (67 mg,

0.25 mmol) was suspended in MeOH (10 mL) and CHCl₃(15 mL),

and (S)-3-amino-1,2-propanediol (48, 56 mg, 0.61 mmol) was added

with stirring. The mixture was heated to reﬂux for 19 h, cooled to

room temperature, and concentrated until ∼1 mL of solvent remained.

The mixture was ﬁltered, and the solid residue was washed with

hexanes (5 mL) to yield 53 (44 mg, 52%) as a red−orange solid: mp

314−315 °C. IR (ﬁlm) 3583, 1734, 1671, 1608, 1534 cm⁻¹. ¹H NMR

(300 MHz, DMSO-d₆) δ 8.66 (dd, J = 8.4, 0.7 Hz, 1 H), 8.54 (dd, J =

1.8, 0.6 Hz, 1 H), 8.22−8.05 (m, 2 H), 7.67−7.46 (m, 3 H), 5.15 (d, J

= 5.0 Hz, 1 H), 5.01 (t, J = 5.6 Hz, 1 H), 4.54 (dd, J = 6.8, 3.6 Hz, 2

H), 3.99 (dt, J = 9.3, 5.0 Hz, 1 H), 3.66−3.52 (m, 2 H). CIMS m/z

(rel intensity) 347 (MH⁺, 100). HRESIMS calcd for C₂₀H₁₃N₂O₄(M

− H⁺) 345.0875, found 345.0865. HPLC purity, 95.12% (MeOH−

H₂O, 90:10).

1

mp 195 °C. IR (ﬁlm) 3401, 1698, 1656, 1647, 1498, 1424 cm⁻¹. H

NMR (300 MHz, DMSO-d₆) δ 8.50 (d, J = 8.6 Hz, 1 H), 8.02 (d, J =

7.5 Hz, 1 H), 7.95 (d, J = 2.1 Hz, 1 H), 7.72 (dd, J = 8.6, 2.2 Hz, 1 H),

7.60−7.37 (m, 3 H), 5.13 (d, J = 5.0 Hz, 1 H), 4.97 (t, J = 5.6 Hz, 1

H), 4.65−4.41 (m, 2 H), 4.07−3.89 (m, 1 H), 3.57 (t, J = 5.3 Hz, 2

H), 2.57 (s, 3 H). CIMS m/z (rel intensity) 368 (MH⁺, 100).

HRESIMS calcd for C₂₀H₁₆NO₄S (M − H⁺) 366.0800, found

366.0794. HPLC purity, 96.39% (MeOH−H₂O, 90:10).

(S)-5,6-Dihydro-6-(2,3-dihydroxypropyl)-3-(methylsulfonyl)-

5,11-dioxo-11H-indeno[1,2-c]isoquinoline (57). Lactone 46 (62

mg, 0.19 mmol) was suspended in MeOH (anhydrous, 10 mL) and

CHCl₃(15 mL), and (S)-3-amino-1,2-propanediol (48, 36 mg, 0.40

mmol) was added with stirring. The mixture was heated to reﬂux for

16 h, cooled to room temperature, and concentrated in vacuo. 80:20

hexanes−EtOAc (10 mL) was added to the residue, and the

suspension was ﬁltered. The product was washed with MeOH (5

mL) to yield 57 (49 mg, 65%) as a red−orange solid: mp 185 °C

1

(dec). IR (ﬁlm) 3468, 1702, 1670, 1647, 1605, 1539 cm⁻¹. H NMR

(300 MHz, DMSO-d₆) δ 8.78 (d, J = 8.6 Hz, 1 H), 8.65 (d, J = 1.9 Hz,

1 H), 8.28 (dd, J = 8.6, 2.1 Hz, 1 H), 8.14 (d, J = 7.4 Hz, 1 H), 7.71−

7.48 (m, 3 H), 5.16 (d, J = 5.0 Hz, 1 H), 5.02 (t, J = 5.5 Hz, 1 H),

4.65−4.51 (m, 2 H), 4.14−3.93 (m, 1 H), 3.61 (t, J = 5.3 Hz, 2 H),

3.30 (s, 3 H). APCIMS m/z (rel intensity) 400 (MH⁺, 100).

HRESIMS calcd for C₂₀H₁₇NO₆SNa (MNa⁺) 422.0674, found

422.0670. HPLC purity, 97.27% (MeOH, 100).

(S)-5,6-Dihydro-6-(2,3-dihydroxypropyl)-3-(methoxycarbon-

yl)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (58). Lactone 47

(42 mg, 0.14 mmol) was dissolved in MeOH/CHCl₃(15:10 mL), and

(S)-3-amino-1,2-propanediol (48, 31 mg, 0.34 mmol) was added. The

mixture was heated at reﬂux under Ar overnight, cooled, and

concentrated. The residue was suspended in CHCl₃/hexanes (10:5

mL), and the suspension was ﬁltered to provide 58 (44 mg, 86%) as

dark-orange solid: mp 174−175 °C (dec). IR (ﬁlm) 3503, 1719, 1637,

1612, 1541, 1438, 1278 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆) δ 8.71

(d, J = 1.8 Hz, 1 H), 8.64 (d, J = 8.5 Hz, 1 H), 8.27 (dd, J = 8.5, 1.9

Hz, 1 H), 8.10 (d, J = 7.3 Hz, 1 H), 7.64−7.44 (m, 3 H), 5.15 (d, J =

5.0 Hz, 1 H), 5.01 (t, J = 5.5 Hz, 1 H), 4.65−4.44 (m, 2 H), 4.08−3.94

(m, 1 H), 3.90 (s, 3 H), 3.60 (t, J = 5.4 Hz, 2 H). CIMS m/z (rel

intensity) 380 (MH⁺, 78). HRCIMS calcd for C₂₁H₁₈NO₆(MH⁺)

380.1129, found 380.1134. HPLC purity, 95.02% (MeOH−H₂O,

85:15).

3-Fluoro-5,6-dihydro-6-(3-(1H-imidazol-1-yl)propyl)-5,11-

dioxo-11H-indeno[1,2-c]isoquinoline (60). Lactone 15 (78 mg,

0.29 mmol) was dissolved in CHCl₃(25 mL) with stirring, and 1-(3-

aminopropyl)imidazole (59, 73 mg, 0.58 mmol) was added. The

mixture was heated to reﬂux for 26 h, cooled, and concentrated to ∼1

mL. Hexanes (5 mL) were added, and the suspension was ﬁltered to

provide 60 (74 mg, 68%) as a red−orange solid: mp 220−222 °C. IR

(ﬁlm) 1698, 1660, 1556, 1509, 1432 cm⁻¹. ¹H NMR (300 MHz,

CDCl₃) δ 8.71 (dd, J = 9.0 Hz, J = 5.3 Hz, 1 H), 7.97 (dd, J = 9.2 Hz, J

= 2.8 Hz, 1 H), 7.64 (s, 1 H), 7.59 (dd, J = 6.8 Hz, J = 1.6 Hz, 1 H),

7.47 (td, J = 8.5 Hz, J = 2.7 Hz, 1 H), 7.40−7.29 (m, 2 H), 7.23−7.17

(m, 1 H), 7.11−7.02 (m, 1 H), 6.66 (d, J = 7.2 Hz, 1 H), 4.58−4.47

(m, 2 H), 4.24 (t, J = 6.4 Hz, 2 H), 2.37 (p, J = 6.7 Hz, 2 H). APCIMS

m/z 374 (MH⁺, 100). HRESIMS calcd for C₂₂H₁₇FN₃O₂(MH⁺)

374.1305, found 374.1307. HPLC purity, 95.21% (1% TFA in

MeOH−H₂O, 85:15).

(S)-2-(Triﬂuoromethyl)-5,6-dihydro-6-(2,3-dihydroxyprop-

yl)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (54). Lactone 42

(66 mg, 0.21 mmol) was suspended in MeOH (10 mL) and CHCl₃

(15 mL), and (S)-3-amino-1,2-propanediol (48, 52 mg, 0.42 mmol)

was added with stirring. The mixture was heated to reﬂux for 14 h,

cooled to room temperature, and concentrated until ∼1 mL of solvent

remained. Precipitation of product occurred upon standing for 30 min,

and the mixture was ﬁltered. The product was washed with hexanes (5

mL) to provide 54 (60 mg, 73%) as a bright-orange solid: mp 214−

215 °C. IR (ﬁlm) 3583, 3294, 1665, 1546, 1314, 1109 cm⁻¹. ¹H NMR

(300 MHz, DMSO-d₆) δ 8.72 (d, J = 8.7 Hz, 1 H), 8.38 (s, 1 H),

8.13−8.07 (m, 2 H), 7.60−7.46 (m, 3 H), 5.13 (d, J = 5.0 Hz, 1 H),

5.00 (t, J = 5.5 Hz, 1 H), 4.62−4.46 (m, 2 H), 3.99 (dt, J = 9.3 Hz, J =

4.9 Hz, 1 H), 3.59 (t, J = 5.5 Hz, 2 H). CIMS m/z (rel intensity) 390

(MH⁺, 100). HRCIMS calcd for C₂₀H₁₅F₃NO₄(MH⁺) 390.0948,

found 390.0951. HPLC purity, 97.24% (MeOH−H₂O, 90:10).

(S)-3-(Triﬂuoromethyl)-5,6-dihydro-6-(2,3-dihydroxyprop-

yl)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (55). Lactone 43

(62 mg, 0.20 mmol) was suspended in MeOH (10 mL) and CHCl₃

(15 mL), and (S)-3-amino-1,2-propanediol (48, 54 mg, 0.43 mmol)

was added with stirring. The mixture was heated to reﬂux for 14 h,

cooled to room temperature, and concentrated until ∼1 mL of solvent

remained. Hexanes (2 mL) were added, and the suspension was

ﬁltered. The product was washed with hexanes (5 mL) to yield 55 (49

mg, 63%) as a red−orange solid: mp 228−230 °C. IR (ﬁlm) 3401,

1

1668, 1559, 1501, 1312, 1128 cm⁻¹. H NMR (300 MHz, DMSO-d₆)

δ 8.87 (s, 1 H), 8.40 (d, J = 8.4 Hz, 1 H), 8.10 (d, J = 7.4 Hz, 1 H),

7.81 (dd, J = 8.5 Hz, J = 1.9 Hz, 1 H), 7.62−7.46 (m, 3 H), 5.15 (d, J =

5.0 Hz, 1 H), 5.00 (t, J = 5.6 Hz, 1 H), 4.59−4.47 (m, 2 H), 4.06−3.95

(m, 1 H), 3.59 (t, J = 5.4 Hz, 2 H). CIMS m/z (rel intensity) 390

N

DOI: 10.1021/acs.jmedchem.5b00303

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

3-Chloro-5,6-dihydro-6-(3-(1H-imidazol-1-yl)propyl)-5,11-

dioxo-11H-indeno[1,2-c]isoquinoline (61). Lactone 30 (82 mg,

0.29 mmol) was dissolved in CHCl₃(25 mL) with stirring, and 1-(3-

aminopropyl)imidazole (59, 73 mg, 0.58 mmol) was added. The

mixture was heated to reﬂux for 20 h, cooled, and concentrated to ∼1

mL. Hexanes (5 mL) were added, and the suspension was ﬁltered to

provide 61 (87 mg, 77%) as a red solid: mp 215−217 °C. IR (ﬁlm)

MHz, DMSO-d₆) δ 8.49 (d, J = 8.5 Hz, 1 H), 7.97 (d, J = 2.1 Hz, 1 H),

7.82 (s, 1 H), 7.74 (dd, J = 8.6, 2.2 Hz, 1 H), 7.62−7.37 (m, 3 H), 7.34

(s, 1 H), 7.22 (d, J = 7.0 Hz, 1 H), 7.01 (s, 1 H), 4.47 (t, J = 7.6 Hz, 2

H), 4.24 (t, J = 6.7 Hz, 2 H), 2.59 (s, 3 H), 2.35−2.15 (m, 2 H). EIMS

m/z (rel intensity) 401 (M⁺, 100). HRESIMS calcd for C₂₃H₁₉N₃O₂S

(MH⁺) 402.1276, found 402.1262. HPLC purity, 95.37% (1% TFA in

MeOH−H₂O, 85:15).

5,6-Dihydro-6-(3-(1H-imidazol-1-yl)propyl)-3-(methylsulfon-

yl)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (66). Lactone 46

(45 mg, 0.14 mmol) was dissolved in CHCl₃(20 mL) with stirring,

and 1-(3-aminopropyl)imidazole (58, 36 mg, 0.29 mmol) was added.

The mixture was heated to reﬂux for 15.5 h, cooled to room

temperature, and concentrated in vacuo. Then 80:20 hexanes−EtOAc

(10 mL) was added to the residue, and the suspension was ﬁltered.

The product was washed with MeOH (5 mL) to provide 66 (34 mg,

57%) as a red−orange solid: mp 142 °C (dec). IR (ﬁlm) 1698, 1668,

1606, 1505, 1304, 1148 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆) δ 8.73

(d, J = 8.6 Hz, 1 H), 8.65 (d, J = 2.0 Hz, 1 H), 8.28 (dd, J = 8.5, 2.1

Hz, 1 H), 7.75 (s, 1 H), 7.66−7.43 (m, 3 H), 7.33−7.22 (m, 2 H), 6.97

(s, 1 H), 4.49 (t, J = 7.7 Hz, 2 H), 4.24 (t, J = 6.7 Hz, 2 H), 3.31 (s, 3

H), 2.34−2.14 (m, 2 H). EIMS m/z 433 (rel intensity) (M⁺, 37), 95

(100). HRESIMS calcd for C₂₃H₁₉N₃O₄SNa (MNa⁺) 456.0994, found

456.0972. HPLC purity, 95.27% (MeOH−H₂O, 90:10).

1

1698, 1664, 1541, 1500, 1317 cm⁻¹. H NMR (300 MHz, CDCl₃) δ

8.62 (d, J = 8.7 Hz, 1 H), 8.28 (d, J = 2.3 Hz, 1 H), 7.80−7.52 (m, 3

H), 7.41−7.28 (m, 2 H), 7.20 (d, J = 1.1 Hz, 1 H), 7.15−7.00 (m, 1

H), 6.64 (d, J = 7.4 Hz, 1 H), 4.51 (t, J = 7.8 Hz, 2 H), 4.24 (t, J = 6.4

Hz, 2 H), 2.35 (p, J = 6.7 Hz, 2 H). ESIMS m/z 390/392 (MH⁺, 100/

37). HRESIMS calcd for C₂₂H₁₇ClN₃O₂(MH⁺) 390.1009, found

390.1009. HPLC purity, 95.04% (1% TFA in MeOH, 100).

5,6-Dihydro-6-(3-(1H-imidazol-1-yl)propyl)-3-iodo-5,11-

dioxo-11H-indeno[1,2-c]isoquinoline (62). Lactone 32 (72 mg,

0.19 mmol) was dissolved in CHCl₃(25 mL) with stirring, and 1-(3-

aminopropyl)imidazole (59, 51 mg, 0.41 mmol) was added. The

mixture was heated to reﬂux for 17 h, cooled, and concentrated to ∼1

mL. Hexanes (5 mL) were added, and the suspension was ﬁltered to

provide 62 (92 mg, 100%) as a red−orange solid: mp 248−250 °C. IR

(ﬁlm) 3401, 1693, 1662, 1601, 1534, 1495, 1424 cm⁻¹. ¹H NMR (300

MHz, CDCl₃) δ 8.68 (d, J = 1.9 Hz, 1 H), 8.42 (d, J = 8.5 Hz, 1 H),

7.99 (dd, J = 8.6 Hz, J = 1.9 Hz, 1 H), 7.90−7.76 (m, 1 H), 7.60 (d, J =

6.5 Hz, 1 H), 7.43−7.29 (m, 2 H, signal obscured by CHCl₃in

CDCl₃), 7.18−7.05 (m, 1 H), 6.75 (d, J = 7.2 Hz, 1 H), 4.64−4.44 (m,

2 H), 4.27 (t, J = 6.5 Hz, 2 H), 2.37 (s, 2 H). ESIMS m/z 482 (MH⁺,

100). HRESIMS calcd for C₂₂H₁₇IN₃O₂(MH⁺) 482.0366, found

482.0364. HPLC purity, 100% (MeOH−H₂O, 85:15).

2-(Triﬂuoromethyl)-5,6-dihydro-6-(3-(1H-imidazol-1-yl)-

propyl)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (63). Lactone

42 (74 mg, 0.23 mmol) was dissolved in CHCl₃(25 mL) with stirring,

and 1-(3-aminopropyl)imidazole (59, 59 mg, 0.47 mmol) was added.

The mixture was heated to reﬂux for 17 h, cooled to room

temperature, and hexanes (5 mL) were added. The suspension was

stirred for 5 min and ﬁltered to give 63 (97 mg, 100%) as a red−

orange solid: mp 220−223 °C. IR (ﬁlm) 1698, 1669, 1516, 1436,

1314, 1122 cm⁻¹. ¹H NMR (300 MHz, CDCl₃) δ 8.81 (d, J = 8.6 Hz,

1 H), 8.61 (s, 1 H), 7.91 (dd, J = 8.6 Hz, J = 2.0 Hz, 1 H), 7.68−7.61

(m, 2 H), 7.45−7.29 (m, 2 H), 7.22 (s, 1 H), 7.08 (s, 1 H), 6.69 (d, J =

7.4 Hz, 1 H), 4.56 (t, J = 8.2 Hz, 2 H), 4.26 (t, J = 6.3 Hz, 2 H), 2.38

(p, J = 6.6 Hz, 1 H). CIMS m/z (rel intensity) 424 (MH⁺, 100).

HRESIMS calcd for C₂₃H₁₇F₃N₃O₂(MH⁺) 424.1273, found 424.1278.

HPLC purity, 95.27% (MeOH−H₂O, 90:10).

3-Fluoro-5,6-dihydro-6-(3-morpholinopropyl)-5,11-dioxo-

11H-indeno[1,2-c]isoquinoline (68). Lactone 15 (88 mg, 0.33

mmol) was dissolved in CHCl₃(25 mL) with stirring, and 3-

morpholinopropylamine (67, 95 mg, 0.66 mmol) was added. The

mixture was heated to reﬂux for 21.5 h, cooled, and concentrated to

∼1 mL. Hexanes (5 mL) were added, and the suspension was ﬁltered

to provide 68 (108 mg, 83%) as a brick-red solid: mp 200−202 °C. IR

1

(ﬁlm) 1693, 1655, 1511, 1456, 1112, 909 cm⁻¹. H NMR (300 MHz,

CDCl₃) δ 8.73 (dd, J = 8.9 Hz, J = 5.3 Hz, 1 H), 7.97 (dd, J = 9.3 Hz, J

= 2.8 Hz, 1 H), 7.74 (d, J = 7.4 Hz, 1 H), 7.63 (dd, J = 6.8 Hz, J = 1.7

Hz, 1 H), 7.51−7.36 (m, 3 H), 4.67−4.55 (m, 2 H), 3.73 (t, J = 4.7

Hz, 4 H), 2.60 (t, J = 6.3 Hz, 2 H), 2.53 (s, 4 H), 2.08 (p, J = 6.5 Hz, 2

H). ESIMS m/z 393 (MH⁺, 100). HRESIMS calcd for C₂₃H₂₂FN₂O₃

(MH⁺) 393.1614, found 393.1612. HPLC purity, 95.05% (1% TFA in

MeOH−H₂O, 90:10).

3-Chloro-5,6-dihydro-6-(3-morpholinopropyl)-5,11-dioxo-

11H-indeno[1,2-c]isoquinoline (69). Lactone 30 (79 mg, 0.28

mmol) was dissolved in CHCl₃(25 mL) with stirring, and 3-

morpholinopropylamine (67, 82 mg, 0.57 mmol) was added. The

mixture was heated to reﬂux for 22 h, cooled, and concentrated to ∼1

mL. Hexanes (5 mL) were added, and the suspension was ﬁltered to

provide 69 (111 mg, 97%) as a red-orange solid: mp 199−200 °C. IR

(ﬁlm) 1698, 1654, 1500, 1427, 1113 cm⁻¹. ¹H NMR (300 MHz,

DMSO-d₆) δ 8.58 (d, J = 8.7 Hz, 1 H), 8.16 (d, J = 2.3 Hz, 1 H),

7.97−7.84 (m, 2 H), 7.70−7.48 (m, 3 H), 4.56 (t, J = 7.5 Hz, 2 H),

3.49 (t, J = 4.3 Hz, 4 H), 2.42−2.24 (m, 4 H), 2.03−1.87 (m, 2 H,

obscured by solvent peak). APCIMS m/z 409/411 (MH⁺, 100/35).

HRESIMS calcd for C₂₃H₂₂ClN₂O₃(MH⁺) 409.1319, found 409.1318.

HPLC purity, 96.24% (1% TFA in MeOH−H₂O, 85:15).

3-(Triﬂuoromethyl)-5,6-dihydro-6-(3-(1H-imidazol-1-yl)-

propyl)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (64). Lactone

43 (76 mg, 0.24 mmol) was dissolved in CHCl₃(25 mL) with stirring,

and 1-(3-aminopropyl)imidazole (59, 63 mg, 0.50 mmol) was added.

The mixture was heated to reﬂux for 17 h, cooled to room

temperature, and hexanes (5 mL) were added. The suspension was

stirred for 5 min and ﬁltered to yield 64 (91 mg, 90%) as a red−orange

solid: mp 194−195 °C. IR (ﬁlm) 1697, 1669, 1561, 1504, 1312, 1128

1

cm⁻¹. H NMR (300 MHz, CDCl₃) δ 8.97 (s, 1 H), 8.43 (d, J = 8.4

5,6-Dihydro-3-iodo-6-(3-morpholinopropyl)-5,11-dioxo-

11H-indeno[1,2-c]isoquinoline (70). Lactone 32 (69 mg, 0.18

mmol) was dissolved in CHCl₃(25 mL) with stirring, and 3-

morpholinopropylamine (67, 53 mg, 0.37 mmol) was added. The

mixture was heated to reﬂux for 22 h, cooled, and concentrated to ∼1

mL. Hexanes (5 mL) were added, and the suspension was ﬁltered to

provide 70 (78 mg, 87%) as a red−orange solid: mp 231−232 °C. IR

(ﬁlm) 1698, 1654, 1493, 1424, 1110 cm⁻¹. ¹H NMR (300 MHz,

CDCl₃) δ 8.66 (d, J = 1.7 Hz, 1 H), 8.41 (d, J = 8.4 Hz, 1 H), 7.96 (dd,

J = 8.5 Hz, J = 1.7 Hz, 1 H), 7.74 (d, J = 6.4 Hz, 1 H), 7.62 (dd, J = 6.7

Hz, J = 1.8 Hz, 1 H), 7.51−7.36 (m, 2 H), 4.66−4.54 (m, 2 H), 3.75

(t, J = 4.4 Hz, 4 H), 2.71−2.44 (m, 6 H), 2.24−1.98 (m, 2 H).

APCIMS m/z 501 (MH⁺, 100). HRESIMS calcd for C₂₃H₂₂IN₂O₃

(MH⁺) 501.0675, found 501.0671. HPLC purity, 96.88% (1% TFA in

MeOH, 100).

Hz, 1 H), 7.74−7.56 (m, 3 H), 7.41−7.30 (m, 2 H), 7.21 (t, J = 1.1

Hz, 1 H), 7.07 (t, J = 1.3 Hz, 1 H), 6.68 (d, J = 7.4 Hz, 1 H),

4.64−4.46 (m, 2 H), 4.26 (t, J = 6.3 Hz, 2 H), 2.47−2.29 (m, 2 H).

CIMS m/z (rel intensity) 424 (MH⁺, 100). HRESIMS calcd for

C₂₃H₁₇F₃N₃O₂(MH⁺) 424.1273, found 424.1266. HPLC purity,

98.31% (1% TFA in MeOH−H₂O, 90:10).

5,6-Dihydro-6-(3-(1H-imidazol-1-yl)propyl)-3-(methylthio)-

5,11-dioxo-11H-indeno[1,2-c]isoquinoline (65). Lactone 45 (64

mg, 0.22 mmol) was dissolved in CHCl₃(20 mL) with stirring, and a

solution of 1-(3-aminopropyl)imidazole (59, 58 mg, 0.46 mmol)

dissolved in CHCl₃(1 mL) was added. The mixture was heated to

reﬂux with stirring under an argon atmosphere for 14.5 h, cooled to

room temperature, and concentrated to a volume of ∼1 mL. Hexanes

(0.5 mL) were added, the mixture was vigorously stirred, and the

mixture was ﬁltered. The solid residue that collected was washed with

hexanes (1 mL) to yield 65 (71 mg, 81%) as a blood-red solid: mp 234

°C (dec). IR (ﬁlm) 1693, 1641, 1573, 1497, 1423 cm⁻¹. ¹H NMR (300

6-Azaphthalide (72). The method of Orlek and co-workers⁶⁴was

followed to yield 72 (1.5 g, 33%) as an oﬀ-white solid: mp 128−130

°C (lit.⁶⁴mp 150−153 °C). IR (KBr) 1764, 1572, 1432, 1005, 756

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1

cm⁻¹. H NMR (CDC1₃, 300 MHz) δ 9.18 (s, 1 H), 8.86 (d, J = 5.2

3-Aza-5,6-dihydro-6-(3-(1H-imidazol-1-yl)propyl)-5,11-

dioxo-11H-indeno[1,2-c]isoquinoline (79). 1-(3-Aminopropyl)-

imidazole (59, 0.037 g, 0.30 mmol) was added to a solution of

lactone 75 (0.050 g, 0.20 mmol) in CHCl₃(30 mL). The solution was

allowed to stir at reﬂux temperature for 15 h, diluted with CHCl₃(50

mL), and washed with H₂O (3 × 20 mL) and brine (20 mL). The

organic layer was dried over sodium sulfate, ﬁltered, and concentrated

to provide a crude solid. The solid was puriﬁed by silica gel column

chromatography, eluting with 97:3 CHCl₃−MeOH, to provide 79

(0.030 mg, 71%) as an orange−red solid: mp 251−252 °C. IR (KBr)

1723, 1678, 767, 685 cm⁻¹. ¹H NMR (CDCl₃, 300 MHz) δ 9.52 (s, 1

H), 8.79 (d, J = 5.6 Hz, 1 H), 8.42 (d, J = 5.5 Hz, 1 H), 7.68 (m, 2 H),

7.46 (t, J = 7.0 Hz, 1 H), 7.36 (t, J = 7.0 Hz, 1 H), 7.21 (s, 1 H), 7.07

(s, 1 H), 6.71 (d, J = 7.4 Hz, 1 H), 4.56 (t, J = 7.8 Hz, 2 H), 4.28 (t, J =

6.2 Hz, 2 H), 2.37 (m, 2 H). ESIMS (m/z relative intensity) 357

(MH⁺, 100). HRESIMS calcd for C₂₁H₁₇N₄O₂357.1352 (MH⁺),

found 357.1351. HPLC purity, 95.74% (1% TFA in MeOH−H₂O,

90:10).

Topoisomerase I-Mediated DNA Cleavage Reactions. A 3′-

[³²P]-labeled 117-bp DNA oligonucleotide was prepared as previously

described. The oligonucleotide contains previously identiﬁed Top1

cleavage sites in 161-bp pBluescript SK(−) phagemid DNA.

Approximately 2 nM radiolabeled DNA substrate was incubated

with recombinant Top1 in 20 μL of reaction buﬀer [10 mM Tris-HCl

(pH 7.5), 50 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, 0.2 mM DTT,

and 15 μg/mL BSA] at 25 °C for 20 min in the presence of various

concentrations of test compounds. The reactions were terminated by

adding SDS (0.5% ﬁnal concentration), followed by the addition of

two volumes of loading dye (80% formamide, 10 mM sodium

hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and 0.1%

bromophenol blue). Aliquots of each reaction mixture were subjected

to 20% denaturing PAGE. Gels were dried and visualized by using a

phosphoimager and ImageQuant software (Molecular Dynamics). For

simplicity, cleavage sites were numbered as previously described in the

161-bp fragment.⁶⁵

Hz, 1 H), 7.50 (d, J = 5.2 Hz, 1 H), 5.35 (s, 2 H). ESIMS m/z (rel

intensity) 136 (MH⁺, 100).

6-Aza-3-bromophthalide (73). Furo[3,4-c]pyridin-3(1H)-one

(72, 1.0 g, 7.4 mmol) was heated at reﬂux with NBS (1.4 g, 8.1

mmol) and AIBN (20 mg) in CCl₄(40 mL) for 2 h. The reaction

mixture was cooled to room temperature, precipitated solids were

ﬁltered oﬀ, and the ﬁltrate was concentrated. The residue was puriﬁed

by silica gel column chromatography, eluting with 3:1 EtOAc−

hexanes, to aﬀord 73 (0.90 g, 57%) as a white solid: mp 96−97 °C. IR

(KBr) 1745, 1587, 668 cm⁻¹. ¹H NMR (CDC1₃, 300 MHz) δ 9.22 (s,

1 H), 8.99 (d, J = 5.2 Hz, 1 H), 7.60 (d, J = 5.2 Hz, 1 H), 7.36 (s, 1 H).

6-Aza-3-hydroxyphthalide (74). Compound 73 (0.8 g, 4 mmol)

was heated at reﬂux in H₂O (40 mL) for 2 h. The obtained mixture

was concentrated to dryness to aﬀord compound 74 (0.5 g, 88%) as a

brown syrup. IR (KBr) 3256, 1734, 1081, 768 cm⁻¹. ¹H NMR

(CDC1₃, 300 MHz) δ 10.62 (s, 0.1 H), 9.16 (s, 1 H), 8.98 (s, 1 H),

7.87 (d, J = 4.4 Hz, 1 H), 6.76 (s, 0.9 H). ESIMS m/z (rel intensity)

152 (MH⁺, 100).

3-Azaindeno[1,2-c]isochromene-5,11-dione (75). Hydroxyph-

thalide 74 (0.5 g, 3 mmol) and phthalide (14, 0.413 g, 3.08 mmol)

were diluted in EtOAc (15 mL). Sodium metal (0.354 g, 15.4 mmol)

was dissolved in MeOH (30 mL), and the solution was added to

reaction mixture. The solution was heated at reﬂux for 24 h, cooled to

room temperature, acidiﬁed with concd HCl (∼4 mL), and

concentrated. The obtained solid was diluted with Ac₂O (20 mL),

and the mixture was heated at reﬂux for 6 h. The solution was

concentrated, diluted with CHCl₃(100 mL), and washed with

saturated NaHCO₃(3 × 50 mL). The organic layer was washed with

brine (75 mL), dried over Na₂SO₄, concentrated, and puriﬁed by silica

gel column chromatography, eluting with 9:1 CHCl₃-hexanes to yield

75 (0.090 g, 11%) as an orange solid: mp 203−205 °C. IR (KBr)

1745, 1706, 1389, 998, 693 cm⁻¹. ¹H NMR (CDCl₃, 300 MHz) δ 9.43

(s, 1 H), 8.89 (d, J = 5.0 Hz, 1 H), 8.15 (d, J = 5.2 Hz, 1 H), 7.66 (m, 1

H), 7.55 (m, 3 H). CIMS (m/z relative intensity) 250 (MH⁺, 100).

HRESIMS calcd for C₁₅H₈NO₃250.0504 (MH⁺), found 250.0495.

3-Aza-5,6-dihydro-6-(3-morpholinopropyl)-5,11-dioxo-11H-

indeno[1,2-c]isoquinoline (76). 3-Morpholinopropylamine (67,

0.043 g, 0.30 mmol) was added to a solution of lactone 75 (0.050

g, 0.20 mmol) in CHCl₃(30 mL). The solution was allowed to stir at

reﬂux temperature for 15 h, diluted with CHCl₃(45 mL), and washed

with H₂O (3 × 25 mL) and brine (25 mL). The organic layer was

dried over sodium sulfate, ﬁltered, and concentrated to provide a crude

solid. The solid was puriﬁed by silica gel column chromatography,

eluting with 1% MeOH in CHCl₃, to provide 76 (0.058 mg, 82%) as

an orange−red solid: mp 210−211 °C. IR (KBr) 1756, 1665, 1189,

Recombinant TDP1 Assay. A 5′-[³²P]-labeled single-stranded

DNA oligonucleotide containing a 3′-phosphotyrosine (N14Y)²⁸was

incubated at 1 nM with 10 pM recombinant TDP1 in the absence or

presence of inhibitor for 15 min at room temperature in the LMP1

assay buﬀer containing 50 mM Tris HCl, pH 7.5, 80 mM KCl, 2 mM

EDTA, 1 mM DTT, 40 μg/mL BSA, and 0.01% Tween-20.³²

Reactions were terminated by the addition of 1 volume of gel loading

buﬀer [99.5% (v/v) formamide, 5 mM EDTA, 0.01% (w/v) xylene

cyanol, and 0.01% (w/v) bromophenol blue]. Samples were subjected

to a 16% denaturing PAGE with multiple loadings at 12 min intervals.

Gels were dried and exposed to a PhosphorImager screen (GE

Healthcare). Gel images were scanned using a Typhoon 8600 (GE

Healthcare), and densitometry analyses were performed using

ImageQuant software (GE Healthcare).

Recombinant TDP2 Assay. TDP2 reactions were carried out as

described previously⁶⁶with the following modiﬁcations. The 19-mer

single-stranded oligonucleotide DNA substrate containing a 5′

phosphotyrosine (Y19, α³²P-cordycepin-3′-labeled) was incubated at

1 nM with 25 pM recombinant human TDP2 in the absence or

presence of inhibitor for 15 min at room temperature in the LMP2

assay buﬀer containing 50 mM Tris-HCl, pH 7.5, 80 mM KCl, 5 mM

MgCl₂, 0.1 mM EDTA, 1 mM DTT, 40 μg/mL BSA, and 0.01%

Tween 20. Reactions were terminated and treated similarly to

recombinant TDP1 reactions (see above).

Molecular Mechanics Calculations. The 1SC7 X-ray crystal

structure ﬁle was prepared for molecular modeling by correcting

several atom types, removing all of the water molecules, and adding

hydrogens. The hydrogens were energy minimized, while the

remaining atoms were frozen in aggregate, in SYBYL.⁶⁷The details

of this minimization, and all of the other minimizations, were set as

follows: Powell method, MMFF94s force ﬁeld,⁶⁸MMFF94 charges,

and 0.05 kcal/mol·Å energy gradient convergence. Selected inden-

oisoquinolines were constructed in SYBYL and energy minimized. The

ligands were docked into the prepared crystal structure using GOLD

3.2 software.⁶⁹The centroid of the binding site was deﬁned by the

756 cm⁻¹. H NMR (CDCl₃, 300 MHz) δ 9.50 (s, 1 H), 8.77 (d, J =

1

5.5 Hz, 1 H), 8.43 (d, J = 5.5 Hz, 1 H), 7.82 (d, J = 6.1 Hz, 1 H), 7.71

(d, J = 6.1 Hz, 1 H), 7.53 (m, 2 H), 4.66 (t, J = 7.6 Hz, 2 H), 3.77 (br

s, 4 H), 2.60 (m, 6 H), 2.12 (m, 2 H). ESIMS (m/z relative intensity)

376 (MH⁺, 100). HRESIMS calcd for C₂₂H₂₂N₃O₃376.1661 (MH⁺),

found 376.1662. HPLC purity, 95.31% (1% TFA in MeOH−H₂O,

90:10).

3-Aza-5,6-dihydro-6-(3-(dimethylamino)propyl)-5,11-dioxo-

11H-indeno[1,2-c]isoquinoline (78). N,N-Dimethyl-1,3-diamino-

propane (77, 0.030 g, 0.30 mmol) was added to a solution of lactone

75 (0.050 g, 0.20 mmol) in CHCl₃(30 mL). The solution was allowed

to stir at reﬂux temperature for 15 h, diluted with CHCl₃(60 mL), and

washed with H₂O (3 × 30 mL) and brine (30 mL). The organic layer

was dried over sodium sulfate, ﬁltered, and concentrated to provide a

crude solid. The solid was puriﬁed by silica gel column

chromatography, eluting with 4% MeOH in CHCl₃, to aﬀord 78

(0.049 g, 72%) as an orange−red solid: mp 168−170 °C. IR (KBr)

1710, 1675, 1567, 765 cm⁻¹. ¹H NMR (CDCl₃, 300 MHz) δ 9.50 (s, 1

H), 8.75 (dd, J = 5.5 Hz, 1 H), 8.40 (d, J = 5.7 Hz, 1 H), 7.79 (m, 1

H), 7.68 (m, 1 H), 7.50 (m, 2 H), 4.61 (m, 2 H), 2.53 (t, J = 6.45 Hz,

2 H), 2.29 (s, 6 H), 2.05 (m, 2 H). CIMS (m/z relative intensity) 334

(MH⁺, 100). HRESIMS calcd for C₂₀H₂₀N₃O₂334.1556 (MH⁺),

found 334.1557. HPLC purity, 99.46% (1% TFA in MeOH−H₂O,

90:10).

P

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

crystallized ligand. Ten GOLD algorithm runs were executed per

ligand. Default parameters were used. The top ten docking poses per

ligand were inspected visually following the docking runs. The docking

poses that had a favorable GOLD score and similar binding mode to

the crystallized ligand were selected for further analysis. Ligand atom

types were inspected and corrected as necessary. Lone pairs added by

GOLD were deleted prior to a subsequent energy minimization of the

docked complexes. Minimization was executed by allowing only the

ligand to move while freezing the surrounding crystal structure as an

aggregate.

ABBREVIATIONS USED

■

APCIMS, atmospheric-pressure chemical ionization mass

spectrometry; CIMS, chemical ionization mass spectrometry;

CPT, camptothecin; DTT, dithiothreitol; EIMS, electron

ionization mass spectrometry; ESIMS, electrospray ionization

mass spectrometry; FDA, U.S. Food and Drug Administration;

TDP1, tyrosyl-DNA phosphodiesterase 1; TDP2, tyrosyl-DNA

phosphodiesterase 2; Top1, human topoisomerase IB

Quantum Mechanics Calculations. The A−T and C−G base

pairs and indenoisoquinoline 91 were individually subjected to

geometry optimization and frequency calculation at the HF/6-

31G** level in Gaussian 09.⁷⁰The original 1SC7 X-ray crystal

structure complex was then replaced with the geometry optimized

parts. The hydrogen in the 3-position of indenoisoquinoline 91 was

replaced with a nitro group (90), ﬂuorine (92), chlorine (93), or

triﬂuoromethyl (94), respectively. The same geometry optimizations

were performed on these molecules before placing them into

complexes with the DNA base pairs. A schematic representation of

the model “indenoisoquinoline−Top1−DNA ternary complex” used

in this study is shown in Figure 6.

After the complexes were assembled, single-point energy calcu-

lations were performed at the MP2/6-31G* level in Gaussian 09. The

basis set superposition error (BSSE) for each complex was calculated

using the counterpoise correction method within the Gaussian 09

package, specifying two fragments: (1) the two ﬂanking DNA base

pairs and (2) the indenoisoquinoline. The base pair−indenoisoquino-

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ASSOCIATED CONTENT

* Supporting Information

SMILES molecular formula strings (CSV). The Supporting

Information is available free of charge on the ACS Publications

website at DOI: 10.1021/acs.jmedchem.5b00303.

■

S

AUTHOR INFORMATION

Corresponding Author

* Phone: 765-494-1465. Fax: 765-494-6970. E-mail: cushman@

purdue.edu.

■

Notes

The authors declare the following competing ﬁnancial

interest(s): Mark Cushman is on the Board of Directors and

is an investor in Linus Oncology, Inc., which has licensed

indenoisoquinoline intellectual property owned by Purdue

University. Neither Linus Oncology, Inc., nor any other

commercial company sponsored or provided other direct

ﬁnancial support to the author or his laboratory for the research

reported in this article. The remaining authors have no

competing and/or relevant ﬁnancial interest(s) to disclose.

ACKNOWLEDGMENTS

■

This work was made possible by the National Institutes of

Health (NIH) through support with Research Grants

U01CA089566 and P30CACA023168. This research was also

supported in part by the Intramural Research Program of the

NIH, National Cancer Institute, Center for Cancer Research. In

vitro cytotoxicity testing was performed by the Developmental

Therapeutics Program at the National Cancer Institute, under

contract NO1-CO-56000. D.E.B. thanks Stephanie Pitman,

Rubayat Khan, Huaping Mo, and Karl Wood for technical

assistance.

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