Discovery and structure-activity relationship of novel 2,3- dihydrobenzofuran-7-carboxamide and 2,3-dihydrobenzofuran-3(2 h)-one-7-carboxamide derivatives as poly(ADP-ribose)polymerase-1 Inhibitors

Article

pubs.acs.org/jmc

Open Access on 06/12/2015

Discovery and Structure−Activity Relationship of Novel 2,3-

Dihydrobenzofuran-7-carboxamide and 2,3-Dihydrobenzofuran-

3(2H)‑one-7-carboxamide Derivatives as Poly(ADP-

ribose)polymerase‑1 Inhibitors

Maulik R. Patel,^†,⊥Aaditya Bhatt,^†,⊥Jamin D. Steﬀen,^‡Adel Chergui,^§Junko Murai,^§Yves Pommier,^§

John M. Pascal,^‡Louis D. Trombetta,^†Frank R. Fronczek,^∥and Tanaji T. Talele*^,†

^†Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, New York 11439,

United States

^‡Department of Biochemistry and Molecular Biology, Kimmel Cancer Center, Thomas Jeﬀerson University, 233 South 10th Street,

Philadelphia, Pennsylvania 19107, United States

^§Developmental Therapeutics Branch, Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer

Institute, National Institutes of Health, 37 Convent Drive, Bethesda, Maryland 20892, United States

^∥Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, United States

S

* Supporting Information

ABSTRACT: Novel substituted 2,3-dihydrobenzofuran-7-carboxamide (DHBF-7-carboxamide) and 2,3-dihydrobenzofuran-

3(2H)-one-7-carboxamide (DHBF-3-one-7-carboxamide) derivatives were synthesized and evaluated as inhibitors of poly(ADP-

ribose)polymerase-1 (PARP-1). A structure-based design strategy resulted in lead compound 3 (DHBF-7-carboxamide; IC₅₀

=

9.45 μM). To facilitate synthetically feasible derivatives, an alternative core was designed, DHBF-3-one-7-carboxamide (36, IC₅₀

= 16.2 μM). The electrophilic 2-position of this scaﬀold was accessible for extended modiﬁcations. Substituted benzylidene

derivatives at the 2-position were found to be the most potent, with 3′,4′-dihydroxybenzylidene 58 (IC₅₀= 0.531 μM) showing a

30-fold improvement in potency. Various heterocycles attached at the 4′-hydroxyl/4′-amino of the benzylidene moiety resulted

in signiﬁcant improvement in inhibition of PARP-1 activity (e.g., compounds 66−68, 70, 72, and 73; IC₅₀values from 0.718 to

0.079 μM). Compound 66 showed selective cytotoxicity in BRCA2-deﬁcient DT40 cells. Crystal structures of three inhibitors

(compounds (−)-13c, 59, and 65) bound to a multidomain PARP-1 structure were obtained, providing insights into further

development of these inhibitors.

number of nuclear target proteins (heteromodiﬁcation) such

as histones, p53, topoisomerase I/II, DNA polymerases, and

DNA ligases.^1,2In response to DNA-strand breaks, PARylation

by PARP-1 is stimulated nearly 500-fold.³PARylation is

involved in a number of important cellular processes including

DNA damage repair, genomic stability, and regulation of

transcription and cell death.^4,5The synthesis of highly

INTRODUCTION

■

Poly(ADP-ribose)polymerase-1 (PARP-1) is an abundant,

ubiquitously expressed nuclear protein that is responsible for

the maintenance and repair of DNA damage.¹PARP-1 is the

founding member of an expanded family of related proteins

with similar enzymatic activity. PARP-1 utilizes NAD⁺as a

substrate to synthesize linear and branched chains of poly-

(ADP-ribose) (PAR). These PAR chains covalently bind to the

acceptor residues. Poly(ADP)ribosylation (PARylation) occurs

mainly on PARP-1 itself (automodiﬁcation) but also on

Received: December 31, 2013

A

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

negatively charged PAR chains leads to chromatin decondensa-

tion and dynamic nucleosome remodeling⁶that facilitates

accessibility of base excision repair (BER) proteins such as

XRCC1, DNA ligase III, and DNA polymerase β (pol β) to

sites of damaged DNA.⁷⁻⁹The presence of PAR is both

transient and dynamic, as hydrolase enzymes such as

poly(ADP-ribose)glycohydrolase (PARG) quickly degrade

PAR into ADP-ribose units.¹⁰

Chart 1. PARP Inhibitors Currently Being Evaluated in the

Clinic

With such prominent and pivotal roles in the maintenance of

genomic stability, PARP-1 inhibitors have been developed to

target certain diseases such as cancer. PARP-1 inhibitors

sensitize tumor cells to certain DNA alkylating agents (e.g.,

temozolomide, cisplatin), topoisomerase I poisons (e.g.,

irinotecan), and other chemotherapeutic agents and are being

used in clinical trials as potentiators of chemo- and radio-

therapy.^5,11,12The ﬁnding that double-strand DNA break

repair-deﬁcient cancer cells carrying defective or mutated

BRCA1 and BRCA2 respond to PARP inhibitors (synthetic

lethality) has paved the way for PARP inhibitors as single

agents therapy in oncology.^13,14Some of the clinically proven

PARP-1 inhibitors such as olaparib have been shown to exhibit

single agent activity for tumors with mutations in BRCA1- and

BRCA2-dependent DNA double-strand break repair mecha-

nisms (homologous recombination, HR).¹⁵⁻¹⁸In addition,

PARP-1 inhibitors may also be eﬀective at targeting other DNA

repair defects in tumors with “BRCAness” phenotypes.¹⁹For

instance, lack of tumor suppressor gene PTEN (phosphatase

and tensin homologue) demonstrate HR defects in human

tumor cell lines, making them responsive to PARP-1

inhibitors.²⁰Taken together, PARP-1 inhibitors oﬀer exciting

therapeutic potential as molecular targeted agents in the ﬁeld of

oncology.

Previously, we reported N-1 substituted indazole-3-carbox-

amide²¹and 2,8-disubstituted quinazolin-4(3H)-one²²deriva-

tives as PARP-1 inhibitors. With the aims of structural novelty

and improved potency of these scaﬀolds, we designed the

previously unexplored pharmacophores 2,3-dihydrobenzofuran-

7-carboxamide (DHBF-7-carboxamide) and 2,3-dihydrobenzo-

furan-3(2H)-one-7-carboxamide (DHBF-3-one-7-carboxamide)

that are targeted to the NAD⁺binding site of PARP-1. We

obtained crystal structure data of these scaﬀolds to help further

drug development. Inhibitor bound crystal structures were of

the PARP-1 multidomain complex with DNA,²³demonstrating

the ﬁrst report of a PARP inhibitor bound to DNA-damage

activated PARP-1.

would establish an intramolecular hydrogen bond leading to the

formation of a 6:5:6 pseudotricyclic ring system with improved

potency.

Similar to other PARP-1 inhibitors, docking of DHBF-7-

carboxamide (compound 3) within the catalytic site of human

PARP-1 (PDB code 4L6S)³²(Figure 1) revealed that the

carbonyl oxygen atom formed a hydrogen bond with the side

chain hydroxyl group of Ser904 (CO···HO-Ser904, 1.91 Å)

and backbone −NH of Gly863 (CO···HN-Gly863, 2.09 Å)

while the carboxamide hydrogen atom entered into hydrogen

bonding interaction with the backbone carbonyl oxygen atom

RESULTS AND DISCUSSION

■

Structure-Based Drug Design. A considerable number of

PARP inhibitors have been described in the literature over the

past several years.^24,25The structures of the selected PARP

inhibitors (ABT-888,²⁶MK-4827,²⁷AG014699,²⁸and AZD-

2281²⁹) in clinical trials are shown in Chart 1. It is well

established that these agents target the NAD⁺binding site by

occupying the nicotinamide pocket. Inhibitors that target this

site contain a cyclic or acyclic carboxamide pharmacophore,

which makes π−π stacking interactions with adjacent tyrosine

residues, Tyr896 and Tyr907, and form three key hydrogen

bonds with Gly863 and Ser904 of the PARP-1 catalytic site.

Optimization eﬀorts that gave rise to these clinical candidates

improved potency by restricting the amide conformation of the

carboxamide through intramolecular hydrogen bonding^27,30,31

or by locking the amide into a ring.^22,28,29With this guiding

concept, we hypothesized that the DHBF-7-carboxamide (3)

Figure 1. XP-Glide predicted binding mode of compound 3 within the

active site of PARP-1, represented in ribbon form, with the interacting

amino acids represented as sticks and atoms colored according to the

following: carbon, beige; hydrogen, white; nitrogen, blue; oxygen, red.

The inhibitor is shown as ball and stick with the same color scheme

depicted above except that carbons are represented as orange. Dotted

red lines indicate intra- and intermolecular hydrogen bonding

interaction, whereas the dotted yellow lines indicate the distance

between the two atoms/groups/centroids in angstroms. The centroids

are marked as green stars. The image was generated using PyMOL,

version 1.6.0.

B

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

of Gly863 (NH···OC-Gly863, 1.92 Å). We observed

formation of an intramolecular hydrogen bond between the

carboxamide hydrogen atoms and the ether oxygen atom of the

DHBF-7-carboxamide that results in the formation of

pseudotricyclic ring and restriction of the carboxamide moiety.

The aromatic ring of DHBF was stabilized through a π−π

stacking interaction with the nearly coplanar electron rich

phenyl ring of Tyr907, as seen with other PARP-1 inhibitors.

To further develop this lead series, we carried out SAR analysis,

stereochemical characterization, and small molecule and

macromolecular X-ray crystallographic analysis of the DHBF-

7-carboxamide and DHBF-3-one-7-carboxamide derivatives.

Further biochemical evaluation of PARP-1 activity and

cytotoxicity analysis of BRCA2-deﬁcient DT40 cells demon-

strated the therapeutic potential of these series as PARP-1

inhibitors.

Synthesis. Dihydrobenzofuran derivatives were synthesized

in good yields with diminutive variations in reported

procedures.³³⁻³⁶The carboxylation at the 7-position of the

2,3-dihydrobenzofuran (DHBF, compound 1) was achieved by

lithiation with n-butyllithium (n-BuLi) in TEMED/hexane at

room temperature under nitrogen atmosphere, followed by

addition of dry ice and acidiﬁcation with concentrated HCl³³

(Scheme 1). The resultant carboxylic acid derivative 2 was

bromine in acetic acid at elevated temperature of 80 °C

provided compound 4.³⁴Nitration³³of 3 with nitric acid and

triﬂuoroacetic acid at low temperature yielded 5 in good yield.

The nitro group of 5 was further reduced to amine using tin

chloride dihydrate in ethyl acetate under reﬂuxing conditions to

yield compound 6.

In order to modify the 2-position of DHBF, we used an

alternative synthetic route to synthesize the 2-substituted and

the 2,5-disubstituted DHBF-7-carboxamide derivatives from the

corresponding salicylic acid derivatives to avoid the low yielding

carboxylation step in the later stages of the synthetic protocol.

First, the salicylic acid derivatives were converted to

corresponding methyl esters using thionyl chloride in the

presence of methanol (Scheme 2). This was followed by O-

allylation, carried out using allyl bromide and potassium

carbonate, to yield compounds 9a−c. The 3,3-sigmatropic

Claisen rearrangement was carried out by heating compounds

9a−c neatly at elevated temperatures to yield ortho-rearranged

products 10a−c.³⁵The furan ring was constructed by reacting

the rearranged product with zirconium chloride in dichloro-

methane to yield the corresponding methyl 2-methyl-DHBF-7-

carboxylate derivatives 11a−c.³⁶Hydrolysis of the methyl ester

using sodium hydroxide provided corresponding acids 12a−c

that were converted to carboxamides 13a−c under mixed

anhydride conditions and aqueous ammonia (as discussed

above). Nitration of compound 13a (where R = H) followed by

reduction was carried out essentially as discussed above to

obtain 14 and 15, respectively.

Scheme 1. Synthesis of 5-Substituted 2,3-

Dihydrobenzofuran-7-carboxamide Derivatives

a

Since the enantiomers bind diﬀerently within the active site,

one of the enantiomers is presumed to be more potent when

compared to the other. Therefore, we decided to resolve the 2-

position enantiomers resulted during the construction of

DHBF ring and evaluate the activity of the pure enantiomers.

The 2-position enantiomers (compounds (+)-13a, (−)-13a,

(+)-13c, and (−)-13c) were resolved by preparative chiral

HPLC, using an amylose based chiral stationary phase

Chiralpak 1A (4.6 mm × 250 mm) at GVK Biosciences Pvt.

Ltd., Hyderabad, India. To determine the absolute conﬁg-

uration at the 2-position of the representative eutomer, the X-

ray crystal structure of 12a was elucidated using the

diastereomeric salt formation approach.³⁷⁻⁴⁰The absolute

conﬁguration from direct determination based on anomalous

scattering from the light atoms was in agreement with the

starting materials. On the basis of the analysis of the crystals of

(−)-enantiomer of 12a with (S)-(−)-α-methylbenzylamine, it

a

Reagents and conditions: (a) (i) n-BuLi, TEMED, hexane, rt, 4 h; (ii)

dry ice, overnight; conc HCl; (b) (i) i-BuOCOCl, NMM, THF, −20

°C, 4 h; (ii) 30% NH₄OH, rt, 1 h; (c) Br₂, CH₃COONa, CH₃COOH,

80 °C, 3 h; (d) TFA, HNO₃, 0 °C to rt, 3 h; (e) SnCl₂·2H₂O, EtOAc,

reﬂux, 4 h; (f) H₂SO₄, MeOH, reﬂux, 3 h.

converted to the carboxamide via a mixed-anhydride method to

get the lead compound 3. Bromination of 3 using a solution of

a

Scheme 2. Synthesis of 2,5-Disubstituted 2,3-Dihydrobenzofuran-7-carboxamides

a

Reagents and conditions: (a) SOCl₂, MeOH, reﬂux, 12 h; (b) allyl bromide, K₂CO₃, NaI, DMF, rt, 12 h; (c) neat, 160−190 °C, 2 h; (d) ZrCl₄,

DCM, rt, 10 h; (e) NaOH, MeOH, reﬂux, 2 h; (f) (i) i-BuOCOCl, NMM, THF, −20 °C, 4 h; (ii) 30% NH₄OH, rt, 1 h; (g) when R = H, TFA,

HNO₃, 0 °C to rt, 3 h; (h) SnCl₂·2H₂O, EtOAc, reﬂux, 4 h.

C

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

was revealed that the (−)-enantiomer bears the R-conﬁguration

(Figure 2). On the basis of this ﬁnding, the (+)-enantiomer was

assigned as the S-enantiomer.

position nitration followed by reduction was performed by the

same procedure described for compound 18a to yield 18b.

Attempts to replace the amino group of compound 18b with a

ﬂuoro group (by the aforementioned method) yielded 5-ﬂuoro-

DHBF-7-carboxylic acid (compound 19b) in a very low yield.

To overcome this, compound 2 was ﬁrst converted to the

corresponding methyl ester 16 by reﬂuxing it in methanol and a

catalytic amount of sulfuric acid as shown in Scheme 1.

Subsequent nitration at the 5-position of 16 followed by

hydrogenation (as mentioned above) yielded methyl 5-amino-

DHBF-7-carboxylate 18c in good yield which was ﬂuorinated

using the aforementioned procedure to get the desired

compound 19c in good yield. Compound 19c was hydrolyzed

to yield 19b, which was then converted to carboxamide using

the mixed-anhydride condition to obtain the desired product

20 (Scheme 3).

The 4-amino-2-methyl-DHBF-7-carboxamide 28 was synthe-

sized using the 4-nitrosalicylic acid 21 as the precursor, since

the direct electrophilic substitution onto the DHBF-7-

carboxamide ring favored the 5-position and attempts to use

4-aminosalicylic acid methyl ester as the starting material

yielded mono-N- and di-N-allylated methyl ester derivatives in

the allylation step (data not shown). The nitro group in

compound 21 acted as a surrogate, which was reduced to an

amino group in the latter steps of the synthetic scheme. The

ring construction was accomplished as outlined in Scheme 4.

The esteriﬁcation of the acid 21 was accomplished by the

aforementioned method wherein the acid was reﬂuxed in the

presence of thionyl chloride and methanol. The resulting ester

22 was allylated onto the phenolic hydroxyl with allyl bromide

utilizing the conditions mentioned previously. The 3,3-

sigmatropic Claisen rearrangement of the allyl derivative 23

was performed using carbitol as a solvent, since the

aforementioned procedure of neat heating of the compound

above 160 °C yielded the rearranged product 24 in a very low

yield. Cyclization of the rearranged nitro analogue followed by

hydrolysis did not yield the desired nitro acid (NiAc) derivative

(Scheme 4). We therefore decided to change the synthetic plan

by reducing the nitro group of 25 to an amine (compound 26)

followed by hydrolysis using sodium hydroxide, yielding acid

intermediate 27. The resulting acid 27 was converted to

carboxamide 28 by modiﬁed mixed-anhydride conditions for

carboxamide synthesis, wherein N-methylmorpholine was

added ﬁrst followed by isobutyl chloroformate to prevent the

N-acylation. The anhydride, once formed, was quickly treated

with dry ammonia gas, resulting in the formation of the target

carboxamide rac-28 (Scheme 4). The rac-28 was resolved by

Figure 2. ORTEP diagram of salt of (R)-(−)-12a with (S)-(−)-α-

methylbenzylamine.

To synthesize the 5-ﬂuoro derivative of the DHBF-7-

carboxamide, we ﬁrst attempted to launch the 5-ﬂuoro

substituent by a reported method⁴¹in which nitration at the

5-position of the DHBF was carried out (as mentioned

previously), followed by its reduction using a Pd/C catalyst, to

yield 5-amino-DHBF compound 18a (Scheme 3). The

diazonium tetraﬂuoroborate salt of 18a was prepared by adding

aqueous sodium nitrite solution into a mixture of 5-amino-

DHBF, hydrochloric acid, and tetraﬂuoroboric acid in THF at

−15 °C. The resulting precipitates were reﬂuxed in xylene to

obtain 5-ﬂuoro-DHBF compound 19a. Unfortunately, the

lithiation-mediated 7-carboxylation of compound 19a did not

yield the desired compound (5-ﬂuoro-DHBF-7-carboxylic acid,

19b). An alternative strategy was applied in which we used

DHBF-7-carboxylic acid 2 as a starting material instead of

DHBF to elude the low yielding carboxylation step. The 5-

a

Scheme 3. Synthesis of 5-Fluoro-2,3-dihydrobenzofuran-7-carboxamide

a

Reagents and conditions: (a) TFA, HNO₃, 0 °C to rt, 3 h; (b) H₂/Pd/C, EtOH, 60 psi, rt, 2−18 h; (c) (i) THF, HCl, HBF₄, rt to −15 °C, NaNO₂,

30 min; (ii) xylene, reﬂux, 2 h; (d) RH, (i) n-BuLi, TEMED, hexane, rt, 4 h; (ii) dry ice, overnight; (e) when R = -COOCH₃; NaOH, MeOH,

reﬂux, 3 h; (f) (i) i-BuOCOCl, NMM, THF, −20 °C, 4 h; (ii) 30% NH₄OH, rt, 1 h.

D

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a

Scheme 4. Synthesis of 4-Amino-2-methyl-2,3-dihydrobenzofuran-7-carboxamide

a

Reagents and conditions: (a) SOCl₂, MeOH, reﬂux, 12 h; (b) allyl bromide, K₂CO₃, NaI, DMF, rt, 12 h; (c) carbitol, 170−180 °C, 2 h; (d) ZrCl₄,

DCM, rt, 10 h; (e) NaOH, MeOH, reﬂux; (f) H₂/Pd/C, EtOH, 60 psi, rt, 4 h; (g) NaOH, MeOH, reﬂux, 18 h; (h) (i) NMM, i-BuOCOCl, THF,

−20 °C, 20 min; (ii) dry NH₃, rt, 45 min.

a

Scheme 5. Synthesis of 3-Oxo-2,3-dihydrobenzofuran-7-carboxamide

a

Reagents and conditions: (a) SOCl₂, MeOH, reﬂux, 12 h; (b) BrCH₂COOEt, K₂CO₃, NaI, DMF, rt, 12 h; (c) KOH, MeOH, reﬂux, 4 h; (d)

KMnO₄, water, reﬂux, 2 h; (e) NaOAc, (CH₃CO)₂O, CH₃COOH, reﬂux, 5 h; (f) HCl (11 N)/H₂O/MeOH, reﬂux, 1 h; (g) (i) i-BuOCOCl, NMM,

THF, −20 °C, 4 h; (ii) 30% NH₄OH, rt, 1 h.

a

Scheme 6. Synthesis of Substituted Benzaldehydes 37−40 and 42

a

Reagents and conditions: (a) K₂CO₃, 4-(2-chloroethyl)morpholine (for compounds 37 and 39) and 4-(2-chloroethyl)piperidine (for compounds

38 and 40), CH₃CN, reﬂux, 6 h; (b) K₂CO₃, KI, bromochloroethane, CH₃CN, reﬂux, 12 h; (c) K₂CO₃, KI, N-methylpiperazine, CH₃CN, reﬂux, 2−6

h.

means of preparative chiral chromatography similar to the

method developed for racemates 13a and 13c.

order to resolve the issue of ring oxidation, which occurred in

the latter case, we decided to oxidize the aromatic methyl group

prior to the ring construction. This was met by ﬁrst esterifying

3-methylsalicylic acid to obtain 30 followed by O-alkylation

with ethyl bromoacetate to yield diester intermediate 31, which

was then hydrolyzed to yield the diacid derivative 32 and was

further oxidized to 2-(carboxymethoxy)isophthalic acid (com-

pound 33). Cyclization of 33 to 3-acetoxybenzofuran-7-

A successful synthesis of DHBF-3-one-7-carboxamide

(compound 36) (Scheme 5) was carried out after trying two

unsuccessful strategies, which involved (i) carboxylation of

DHBF-3-one and (ii) oxidation of 7-methyl group of 7-methyl-

DHBF-3-one, none of which yielded the desired penultimate

intermediate DHBF-3-one-7-carboxylic acid 35. Finally, in

E

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a

Scheme 7. Synthesis of Substituted Benzaldehydes 44 and 46−49

a

Reagents and conditions: (a) K₂CO₃, bromochloropropane, CH₃CN, reﬂux, 3 h; (b) K₂CO₃, morpholine, CH₃CN, reﬂux, 6 h; (c) chloroacetyl

chloride, DCM, triethylamine, 0 °C to rt, 3 h; (d) K₂CO₃, 4-hydroxybenzaldehyde, CH₃CN, reﬂux, 6 h; (e) epibromhydrin, K₂CO₃, CH₃CN, reﬂux,

6 h; (f) morpholine, K₂CO₃, CH₃CN, reﬂux, 6 h; (g) 4-ﬂuorophenylpiperazine, K₂CO₃, CH₃CN, reﬂux, 48 h.

carboxylic acid (34) was accomplished by treating 33 with the

1:3:5⁴²proportions of sodium acetate, acetic acid, and acetic

anhydride, respectively, under reﬂuxing conditions. The

resulting compound was hydrolyzed using a mixture of

1:10:40 ⁴²proportions of hydrochloric acid (11 N)/water/

methanol, generating a 3-oxo derivative 35. The resulting acid

was ﬁnally converted to an amide 36 by the aforementioned

mixed-anhydride technique.

Knoevenagel condensation⁴⁵of compound 36 with various

commercially available and in house synthesized benzaldehyde

derivatives under reﬂuxing toluene in the presence of

ammonium acetate yielded target compounds 50−73, which

were ﬁnally washed with water (to remove excess of

ammonium acetate catalyst), dried, and obtained in moderate

yields as shown in Scheme 8.

a

The benzaldehydes required for the synthesis of target

compounds 50−71 were either commercially available or

prepared (37−49) as shown in Schemes 6 and 7. The 3- or 4-

hydroxybenzaldehydes were alkylated with commercially

available 2-chloroethylmorpholine/piperidine in the presence

of potassium carbonate under reﬂuxing conditions to obtain

37−40 (Scheme 6). The 4-(2-chloroethoxy)benzaldehyde 41

was synthesized by reacting 4-hydroxybenzaldehyde with

bromochloroethane in the presence of potassium carbonate

and reﬂuxing acetonitrile. Compound 41 was then reacted with

N-methylpiperazine to yield the desired benzaldehyde 42

(Scheme 6). Benzaldehyde derivative 43 was prepared similar

to that of 41 using bromochloropropane instead of

bromochloroethane (Scheme 7). Compound 43 was then

treated with morpholine to obtain the desired aldehyde

intermediate 44. The acyl derivative 46 was synthesized by

performing diminutive variations in the reported conditions

(Scheme 7).^43,44First, the morpholine was reacted with

chloroacetyl chloride in the presence of triethylamine in

dichoromethane at 0 °C, which generated N-2-chloroacetyl-

morpholine (45). Compound 45 was then treated with 4-

hydroxybenzaldehyde by following the aforementioned alkyla-

tion technique (using potassium carbonate as a base) to yield

46. The aldehyde 47 was synthesized by alkylating 4-

hydroxybenzaldehyde with epibromhydrin under alkylation

conditions (Scheme 7). Compound 48 was synthesized by

epoxide ring-opening, which was mediated by nucleophilic

attack of morpholine onto the electropositive methylene group

of the epoxide ring of 47 (Scheme 7). Compound 49 was

synthesized by alkylating commercially available 1-(4-

ﬂuorophenyl)piperazine with compound 41 using potassium

carbonate as a base (Scheme 7).

Scheme 8. Synthesis of Target Compounds 50−73

a

Reagents and conditions: (a) NH₄OAc, commercially available/

synthesized R-CHO, toluene, reﬂux, 1−24 h.

The benzaldehydes required for making the target

sulfonamide analogues 72 and 73 were synthesized by reacting

72a (obtained by reducing 4-nitrobenzaldehyde using tin

chloride dihydrate in ethyl acetate) with chloroethanesulfonyl

chloride (to obtain compound 72b) and chloropropanesulfonyl

chloride (to obtain compound 72c) at 0 °C in the presence of

triethylamine in dichloromethane. The resulting benzaldehydes

72b and 72c were then reacted with N-methylpiperazine to

yield intermediates 72d and 72e, respectively (Scheme 9).

Structure−Activity Relationship. All synthesized ana-

logues were evaluated for PARP-1 inhibitory activity. The

clinical candidates, veliparib (ABT-888) and olaparib (AZD-

2281), were used as reference standards in the PARP-1 enzyme

inhibitory assay. Our initial lead compound 3 demonstrated

moderate activity with an IC₅₀value of 9.45 μM (Table 1).

From the docking model of 3 into the PARP-1 active site

(Figure 1), it was observed that the carboxamide group

undergoes the aforementioned interactions with Gly863 (NH···

OC-Gly863, 1.92 Å; CO···HN-Gly863, 2.09 Å) and

Ser904 (CO···HO-Ser904, 1.91 Å), and the aromatic ring

was stabilized through π−π stacking interaction with the nearly

coplanar electron rich phenyl ring of Tyr907 (centroid_ligand

−

F

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Scheme 9. Synthesis of Substituted Benzaldehyde Intermediates 72d and 72e Required for Respective Synthesis of Target

a

Compounds 72 and 73

a

Reagents and conditions: (a) SnCl₂·2H₂O, ethyl acetate, reﬂux, 4 h; (b) chloroethanesulfonyl chloride (for compound 72b) or

chloropropanesulfonyl chloride (for compound 72c), DCM, triethylamine, 0 °C to rt, 6 h; (c) N-methylpiperazine, K₂CO₃, CH₃CN, reﬂux, 6 h.

Table 1. Structures and PARP-1 Inhibition Data for the

Synthesized Compounds 3−28

values (6.34 and 8.44 μM, respectively). The 5-methyl, 5-nitro,

and 5-amino derivatives (compounds 13b, 14, and 15,

respectively) proved to be inactive, similar to the earlier series

of compounds (4, 5, and 6). Collectively, this indicates that the

active site region around the 5-position of the DHBF scaﬀold

bears steric restrictions (e.g., compounds 13a vs 13b, Table 1).

Examination of previous X-ray crystallography data revealed

that small substituents, e.g., ﬂuoro, are well tolerated at the 6-

position (5-position equivalent in the DHBF scaﬀold) of the

benzimidazole-4-carboxamide scaﬀold.¹²Similarly, addition of a

ﬂuoro group at the 5-position of 2H-indazole-7-carboxamide⁴⁶

and at the 8-position of 1,3,4,5-tetrahydrobenzonaphthyridin-6-

one⁴⁷(analogous to the 5-position in the DHBF scaﬀold)

showed 2- to 3-fold improvement in activity. To obtain the 5-

ﬂuoro substituted derivative of compound 3 (5-ﬂuoro-DHBF-

7-carboxamide, compound 20), we developed a new synthetic

route (Scheme 3). As expected from the previous ﬁnding, the 5-

ﬂuoro derivative 20 (IC₅₀= 2.12 μM) was found to be ∼5-fold

more potent than our initial lead 3 (Table 1). To examine the

eﬀect of the 5-ﬂuoro substitution on 2-methyl-DHBF-7-

carboxamide (13a), we synthesized rac-13c (IC₅₀= 2.45 μM)

and found it to be 4-fold more potent than 13a. The compound

rac-13c was resolved and enantiomers were evaluated in vitro

for PARP-1 inhibition. The (+)-13c enantiomer had an IC₅₀

value of 3.62 μM, whereas (−)-13c exhibited an IC₅₀value of

1.53 μM. The electron deﬁcit phenyl ring of compound

(−)-13c (contributed to by the electronegative nature of the

ﬂuorine group) was predicted to generate a stronger π−π

stacking interaction with the phenyl ring of Tyr907 compared

to the unsubstituted analogue 13a (Table 1).

To further develop this scaﬀold, we obtained cocrystal

structure data of (−)-13c bound to the catalytic domain of

PARP (Figure 3E). From this structure it appeared the 4-

position of (−)-13c was in proximity to the γ-carboxylate group

of Glu988 (an essential residue involved in PAR synthesis).

Therefore, we chose to synthesize the 4-amino derivative rac-28

to capture electrostatic interactions with the carboxylate group

of Glu988. The 4-amino-2-methyl-DHBF-7-carboxamide rac-28

(IC₅₀= 4.65 μM) had a 2-fold increase in inhibition over rac-

13a, thus underscoring the positive role of the 4-amino group.

Chiral resolution of this racemate yielded individual enan-

tiomers (compounds 28¹and 28²) that were discriminated by

enzymatic activity as evident from their respective IC₅₀values

of 2.43 and 5.33 μM (Table 1).

b

compd

R₁

-H

R₂

-H

R₃

-H

IC₅₀(μM)

3

4

5

6

9.45 0.25

>25

-Br

-H

-NO₂

-NH₂

-H

>25

-H

>25

rac-13a

(−)-13a

(+)-13a

13b

-CH₃

-H

10.44 0.96

6.34 1.08

8.44 1.05

>25

-H

-CH₃

-F

-H

rac-13c

(−)-13c

(+)-13c

14

-H

2.45 0.65

1.53 0.12

3.62 0.76

>25

-F

-H

-F

-H

-NO₂

-NH₂

-F

-H

15

-H

>25

20

-H

2.12 0.37

4.65 0.25

2.43 0.42

5.33 0.16

6.80 0.57

0.006 0.001

0.007 0.001

rac-28

-H

-CH₃

-NH₂

a

28¹

-H

a

28²

-H

c

ILC

c

ABT-888

c

AZD-2281

a

28¹and 28²represent the resolved enantiomers of the racemate 28

b

by chiral HPLC technique. IC₅₀values were determined by at least

two independent experiments done in triplicate. Indazole lead

c

compound (ILC) reported in ref 21 and clinical candidates ABT-

888 and AZD-2281 were included as reference standards for

comparative purposes.

centroid_Tyr907= 4.3 Å, Figure 1). Initially, we synthesized

derivatives of 3 modiﬁed at the synthetically feasible 5-position.

Substitution with bromo, nitro, and amino at the 5-position of

3 yielded compounds 4, 5, and 6, which were concluded to be

inactive with IC₅₀values above 25 μM. Because of the

unfavorable contribution of substituents at the 5-position of 3,

we next focused onto the 2-position of the 2,3-DHBF scaﬀold.

To probe the 2-position of 3, we followed a new synthetic route

as depicted in Scheme 2. The 2-methyl analogue rac-13a was

obtained in racemic form and showed an IC₅₀value of 10.44

μM. Compound rac-13a oﬀered an opportunity to investigate

PARP-1 inhibitory activity of each enantiomer. The rac-13a was

resolved into pure enantiomers by preparative chiral

chromatography (GVK Biosciences Pvt. Ltd., India). Both

(R)-(−) and (S)-(+) isomers of 13a exhibited comparable IC₅₀

Extensive analysis of the substituent eﬀect onto the 2-, 4- and

5-positions of the DHBF-7-carboxamide core revealed that the

2-position was the most promising site for various substitutions

owing to the steric restriction at the 5-position of DHBF-7-

carboxamide. Cocrystal structure data (Figure 3E) revealed an

empty pocket adjacent to the 2-position of the DHBF scaﬀold,

which could likely tolerate large substitutions. Since sub-

stitution at the 2-position of the DHBF scaﬀold presented

G

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 3. Crystal structure of inhibitors in the active site of PARP-1. (A) Structural representation of the PARP-1 activated complex, which highlights

the NAD⁺binding site (active site) where most PARP inhibitors bind. (B−D) F_o− F_celectron density diﬀerence map (blue mesh) of PARP-1

crystals soaked with 1−5 mM (−)-13c, 59, or 65 (PBD code 4OPX, 4OQA, or 4OQB, respectively), contoured to 2.5σ in which density is calculated

in the absence of ligand. (E−G) Compounds bound to the NAD⁺site of PARP-1 make π−π stacking interactions between Tyr907 and Tyr896 and

H-bond interactions with the backbone of Gly863 and Ser904 consistent with the traditional benzamide pharmacophore of most PARP inhibitors.

Compounds 59 and 65 extend out of the nicotinamide pocket and make further interactions in the ADP-ribose pocket of the NAD⁺binding site.

signiﬁcant synthetic challenges, we proposed an alternative

core, DHBF-3-one-7-carboxamide (compound 36, IC₅₀= 16.2

μM, Table 2), which is analogous to the DHBF-7-carboxamide

with a robust synthetic advantage of presenting an electrophilic

2-position (being connected to the carbonyl group on one side

and ethereal oxygen atom on the other side) amenable to

Knoevenagel condensation with benzaldehydes. Knoevenagel

condensation of compound 36 with substituted benzaldehydes

resulted in the desired 2-position substituted compounds

(Table 2). The exocyclic double bond of the benzylidene group

in these target compounds (Table 2) is stereogenic; however, it

has previously been reported that condensation at the 2-

position of DHBF-3-one with diﬀerent aldehydes yields the Z-

isomer.^42,48In agreement with literature NMR reports, the

oleﬁnic β-proton in these compounds was found to be around

7.03 ppm, which indicates the formation of the Z-isomer

(calculated chemical shift for the same proton of the E-isomer

was found to be ∼6.19 ppm).

In order to study the steric compliance at the 2-position of

the DHBF-3-one-7-carboxamide scaﬀold, initial compounds

probed the active site pocket by substituting hydrophobic

groups such as phenyl (compound 50), 3-phenoxyphenyl

(compound 51), and p-chlorophenyl (compound 52). PARP-1

inhibitory data of this series revealed that extensive hydro-

phobic substitution at the 2-position marginally improved

potency (IC₅₀values of 12.02, 4.65, and 6.09 μM, respectively;

Table 2). However, the added hydrophobicity contributed by

these substitutions inevitably increased the magnitude of log P,

diminishing the druglike properties of the molecules. Structural

data revealed that the benzylidene ring of the DHBF-3-one-7-

carboxamide scaﬀold was located in proximity to polar side

groups, namely, Glu763, Asp766, and Tyr889 (Figure 3F). This

indicated that the polar substituents on the benzylidene ring

could in turn favor PARP-1 inhibition. With this hypothesis, we

aimed at synthesizing analogues bearing polar groups on the

benzylidene ring. Compound 53 with a vanillyl (4′-hydroxy-3′-

H

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 2. Structures and PARP-1 Inhibition Data for Target Compounds 36 and 50−73

a

b

IC₅₀values were determined by at least two independent experiments done in triplicate. Indazole lead compound (ILC) reported in ref 21 and

clinical candidates ABT-888 and AZD-2281 were included as reference standards for comparative purposes.

I

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 4. Predicted binding mode of compounds 66 (A) and 72 (B) within the active site of PARP-1. The color scheme for the active site amino

acid residues and the inhibitor is the same as in Figure 1. The centroids are generated as green stars. The yellow dotted lines indicate the distance

between the two atoms/groups/centroids (in angstroms). The image was generated in PyMOL, version 1.6.0.

methoxyphenyl) group was found to exhibit an IC₅₀value of

3.62 μM (Table 2). The moderate inhibition by compound 54

(IC₅₀= 3.30 μM) also suggested that substitutions made onto

the 4′-position of the benzylidene ring were well tolerated.

Structural data suggested that a nearby vacant pocket was likely

accessible from the 4′-position of the benzylidene ring, without

any potential for a steric clash (Figure 3F). In order to further

delineate the contribution of the hydroxyl group at each

position of the benzylidene moiety, we synthesized analogues

bearing hydroxyl groups at 2′-position (55), 3′-position (56),

and 4′-position (57). Among them, 56 and 57 were found to

have improved potency with IC₅₀values of 2.14 and 0.813 μM,

respectively (Table 2). Poor inhibition of PARP-1 by

compound 55 also corroborated the importance of 4′-position

substitution onto the benzylidene ring. This further suggested

that the hydroxyl group at the 4′-position of the benzylidene

ring is important to increase potency. We also synthesized

dihydroxy substituted analogues, bearing hydroxyl groups at the

3′-position and either the 4′- (compound 58) or 5′-position

(compound 60). Among the dihydroxy analogues (compounds

58, 59, 60, and 61), compound 58 bearing 3′,4′-dihydroxy

substitution and compound 59 with 2′,4′-dihydroxy groups

demonstrated IC₅₀values of 0.531 and 0.753 μM, respectively.

Structural data of 59-PARP-1 revealed that the 4′-hydroxyl

group substituted onto the benzylidene ring was directed

toward the side chain of Asp766 (Figure 3F). In addition,

methylation of the 4′-hydroxyl group of compound 57 led to

compound 54 which showed 4-fold reduced inhibitory activity.

Collectively, these data clearly underline that polar hydroxyl

groups at the 4′-position are beneﬁcial for PARP-1 inhibition.

Interestingly, poor inhibition by compounds 60 and 61

indicated that neither the 3′-position nor 5′-position was a

signiﬁcant substituent position for PARP-1 inhibition.

According to the structural data, the 4′-hydroxyl group of

compound 59 was found to point toward the vacant

hydrophilic pocket formed by the side chains of Asp766,

Asn767, Asp770, Ser864, Asn868, and Arg878 (Figure 3F).

With an intention to extend substitutions into this pocket, we

decided to attach a basic nitrogen heterocycle at this position,

wherein the hydroxyl group would act as a good handle for

synthetic feasibility. Toward this end, we synthesized an

analogue bearing piperidine (compound 64) linked to the 4′-

position of 57 by an ethyl linker. Our next strategy was aimed

at incorporation of an electronegative oxygen atom at the C₄-

position of the piperidine ring, which involved replacing the

piperidine ring (64) with a morpholine (65). However, this

approach did not lead to the desired result, since compound 65

(IC₅₀= 2.07 μM) was found to be 4-fold less potent compared

to 64 (IC₅₀= 0.544 μM). However, a potential interaction

between the morpholine ring oxygen atom and the side chain

of Arg878 was suggested by studying the X-ray cocrystal

structure of compound 65 bound to the catalytic domain in the

PARP-1 (Figure 3G). In the case of 66 (IC₅₀= 0.114 μM), the

electron rich N-methylpiperazine ring was observed to be in

proximity to the side chain of Arg878 based on molecular

docking (Figure 4A). Further, the terminal nitrogen atom of

the piperazine moiety was predicted to undergo an electrostatic

interaction with the side chain carboxyl group of Asp770

(NH⁺···⁻OOC-Asp770, 1.89 Å). These data hinted toward a

favorable role of basic groups at the 4′-substitued saturated

heterocycles (Figure 4A). Additionally, we wanted to test if the

above substituents at the 3′-position of the benzylidene ring

could also bind favorably to this hydrophilic pocket. This was

experimentally veriﬁed by synthesizing analogues bearing a

morpholine ring (compound 62) or piperidine ring (compound

63) linked by an ethyl linker to the 3′-position of compound

56. However, substitution at the 3′-position was found to be

J

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

detrimental for PARP-1 inhibition (compounds 62 and 63 vs

64 and 65, respectively).

Selective Killing of BRCA2-Deﬁcient Cells. Since

catalytic PARP inhibition is known to induce synthetic lethality

in BRCA1- or BRCA2-deﬁcient cells, we tested one of the

potent inhibitors (compound 66) for sensitivity against wild-

type, PARP-1-deﬁcient, and BRCA2-deﬁcient DT40 cells. This

compound was compared to veliparib, a leading clinical PARP

inhibitor with high potency. Consistent with previous reports,⁵⁰

veliparib was not cytotoxic in wild-type and PARP-1-deﬁcient

cells up to 10 μM, while it was selectively cytotoxic in BRCA2-

deﬁcient cells with an IC₉₀of 5.0 μM (Figure 5A). Compound

Up to this point, we tested the eﬃcacy of inhibitors bearing

ethyl linkers at diﬀerent positions of benzylidene (compounds

62−66). In an attempt to evaluate the eﬀect of diﬀerent linkers,

we synthesized analogues 67, 68, and 70. With an intention to

extend the substitution deeper into the hydrophilic pocket, we

extended the linker by one carbon atom (compound 67) to

bring the oxygen of the morpholine ring in compound 65

closer to the side chain of Arg878. Compound 67 was found to

be nearly 3 times more potent (IC₅₀= 0.718 μM) compared to

the corresponding ethyl analogue 65 (IC₅₀= 2.07 μM), likely

owing to the stronger interaction with the guanidine group of

Arg878 (Table 2). In order to increase the ligand−PARP-1

interactions, we aimed to introduce polar groups onto the

linker positions, with the potential for interaction with the polar

amino acid residues present in the hydrophilic pocket.

Introduction of a hydroxyl group onto the second carbon

atom of the propyl linker (compound 70) led to a signiﬁcant

improvement in the potency with an IC₅₀of 0.176 μM. Another

advantage of polar substitutions is that they also improved the

solubility properties of this compound. The replacement of the

ethyl group of compound 65 with the acetyl group (compound

68) led to a 9-fold improvement in potency (IC₅₀= 0.223 μM).

This observation revealed that polar groups incorporated onto

the linker signiﬁcantly improved potency. In order to

substantiate our hypothesis that substituted nitrogen containing

heterocycles play a signiﬁcant role in PARP-1 inhibition, we

synthesized and tested a truncated analogue with an

oxiranylmethoxy moiety (compound 69, IC₅₀= 1.21 μM). It

did not completely lose potency owing to the contribution by

polar epoxide ring (Table 2).

Several fragments have been linked to nicotinamide

mimicking pharmacophores and are widely reported in the

literature as signiﬁcantly improving PARP-1 inhibition.⁴⁹In an

attempt to check the usefulness of one such fragment known to

give signiﬁcant inhibition, we tested the impact of linking 1-(4-

ﬂuorophenyl)piperazine via ethyl bridge at the 4′-position of

the benzylidene moiety. This analogue (compound 71) gave a

signiﬁcant inhibition with an IC₅₀value of 0.445 μM,

demonstrating that our studies correlate with literature reports.

In an attempt to improve the enzyme inhibitory activity of

66, we have successfully replaced the ethoxy linker in 66 with

ethanesulfonamide (compound 72, IC₅₀= 0.079 μM) and

propanesulfonamide (compound 73, IC₅₀= 0.113 μM) linkers.

It was anticipated that the sulfonamide groups will provide a

sharp angle, mimic the phosphate group of NAD⁺(substrate of

PARP), and place the piperazine moiety deeper into the

adenine-ribose binding pocket of PARP-1 active site. Two of

the most potent inhibitors 66 and 72 not only are water-soluble

but also present a group amenable for the preparation of

pharmaceutical salts. Predicted binding model of 72 within the

active site of PARP-1 is shown in Figure 4B. In contrast to

compound 66, compound 72 showed three additional hydro-

gen bonds between the sulfonamide group and the side chains

of Asp766, Ser864, and Asn868. Similar to compound 66,

sulfonamide analogue 72 was able to reach the adenine-ribose

binding pocket, as evident from the docking studies (Figure

4B), wherein the N-methylpiperazine moiety of 72 is in the

vicinity of adenine-ribose binding residues Asp770 and Arg878.

Taken together, successful replacement of ethoxy linker with

ethanesulfonamide further showcases the potential of develop-

ing highly active DHBF-3-one scaﬀold inhibitors.

Figure 5. Cell viability of DT40 cells after continuous exposure to (A)

veliparib or (B) compound 66 for 72 h. Cellular ATP concentration

was used to measure cellular viability. The viability of untreated cells

was set as 100%. Error bars represent standard deviation (n = 3).

Circle: wild-type cell. Triangle: PARP1-deﬁcient cells. Square: BRCA2-

deﬁcient cells. Invisible error bars are encompassed within the symbol

sizes.

66 also selectively killed BRCA2-deﬁcient cells with an IC₉₀of

5.2 μM while being minimally cytotoxic in wild-type and PARP-

1-deﬁcient cells (Figure 5B). The weak cytotoxicity of

compound 66 in wild-type and PARP-1-deﬁcient cells

compared to veliparib suggests oﬀ-target eﬀects beside PARP-

1. PARP inhibitors can have promiscuous inhibitory activity

extending to PARP-1−4 and tankyrases²⁵and can have PARP-

1-independent cytotoxicity.^51,52Hence, we conclude that

compound 66 has similar cellular potency as veliparib in

BRCA2-deﬁcient cells.

Structural Studies. To further validate our SAR approach

and docking studies, we obtained crystal structure data of both

scaﬀolds bound to the recent structure of PARP-1 in complex

with DNA (Figure 3A).²³The diﬀraction limit of these crystals

restricts the level of detail obtained from the PARP-1/

compound complexes as a result of large multidomain protein;

however, the data allowed us to conﬁdently model the major

features of their binding poses within the catalytic domain.

Furthermore, we obtained data with three compounds of

various sizes but based on the same scaﬀold, which helped

conﬁrm the placement of the inhibitors (Figure 3A). Consistent

K

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

in open capillary tube on a Thomas-Hoover capillary melting point

apparatus and reported as uncorrected values. H NMR spectra were

with the docking studies, the benzamide portion of the DHBF

scaﬀold stacks between two tyrosine residues and makes

hydrogen bonding interactions with Gly863 and Ser904 (Figure

3E). Compound 59 appears to reach outside of the traditional

nicotinamide pocket with its benzylidene modiﬁcation to

further interact with Tyr889 (Figure 3F). It is interesting to

note that modiﬁcation of the 5′-position of the benzylidene ring

would cause a steric clash with Tyr889, which is consistent with

the complete loss of potency observed with compounds

containing a 5′-position modiﬁcation (60 and 61). Scaﬀolds

with larger modiﬁcations reach deeper into the adenine-ribose

binding region of the active site, as seen with compound 65

(Figure 3G). The observed interaction of 65 with Arg878 is

only speculative because of the poor density in this region.

However, the structures clearly explain why the 4′-position

modiﬁcations are superior in potency compared to the 3′-

position modiﬁcations, since the 3′-position would lead to

signiﬁcant steric clash in the NAD⁺binding site.

1

recorded on a Bruker AM-400 spectrometer. Chemical shifts are

reported as δ (ppm) relative to the tetramethylsilane as an internal

standard. Coupling constants (J) are expressed in hertz (Hz). Proton

peak multiplicity is reported as s (singlet), d (doublet), t (triplet), q

(quintet), sext (sextet), dd (doublet of doublets), ddt (doublet of

doublet of triplet), td (triplet of doublets), m (multiplet), and bs

(broad singlet). All compounds were routinely checked by thin layer

1

chromatography (TLC) and H NMR. TLC was performed on silica

gel ﬂuorescent coated plates obtained from Analtech, Inc., Newark,

DE. Flash chromatography puriﬁcation was performed using silica gel

(0.060−0.200 mm) obtained from Dynamic Adsorbents, Norcross,

GA. Optical rotation of the chiral compounds dissolved in methanol

was measured using PerkinElmer 241 polarimeter. Elemental analysis

(C, H, N) was performed by Atlantic Microlab, Inc., (Norcross, GA)

for all compounds screened in the biological assays, and the results are

within 0.40% of theoretical values. The purity of the synthesized

target compounds was determined to be ≥95% by elemental analysis.

Chiral HPLC Analysis. The chiral column used to resolve the

enantiomers (CHIRALPACK 1A) had amylose tris(3,5-dimethylphe-

nylcarbamate) as the chiral auxiliary. Optimum separation was

achieved for both compounds when the ﬂow rate of the mobile

phase was set to 1.0 mL/min. The samples (compounds 13a and 13c)

were dissolved in ethanol and eluted using an isocratic mobile phase

(n-hexane/ethanol 85:15) at an ambient temperature of 25 °C.

Compound 28 was resolved by using an isocratic mobile phase (n-

hexane/isopropyl alcohol 70:30). Retention times for the individual

enantiomers were reported as t_R(in minutes), whereas the ee

(enantiomeric excess) values were calculated based on the UV

absorption (254 nm) areas for the two enantiomers, respectively. The

purity of the resolved enantiomers was determined by measuring the

area under the curve (AUC) of the LC−MS spectra of the individual

enantiomers and represented as percent AUC of the molecular ion

peak relative to the other peaks in the spectra.

Synthesis of 2,3-Dihydrobenzofuran-7-carboxylic Acid (2).

To a solution of n-butyllithium (2.5 M in hexane, 2 mL, 5.0 mmol) in

5 mL of hexane at room temperature was added (0.58 g, 5.0 mmol)

N,N,N′,N′-tetramethylethylenediamine (TEMED), followed by a

solution of 2,3-dihydrobenzofuran (1, 0.3 g, 2.5 mmol) in hexane (5

mL). The mixture was stirred under nitrogen at room temperature for

4 h and then poured into dry ice. After being stirred at room

temperature overnight, the mixture was diluted with 15 mL of water

and the layers were separated. The aqueous layer was acidiﬁed with

hydrochloric acid to pH 1, cooled, and the precipitates were collected

on a ﬁlter. The crude product was reprecipitated using ethyl acetate/

hexane to give compound 2 (0.21 g, yield 52%) as white solid: mp

163−165 °C (lit. 167−169 °C);³³¹H NMR (400 MHz, DMSO-d₆,

TMS) δ 3.18 (t, J = 8.8 Hz, 2H), 4.59 (t, J = 8.8 Hz, 2H), 6.86 (t, J =

8.8 Hz, 1H), 7.41 (d, J = 7.4 Hz, 1H), 7.56 (d, J = 7.9 Hz, 1H), 12.62

(s, 1H).

Synthesis of 2,3-Dihydrobenzofuran-7-carboxamide (3). To

a solution of compound 2 (0.3 g, 1.83 mmol) in 7 mL of anhydrous

tetrahydrofuran (THF) were added isobutyl chloroformate (0.3 g, 2.2

mmol) and N-methylmorpholine (NMM) (0.22 g, 2.2 mmol) under

nitrogen atmosphere at −20 °C, and the mixture was stirred for 4 h.

Then to this mixture was added 5 mL of aqueous ammonia (30%), and

the mixture was stirred at room temperature for 1 h. The organic layer

was separated, and the aqueous layer was extracted with (2 × 10 mL)

of THF. The combined organic layers were dried over sodium sulfate

and concentrated under vacuum. The resulting residue was puriﬁed by

column chromatography using ethyl acetate/methanol (95:5) as an

eluent, and the product was obtained as white crystals (0.23 g, yield

75%): mp 185−187 °C; ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 3.23

(t, J = 8.6 Hz, 2H), 4.68 (t, J = 8.8 Hz, 2H), 6.92 (t, J = 7.5 Hz, 1H),

7.29 (s, 1H), 7.39 (d, J = 7.2 Hz, 1H), 7.58 (s, 1H), 7.61 (d, J = 7.9

Hz, 1H). Anal. Calcd for C₉H₉NO₂·¹/₆CH₃OH: C, 65.34; H, 5.74; N,

8.31. Found: C, 65.52; H, 5.51; N, 8.41.

CONCLUSIONS

■

A novel series of DHBF-7-carboxamide and DHBF-3-one-7-

carboxamide derivatives were designed, synthesized, and

evaluated for PARP-1 inhibition. Substituents larger than

ﬂuorine at the 5-position of the DHBF scaﬀold were found

to be detrimental for PARP-1 inhibition. The 2-position methyl

substitution is well tolerated in the DHBF-7-carboxamide

scaﬀold, yielding enantiomers that bind diﬀerently in the active

site. The molecules were resolved and tested for PARP-1

inhibitory activity concluding levorotatory analogues to be the

eutomers ((−)-13a and (−)-13c). Synthesizing the DHBF-3-

one-7-carboxamide derivatives demonstrated an added advant-

age of an ease of substitution at the electrophilic 2-position. An

initial set of lead compounds 57, 58, and 59 revealed that

substituting the hydrophilic groups onto the 4′-position of the

benzylidene ring was important for potency. Alkylating the 4′-

hydroxyl group of compound 57 with the basic heterocycles

linked by a two-carbon spacer generated compounds 64 and 66

with signiﬁcantly improved PARP-1 inhibitory activity. Crystal

structure determination conﬁrmed that these compounds target

the nicotinamide binding pocket of the active site and reach out

into the adenine-ribose binding region, resulting in increased

potency. Extending the side chain on the 4′-position of the

benzylidene ring as well as modiﬁcation of the linker proved to

have a signiﬁcant eﬀect on PARP-1 inhibition, as evident from

the inhibition by compounds 67−71. Also, signiﬁcant inhibition

by 71 highlighted that our studies corroborated with literature

reports.⁴⁹The replacement of ethoxy linker in 66 with

aminosulfonylethyl and aminosulfonylpropyl linkers, respec-

tively, resulted in improved inhibitors 72 and 73. Compound

66 was selectively active in BRCA2-deﬁcient cells and

comparable to veliparib. Overall, compound 66 was identiﬁed

as one of the potent compounds in the series with an IC₅₀of

0.114 μM in an enzyme assay and an IC₉₀of 5.2 μM against

BRCA2-deﬁcient DT40 cells. Compounds 66 and 72 will serve

as promising leads for future SAR studies.

EXPERIMENTAL SECTION

■

Chemistry. Synthesis: General. All chemicals and solvents were

purchased from Sigma−Aldrich (St. Louis, MO), AK Scientiﬁc (Union

City, CA), Oakwood Laboratories (West Columbia, SC), and Alfa

Aesar (Ward Mill, MA) and were used as received. The clinical

candidates ABT-888 and AZD-2281 were purchased from the

Selleckchem library (Houston, TX). Melting points were determined

Synthesis of 5-Bromo-2,3-dihydrobenzofuran-7-carboxa-

mide (4). To a mixture of DHBF-7-carboxamide 3 (0.2 g, 1.23

L

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

mmol) and sodium acetate (0.18 g, 2.21 mmol) in 3 mL of acetic acid

was added bromine solution (0.25 g, 1.60 mmol) in 2 mL of acetic

acid. The reaction mixture was heated at 80 °C for 3 h and then

poured onto ice and ﬁltered. Reprecipitation from ethyl acetate

aﬀorded the desired compound 4 as a white solid (0.24 g, yield 80%):

mp 195−197 °C; ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 3.25 (t, J =

8.8 Hz, 2H), 4.71 (t, J = 8.8 Hz, 2H), 7.26 (s, 1H), 7.57 (d, J = 1.9 Hz,

1H), 7.66 (d, J = 2.3 Hz, 1H), 7.73 (s, 1H). Anal. Calcd for

C₉H₈BrNO₂·¹/₃CH₃OH: C, 44.32; H, 3.69; N, 5.54. Found: C, 44.52;

H, 3.43; N, 5.24.

Synthesis of 5-Nitro-2,3-dihydrobenzofuran-7-carboxamide

(5). To an ice-cooled solution of DHBF-7-carboxamide 3 (0.2 g, 1.23

mmol) in 5 mL of triﬂuoroacetic acid was added 0.4 mL of nitric acid

dropwise. After 30 min, the ice bath was removed and the mixture was

stirred at room temperature for 3 h, and then the mixture was poured

into ice−water. The resulting precipitates were collected on a ﬁlter to

give a crude product which was reprecipitated from ethyl acetate to

yield compound 5 (0.17 g, yield 65%) as a pale yellow solid: mp 239−

241 °C; ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 3.35 (t, J = 8.4 Hz,

2H), 4.48 (t, J = 8.6 Hz, 2H), 7.42 (s, 1H), 7.92 (s, 1H), 8.26 (s, 1H),

8.47 (s, 1H). Anal. Calcd for C₉H₈N₂O₄·¹/₆H₂O: C, 51.18; H, 3.79; N,

13.27. Found: C, 51.37; H, 3.76; N, 13.10.

Synthesis of 5-Amino-2,3-dihydrobenzofuran-7-carboxa-

mide (6). To a solution of compound 5 (0.2 g, 0.96 mmol) in 7

mL of ethyl acetate was added tin chloride dihydrate (1.3 g, 5.77

mmol), and the mixture was reﬂuxed for 4 h. After completion of the

reaction, mixture was diluted (add 15 mL of ethyl acetate), cooled, and

poured into the saturated sodium bicarbonate solution (20 mL). The

organic layer was separated and the aqueous layer was washed with

ethyl acetate (2 × 20 mL). The combined organic layers were dried

over sodium sulfate and evaporated to give crude product, which was

puriﬁed by column chromatography (ethyl acetate/methanol 95:5) to

yield compound 6 as a pale yellow solid (0.09 g, yield 55%): mp 141−

143 °C; ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 3.11 (t, J = 8.2 Hz,

2H), 4.55 (t, J = 8.8 Hz, 2H), 4.76 (bs, 2H), 6.67 (d, J = 1.4 Hz, 1H),

6.87 (d, J = 2.2 Hz, 1H), 7.17 (s, 1H), 7.42 (s, 1H). Anal. Calcd for

C₉H₁₀N₂O₂: C, 60.66; H, 5.66; N, 15.72. Found: C, 60.76; H, 5.76; N,

15.57.

17.4, 10.7, 4.7 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 7.13 (d, J = 8.3 Hz,

1H), 7.51 (td, J = 7.8, 1.7 Hz, 1H), 7.63 (dd, J = 7.7, 1.6 Hz, 1H).

Synthesis of Methyl 2-(Allyloxy)-5-methylbenzoate (9b). 9b

was synthesized according to compound 9a using compound 8b (3.25

g, 19.6 mmol) as the starting material, as pale yellow oil (3.51 g, yield

1

87%): H NMR (400 MHz, CDCl₃, TMS) δ 2.30 (s, 3H), 3.89 (s,

3H), 4.58 (m, 2H), 5.29 (dd, J = 10.6, 1.2 Hz, 1H), 5.50 (dd, J = 17.2,

1.4 Hz, 1H), 6.06 (ddt, J = 17.2, 10.6, 4.8 Hz, 1H), 6.85 (d, 1H), 7.23

(dd, J = 8.4, 2.2 Hz, 1H), 7.62 (s, 1H).

Synthesis of Methyl 2-(Allyloxy)-5-ﬂuorobenzoate (9c). 9c

was synthesized according to compound 9a using compound 8c (3.06

g, 18.0 mmol) as the starting material, as colorless-light yellow oil

1

(3.21 g, yield 85%): H NMR (400 MHz, CDCl₃, TMS) δ 3.89 (s,

3H), 4.58 (m, 2H), 5.29 (dd, J = 11.0, 1.8 Hz, 1H), 5.50 (dd, J = 17.4,

1.8 Hz, 1H), 6.05 (ddt, J = 17.4, 10.6, 4.6 Hz, 1H), 6.93 (dd, J = 7.8,

1.8 Hz, 1H), 7.14 (dd, J = 7.8, 1.8 Hz, 1H), 7.51 (dd, J = 7.8, 1.8 Hz,

1H).

Synthesis of Methyl 3-Allyl-2-hydroxybenzoate (10a).

Compound 9a (3.00 g, 15.62 mmol) was neatly heated at 160 °C

for 2 h, which resulted in the crude brownish oil that was puriﬁed by

ﬂash chromatography (n-hexane/ethyl acetate 95:5) to get compound

10a as pale yellow oil (2.61 g, yield 87%): ¹H NMR (400 MHz,

DMSO-d₆, TMS) δ 3.36 (d, J = 6.5 Hz, 2H), 3.90 (s, 3H), 5.04 (m,

2H), 6.02 (ddt, J = 16.8, 10.2, 6.5 Hz, 1H), 6.90 (t, J = 7.8 Hz, 1H),

7.39 (d, J = 7.3 Hz, 1H), 7.67 (dd, J = 7.8, 1.7 Hz, 1H), 10.92 (s, 1H).

Synthesis of Methyl 3-Allyl-2-hydroxy-5-methylbenzoate

(10b). Compound 9b (2.09 g, 10.19 mmol) was neatly heated at

190 °C for 4 h to obtain crude brownish oil that was further puriﬁed

by ﬂash chromatography to get compound 10b as pale yellow oil (1.73

g, yield 83%): ¹H NMR (400 MHz, CDCl₃, TMS) δ 2.25 (s, 3H), 3.39

(d, J = 6.8 Hz, 2H), 3.93 (s, 3H), 5.06 (m, 1H), 5.09 (m, 1H), 6.00

(ddt, J = 16.0, 10.0, 6.6 Hz, 1H), 7.14 (s, 1H), 7.51 (s, 1H), 10.85 (s,

1H).

Synthesis of Methyl 3-Allyl-5-ﬂuoro-2-hydroxybenzoate

(10c). Compound 9c (2.6 g, 12.38 mmol) was neatly heated at 190

°C for 4 h to obtain crude brownish oil that was further puriﬁed by

ﬂash chromatography to get compound 10c as pale yellow oil (2.13 g,

yield 82%): ¹H NMR (400 MHz, CDCl₃, TMS) δ 3.41 (d, J = 6.6 Hz,

2H), 3.94 (s, 3H), 5.10 (m, 1H), 5.13 (m, 1H), 6.05 (ddt, J = 17.0,

10.4, 6.6 Hz, 1H), 7.09 (dd, J = 8.8, 3.3 Hz, 1H), 7.38 (dd, J = 8.6, 3.3

Hz, 1H), 10.83 (s, 1H).

Synthesis of Methyl 5-Methylsalicylate (8b). To a solution of

5-methylsalicylic acid (3.0 g, 19.7 mmol) in methanol was added

dropwise thionyl chloride (2.81 g, 23.70 mmol), and then the reaction

mixture was reﬂuxed for 12 h. The reaction mixture was then cooled to

room temperature, and the solvent was evaporated under vacuum. The

resulting oil was diluted with water (20 mL) followed by extracting it

with ethyl acetate (3 × 20 mL). The organic layers were combined,

dried over sodium sulfate, and evaporated under vacuum to yield a

yellow liquid, which was further puriﬁed by column chromatography

(n-hexane/ethyl acetate 95:5) to give the desired product 8b as a pale

Synthesis of Methyl 2-Methyl-2,3-dihydrobenzofuran-7-

carboxylate (11a). To a solution of compound 10a (2.0 g, 10.41

mmol) in dichloromethane (20 mL), cooled to 0 °C, zirconium(IV)

chloride (2.91 g, 12.48 mmol) was added in portions, and the reaction

mixture was stirred at room temperature for 10 h. After completion,

the reaction was quenched by addition of cold water (10 mL) and the

organic layer was separated. The aqueous layer was extracted with (3 ×

10 mL) dichloromethane. The combined organic phase was washed

with water, dried over sodium sulfate, and concentrated to give the

crude product, which was puriﬁed by ﬂash chromatography (n-hexane/

ethyl acetate 80:20) to get the desired product 11a as pale yellow oil

(1.70 g, yield 85%): ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 1.40 (d,

J = 6.3 Hz, 3H), 2.78 (dd, J = 15.7, 7.6 Hz, 1H), 3.32 (dd, J = 15.7, 9.0

Hz, 1H), 3.78 (s, 3H), 5.0 (sext, J = 7.0 Hz, 1H), 6.87 (t, J = 7.3 Hz,

1H), 7.40 (dd, J = 7.3, 1.3 Hz, 1H), 7.57 (d, J = 7.8 Hz, 1H).

Synthesis of Methyl 2,5-Dimethyl-2,3-dihydrobenzofuran-7-

carboxylate (11b). 11b was synthesized according to the above

procedure for 11a using compound 10b (1.5 g, 7.28 mmol) as starting

1

yellow oil (2.95 g, yield 90%): H NMR (400 MHz, CDCl₃, TMS) δ

2.26 (s, 3H), 3.92 (s, 3H), 6.87 (d, J = 8.5 Hz, 1H), 7.24 (d, J = 8.7

Hz, 1H), 7.61 (s, 1H), 10.57 (s, 1H).

Synthesis of Methyl 5-Fluorosalicylate (8c). 8c was obtained

by the same procedure as mentioned for compound 8b using 5-

ﬂuorosalicylic acid (2.96 g, 19.0 mmol) as starting material, as pale

yellow crystalline solid (2.74 g, yield 85%): ¹H NMR (400 MHz,

CDCl₃, TMS) δ 3.96 (s, 3H), 6.94 (dd, J = 9.0, 4.6 Hz, 1H), 7.18 (td, J

= 8.5, 3.3 Hz, 1H), 7.50 (dd, J = 8.8, 3.3 Hz, 1H), 10.52 (s, 1H).

Synthesis of Methyl 2-(Allyloxy)benzoate (9a). To a solution

of methyl salicylate (3.0 g, 19.7 mmol) in 10 mL of DMF were added

allyl bromide (2.63 g, 21.7 mmol), potassium carbonate (2.99 g, 21.7

mmol), and sodium iodide (3.25 g, 21.7 mmol). The reaction mixture

was stirred for 12 h at room temperature and then poured into water

(50 mL) and extracted with ethyl acetate (3 × 50 mL). The organic

layers were combined, dried over sodium sulfate, and concentrated

under vacuum to yield yellowish brown oil which was further puriﬁed

by column chromatography (n-hexane/ethyl acetate 90:10) to get the

1

material, as colorless oil (1.24 g, yield 83%): H NMR (400 MHz,

CDCl₃, TMS) δ 1.50 (d, J = 6.2 Hz, 3H), 2.27 (s, 3H), 2.77 (dd, J =

15.6, 7.4 Hz, 1H), 3.28 (dd, J = 15.6, 8.8 Hz, 1H), 3.88 (s, 3H), 5.04

(sext, J = 7.0 Hz, 1H), 7.12 (s, 1H), 7.50 (s, 1H).

Synthesis of Methyl 5-Fluoro-2-methyl-2,3-dihydrobenzo-

furan-7-carboxylate (11c). 11c was synthesized according to 11a

using compound 10c (1.2 g, 5.71 mmol) as starting material and

1

obtained as colorless oil (1.0 g, yield 83%): H NMR (400 MHz,

1

desired product 9a as pale yellow oil (3.52 g, yield 92%): H NMR

CDCl₃, TMS) δ 1.52 (d, J = 6.2 Hz, 3H), 2.82 (dd, J = 16.0, 7.4 Hz,

1H), 3.32 (dd, J = 16.0, 9.0 Hz, 1H), 3.89 (s, 3H), 5.09 (sext, J = 7.0

Hz, 1H), 7.04 (d, J = 7.4 Hz, 1H), 7.39 (dd, J = 9.6, 2.8 Hz, 1H).

(400 MHz, DMSO-d₆, TMS) δ 3.79 (s, 3H), 4.63 (m, 2H), 5.25 (dd, J

= 10.6, 1.7 Hz, 1H), 5.46 (dd, J = 17.2, 1.7 Hz, 1H), 6.02 (ddt, J =

M

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Synthesis of 2-Methyl-2,3-dihydrobenzofuran-7-carboxylic

Acid (12a). To a solution of compound 11a (1.50 g, 7.81 mmol) in

methanol was added sodium hydroxide (0.94 g, 23.43 mmol), and the

mixture was reﬂuxed for 2 h. Upon completion, the mixture was

cooled and the solvent was evaporated under reduced pressure. The

resulting white solid was dissolved in a small amount of water and

cooled to 0 °C. To this, concentrated hydrochloric acid was added

dropwise until pH 1 to get white precipitates, which were ﬁltered and

dried under vacuum to get the desired compound rac-12a as white

solid (1.17 g, yield 85%): mp 121−123 °C (lit. 125−127 °C);⁵³¹H

NMR (400 MHz, DMSO-d₆, TMS) δ 1.40 (d, J = 6.2 Hz, 3H), 2.78

(dd, J = 16.0, 7.8 Hz, 1H), 3.31 (dd, J = 15.9, 8.8 Hz, 1H), 4.97 (sext, J

= 7.0 Hz, 1H), 6.85 (t, J = 7.6 Hz, 1H), 7.37 (dd, J = 7.1, 1.1 Hz, 1H),

7.55 (d, J = 7.7 Hz, 1H), 12.56 (bs, 1H).

material to yield a white solid (0.74 g, yield 75%): mp 152−153 °C;

¹H NMR (400 MHz, DMSO-d₆, TMS) δ 1.42 (d, J = 6.3 Hz, 3H),

2.24 (s, 3H), 2.95 (dd, J = 15.9, 7.4 Hz, 1H), 3.32 (dd, J = 15.7, 8.8

Hz, 1H), 5.04 (sext, J = 7.0 Hz, 1H), 7.17 (s, 1H), 7.21 (s, 1H), 7.42

(s, 1H), 7.54 (s, 1H). Anal. Calcd for C₁₁H₁₃NO₂: C, 69.09; H, 6.85;

N, 7.32. Found: C, 68.81; H, 6.97; N, 7.20.

Synthesis of 5-Fluoro-2-methyl-2,3-dihydrobenzofuran-7-

carboxamide (13c). 13c was synthesized by mixed-anhydride

method according to compound 3 using rac-12c (1.30 g, 6.63

mmol) as starting material to yield a white solid (0.90 g, yield 70%):

1

mp 137−139 °C; H NMR (400 MHz, DMSO-d₆, TMS) δ 1.44 (d, J

= 6.1 Hz, 3H), 2.84 (dd, J = 16.2, 8.1 Hz, 1H), 3.37 (dd, J = 16.2, 8.8

Hz, 1H), 5.10 (sext, J = 7.0 Hz, 1H), 7.29 (m, 3H), 7.76 (s, 1H). Anal.

Calcd for C₁₀H₁₀FNO₂·¹/₆CH₃OH: C, 60.89; H, 5.32; N, 6.98. Found:

C, 60.62; H, 5.32; N, 6.98.

The 2-methyl enantiomers obtained were resolved to elucidate the

absolute stereochemistry at the 2-position of 12a, wherein the acid

derivative was heated with equimolar proportion of either of the

(−)-brucine dihydrate in acetone or (S)-(−)-α-methylbenzylamine or

(R)-(+)-α-methylbenzylamine in methanol for crystallization. The

superior quality crystals obtained were used to elucidate the absolute

stereochemistry at Department of Chemistry, Louisiana State

University, Baton Rouge, LA, by X-ray crystallographic analysis.

Furthermore, the absolute stereochemistry of the compound was

correlated with the optical rotation performed by quenching the

crystals (having stereochemistry determined) with diluted hydro-

chloric acid. The quenched fractions, upon extraction in ethyl acetate,

were subjected to optical rotation using a polarimeter at a

concentration of 0.1 g/mL in methanol. The speciﬁc rotation for

each enantiomer was calculated from the observed rotation, which was

The amide enantiomers were resolved by means of chiral

chromatography technique at GVK Biosciences Pvt. Ltd., Hyderabad,

India. Optical rotations of the individual enantiomers measured were

found to be [α]²⁵+14.2°, −12.3°. t_R= 7.02 min (dextro) and 6.56

D

min (levo); LC−MS purity = 99.48% (dextro), 99.69% (levo); ee =

90.38% (dextro), 93.25% (levo).

Synthesis of 2-Methyl-5-nitro-2,3-dihydrobenzofuran-7-car-

boxamide (14). 14 was synthesized according to compound 5 using

rac-13a (1.20 g, 6.78 mmol) as starting material to give a pale yellow

1

solid (0.98 g, yield 65%): mp 204−205 °C; H NMR (400 MHz,

DMSO-d₆, TMS) δ 1.50 (d, J = 6.5 Hz, 3H), 2.95 (dd, J = 16.4, 7.6 Hz,

1H), 3.49 (dd, J = 16.4, 8.8 Hz, 1H), 5.29 (sext, J = 7.0 Hz, 1H), 7.91

(s, 1H), 7.39 (s, 1H), 8.23 (s, 1H), 8.48 (d, J = 2.6 Hz, 1H). Anal.

Calcd for C₁₀H₁₀N₂O₄: C, 54.05; H, 4.54; N, 12.61. Found: C, 53.89;

H, 4.48; N, 12.46.

found to be [α]²⁵+14.3° and −13.8°, respectively. The X-ray

D

Synthesis of 5-Amino-2-methyl-2,3-dihydrobenzofuran-7-

carboxamide (15). 15 was obtained according to the procedure for

compound 6 using 14 (0.60 g, 2.70 mmol) as starting material,

crystallographic analysis of the diastereomeric salt of the (−)-enan-

tiomer of 12a with (S)-(−)-α-methylbenzylamine revealed that the

(−)-enantiomer bears the R-conﬁguration (Figure 2). On the basis of

this ﬁnding, the (+)-enantiomer was assigned as the S-enantiomer.

Synthesis of 2,5-Dimethyl-2,3-dihydrobenzofuran-7-carbox-

ylic Acid (12b). 12b was prepared according to the procedure used to

synthesize compound 12a using compound 11b (1.50 g, 7.28 mmol)

as starting material and obtained as a white solid (1.21 g, yield 87%):

mp 146−148 °C (lit. 149−150 °C);⁵⁴¹H NMR (400 MHz, DMSO-d₆,

TMS) δ 1.38 (d, J = 6.1 Hz, 3H), 2.22 (s, 3H), 2.73 (dd, J = 16.0, 7.6

Hz, 1H), 3.27 (dd, J = 15.6, 8.9 Hz, 1H), 4.93 (sext, J = 7.0 Hz, 1H),

7.19 (s, 1H), 7.35 (s, 1H), 12.48 (s, 1H).

1

yielding a pale yellow solid (0.26 g, yield 50%): mp 152−154 °C; H

NMR (400 MHz, DMSO-d₆, TMS) δ 1.39 (d, J = 6.2 Hz, 3H), 2.71

(dd, J = 15.7, 7.6 Hz, 1H), 3.22 (dd, J = 15.7, 8.6 Hz, 1H), 4.75 (s,

2H), 4.92 (sext, J = 7.0 Hz, 1H), 6.62 (s,1H), 6.86 (s, 1H), 7.18 (s,

1H), 7.42 (s, 1H). Anal. Calcd for C₁₀H₁₂N₂O₂: C, 62.49; H, 6.29; N,

14.57. Found: C, 62.27; H, 6.45; N, 13.56.

Synthesis of Methyl 2,3-Dihydrobenzofuran-7-carboxylate

(16). To a suspension of 2 (3.00 g, 18.29 mmol) in methanol was

added a catalytic amount of sulfuric acid, and the mixture was reﬂuxed

for 3 h. After completion of the reaction, the reaction mixture was

cooled and concentrated under vacuum. To the crude mass, water (20

mL) was added and compound was extracted in ethyl acetate (3 × 20

mL). The combined organic layers were dried over sodium sulfate and

concentrated under reduced pressure to get crude solid, which was

puriﬁed by ﬂash chromatography to get desired product 16 as white

Synthesis of 5-Fluoro-2-methyl-2,3-dihydrobenzofuran-7-

carboxylic Acid (12c). 12c was prepared according to the procedure

used to synthesize compound 12a using compound 11c (1.30 g, 6.19

mmol) as starting material and obtained as pale yellow solid (1.03 g,

yield 85%): mp 128−130 °C (lit. 129−131 °C);⁵³¹H NMR (400

MHz, DMSO-d₆, TMS) δ 1.39 (d, J = 6.3 Hz, 3H), 2.95 (dd, J = 16.4,

7.9 Hz, 1H), 3.32 (dd, J = 15.9, 9.0 Hz, 1H), 5.00 (sext, J = 7.0 Hz,

1H), 7.24 (dd, J = 9.9, 2.9 Hz, 1H), 7.32 (dd, J = 8.0, 3.0 Hz, 1H),

12.89 (s, 1H).

1

crystalline solid (2.93 g, yield 90%): H NMR (400 MHz, CDCl₃,

TMS) δ 3.21 (t, J = 8.8 Hz, 2H), 3.89 (s, 3H), 4.70 (t, J = 8.8 Hz, 2H),

6.86 (t, J = 7.6 Hz, 1H), 7.33 (d, J = 7.0 Hz, 1H), 7.71 (d, J = 7.6 Hz,

1H).

Synthesis of 5-Nitro-2,3-dihydrobenzofuran (17a). 17a was

synthesized according to compound 5 using compound 1 (1.10 g, 9.16

mmol) as starting material, as pale yellow solid (1.18 g, yield 78%): ¹H

NMR (400 MHz, DMSO-d₆, TMS) δ 3.28 (t, J = 8.8 Hz, 2H), 4.72 (t,

J = 8.8 Hz, 2H), 6.94 (d, J = 8.8 Hz, 1H), 8.06 (dd, J = 8.8, 2.5 Hz,

1H), 8.13 (s, 1H).

Synthesis of 2-Methyl-2,3-dihydrobenzofuran-7-carboxa-

mide (13a). The acid rac-12a (1.10 g, 6.18 mmol) was converted

to carboxamide using mixed-anhydride method as followed for

compound 3 to yield the amide as white solid (0.87 g, yield 80%):

1

mp 124−125 °C; H NMR (400 MHz, DMSO-d₆, TMS) δ 1.43 (d, J

= 6.1 Hz, 3H), 2.83 (dd, J = 15.4, 7.1 Hz, 1H), 3.36 (dd, J = 15.4, 9.0

Hz, 1H), 5.08 (sext, J = 7.0 Hz, 1H), 7.25 (s,1H), 7.35 (d, J = 6.8 Hz,

1H), 7.57 (s, 1H), 7.62 (d, J = 7.8 Hz, 1H). Anal. Calcd for

C₁₀H₁₁NO₂: C, 67.78; H, 6.26; N, 7.90. Found: C, 67.56; H, 6.37; N,

7.80.

Synthesis of 5-Nitro-2,3-dihydrobenzofuran-7-carboxylic

Acid (17b). 17b was synthesized according to compound 5 using

compound 2 (1.20 g, 7.32 mmol) as starting material, as pale yellow

solid (1.07 g, yield 70%): mp 245−247 °C (lit. 249−251.5 °C);³³¹H

NMR (400 MHz, DMSO-d₆, TMS) δ 3.30 (t, J = 8.6 Hz, 2H), 4.81 (t,

J = 8.6 Hz, 2H), 8.29 (s, 1H), 8.44 (s, 1H), 13.37 (bs, 1H).

Synthesis of Methyl 5-Nitro-2,3-dihydrobenzofuran-7-car-

boxylate (17c). 17c was synthesized according to compound 5 using

compound 16 (1.60 g, 8.99 mmol) as starting material, yielding yellow

The racemate amide (rac-13a) was resolved by means of preparative

chiral chromatography technique at GVK Biosciences Pvt. Ltd.,

Hyderabad, India. Optical rotations of the individual enantiomers

measured were found to be [α]²⁵+8.3°, −8.1°. t_R= 7.68 min

D

(dextro), 7.04 min. (levo); LC−MS purity = 99.79% (dextro), 98.42%

(levo); ee = 90.22% (dextro), 97.30% (levo).

1

solid (1.52 g, yield 76%): H NMR (400 MHz, DMSO-d₆, TMS) δ

Synthesis of 2,5-Dimethyl-2,3-dihydrobenzofuran-7-carbox-

amide (13b). 13b was synthesized by mixed-anhydride method

according to compound 3 using 12b (1.00 g, 5.21 mmol) as starting

3.32 (t, J = 8.6 Hz, 2H), 3.84 (s, 1H), 4.84 (t, J = 8.8 Hz, 2H), 8.31 (s,

1H), 8.43 (s, 1H).

N

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Synthesis of 5-Amino-2,3-dihydrobenzofuran (18a). To a

suspension of 17a (3.00 g, 18.18 mmol) in ethanol (50 mL) was added

10% palladium on carbon (0.20 g), and the mixture was stirred under

hydrogen atmosphere at 60 psi for 18 h. The mixture was then ﬁltered

through Celite bed and concentrated to aﬀord a gray solid, which was

further puriﬁed by column chromatography to get the desired

compound 18a as brownish-black solid (2.17 g, yield 90%): mp 77−

79 °C (lit. 80−81 °C);⁵⁴¹H NMR (400 MHz, CDCl₃, TMS) δ 3.11

(t, J = 8.6 Hz, 2H), 3.30 (bs, 2H), 4.48 (t, J = 8.6 Hz, 2H), 6.45 (d, J =

8.2 Hz, 1H), 6.59 (m, 2H).

and obtained as pale yellow solid (0.12 g, yield 80%): mp 188−190

°C; ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 3.25 (t, J = 8.8 Hz, 2H),

4.71 (t, J = 8.8 Hz, 2H), 7.30 (m, 3H), 7.74 (s, 1H). Anal. Calcd for

C₉H₈FNO₂: C, 59.67; H, 4.45; N, 7.73. Found: C, 59.67; H, 4.51; N,

7.55.

Synthesis of Methyl 4-Nitrosalicylate (22). 22 was synthesized

using 4-nitrosalicylic acid as the starting material as per the

aforementioned method for compound 8b (3.00 g, 16.40 mmol)

and obtained as bright yellow solid (2.90 g, yield 90%): ¹H NMR (400

MHz, DMSO-d₆, TMS) δ 3.95 (s, 3H), 5,87 (s, 1H), 7.43 (d, J = 8.7

Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 11.31 (s, 1H).

Synthesis of Methyl 2-(Allyloxy)-4-nitrobenzoate (23). 23

was synthesized using compound 22 (2.50 g, 12.70 mmol) following

the same protocol as that of compound 9a, as a bright yellow solid

(2.62 g, yield 87%): ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 3.86 (s,

3H), 4.81 (d, J = 6.9 Hz, 2H), 5.30 (dd, J = 13.5, 4.2 Hz, 1H), 5.47

(dd, J = 11.3, 5.6 Hz, 1H), 6.04 (ddt, J = 17.0, 10.4, 6.8 Hz, 1H), 7.87

(s, 2H), 7.89 (s, 1H).

Synthesis of 5-Amino-2,3-dihydrobenzofuran-7-carboxylic

Acid (18b). 18b was obtained by the aforementioned procedure for

compound 18a using 17b (1.3 g, 6.22 mmol) as starting material

(reaction completed in 2 h), as brownish black solid (1.24 g, yield

1

82%): H NMR (400 MHz, DMSO-d₆, TMS) δ 3.05 (t, J = 8.6 Hz,

2H), 4.44 (t, J = 8.6 Hz, 2H), 4.60 (bs, 2H), 6.72 (s, 1H), 6.80 (s, 1H),

12.39 (bs, 1H).

Synthesis of Methyl 5-Amino-2,3-dihydrobenzofuran-7-

carboxylate (18c). 18c was obtained by the aforementioned

procedure for compound 18a using 17c (1.2 g, 5.40 mmol) as

starting material (reaction completed in 2 h), as crystalline pale yellow

Synthesis of Methyl 3-Allyl-2-hydroxy-4-nitrobenzoate (24).

24 was synthesized using compound 23 (1.00 g, 4.22 mmol) with the

diminutive variation in the procedure of compound 10a where the O-

allylated product was heated in carbitol at an elevated temperature of

170−180 °C for 2 h and obtained as a dark brown oil (0.30 g, yield

1

solid (0.87 g, yield 84%): H NMR (400 MHz, CDCl₃, TMS) δ 3.16

(t, J = 8.7 Hz, 2H), 3.49 (bs, 2H), 3.89 (s, 3H), 4.65 (t, J = 8.7 Hz,

2H), 6.79 (s, 1H), 7.06 (s, 1H).

1

30%): H NMR (400 MHz, DMSO-d₆, TMS) δ 3.49 (d, J = 6.1 Hz,

Synthesis of 5-Fluoro-2,3-dihydrobenzofuran (19a). To a

solution compound 18a (2.00 g, 14.49 mmol) in THF (50 mL) was

added 1.5 mL of concentrated aqueous hydrochloric acid in several

portions. To the resulting white precipitates was added dropwise

tetraﬂuoroboric acid (2.7 mL). The mixture was then chilled (−15

°C), and to it was added sodium nitrite (1.10 g, 15.94 mmol) in water

(5 mL). Initially the suspension turned deep gray and was

homogenized, followed by precipitation after some time. The mixture

was stirred for 30 min at −15 °C, and then the solid was collected by

ﬁltration and washed with cold water, cold ethanol, and cold ether.

The solid was dried by vacuum ﬁltration to aﬀord 5-diazonium-2,3-

DHBF tetraﬂuoroborate salt, which was used without further

puriﬁcation.

A suspension of the above diazonium tetraﬂuoroborate salt in

xylene (10 mL) was reﬂuxed for 2 h. The mixture was then cooled and

diluted with 20 mL of saturated aqueous sodium bicarbonate and then

extracted with 3 × 20 mL of ethyl acetate. The combined organic

layers were washed with 50 mL of aqueous sodium bicarbonate, 50 mL

of brine, dried over sodium sulfate, ﬁltered, and concentrated under

vacuum to aﬀord crude oil. The crude oil was puriﬁed by gradient ﬂash

chromatography (n-hexane/ethyl acetate 100:0 and than 99.5:0.5) to

aﬀord compound 19a as pale yellow oil (0.61 g, yield 30%): ¹H NMR

(400 MHz, CDCl₃, TMS) δ 3.16 (t, J = 8.6 Hz, 2H), 4.55 (t, J = 8.6

Hz, 2H), 6.66 (dd, J = 8.6, 4.2 Hz, 1H), 6.76 (t, J = 9.0 Hz, 1H), 6.87

(d, J = 7.8 Hz, 1H).

Synthesis of Methyl 5-Fluoro-2,3-dihydrobenzofuran-7-

carboxylate (19c). 19c was obtained by the aforementioned

procedure for compound 19a using 18c (2.10 g, 10.88 mmol) as

starting material with a small modiﬁcation (instead of ﬁltering oﬀ the

diazonium tetraﬂuoroborate salt). The mixture was concentrated

under reduced pressure and the resulting mass was reﬂuxed in xylene

to yield compound 19c as colorless solid (0.30 g, yield 14%): ¹H NMR

(400 MHz, CDCl₃, TMS) δ 3.23 (t, J = 8.8 Hz, 2H), 3.91 (s, 3H), 4.73

(t, J = 8.8 Hz, 2H), 7.08 (d, J = 7.3 Hz, 1H), 7.38 (dd, J = 9.6, 2.7 Hz,

1H).

Synthesis of 5-Fluoro-2,3-dihydrobenzofuran-7-carboxylic

Acid (19b). 19b was obtained from ester hydrolysis of compound 19c

using sodium hydroxide (0.25 g, 1.27 mmol) as mentioned for

synthesis of compounds 12a−c; however, the mixture was reﬂuxed for

3 h (instead of 2 h), and the product was obtained as white solid (0.20

g, yield 87%): ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 3.20 (t, J = 8.8

Hz, 2H), 4.62 (t, J = 8.8 Hz, 2H), 7.25 (dd, J = 9.8 Hz, 3.0 Hz, 1H),

7.35 (d, J = 7.8 Hz, 1H), 12.91 (bs, 1H).

2H), 3.95 (s, 3H), 4.99 (dd, J = 11.30, 6.74 Hz, 2H), 5.88 (ddt, J =

17.0, 10.4, 6.8 Hz, 1H), 7.43 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 8.8 Hz,

1H), 11.27 (s, 1H).

Synthesis of Methyl 2-Methyl-4-nitro-2,3-dihydrobenzofur-

an-7-carboxylate (25). 25 was obtained from compound 24 (0.85 g,

3.58 mmol) as per the procedure mentioned for compound 11a in

1

form of pale brown solid (0.55 g, yield 65%): H NMR (400 MHz,

DMSO-d₆, TMS) δ 1.46 (d, J = 6.2 Hz, 3H), 3.26 (dd, J = 11.3, 5.1 Hz,

1H), 3.81 (dd, J = 10.1, 6.5 Hz, 1H), 3.84 (s, 3H), 5.18 (sext, J = 7.0

Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H).

Synthesis of Methyl 4-Amino-2-methyl-2,3-dihydrobenzo-

furan-7-carboxylate (26). 26 was obtained from compound 25

(1.10 g, 4.64 mmol) using the exact procedure as mentioned for

compound 18a and obtained as bright yellow powder (0.82 g, yield

1

85%): H NMR (400 MHz, DMSO-d₆, TMS) δ 1.40 (d, J = 6.2 Hz,

3H), 2.48 (dd, J = 15.1, 7.8 Hz, 1H), 3.02 (dd, J = 15.3, 9.2 Hz, 1H),

3.71 (s, 3H), 4.95 (sext, J = 7.1 Hz, 1H), 5.62 (s, 2H), 6.2 (d, J = 8.5

Hz, 1H), 7.40 (d, J = 8.6 Hz. 1H).

Synthesis of 4-Amino-2-methyl-2,3-dihydrobenzofuran-7-

carboxylic Acid (27). 27 was synthesized by hydrolyzing compound

26 (0.60 g, 2.89 mmol) in the same way as that the method followed

for compound 12a and obtained in form of pale brown solid (0.41 g,

1

yield 73%): H NMR (400 MHz, DMSO-d₆, TMS) δ 1.38 (d, J = 6.2

Hz, 3H), 2.54 (dd, J = 15.1, 7.8 Hz, 1H), 3.07 (dd, J = 15.3, 9.2 Hz,

1H), 4.94 (sext, J = 7.1 Hz, 1H), 5.66 (s, 2H), 6.16 (d, J = 8.4 Hz, 1H),

7.35 (d, J = 8.6 Hz, 1H), 13.1 (s, 1H).

Synthesis of 4-Amino-2-methyl-2,3-dihydrobenzofuran-7-

carboxamide (28). To a well stirred solution of compound 27

(0.30 g, 1.55 mmol) in anhydrous THF was added sequentially N-

methylmorpholine (0.3 mL) followed by isobutyl chloroformate (0.3

mL) at −20 °C under nitrogen atmosphere. The resulting reaction

mixture was then allowed to stir for 30 min to yield the anhydride

through which was passed dry ammonia gas under anhydrous

condition. The mixture was then brought to room temperature with

intermittent monitoring by thin layer chromatography. Upon

completion, the reaction mixture was puriﬁed by column chromatog-

raphy (ethyl acetate/methanol 95:5), resulting in a pale yellow solid

(0.12 g, yield 40%): mp 245−248 °C; ¹H NMR (400 MHz, DMSO-d₆,

TMS) δ 1.42 (d, J = 6.3 Hz, 3H), 2.52 (dd, J = 15.1, 9.2 Hz, 1H), 3.09

(dd, J = 15.6, 9.1 Hz, 1H), 5.03 (sext, J = 7.1 Hz, 1H), 5.63 (s, 2H),

6.15 (d, J = 8.6 Hz, 1H), 6.91 (s, 1H), 7.06 (s, 1H), 7.36 (d, J = 8.6

Hz, 1H).

The amide was resolved by means of preparative chiral

chromatography technique at GVK Biosciences Pvt. Ltd., Hyderabad,

India. Individual enantiomers were resolved and characterized for

purity and retention times, with t_R= 6.93 min (compound 28¹) and

Synthesis of 5-Fluoro-2,3-dihydrobenzofuran-7-carboxa-

mide (20). 20 was synthesized from compound 19b (0.15 g, 0.82

mmol) following the exact procedure used to synthesize compound 3

O

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

8.12 min (compound 28²); LC−MS purity = 99.07% (compound

28¹), 96.58% (compound 28²); ee = 94.00% (compound 28¹), 94.46%

(compound 28²).

1H), 8.14 (d, J = 7.4 Hz, 1H). Anal. Calcd for C₉H₇NO₃: C, 61.02; H,

3.98; N, 7.91. Found: C, 61.35; H, 3.62; N, 7.86.

Synthesis of 3-(2-Morpholin-4-ylethoxy)benzaldehyde (37).

To a well stirred solution of 3-hydroxybenzaldehyde (0.5 g, 4.09

mmol) in acetonitrile were added 4-(2-chloroethyl)morpholine (0.61

g, 4.09 mmol) and anhydrous potassium carbonate (0.56 g, 4.09

mmol), and the mixture was reﬂuxed for 6 h. Upon completion, the

reaction mixture was partitioned between ethyl acetate and water. The

organic layer was dried over anhydrous magnesium sulfate and

concentrated under vacuum. The resulting crude product obtained

upon workup was ﬁnally puriﬁed by column chromatography

(employing a mobile phase of DCM/methanol/2 M methanolic

ammonia 90:10:2) to yield the desired compound as dark brown oil

Synthesis of Methyl 2-Hydroxy-3-methylbenzoate (30). 30

was obtained by the same procedure mentioned for compound 8b

using compound 29 (3.00 g, 19.73 mmol) as starting material and

methanol as a solvent to yield compound 30 as pale yellow oil (2.00 g,

yield 60%): ¹H NMR (400 MHz, CDCl₃, TMS) δ 1.40 (t, J = 7.2 Hz,

3H), 2.26 (s, 3H), 4.39 (q, J = 7.1 Hz, 2H), 6.77 (t, J = 7.6 Hz, 1H),

7.30 (d, J = 7.2 Hz, 1H), 7.70 (d, J = 7.6 Hz, 1H), 11.10 (s, 1H).

Synthesis of Methyl 2-(2-Ethoxy-2-oxoethoxy)-3-methyl-

benzoate (31). To a solution of 30 (3.0 g, 18.0 mmol) in 10 mL

of DMF were added ethyl bromoacetate (4.51 g, 27.0 mmol),

potassium carbonate (2.72 g, 19.8 mmol), and sodium iodide (2.96 g,

19.8 mmol). The reaction mixture was stirred for 12 h at room

temperature and then poured into water (50 mL) and extracted with

ethyl acetate (3 × 50 mL). The organic layers were combined, dried

over sodium sulfate, and concentrated under vacuum to yield yellowish

brown oil which was further puriﬁed by column chromatography (n-

hexane/ethyl acetate 90:10) to get the desired product 31 as pale

1

(0.59 g, yield 62%): H NMR (400 MHz, CDCl₃, TMS) δ 2.17−2.32

(m, 4H), 2.47 (t, J = 5.4 Hz, 2H), 3.35−3.39 (m, 4H), 3.82 (t, J = 5.5

Hz, 2H), 6.87 (s, 1H), 7.02−7.09 (m, 1H), 7.09−7.25 (m, 2H), 9.62

(s, 1H).

Synthesis of 3-(2-Piperidin-1-ylethoxy)benzaldehyde (38).

38 was synthesized by following the same protocol as for compound

37 by reacting 3-hydroxybenzaldeyde (0.3 g, 2.45 mmol) and 1-(2-

chloroethyl)piperidine (0.36 g, 2.45 mmol) using potassium carbonate

(0.50 g, 3.70 mmol) as a base under reﬂuxing conditions for 6 h. The

resulting crude product was puriﬁed by column chromatography

(employing a mobile phase of DCM/methanol/2 M methanolic

1

yellow oil (2.20 g, yield 92%): H NMR (400 MHz, CDCl₃, TMS) δ

1.32 (t, J = 7.2 Hz, 3H), 1.37 (t, J = 7.2 Hz, 3H), 2.35 (s, 2H), 4.29 (q,

J = 7.5 Hz, 2H), 4.35 (q, J = 7.5 Hz, 2H), 4.58 (s, 2H), 7.08 (t, J = 7.6

Hz, 1H), 7.35 (d, J = 7.4 Hz, 1H), 7.66 (d, J = 7.6 Hz, 1H).

Synthesis of 2-(Carboxymethoxy)-3-methylbenzoic Acid

(32). 32 was obtained in the same way as the protocol followed for

compound 12a using 3 equiv of potassium hydroxide when added to

compound 31 under reﬂuxing conditions, yielding 32 as pale yellow

solid (1.34 g, yield 80%): mp 202−204 °C (lit. 207 °C);⁵⁵¹H NMR

(400 MHz, DMSO-d₆, TMS) δ 2.28 (s, 3H), 4.49 (s, 2H), 7.10 (t, J =

7.6 Hz, 1H), 7.40 (d, J = 7.7 Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 12.97

(s, 2H).

1

ammonia 90:10:2) to yield a pale yellow oil (0.39 g, yield 72%): H

NMR (400 MHz, CDCl₃, TMS) δ 1.24−1.43 (m, 5H), 2.01−2.34 (m,

5H), 2.47 (t, J = 6.0 Hz, 2H), 3.83 (t, J = 6.0 Hz, 2H), 6.89 (s, 1H),

7.05−7.29 (m, 3H), 9.65 (s, 1H).

Synthesis of 4-(2-Morpholin-4-ylethoxy)benzaldehyde (39).

39 was synthesized in the same way as mentioned for compound 37,

by mixing equimolar proportions of 4-hydroxybenzaldehyde (0.5 g,

4.09 mmol) and 4-(2-chloroethyl)morpholine (0.61 g, 4.09 mmol)

under reﬂuxing conditions in the presence of potassium carbonate

(0.56 g, 4.09 mmol). The mixture was reﬂuxed for 6 h. The resulting

crude product obtained upon workup was puriﬁed by column

chromatography (employing a mobile phase of DCM/methanol/2

M methanolic ammonia 90:10:2) to provide pale brown oil (0.67 g,

yield 70%): bp >300 °C (lit. 394 °C);⁵⁶¹H NMR (400 MHz, CDCl₃,

TMS) δ 2.36−2.50 (m, 4H), 2.65 (t, J = 5.3 Hz, 2H), 3.55 (t, J = 3.9

Hz, 4H), 4.01 (t, J = 5.3 Hz, 2H), 6.84 (d, J = 8.0 Hz, 2H), 7.65 (d, J =

7.8 Hz, 2H), 9.69 (s, 1H).

Synthesis of 2-(Carboxymethoxy)isophthalic Acid (33). To a

suspension of compound 32 (2.0 g, 9.5 mmol) in 20 mL of water was

added KMnO₄(7.57 g, 47.6 mmol) gradually, and the resulting

reaction mixture was reﬂuxed for 2 h. Upon completion, the reaction

mixture was ﬁltered and ﬁltrate was concentrated under vacuum. The

resulting white solid was dissolved in minimum amount of water and

cooled to 0 °C. To this solution, concentrated HCl (11 N) was added

dropwise until pH 1 was obtained. The resulting precipitates were

ﬁltered and dried to get the desired compound 33 as white solid (0.68

1

Synthesis of 4-(2-Piperidin-1-ylethoxy)benzaldehyde (40).

40 was synthesized by following the same method as mentioned for

compound 37 by reacting 4-hydroxybenzaldehyde (0.3 g, 2.45 mmol)

and 1-(2-chloroethyl)piperidine (0.36 g, 2.45 mmol) in reﬂuxing

conditions under the inﬂuence of potassium carbonate (0.50 g, 3.70

mmol) as a base. The mixture was reﬂuxed for 6 h. The resulting crude

product was puriﬁed by column chromatography (employing a mobile

phase of DCM/methanol/2 M methanolic ammonia 90:10:2) to yield

pale yellow oil (0.37 g, yield 68%): bp >300 °C (lit. 378 °C);⁵⁶¹H

NMR (400 MHz, CDCl₃, TMS) δ 1.38 (q, J = 6.3 Hz, 2H), 1.46 (J =

5.7 Hz, 2H), 1.59 (q, J = 5.5 Hz. 4H), 1.66−1.80 (m, 2H), 2.71 (t, J =

6.0 Hz, 2H), 3.70 (t, J = 6.8 Hz, 2H), 7.70 (d, J = 8.1 Hz, 2H), 7.00 (d,

J = 8.2 Hz, 2H), 9.80 (s, 1H).

Synthesis of 4-(2-Chloroethoxy)benzaldehyde (41). To a well

stirred solution of 4-hydroxybenzaldehyde (0.3 g, 2.45 mmol) in

acetonitrile was added bromochloroethane (0.35 g, 2.45 mmol),

followed by subsequent addition of potassium carbonate (0.41 g, 3.0

mmol) and potassium iodide (0.41 g, 2.45 mmol) under reﬂux. The

reaction was allowed to run for 12 h. The reaction progress was

monitored by thin layer chromatography. Upon completion, the

reaction mass was diluted with water and extracted thrice with ethyl

acetate (3 × 50 mL). The combined organic layers were dried over

magnesium sulfate and concentrated under vacuum to aﬀord a crude

product, which was further puriﬁed by column chromatography (n-

hexane/ethyl acetate 90:10) to aﬀord compound 41 as a pale yellow

oil which upon prolonged standing turned solid (0.32 g, yield 72%):

mp 29−32 °C (lit. 31 °C);⁵⁷¹H NMR (400 MHz, CDCl₃, TMS) δ

3.75 (t, J = 5.6 Hz, 2H), 4.19 (t, J = 5.6 Hz, 2H), 6.90 (d, J = 8.3 Hz,

2H), 7.72 (d, J = 8.4 Hz, 2H), 9.76 (s, 1H).

g, yield 30%): H NMR (400 MHz, DMSO-d₆, TMS) δ 4.56 (s, 2H),

7.29 (t, J = 7.6 Hz, 1H), 7.84 (d, J = 7.6 Hz, 2H), 13.45 (bs, 3H).

Synthesis of 3-Acetoxybenzofuran-7-carboxylic Acid (34).

To a mixture of 33 (1.0 g, 4.17 mmol) and sodium acetate (0.34 g,

4.17 mmol) were added 3 mL of acetic acid and 5 mL of acetic

anhydride, and the resulting mixture was reﬂuxed for 5 h. Upon

completion, the reaction mixture was extracted with ethyl acetate (3 ×

15 mL). The combined organic layers were dried over magnesium

sulfate and concentrated under vacuum to obtain a yellow mass, which

was puriﬁed by column chromatography (n-hexane/ethyl acetate

90:10) to get the desired compound 34 as pale yellow solid (0.55 g,

1

yield 60%): H NMR (400 MHz, DMSO-d₆, TMS) δ 2.39 (s, 3H),

7.39 (t, J = 7.6 Hz, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.89 (d, J = 7.5 Hz,

1H), 8.29 (s, 1H), 13.38 (bs, 1H).

Synthesis of 3-Oxo-2,3-dihydrobenzofuran-7-carboxylic

Acid (35). Compound 34 (0.5 g, 2.27 mmol) was dissolved in a 10

mL mixture of HCl/H₂O/MeOH (1:10:40) and was reﬂuxed for 1 h.

Upon completion, the reaction mixture was concentrated under

vacuum. The resulting solid obtained was ﬁltered oﬀ and washed with

water to yield the desired compound as an orange solid (0.24 g, yield

60%): ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 4.89 (s, 2H), 7.23 (t, J

= 7.6 Hz, 1H), 7.87 (d, J = 7.5 Hz, 1H), 8.18 (d, J = 7.7 Hz, 1H), 13.24

(bs, 1H).

Synthesis of 3-Oxo-2,3-dihydrobenzofuran-7-carboxamide

(36). 36 was obtained by the same procedure mentioned for

compound 3 using compound 35 (2.00 g, 11.23 mmol) as a starting

material to yield compound 36 as yellow solid (0.70 g, yield 35%): mp

1

194−196 °C; H NMR (400 MHz, DMSO-d₆, TMS) δ 4.93 (s, 2H),

7.26 (t, J = 7.6 Hz, 1H), 7.44 (s, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.86 (s,

P

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Synthesis of 4-[2-(4-Methylpiperazin-1-yl)ethoxy]-

benzaldehyde (42). To a suspension of potassium carbonate (0.21

g, 1.5 mmol) and potassium iodide (0.18 g, 1.1 mmol) in acetonitrile

were added compound 41 (0.18 g, 1.0 mmol) and N-methylpiperazine

(0.15 g, 1.5 mmol) under reﬂuxing condition. The reaction mass was

reﬂuxed for 6 h and worked up by diluting with a small quantity of

water. The diluted mass was extracted thrice with ethyl acetate (3 × 20

mL). The combined organic layers were dried over magnesium sulfate

and concentrated under vacuum to obtain a crude mass. The crude

product was ﬁnally puriﬁed by preparative TLC (employing a mobile

phase of DCM/methanol/2 M methanolic ammonia 90:10:2) to yield

mobile phase of n-hexane/ethyl acetate 70:30) to yield compound 47

as pale brown oil (0.44 g, yield 60%): bp >300 °C (lit. 327 °C);⁵⁹¹H

NMR (400 MHz, CDCl₃, TMS) δ 2.58−2.63 (m, 1H), 2.74 (t, J = 4.6

Hz, 1H), 3.17−3.25 (m, 1H), 3.78 (dd, J = 11.2, 6.2 Hz, 1H), 4.20

(dd, J = 11.2, 3.2 Hz, 1H), 6.84 (d, J = 8.5 Hz, 2H), 7.64 (d, J = 8.5

Hz, 2H), 9.69 (s, 1H).

Synthesis of 4-(2-Hydroxy-3-morpholin-4-ylpropoxy)-

benzaldehyde (48). This compound was synthesized by the

procedure used for compound 37, by alkylating morpholine (0.3 g,

3.44 mmol) with intermediate 47 (0.61 g, 3.44 mmol) utilizing

potassium carbonate (0.70 g, 5.16 mmol) as a base. The reaction mass

was reﬂuxed for 6 h. It was then ﬁltered and concentrated under

vacuum. The resulting crude product was puriﬁed by column

chromatography (employing a mobile phase of DCM/methanol/2

M methanolic ammonia 90:10:2) to yield compound 48 as a pale

1

compound 42 in the form of a pale yellow oil (0.17 g, yield 70%): H

NMR (400 MHz, CDCl₃, TMS) δ 2.41 (s, 3H), 2.63−2.83 (m, 8H),

2.86 (t, J = 5.3 Hz, 2H), 4.17 (t, J = 5.9 Hz, 2H), 6.98 (d, J = 8.6 Hz,

2H), 7.80 (d, J = 8.6 Hz, 2H), 9.86 (s, 1H).

1

Synthesis of 4-(3-Chloropropoxy)benzaldehyde (43). 43 was

synthesized in the same manner as that for compound 37 by using

equimolar proportions of 4-hydroxybenzaldehyde (2.00 g, 16.4 mmol)

and bromochloropropane (2.62 g, 16.4 mmol) using potassium

carbonate (3.40 g, 24.6 mmol) as a base. The mixture was reﬂuxed for

3 h. The crude mass obtained upon workup was puriﬁed by column

chromatography employing a mobile phase of n-hexane/ethyl acetate

90:10 to yield yellow oil which eventually turned solid upon prolonged

standing (2.00 g, yield 61%): mp 28−31 °C (lit. 29 °C);⁵⁸¹H NMR

(400 MHz, CDCl₃, TMS) δ 2.40 (t, J = 7.4 Hz, 2H), 3.59 (t, J = 4.7

Hz, 2H), 3.99 (t, J = 6.1 Hz, 2H), 6.88 (d, J = 8.9 Hz, 2H), 7.70 (d, J =

8.8 Hz, 2H), 9.74 (s, 1H).

viscous brown oil (0.53 g, yield 58%): H NMR (400 MHz, CDCl₃,

TMS) δ 2.42−2.75 (m, 6H), 3.61−3.87 (m, 5H), 4.03−4.23 (m, 3H),

7.03 (d, J = 8.7 Hz, 2H), 7.82 (d, J = 8.7 Hz, 2H), 9.86 (s, 1H).

Synthesis of 4-{2-[4-(4-Fluorophenyl)piperazin-1-yl]ethoxy}-

benzaldehyde (49). 49 was synthesized in the same manner as that

for compound 37 by using equimolar proportions of compound 41

(0.46 g, 2.50 mmol) and 4-ﬂuorophenylpiperazine (0.45 g, 2.50 mmol)

utilizing potassium carbonate (0.52 g, 3.75 mmol) as a base. The

reaction was reﬂuxed for 48 h and the product was puriﬁed by column

chromatography (utilizing a mobile phase of n-hexane/ethyl acetate

70:30) to obtain compound 49 as a pale brown oil (0.50 g, yield 61%):

¹H NMR (400 MHz, CDCl₃, TMS) δ 2.53−2.76 (m, 4H), 2.79 (t, J =

Synthesis of 4-(3-Morpholin-4-ylpropoxy)benzaldehyde

(44). 44 was synthesized in the same manner as that for compound

37 by using equimolar proportions of compound 43 (2.00 g, 10.1

mmol) and morpholine (0.90 g, 10.1 mmol) utilizing potassium

carbonate (2.10 g, 15.15 mmol) as a base. The mixture was reﬂuxed for

6 h and the product was puriﬁed by column chromatography

(employing a mobile phase of ethyl acetate/methanol/2 M methanolic

ammonia 90:10:2) to yield 44 as dark brown viscous oil (2.10 g, yield

82%): ¹H NMR (400 MHz, CDCl₃, TMS) δ 1.99 (q, J = 6.5 Hz, 2H),

2.37−2.50 (m, 4H), 2.52 (t, J = 7.3 Hz, 2H), 3.62−3.78 (s, 4H), 4.10

(t, J = 6.3 Hz, 2H), 6.99 (d, J = 8.7 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H),

9.85 (s, 1H).

5.4 Hz, 2H), 2.98−3.21 (m, 4H), 4.11 (t, J = 5.4 Hz, 2H), 6.71−6.82

(m, 2H), 6.87 (t, J = 8.7 Hz, 2H), 6.93 (d, J = 8.7 Hz, 2H), 7.73 (d, J =

8.4 Hz, 2H), 9.77 (s, 1H).

General Procedure for Synthesis of Compounds 50−73. To a

well stirred suspension having equimolar proportions of compound 36

and the corresponding benzaldehyde (commercially available or

synthesized) in toluene (3−10 mL) was added ammonium acetate

(1−1.5 equiv). The reaction mass was reﬂuxed until completion (1−

24 h) and intermittently monitored using TLC. Upon completion of

the reaction, the toluene was evaporated under vacuum and the

ensuing mass was puriﬁed by either column chromatography or

preparative TLC to obtain the desired compound. The isolated

compound was washed with water and dried under vacuum to yield

the title compounds 50−73.

Synthesis of (Z)-2-Benzylidene-3-oxo-2,3-dihydrobenzofur-

an-7-carboxamide (50). The title compound was prepared

according to general procedure using 36 (0.07 g, 0.39 mmol),

ammonium acetate (0.03 g, 0.39 mmol), and benzaldehyde (0.04 g,

0.39 mmol), and the mixture was reﬂuxed for 2 h. It was puriﬁed by

column chromatography (ethyl acetate/methanol 95:5) and obtained

as a pale yellow solid (0.07 g, yield 70%): mp 212−214 °C; ¹H NMR

(400 MHz, DMSO-d₆, TMS) δ 7.05 (s, 1H), 7.24 (t, J = 7.3 Hz, 1H,),

7.40 (t, J = 7.7 Hz, 1H,), 7.50 (m, 3H), 7.87 (s, 1H), 7.95 (m, 2H),

8.06 (m, 2H). Anal. Calcd for C₁₆H₁₁NO₃·1H₂O: C, 67.84; H, 4.63; N,

4.94. Found: C, 67.56; H, 4.51; N, 4.80.

Synthesis of (Z)-3-Oxo-2(3′-phenoxybenzylidene)-2,3-dihy-

drobenzofuran-7-carboxamide (51). The title compound was

obtained following the general procedure using 36 (0.1 g, 0.56 mmol),

ammonium acetate (0.06 g, 0.78 mmol), and 3-phenoxybenzaldehyde

(0.11 g, 0.56 mmol). The reaction mass was reﬂuxed for 24 h and

puriﬁed by column chromatography (ethyl acetate/methanol 95:5) to

yield pale yellow solid (0.13 g, yield 65%): mp 195−198 °C; ¹H NMR

(400 MHz, DMSO-d₆, TMS) δ 7.05 (s,1H), 7.06−7.10 (m, 3H), 7.17

(t, J = 7.4 Hz, 1H), 7.37−7.44 (m, 3H), 7.50 (t, J = 8.1 Hz, 1H), 7.64

(s, 1H), 7.79 (s, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.91 (s, 1H), 7.93 (dd, J

= 7.6, 1.4 Hz, 1H), 8.04 (dd, J = 7.6, 1.3 Hz, 1H). Anal. Calcd for

C₂₂H₁₅NO₄·¹/₂H₂O: C, 72.12; H, 4.23; N, 3.92. Found: C, 71.98; H,

4.76; N, 3.49.

Synthesis of 2-Chloro-1-morpholin-4-ylethanone (45). 45

was synthesized by performing diminutive changes in the reported

procedure.^43,44A solution of morpholine (0.87 g, 10.0 mmol) in DCM

was added dropwise to an ice cold vigorously stirred solution of

equimolar proportions of chloroacetyl chloride (1.13 g, 10.0 mmol)

and triethylamine (1.50 g, 15.0 mmol) in DCM. The mixture was

brought to room temperature after 30 min and allowed to stir for

additional 3 h. After 3 h, the reaction mass was evaporated under

reduced pressure and the resulting crude product was puriﬁed by

column chromatography (employing a mobile phase of DCM/

methanol/2 M methanolic ammonia 90:10:2), yielding the desired

product 45 as dark brown oil (1.45 g, yield 89%): bp 292−296 °C (lit.

294 °C);⁴⁴¹H NMR (400 MHz, CDCl₃, TMS) δ 3.25−3.37 (m, 4H),

3.42 (t, J = 4.8 Hz, 2H), 3.45 (t, J = 4.8 Hz, 2H), 3.89 (s, 2H).

Synthesis of 4-(2-Morpholin-4-yl-2-oxoethoxy)-

benzaldehyde (46). 46 was synthesized in the same manner as

that for compound 37 by using equimolar proportions of 4-

hydroxybenzaldehyde (2.00 g, 16.4 mmol) and 45 (2.67 g, 16.4

mmol) utilizing potassium carbonate (3.40 g, 24.6 mmol) as a base.

The reaction mixture was reﬂuxed for 6 h and the product was puriﬁed

by column chromatography (employing a mobile phase of ethyl

acetate/methanol/2 M methanolic ammonia 90:10:2) to yield 46 as

1

dark brown oil (3.10 g, yield 76%): H NMR (400 MHz, CDCl₃,

TMS) δ 3.40−3.52 (m, 4H), 3.53−3.68 (m, 4H), 5.01 (s, 2H), 7.11

(d, J = 8.6 Hz, 2H), 7.86 (d, J = 8.6 Hz, 2H), 9.87 (s, 1H).

Synthesis of 4-Oxiranylmethoxybenzaldehyde (47). 47 was

synthesized in the same manner as that for compound 37 by using

equimolar proportions of 4-hydroxybenzaldehyde (0.50 g, 4.09 mmol)

and epibromhydrin (0.56, 4.09 mmol) utilizing potassium carbonate

(0.85 g, 6.14 mmol) as a base. The reaction mass was reﬂuxed for 6 h

and the product was puriﬁed by column chromatography (utilizing a

Synthesis of (Z)-2-(4′-Chlorobenzylidene)-3-oxo-2,3-dihy-

drobenzofuran-7-carboxamide (52). The title compound was

synthesized as per the general procedure using 36 (0.1 g, 0.56 mmol),

ammonium acetate (0.05 g, 0.6 mmol), and 4-chlorobenzaldehyde

(0.08 g, 0.56 mmol), and the mixture was reﬂuxed for 4 h. It was

Q

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

puriﬁed by column chromatography (ethyl acetate/methanol 95:5) to

yield a pale yellow solid (0.11 g, yield 62%): mp 290−293 °C; H

NMR (400 MHz, DMSO-d₆, TMS) δ 8.06 (d, J = 7.1 Hz, 3H), 7.94

(d, J = 7.1 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.39 (t, J = 7.6 Hz, 1H),

7.07 (s, 1H), 7.82 (s, 1H). Anal. Calcd for C₁₆H₁₀ClNO₃: C, 64.12; H,

3.36; N, 4.67. Found: C, 64.56; H, 3.43; N, 4.30.

mmol), ammonium acetate (0.05 g, 0.60 mmol), and 3,4-

dihydroxybenzaldehyde (0.05 g, 0.39 mmol), and the mixture was

reﬂuxed for 4 h. The reaction mixture was puriﬁed by column

chromatography (ethyl acetate/methanol 95:5) to obtain a dark brown

1

solid (0.17 g, yield 56%): mp 257−259 °C; H NMR (400 MHz,

DMSO-d₆, TMS) δ 6.81−6.94 (m, 2H), 7.30−7.49 (m, 2H), 7.77−

7.88 (m, 2H), 7.90 (d, J = 7.5 Hz, 1H), 8.02 (d, J = 7.3 Hz, 1H), 9.23

(bs, 1H), 9.92 (bs, 1H). Anal. Calcd for C₁₆H₁₁NO₅·1H₂O: C, 61.15;

H, 3.83; N, 4.46. Found: C, 61.52; H, 3.91; N, 5.14.

Synthesis of (Z)-2-(4′-Hydroxy-3′-methoxybenzylidene)-3-

oxo-2,3-dihydrobenzofuran-7-carboxamide (53). The title com-

pound was prepared according to the general procedure using 36 (0.1

g, 0.56 mmol), ammonium acetate (0.06 g, 0.78 mmol), and 4-

hydroxy-3-methoxybenzaldehyde (0.09 g, 0.56 mmol), and the mixture

was reﬂuxed for an hour. The product obtained was puriﬁed by

column chromatography (ethyl acetate/methanol 95:5) to yield bright

Synthesis of (Z)-2-(2′,4′-Dihydroxybenzylidene)-3-oxo-2,3-

dihydrobenzofuran-7-carboxamide (59). The target compound

was prepared according to the general procedure using 36 (0.07 g,

0.39 mmol), ammonium acetate (0.03 g, 0.39 mmol), and 2,4-

dihydroxybenzaldehyde (0.05 g, 0.39 mmol). The reaction lasted for

almost 10 h and the mixture was puriﬁed by column chromatography

(ethyl acetate/methanol 95:5) to obtain a brick red solid (0.19 g, yield

1

yellow solid (0.14 g, yield 80%): mp 268−270 °C; H NMR (400

MHz, DMSO-d₆, TMS) δ 3.87 (s, 3H), 6.88 (d, J = 7.9 Hz, 1H,), 6.98

(s, 1H), 7.34−7.41 (m, 2H), 7.85 (s, 1H), 7.91 (d, J = 7.2 Hz, 1H,),

7.96 (s, 2H), 8.05 (d, J = 7.4 Hz, 1H,), 9.94 (s, 1H). Anal. Calcd for

C₁₇H₁₃NO₅·²/₃H₂O: C, 63.16; H, 4.47; N, 4.33. Found: C, 63.26; H,

4.77; N, 4.41.

1

65%): mp >300 °C; H NMR (400 MHz, DMSO-d₆, TMS) δ 6.81−

6.94 (m, 2H), 7.30−7.49 (m, 2H), 7.77−7.88 (m, 2H), 7.90 (d, J = 7.5

Hz, 1H), 8.02 (d, J = 7.3 Hz, 1H), 9.23 (bs, 1H), 9.92 (bs, 1H). Anal.

Calcd for C₁₆H₁₁NO₅: C, 64.65; H, 3.73; N, 4.71. Found: C, 64.76; H,

3.98; N, 4.60.

Synthesis of (Z)-2-(4′-Methoxybenzylidene)-3-oxo-2,3-dihy-

drobenzofuran-7-carboxamide (54). The title compound was

synthesized by following the general procedure using 36 (0.15 g, 0.84

mmol), ammonium acetate (0.07 g, 0.84 mmol), and 4-methox-

ybenzaldehyde (0.11 g, 0.84 mmol). The reaction mass was reﬂuxed

for 1.5 h and puriﬁed by column chromatography (ethyl acetate/

methanol 95:5) to yield a bright yellow solid (0.20 g, yield 80%): mp

Synthesis of (Z)-2-(3′,5′-Dihydroxybenzylidene)-3-oxo-2,3-

dihydrobenzofuran-7-carboxamide (60). The title compound was

synthesized according to the general procedure using 36 (0.07 g, 0.39

mmol), ammonium acetate (0.03 g, 0.39 mmol), and 3,5-

dihydroxybenzaldehyde (0.05 g, 0.39 mmol). The mixture was

reﬂuxed for 18 h and the title compound was puriﬁed by column

chromatography (ethyl acetate/methanol 95:5) to obtain a light brown

solid (0.19 g, yield 65%): mp >300 °C; ¹H NMR (400 MHz, DMSO-

d₆, TMS) δ 6.39 (s, 1H), 6.81 (s, 1H), 6.87 (s, 2H), 7.38 (t, J = 7.4 Hz,

1H), 7.76 (s, 1H), 7.87 (s, 1H), 7.92 (d, J = 6.8 Hz, 1H), 8.05 (d, J =

7.6 Hz, 1H), 9.54 (s, 2H). Anal. Calcd for C₁₆H₁₁NO₅: C, 64.65; H,

3.73; N, 4.71. Found: C, 64.52; H, 3.43; N, 5.14.

Synthesis of (Z)-2-(2′,5′-Dihydroxybenzylidene)-3-oxo-2,3-

dihydrobenzofuran-7-carboxamide (61). The target compound

was synthesized following the general procedure using 36 (0.07 g, 0.39

mmol), ammonium acetate (0.03 g, 0.39 mmol), and 2,5-

dihydroxybenzaldehyde (0.05 g, 0.39 mmol), and the mixture was

reﬂuxed for 21 h. The title compound was puriﬁed by column

chromatography (ethyl acetate/methanol 95:5) to obtain a dark brown

solid (0.16 g, yield 55%): mp >300 °C; ¹H NMR (400 MHz, DMSO-

d₆, TMS) δ 6.78 (s, 1H), 6.80 (s, 1H), 7.25 (s, 1H), 7.38 (t, J = 7.4 Hz,

1H), 7.54 (s, 1H), 7.78 (s, 1H), 7.87 (s, 1H), 7.92 (d, J = 7.5 Hz, 1H),

8.05 (d, J = 7.5 Hz, 1H), 9.04 (s, 1H), 9.85 (s, 1H). Anal. Calcd for

C₁₆H₁₁NO₅: C, 64.65; H, 3.73; N, 4.71. Found: C, 64.90; H, 4.03; N,

4.59.

Synthesis of (Z)-2-[3′-(2-Morpholin-4-ylethoxy)-

benzylidene]-3-oxo-2,3-dihydrobenzofuran-7-carboxamide

(62). The title compound was synthesized according to the general

procedure using 36 (0.07 g, 0.39 mmol), ammonium acetate (0.03 g,

0.39 mmol), and compound 37 (0.09 g, 0.39 mmol), and the mixture

was reﬂuxed for 6 h and puriﬁed by ﬂash chromatography (employing

a mobile phase of ethyl acetate/methanol/2 M methanolic ammonia

90:10:2) to yield a bright yellow solid (0.11 g, yield 68%): mp 189−

193 °C; ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 2.55−2.62 (m, 4H),

2.75 (t, J = 5.6 Hz, 2H), 3.59 (t, J = 4.0 Hz, 4H), 4.18 (t, J = 5.6 Hz,

2H), 7.02 (s, 1H), 7.06 (d, J = 8.4 Hz, 1H), 7.40 (t, J = 7.8 Hz, 2H),

7.59 (d, J = 7.5 Hz, 1H), 7.72 (s, 1H), 7.86 (s, 1H), 7.93 (d, J = 8.3

Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 7.4 Hz, 1H). Anal.

Calcd for C₂₂H₂₂N₂O₅·³/₄H₂O: C, 64.77; H, 5.81; N, 6.87: found: C,

65.12; H, 5.74; N, 6.90.

1

248−250 °C; H NMR (400 MHz, DMSO-d₆, TMS) δ 3.84 (s, 3H),

7.04 (s, 1H), 7.07 (d, J = 7.6 Hz, 2H), 7.37 (t, J = 7.5 Hz, 1H), 7.82 (s,

1H), 7.87−7.99 (m, 2H), 8.03 (d, J = 7.2 Hz, 3H). Anal. Calcd for

C₁₇H₁₃NO₄·¹/₂H₂O: C, 67.10; H, 4.64; N, 4.60. Found: C, 67.29; H,

4.45; N, 4.54.

Synthesis of (Z)-2-(2′-Hydroxybenzylidene)-3-oxo-2,3-dihy-

drobenzofuran-7-carboxamide (55). The compound was obtained

by following the general procedure using 36 (0.13 g, 0.73 mmol),

ammonium acetate (0.06 g, 0.78 mmol), and 2-hydroxybenzaldehyde

(0.09 g, 0.73 mmol). The mixture was reﬂuxed for 6 h and puriﬁed by

column chromatography (ethyl acetate/methanol 95:5) to yield a pale

1

yellow solid (0.08 g, yield 56%): mp 239−241 °C; H NMR (400

MHz, DMSO-d₆, TMS) δ 6.92 (t, J = 7.5 Hz, 1H), 6.97 (d, J = 8.0 Hz,

1H), 7.28−7.35 (m, 2H), 7.38 (t, J = 7.5 Hz, 1H), 7.84−7.90 (m, 2H),

7.92 (d, J = 7.8 Hz, 1H), 8.04 (d, J = 7.5 Hz, 1H), 8.20 (d, J = 7.5 Hz,

1H), 10.58 (s, 1H). Anal. Calcd for C₁₆H₁₁NO₄·¹/₂H₂O: C, 66.20; H,

4.17; N, 4.83. Found: C, 66.29; H, 4.43; N, 4.52.

Synthesis of (Z)-2-(3′-Hydroxybenzylidene)-3-oxo-2,3-dihy-

drobenzofuran-7-carboxamide (56). The title compound was

prepared according to the general procedure using 36 (0.10 g, 0.56

mmol), ammonium acetate (0.05 g, 0.60 mmol), and 3-hydrox-

ybenzaldehyde (0.07 g, 0.56 mmol), and the mixture was reﬂuxed for 4

h. It was puriﬁed by column chromatography (ethyl acetate/methanol

95:5) to obtain a pale yellow solid (0.09 g, yield 61%): mp 238−240

°C; ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 6.90 (d, J = 8.0 Hz, 1H),

6.94 (s, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.35−7.42 (m, 2H), 7.51 (d, J =

7.6 Hz, 1H), 7.83−7.90 (m, 2H), 7.93 (d, J = 7.6 Hz, 1H), 8.05 (d, J =

7.3 Hz, 1H), 9.75 (s, 1H). Anal. Calcd for C₁₆H₁₁NO₄: C, 64.21; H,

4.38; N, 4.68. Found: C, 64.10; H, 4.43; N, 4.78.

Synthesis of (Z)-2-(4′-Hydroxybenzylidene)-3-oxo-2,3-dihy-

drobenzofuran-7-carboxamide (57). The target compound was

prepared by following the general procedure using 36 (0.10 g, 0.56

mmol), ammonium acetate (0.05 g, 0.60 mmol), and 4-hydrox-

ybenzaldehyde (0.07 g, 0.56 mmol). The reaction mass was reﬂuxed

for 4 h and puriﬁed by column chromatography (ethyl acetate/

methanol 95:5) to obtain a pale yellow solid (0.10 g, yield 67%): mp

298−300 °C; ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 6.89 (d, J = 8.6

Hz, 2H), 6.99 (s, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.85 (s, 1H), 7.88−

7.94 (m, 4H), 8.02 (d, J = 7.6 Hz, 1H), 10.35 (s, 1H). Anal. Calcd for

C₁₆H₁₁NO₄: C, 64.21; H, 4.38; N, 4.68. Found: C, 64.65; H, 4.43; N,

4.90.

Synthesis of (Z)-3-Oxo-2-[3′-(2-piperidine-1-ylethoxy)-

benzylidene]-2,3-dihydrobenzofuran-7-carboxamide (63). The

target compound was prepared by following the general procedure

using 36 (0.09 g, 0.50 mmol), ammonium acetate (0.04 g, 0.50 mmol),

and compound 38 (0.12 g, 0.50 mmol). The mixture was reﬂuxed for 4

h and puriﬁed by ﬂash chromatography (employing a mobile phase of

ethyl acetate/methanol/2 M methanolic ammonia 90:10:2) to obtain

Synthesis of (Z)-2-(3′,4′-Dihydroxybenzylidene)-3-oxo-2,3-

dihydrobenzofuran-7-carboxamide (58). The title compound was

synthesized according to the general procedure using 36 (0.07 g, 0.39

1

bright yellow solid (0.12 g, yield 61%): mp 187−191 °C; H NMR

(400 MHz, DMSO-d₆, TMS) δ 1.09−1.28 (m, 2H), 1.33−1.58 (m,

R

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

8H), 2.74 (t, J = 5.4 Hz, 2H), 4.18 (t, J = 5.6 Hz, 2H), 7.03 (s, 1H),

7.05 (d, J = 8.0 Hz, 1H), 7.40 (t, J = 8.0 Hz, 2H), 7.60 (d, J = 6.9 Hz,

1H), 7.71 (s, 1H), 7.92 (s,1H), 7.96 (d, J = 8.0 Hz, 2H), 8.06 (d, J =

6.9 Hz, 1H). Anal. Calcd for C₂₃H₂₄N₂O₄: C, 70.39; H, 6.16; N, 7.14.

Found: C, 70.26; H, 6.39; N, 7.49.

TMS) δ 3.41−3.51 (m, 4H), 3.51−3.68 (m, 4H), 4.98 (s, 2H), 7.04 (s,

1H), 7.06 (d, J = 7.8 Hz, 2H), 7.38 (t, J = 7.7 Hz, 1H), 7.83 (s, 1H),

7.90−8.06 (m, 5H). Anal. Calcd for C₂₂H₂₀N₂O₆: C, 64.70; H, 4.94;

N, 6.86. Found: C, 65.06; H, 4.81; N, 6.80.

Synthesis of (Z)-2-(4′-Oxiranylmethoxybenzylidene)-3-oxo-

2,3-dihydrobenzofuran-7-carboxamide (69). The title compound

was obtained according to the general procedure using 36 (0.39 g, 2.20

mmol), ammonium acetate (0.15 g, 2.20 mmol), and 47 (0.39 g, 2.20

mmol). The reaction mixture was reﬂuxed for 8 h and puriﬁed by

column chromatography (employing a mobile phase of n-hexane/ethyl

acetate 60:40) to yield compound 69 as a yellowish brown solid (0.44

Synthesis of (Z)-3-Oxo-2-[4′-(2-piperidine-1-ylethoxy)-

benzylidene]-2,3-dihydrobenzofuran-7-carboxamide (64). The

title compound was synthesized according to the general procedure

using 36 (0.11 g, 0.60 mmol), ammonium acetate (0.05 g, 0.60 mmol),

and 40 (0.14 g, 0.60 mmol). The reaction mixture was reﬂuxed for 10

h and puriﬁed by ﬂash chromatography (employing a mobile phase of

ethyl acetate/methanol/2 M methanolic ammonia 90:10:5) to yield a

1

g, yield 59%): mp 167−170 °C; H NMR (400 MHz, DMSO-d₆,

1

bright yellow solid (0.13 g, yield 66%): mp 210−213 °C; H NMR

TMS) δ 2.71−2.76 (m, 1H), 2.88 (t, J = 4.6 Hz, 1H), 3.36−3.39 (m,

1H), 3.94 (dd, J = 11.2, 4.2 Hz, 1H), 4.46 (dd, J = 11.2, 3.2 Hz, 1H),

7.05 (s, 1H), 7.11 (d, J = 8.8 Hz, 2H), 7.39 (t, J = 8.2 Hz, 1H), 7.84 (s,

1H), 7.93 (d, J = 7.1 Hz, 2H), 7.96 (s, 1H), 8.04 (d, J = 8.2 Hz, 2H).

Anal. Calcd for C₁₉H₁₅NO₅: C, 67.65; H, 4.48; N, 4.15. Found: C,

67.41; H, 4.57; N, 4.13.

(400 MHz, DMSO-d₆, TMS) δ 1.33−1.41 (m, 2H), 1.49 (q, J = 5.6

Hz, 4H), 2.43 (s, 4H), 2.67 (t, J = 5.4 Hz, 2H), 4.16 (t, J = 5.9 Hz,

2H), 7.03 (s, 1H), 7.06 (d, J = 8.7 Hz, 2H), 7.37 (t, J = 7.6 Hz, 1H),

7.82 (s, 1H), 7.91 (d, J = 7.3 Hz, 1H), 7.93 (s, 1H), 8.01 (d, J = 8.3

Hz, 2H), 8.04 (s, 1H). Anal. Calcd for C₂₃H₂₄N₂O₄·¹/₂H₂O: C, 68.81;

H, 6.28; N, 6.98. Found: C, 68.96; H, 6.28; N, 6.98.

Synthesis of (Z)-2-[4′-(2-Hydroxy-3-morpholin-4-

ylpropoxy)benzylidene]-3-oxo-2,3-dihydrobenzofuran-7-car-

boxamide (70). The target compound was prepared by following the

general procedure using 36 (0.39 g, 2.20 mmol), ammonium acetate

(0.15 g, 2.20 mmol), and compound 48 (0.58 g, 2.20 mmol). The

reaction mass was reﬂuxed for 18 h and puriﬁed by column

chromatography (employing a mobile phase of DCM/methanol/2

M methanolic ammonia 90:10:2) to yield 70 as a yellowish brown

Synthesis of (Z)-2-[4′-(2-Morpholin-4-ylethoxy)-

benzylidene]-3-oxo-2,3-dihydrobenzofuran-7-carboxamide

(65). The title compound was prepared according to the general

procedure using 36 (0.12 g, 0.70 mmol), ammonium acetate (0.06 g,

0.70 mmol), and compound 39 (0.17 g, 0.70 mmol), and the mixture

was reﬂuxed for 5 h. It was puriﬁed by ﬂash chromatography

(employing a mobile phase of ethyl acetate/methanol/2 M methanolic

ammonia 90:10:2) to obtain a bright yellow solid (0.17 g, yield 64%):

mp 216−220 °C; ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 2.46−2.48

(m, 4H), 2.71 (t, J = 5.3 Hz, 2H), 3.58 (t, J = 4.4 Hz, 4H), 4.19 (t, J =

5.5 Hz, 2H), 7.03 (s, 1H), 7.08 (d, J = 8.4 Hz, 2H), 7.37 (t, J = 7.6 Hz,

1H), 7.82 (s, 1H), 7.91 (d, J = 7.8 Hz, 1H), 7.94 (s, 1H), 8.01 (d, J =

8.4 Hz, 2H), 8.04 (s, 1H). Anal. Calcd for C₂₂H₂₂N₂O₅·¹/₂H₂O: C,

65.50; H, 5.75; N, 6.94: found: C, 65.73; H, 5.74; N, 6.90.

1

solid (0.48 g, yield 52%): mp 210−212 °C; H NMR (400 MHz,

DMSO-d₆, TMS) δ 2.34−2.47 (m, 6H), 3.57 (t, J = 4.3 Hz, 4H),

3.93−4.05 (m, 2H), 4.10 (d, J = 7.2 Hz, 1H), 4.97 (s, 1H), 7.04 (s,

1H), 7.08 (d, J = 8.5 Hz, 2H), 7.38 (t, J = 7.7 Hz, 1H), 7.83 (s, 1H),

7.92 (d, J = 7.6 Hz, 1H), 7.95 (s, 1H), 8.00−8.07 (m, 3H). Anal. Calcd

for C₂₃H₂₄N₂O₆·1H₂O: C, 62.43; H, 5.92; N, 6.33: found: C, 62.19; H,

5.74; N, 6.59.

Synthesis of (Z)-2-{4′-[2-(4-Methylpiperazin-1-yl)ethoxy]-

benzylidene}-3-oxo-2,3-dihydrobenzofuran-7-carboxamide

(66). The target compound was synthesized following the general

procedure using 36 (0.08 g, 0.45 mmol), ammonium acetate (0.03 g,

0.45 mmol), and 42 (0.13 g, 0.45 mmol), and the mixture was reﬂuxed

for 12 h. It was puriﬁed by preparative TLC (employing a mobile

phase of DCM/methanol/2 M methanolic ammonia 90:10:2) to yield

compound 66 as a yellowish brown solid (0.11 g, yield 62%): mp

Synthesis of (Z)-2-(4′-{2-[4-(4-Fluorophenyl)piperazin-1-yl]-

ethoxy}benzylidene)-3-oxo-2,3-dihydrobenzofuran-7-carbox-

amide (71). The title compound was prepared as per the general

procedure using 36 (0.39 g, 2.20 mmol), ammonium acetate (0.15 g,

2.20 mmol), and 49 (0.72 g, 2.20 mmol), and the mixture was reﬂuxed

for 8 h and puriﬁed by column chromatography employing a mobile

phase of n-hexane/ethyl acetate 50:50 to yield compound 71 as a

1

bright yellow solid (0.62 g, yield 58%): mp 206−208 °C; H NMR

1

210−212 °C; H NMR (400 MHz, DMSO-d₆, TMS) δ 2.24 (s, 3H),

(400 MHz, DMSO-d₆, TMS) δ 2.65 (t, J = 4.9 Hz, 4H), 2.79 (t, J = 5.8

Hz, 2H), 3.09 (t, J = 4.4 Hz, 4H), 4.24 (t, J = 6.0 Hz, 2H), 6.90−7.13

(m, 8H), 7.38 (t, J = 7.7 Hz, 1H), 7.83 (s, 1H), 7.92 (d, J = 7.1 Hz,

1H), 7.95 (s, 1H), 8.00−8.03 (m, 2H). Anal. Calcd for C₂₈H₂₆FN₃O₄·

1H₂O: C, 65.55; H, 5.40; N, 8.19. Found: C, 65.15; H, 5.62; N, 7.98.

Synthesis of (Z)-2-{4-[2-(4-Methylpiperazin-1-yl)ethane-

sulfonylamino]benzylidene}-3-oxo-2,3-dihydrobenzofuran-7-

carboxamide (72). Compound 72 was prepared by following the

general procedure using 36 (0.39 g, 2.20 mmol), ammonium acetate

(0.15 g, 2.20 mmol), and compound 72d (0.68 g, 2.20 mmol). The

reaction mass was reﬂuxed for 6 h and puriﬁed by column

chromatography (mobile phase of DCM/methanol/2 M methanolic

ammonia 90:10:2) to yield 72 as a yellowish brown solid (0.55 g,

52%): mp 265−268 °C; ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 1.88

(s, 3H), 2.43−2.47 (m, 4H), 2.59−2.68 (m, 4H), 3.16 (t, J = 7.1 Hz,

2H), 4.02 (t, J = 7.7 Hz, 2H), 7.01 (s, 1H), 7.11 (d, J = 8.1 Hz, 2H),

7.37 (t, J = 8.0 Hz, 1H), 7.80−7.93 (m, 4H), 7.96−8.16 (m, 3H). Anal.

Calcd for C₂₃H₂₆N₄O₅S·1.5H₂O: C, 57.25; H, 5.71; N, 11.61. Found:

C, 57.51; H, 5.62; N, 11.87.

2.30−2.47 (m, 5H), 2.52−2.66 (m, 3H), 2.72 (t, J = 5.1 Hz, 2H), 4.17

(t, J = 5.4 Hz, 2H), 7.03 (s, 1H), 7.07 (d, J = 8.4 Hz, 2H), 7.37 (t, J =

7.5 Hz, 1H), 7.82 (s, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.94 (s, 1H),

7.99−8.06 (m, 3H). Anal. Calcd for C₂₃H₂₅N₃O₄·¹/₂H₂O: C, 66.40; H,

6.29; N, 10.09. Found: C, 66.76; H, 6.55; N, 9.74.

Synthesis of (Z)-2-[4′-(3-Morpholin-4-ylpropoxy)-

benzylidene]-3-oxo-2,3-dihydrobenzofuran-7-carboxamide

(67). The target compound was prepared as per the general procedure

using 36 (0.39 g, 2.20 mmol), ammonium acetate (0.15 g, 2.20 mmol),

and 44 (0.55 g, 2.20 mmol). The reaction mass was reﬂuxed for 4 h

and puriﬁed by column chromatography (employing a mobile phase of

DCM/methanol/2 M methanolic ammonia 90:10:2) to yield

compound 67 as a bright yellow solid (0.61 g, yield 68%): mp

186−190 °C; ¹H NMR (400 MHz, DMSO-d₆, TMS) δ 1.91 (q, J = 7.0

Hz, 2H), 2.32−2.40 (m, 4H), 2.43 (t, J = 7.4 Hz, 2H), 3.58 (t, J = 4.3

Hz, 4H), 4.12 (t, J = 6.0 Hz, 2H), 7.04 (s, 1H), 7.06 (d, J = 8.0 Hz,

2H), 7.38 (t, J = 7.6 Hz, 1H), 7.83 (s, 1H), 7.92 (d, J = 8.2 Hz, 1H),

7.95 (s, 1H), 7.99−8.06 (m, 3H). Anal. Calcd for

C₂₃H₂₄N₂O₅·¹/₂H₂O: C, 66.17; H, 6.04; N, 6.71. Found: C, 65.91;

H, 6.01; N, 6.69.

Synthesis of (Z)-2-[4′-(2-Morpholin-4-yl-2-oxoethoxy)-

benzylidene]-3-oxo-2,3-dihydrobenzofuran-7-carboxamide

(68). The target compound was synthesized according to the general

procedure using 36 (0.39 g, 2.20 mmol), ammonium acetate (0.15 g,

2.20 mmol), and compound 46 (0.55 g, 2.20 mmol), and the mixture

was reﬂuxed for 15 h and puriﬁed by column chromatography

(employing a mobile phase of DCM/methanol/2 M methanolic

ammonia 90:10:2) to yield compound 68 as a yellowish brown solid

(0.68 g, yield 76%): mp 258−260 °C; ¹H NMR (400 MHz, DMSO-d₆,

Synthesis of (Z)-2-(4-(3-(4-Methylpiperazin-1-yl)-

propylsulfonamido)benzylidene)-3-oxo-2,3-dihydrobenzofur-

an-7-carboxamide (73). The title compound was prepared by

following the general procedure using 36 (0.39 g, 2.20 mmol),

ammonium acetate (0.15 g, 2.20 mmol), and compound 72e (0.71 g,

2.20 mmol). The reaction mass was reﬂuxed for 8 h and puriﬁed by

column chromatography (mobile phase of DCM/methanol/2 M

methanolic ammonia 90:10:2) to yield 73 as a yellowish brown solid

1

(0.60 g, 55%): mp 287−290 °C; H NMR (400 MHz, DMSO-d₆,

TMS) δ 1.85 (s, 3H), 2.36−2.47 (m, 4H), 3.29−3.41 (m, 4H), 3.60

(q, J = 7.6 Hz, 2H), 3.84 (t, J = 7.4 Hz, 2H), 4.13 (t, J = 7.1 Hz, 2H),

S

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

7.03 (s, 1H), 7.24−7.32 (m, 2H), 7.39 (t, J = 8.2 Hz, 1H), 7.84 (s,

1H), 7.86−7.98 (m, 3H), 8.06 (d, J = 8.3 Hz, 3H). Anal. Calcd for

C₂₄H₂₈N₄O₅S·1H₂O: C, 57.36; H, 6.02; N, 11.15. Found: C, 57.46; H,

5.75; N, 11.46.

paper towels. Care was taken to avoid the splashing of the sample back

into wells. The paper towels were discarded after each tapping to

ensure thorough drying before proceeding with the assay. In the ﬁrst

phase of the assay, the ribosylation reaction was initiated into the wells

by adding 10 μL of the inhibitor solution, 15 μL of diluted PARP-1

enzyme (providing 0.5 unit/well), and 25 μL of PARP cocktail

(containing of biotinylated NAD⁺, activated DNA in Tris-Cl, pH 8.0,

and EDTA). They were serially added into each well (PARP buﬀer was

added instead of the inhibitor solution and enzyme solution in the case

of the blank reading, while PARP buﬀer was added instead of the

inhibitor solution in the case of the negative control). 3-Amino-

benzamide (3-AB), veliparib, and olaparib were used as positive

controls. The strip wells were then incubated at room temperature for

60 min and then washed twice with 200 μL of a 0.1% Triton solution

in phosphate buﬀered saline (PBS: Na₂HPO₄, NaH₂PO₄, and NaCl)

and twice with 200 μL of PBS. The wells were dried thoroughly as

mentioned before and then a 50 μL of diluted Strep-HRP (containing

a mixture of 1× Strep diluent and Strep-HRP enzyme in distilled

water, respectively) was added to each well, and the strips were further

incubated at room temperature for 60 min to detect the degree of

ribosylation. After the wells were washed twice with 200 μL of 0.1%

Triton solution in PBS and 200 μL of PBS, the wells were dried and 50

μL of TACS-Sapphire colorimetric substrate was added to each well

and allowed to stand in the dark for 10−15 min. The intensity of the

blue color that developed in each well was measured on a microplate

reader at 630 nm. Reaction was stopped by the addition of 50 μL of

5% phosphoric acid to each well, and the absorbance was measured at

450 nm. All samples were tested in triplicate. Compounds that

inhibited PARP-1 activity by ≥50% at a concentration of 25 μM (in

the case of DHBF-7-carboxamide derivatives) and 10 μM (in the case

of DHBF-3-one-7-carboxamide derivatives) were further tested to

obtain IC₅₀values. To determine the IC₅₀value for each inhibitor,

dose−response curves were generated with ﬁve to six diﬀerent

concentrations, averaging absorbance values of each inhibitor

concentration (values correlated after deducting the blank reading).

The data were plotted against the log of the concentration of each

respective inhibitor (semilog plot), and the IC₅₀value for each plot

was obtained using regression wizard from Sigma Plot, version 10.0

(San Jose, CA). Data presented are the results of at least two

independent experiments done in triplicate. The results of these

studies are presented as the mean standard deviation (SD).

Cell Lines. Chicken DT40 B cell lines used in this study were

obtained from the laboratory of Dr. Takeda; Laboratory of Radiation

Genetics, Graduate School of Medicine, Kyoto University (Kyoto,

Japan).

Synthesis of 4-Aminobenzaldehyde (72a). 72a was synthesized

by following the same protocol as that of compound 6 by treating 4-

nitrobenzaldehyde (1.0 g, 6.6 mmol) with tin chloride dihydrate (4.5 g,

19.8 mmol) in reﬂuxing ethyl acetate for 4 h. The reaction was worked

up as per the above-mentioned procedure (for compound 6) and the

sample puriﬁed by column chromatography (n-hexane/ethyl acetate

50:50) to yield compound 72a as pale yellow solid (0.46 g, yield 58%):

mp 69−72 °C (lit. 70−72 °C);⁶⁰¹H NMR (400 MHz, DMSO-d₆,

TMS) δ 5.91 (s, 2H), 6.78 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.4 Hz,

2H), 9.76 (s,1H).

Synthesis of 2-Chloro-N-(4-formylphenyl)ethane-

sulfonamide (72b). 72b was synthesized by following the same

procedure as for compound 45 by using equimolar proportions of 72a

(1.21 g, 10.0 mmol), chloroethanesulfonyl chloride (1.63 g, 10.0

mmol), and triethylamine (1.0 g, 10.0 mmol). The mixture was

allowed to stir at room temperature for 6 h and the product was

puriﬁed by column chromatography (n-hexane/ethyl acetate 80:20) to

1

yield compound 72b as a pale brown solid (1.70 g, 70%): H NMR

(400 MHz, DMSO-d₆, TMS) δ 3.35 (t, J = 7.1 Hz, 2H), 3.52 (t, J = 7.0

Hz, 2H), 7.45 (d, J = 8.8 Hz, 2H), 7.72 (s, 1H), 8.05 (d, J = 8.9 Hz,

2H), 9.95 (s, 1H).

Synthesis of 3-Chloro-N-(4-formylphenyl)propane-1-sulfo-

namide (72c). The title compound was synthesized in the same way

as that of compound 45 using equimolar proportions of 72a (1.21 g,

10.0 mmol), chloropropanesulfonyl chloride (1.77 g, 10.0 mmol), and

triethylamine (1.0 g, 10.0 mmol). The mixture was allowed to stir at

room temperature for 6 h and the product was puriﬁed by column

chromatography (n-hexane/ethyl acetate 80:20) to yield compound

1

72c as a white solid (1.69 g, 65%): H NMR (400 MHz, CDCl₃,

TMS) δ 3.78 (q, J = 7.1 Hz, 2H), 3.95 (t, J = 7.2 Hz, 2H), 4.35 (t, J =

7.1 Hz, 2H), 7.31 (d, J = 7.5 Hz, 2H), 7.80 (s, 1H), 8.09 (d, J = 7.5 Hz,

2H), 9.90 (s, 1H).

Synthesis of N-(4-Formylphenyl)-2-(4-methylpiperazin-1-yl)-

ethanesulfonamide (72d). 72d was obtained in the same way as

that of compound 37 using equimolar proportions of 72b (1.20 g, 5.0

mmol), N-methylpiperazine (0.5 g, 5.0 mmol), and potassium

carbonate (1.0 g, 7.5 mmol) when reﬂuxed for 6 h, and the product

was puriﬁed by column chromatography (mobile phase of DCM/

methanol/2 M methanolic ammonia 90:10:2) to yield 72d as a pale

1

brown solid (0.96 g, 62%): H NMR (400 MHz, DMSO-d₆, TMS) δ

2.10 (s, 3H), 2.67−2.89 (m, 6H), 3.15−3.21 (m, 4H), 3.46 (t, J = 7.1

Hz, 2H), 7.39 (d, J = 8.8 Hz, 2H), 7.69 (s, 1H), 7.90 (d, J = 8.9 Hz,

2H), 9.91 (s, 1H).

Measurement of Cellular Sensitivity to Drugs. To measure

drug cytotoxicity, cells were continuously exposed to various drug

concentrations for 72 h in triplicate. Two-hundred DT40 cells were

seeded into 384-well white plate (no. 6007680 PerkinElmer Life

Sciences) in 40 μL of medium per well. Cell viability was determined

using the ATPlite one-step kit (PerkinElmer). A 20 μL ATPlite

solution was added to each well. After 5 min, luminescence was

measured by EnVision 2104 multilabel reader (PerkinElmer). The

ATP level in untreated cells was deﬁned as 100%. Percent viability of

treated cells was deﬁned as (ATP level of treated cell/ATP level of

untreated cells) × 100. Further information on this assay is described

in ref 61.

Molecular Modeling. Computational work was carried out on a

Dell Precision 490 dual processor workstation with the Linux

operating system (Ubuntu 12.04 LTS). The initial protein structure

of PARP-1 bound with benzoxazinone inhibitor (PDB code 4L6S)³²

was reﬁned by default parameters in Protein Preparation Wizard

implemented in Maestro, version 9.5, and the Impact program, version

Synthesis of N-(4-Formylphenyl)-3-(4-methylpiperazin-1-yl)-

propane-1-sulfonamide (72e). Compound 72e was obtained in the

same way as that of compound 37 using equimolar proportions of 72c

(2.61 g, 10.0 mmol), N-methylpiperazine (1.0 g, 10.0 mmol), and

potassium carbonate (2.1 g, 15.0 mmol) when reﬂuxed for 6 h, and the

product was puriﬁed by column chromatography (mobile phase of

DCM/methanol/2 M methanolic ammonia 90:10:2) to yield 72e as a

pale brown solid (1.75 g, 54%): ¹H NMR (400 MHz, CDCl₃, TMS) δ

1.73−1.95 (m, 4H), 2.06−2.40 (m, 7H), 3.62 (q, J = 7.2 Hz, 2H), 3.85

(t, J = 7.5 Hz, 2H), 4.12 (t, 7.3 Hz, 2H), 7.33 (d, J = 7.5 Hz, 2H), 7.69

(s, 1H), 7.92 (d, J = 7.5 Hz, 2H), 9.90 (s, 1H).

Biological Methods. PARP-1 Inhibition Assay. Inhibitory

activity of the synthesized PARP-1 inhibitors was determined using

a commercially available 96-well assay kit (catalog no. 4677-096-K;

universal colorimetric PARP assay kit from Trevigen, Inc.,

Gaithersburg, MD) according to the instructions provided by the

manufacturer (with diminutive modiﬁcations) and our prior

reports.^21,22Brieﬂy, the stock solutions of the various test compounds

were made in dimethylsulfoxide (DMSO) and then serially diluted

with distilled water to the required concentrations. In the beginning

the strip wells were rehydrated by adding 50 μL of 1× PARP buﬀer

into each well followed by incubation for 30 min. After 30 min, the

microplate strip plate was tapped a few times upside down onto the

6.0 (Schrodinger, LLC, New York, NY, 2013), in which the

̈

protonation states of the ionizable residues were treated to their

dominant ionic forms at physiological pH. Reﬁned PARP structure was

further used to generate a deﬁned grid centered on a bound

benzoxazinone inhibitor. Proposed and synthesized inhibitors were

docked using the bound inhibitor grid. The inhibitory structures were

built using the build tool in Maestro, version 9.5, and energy-

T

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

minimized by the Macromodel program, version 10.1 (Schrodinger,

̈

S2−S8). This material is available free of charge via the Internet

LLC., New York, NY, 2013), using the steepest descent followed by

truncated Newton conjugate gradient protocol. LigPrep tool, version

2.7, was used to obtain the low-energy 3D structures of all inhibitors.

Default settings were used, but the “Generate Tautomers” option was

not selected. Resultant inhibitor structures were docked within the

active site of PARP-1 using the “Extra Precision” (XP) mode of Glide

docking program, version 6.0, and the default parameters. The images

were generated using PYMOL, version 1.6.

Cloning and Protein Production. The Zn1/Zn3 (a fusion of

residues 1−96 and residues 207−366) and the WGR-CAT (residues

518−1014) fragments of PARP-1 used for crystallography were

expressed from the pET24 expression vector with a C-terminal

hexahistidine tag as previously described.⁶²PARP-1 proteins were

expressed in Escherichia coli and puriﬁed as previously described using

a three-column puriﬁcation protocol.⁶³

at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*E-mail: talelet@stjohns.edu. Phone: +1-718-990-5405. Fax:

+1-718-990-1877.

■

Author Contributions

^⊥M.R.P. and A.B. contributed equally to this work.

All authors have given approval to the ﬁnal version of the

manuscript.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

Crystallization of the PARP-1/DNA Complex. Crystals of the

activated PARP-1 complex were grown as previously published with

minor modiﬁcation.²³In brief, crystals were grown in sitting drop

vapor diﬀusion at 20 °C in 3.0 μL droplets that combined a 2.1 μL

mixture of protein (300 μM Zn1/Zn3 and WGR-CAT in 25 mM

Hepes, pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.1 mM TCEP)

with 475 μM 26-bp palindromic DNA duplex (5′ GCCTACC-

GGTTCGCGAACCGGTAGGC 3′) and 0.9 μL of well solution

(6.8% PEG 3350, 2% ethylene glycol, and 100 mM Hepes, pH 7.5).

Crystals were grown to their largest size after approximately 3−4

weeks, upon which they were collected and soaked with inhibitors by

hanging drop diﬀusion at 22 °C in 7 μL droplets consisting of 5 mM

inhibitor in well solution (6.5% PEG 3350, 1.9% ethylene glycol, 5%

DMSO, 75 mM NaCl, 0.5 mM EDTA, 0.05 mM TCEP, and 95 mM

Hepes, pH 7.5). After 1−7 days of soaking, crystals were quickly

transferred to a cryo solution (3% PEG 3350, 25% ethylene glycol, 25

mM NaCl, 0.05 mM TCEP, 25 mM Hepes, pH 7.5, and 5 mM

inhibitor in 5% DMSO) prior to ﬂash-cooling in liquid nitrogen. X-ray

diﬀraction data were collected at beamline X29A of the National

Synchrotron Light Source (NSLS, Brookhaven National Laboratory),

and processed using Xia2 (Supporting Information Table S1).⁶⁴

Structure Determination and Reﬁnement. The structure of

activated PARP-1 (PDB code 4DQY)²³was used as a starting model.

The weighted F_o− F_celectron density maps calculated after rigid body

reﬁnement in REFMAC5 from the CCP4 suite resulted in signiﬁcant

diﬀerence density in one catalytic domain of the two present in the

asymmetric unit, which was used to position the inhibitor. As with the

original structure, the second copy of the PARP-1 complex is not as

well-deﬁned, presumably because of ﬂexibility. Thus, the bound

compounds were only modeled in one copy of the catalytic domain.

The inhibitors were placed according to their benzamide portion of

DHBF scaﬀold, which has a well-established binding site. The

constructed model was reﬁned in REFMAC5 using restrained and

TLS reﬁnement.⁶⁵The reﬁned models exhibit good geometry and

agree well with the original structure 4DQY, and the bound

compounds are well represented in the ﬁnal electron density maps

(Figure 3). Coordinates and structure factors have been deposited in

the Protein Data Bank (see Table S1 for the PDB entries).

■

This research was supported by the Department of

Pharmaceutical Sciences of St. John’s University and St.

John’s University Seed Grant 579-1110 to T.T.T. and by

funds from the NIH (Grant R01 GM087282) to J.M.P. J.D.S. is

supported by a Ruth L. Kirschstein National Research Service

Award. Y.P., A.C. and J.M. were supported by the Intramural

Program, Center for Cancer Research, National Cancer

Institute, NIH (Grant Z01 BC006150).

ABBREVIATIONS USED

■

3-AB, 3-aminobenzamide; ATP, adenosine triphosphate; AUC,

area under the curve; BER, base excision repair; BRCA, breast

cancer; CCDC, Cambridge Crystallographic Data Centre;

CCP4, Collaborative Computing Project Number 4; DHBF,

dihydrobenzofuran; DMF, N,N-dimethylformamide; DMSO,

dimethylsulfoxide; EDTA, ethylenediaminetetraacetic acid;

Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;

HPLC, high performance liquid chromatography; HR,

homologous recombination; NAD⁺, nicotinamide adenine

dinucleotide; NMM, N-methylmorpholine; PAR, poly(ADP-

ribose); PARG, poly(ADP-ribose)glycohydrolase; PARP, poly-

(ADP-ribose)polymerase; PBS, phosphate buﬀered saline;

PDB, Protein Data Bank; PEG, polyethylene glycol; PTEN,

phosphatase and tensin homologue; SAR, structure−activity

relationship; TCEP, tris(2-carboxyethyl)phosphine hydrochlor-

ide; TEMED, N,N,N′,N′-tetramethylethylenediamine; THF,

tetrahydrofuran; TLC, thin-layer chromatography; TLS, trans-

lation libration screw-motion; TMS, tetramethylsilane; XRCC,

X-ray cross-complementary protein

REFERENCES

■

(1) Gibson, B. A.; Kraus, W. L. New insights into the molecular and

cellular functions of poly(ADPribose) and PARPs. Nat. Rev. Mol. Cell

Biol. 2012, 13, 411−424.

(2) Luo, X.; Kraus, W. L. On PAR with PARP: cellular stress

signaling through poly(ADP-ribose) and PARP-1. Genes Dev. 2012, 26,

417−432.

Absolute Conﬁguration Determination for 12a. The absolute

structure of (R)-(−)-12a with (S)-(−)-α-methylbenzylamine was

conﬁrmed based on 1215 Bijvoet pairs measured with Cu Kα radiation

on a Bruker Kappa Apex-II DUO diﬀractometer. Reﬁnement of the

Flack parameter⁶⁶resulted in X = −0.12(15), and the Hooft

parameter⁶⁷was Y = −0.09(7), corresponding to a probability of

1.000 that the conﬁguration shown in Figure 2 is correct, in agreement

with the known (S)-conﬁguration of the amine. (CCDC 977479; full

details are in Supporting Information Tables S2−S8).

(3) Jankevicius, G.; Hassler, M.; Golia, B.; Rybin, V.; Zacharias, M.;

Timinszky, G.; Ladurner, A. G. A family of macrodomain proteins

reverses cellular mono-ADP-ribosylation. Nat. Struct. Mol. Biol. 2013,

20, 508−514.

(4) Hottiger, M. O.; Hassa, P. O.; Luscher, B.; Schuler, H.; Koch-

Nolte, F. Toward a unified nomenclature for mammalian ADP ribosyl

transferases. Trends Biochem. Sci. 2010, 35, 208−219.

(5) Rouleau, M.; Patel, A.; Hendzel, M. J.; Kaufmann, S. H.; Poirier,

G. G. PARP inhibition: PARP1 and beyond. Nat. Rev. Cancer 2010, 10,

293−301.

ASSOCIATED CONTENT

■

S

* Supporting Information

Summary of the cocrystal data of compounds (−)-13c, 59, and

65 (Table S1) and crystallographic data of (R)-(−)-12a (Table

(6) Wacker, D. A.; Frizzell, K. M.; Zhang, T.; Kraus, W. L. Regulation

of chromatin structure and chromatin dependent transcription by

U

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

poly(ADP-ribose) polymerase-1: possible targets for drug-based

therapies. Subcell. Biochem. 2007, 41, 45−69.

(7) El-Khamisy, S. F.; Masutani, M.; Suzuki, H.; Caldecott, K. W. A

requirement for PARP-1 for the assembly or stability of XRCC1

nuclear foci at sites of oxidative DNA damage. Nucleic Acids Res. 2003,

31, 5526−5533.

(8) Lan, L.; Nakajima, S.; Oohata, Y.; Takao, M.; Okano, S.;

Masutani, M.; Wilson, S. H.; Yasui, A. In situ analysis of repair

processes for oxidative DNA damage in mammalian cells. Proc. Natl.

Acad. Sci. U.S.A. 2004, 101, 13738−13743.

(9) Okano, S.; Lan, L.; Tomkinson, A. E.; Yasui, A. Translocation of

XRCC-1 and DNA ligase III-alpha from centrosomes to chromosomes

in response to DNA damage in mitotic human cells. Nucleic Acids Res.

2005, 33, 422−429.

(25) Steffen, J. D.; Brody, J. R.; Armen, R. S.; Pascal, J. M. Structural

implications for selective targeting of PARPs. Front. Oncol. 2013, 3,

301−315.

(26) Donawho, C. K.; Luo, Y.; Penning, T. D.; Bauch, J. L.; Bouska, J.

J.; Bontcheva-Diaz, V. D.; Cox, B. F.; DeWeese, T. L.; Dillehay, L. E.;

Ferguson, D. C.; Ghoreishi-Haack, N. S.; Grimm, D. R.; Guan, R.;

Han, E. K.; Holley-Shanks, R. R.; Hristov, B.; Idler, K. B.; Jarvis, K.;

Johnson, E. F.; Kleinberg, L. R.; Klinghofer, V.; Lasko, L. M.; Liu, X.;

Marsh, K. C.; McGonigal, T. P.; Meulbroek, J. A.; Olson, A. M.; Palma,

J. P.; Rodriguez, L. E.; Shi, Y.; Stavropoulos, J. A.; Tsurutani, A. C.;

Zhu, G. D.; Rosenberg, S. H.; Giranda, V. L.; Frost, D. J. ABT-888, an

orally active poly(ADP-ribose) polymerase inhibitor that potentiates

DNA-damaging agents in preclinical tumor models. Clin. Cancer Res.

2007, 13, 2728−2737.

(27) Jones, P.; Altamura, S.; Boueres, J.; Ferrigno, F.; Fonsi, M.;

Giomini, C.; Lamartina, S.; Monteagudo, E.; Ontoria, J. M.; Orsale, M.

V.; Palumbi, M. C.; Pesci, S.; Roscilli, G.; Scarpelli, R.; Schultz-

Fademrecht, C.; Toniatti, C.; Rowley, M. Discovery of 2-{4-[(3S)-

piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide (MK-4827): a

novel oral poly(ADP-ribose)polymerase (PARP) inhibitor efficacious

in BRCA-1 and -2 mutant tumors. J. Med. Chem. 2009, 52, 7170−

7185.

(28) Thomas, H. D.; Calabrese, C. R.; Batey, M. A.; Canan, S.;

Hostomsky, Z.; Kyle, S.; Maegley, K. A.; Newell, D. R.; Skalitzky, D.;

Wang, L. Z.; Webber, S. E.; Curtin, N. J. Preclinical selection of a novel

poly(ADP-ribose) polymerase inhibitor for clinical trial. Mol. Cancer

Ther. 2007, 6, 945−956.

(29) Menear, K. A.; Adcock, C.; Boulter, R.; Cockcroft, X. L.;

Copsey, L.; Cranston, A.; Dillon, K. J.; Drzewiecki, J.; Garman, S.;

Gomez, S.; Javaid, H.; Kerrigan, F.; Knights, C.; Lau, A.; Loh, V. M.,

Jr.; Matthews, I. T.; Moore, S.; O’Connor, M. J.; Smith, G. C.; Martin,

N. M. 4-[3-(4-Cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluoro-

benzyl]-2H-phthalazin-1-one: a novel bioavailable inhibitor of poly-

(ADP-ribose) polymerase-1. J. Med. Chem. 2008, 51, 6581−6591.

(30) Penning, T. D.; Zhu, G.; Gandhi, V. B.; Gong, J.; Thomas, S.;

Lubisch, W.; Grandel, R.; Wernet, W.; Park, C. H.; Fry, E. H.; Liu, X.;

Shi, Y.; Klinghofer, V.; Johnson, E. F.; Donawho, C. K.; Frost, D. J.;

Bontcheva-Diaz, V.; Bouska, J. J.; Olson, A. M.; Marsh, K. C.; Luo, Y.;

Rosenberg, S. H.; Giranda, V. L. Discovery and SAR of 2-(1-

propylpiperidin-4-yl)-1H-benzimidazole-4-carboxamide: a potent in-

hibitor of poly(ADP-ribose) polymerase (PARP) for the treatment of

cancer. Bioorg. Med. Chem. 2008, 16, 6965−6975.

(31) Griﬃn, R. J.; Calvert, A. H.; Curtin, N. J.; Newell, D. R.;

Golding, B. T. Benzoxazole-4-carboxamides and their use in inhibiting

poly(ADP-ribose) polymerase activity and improving cytotoxic

eﬀectiveness of cytotoxic drugs or radiotherapy. PCT. Int. Appl. WO

9524379 A1 19950914, 1995.

(32) Gangloff, A. R.; Brown, J.; de Jong, R.; Dougan, D. R.;

Grimshaw, C. E.; Hixon, M.; Jennings, A.; Kamran, R.; Kiryanov, A.;

O’Connell, S.; Taylor, E.; Vu, P. Discovery of novel benzo[b][1,4]-

oxazin-3(4H)-ones as poly(ADP-ribose)polymerase inhibitors. Bioorg.

Med. Chem. Lett. 2013, 23, 4501−4505.

(33) Lednicer, D.; Sun, J. H. Preparation of benzo[b]furan-7-

carboxamides as antiemetic or antipsychotic agents. Eur. Pat. Appl.

234872, 1987.

(10) Rossi, L.; Denegri, M.; Torti, M.; Poirier, G. G.; Scovassi, A. I.

Poly(ADP-ribose) degradation by post-nuclear extracts from human

cells. Biochimie 2002, 84, 1229−1235.

(11) Mangerich, A.; Burkle, A. How to kill tumor cells with inhibitors

of poly(ADP-ribosyl)ation. Int. J. Cancer 2011, 128, 251−265.

(12) Penning, T.; Zhu, G.; Gandhi, V. B.; Gong, J.; Liu, X.; Shi, Y.;

Klinghofer, V.; Johnson, E. F.; Donawho, C. K.; Frost, D. J.;

Bontcheva-Diaz, V.; Bouska, J. J.; Osterling, D. J.; Olson, A. M.; Marsh,

K. C.; Luo, Y.; Giranda, V. L. Discovery of the poly(ADP-ribose)

polymerase (PARP) inhibitor 2-[(R)-2-methylpyrrolidin-2-yl]-1H-

benzimidazole-4-carboxamide (ABT-888) for the treatment of cancer.

J. Med. Chem. 2009, 52, 514−523.

(13) Bryant, H. E.; Schultz, N.; Thomas, H. D.; Parker, K. M.;

Flower, D.; Lopez, E.; Kyle, S.; Meuth, M.; Curtin, N. J.; Helleday, T.

Specific killing of BRCA2-deficient tumors with inhibitors of

poly(ADP-ribose) polymerase. Nature 2005, 434, 913−917.

(14) Farmer, H.; McCabe, N.; Lord, C. J.; Tutt, A. N. J.; Johnson, D.

A.; Richardson, T. B.; Santarosa, M.; Dillon, K. J.; Hickson, I.; Knights,

C.; Martin, N. M. B.; Jackson, S. P.; Smith, G. C. M.; Ashworth, A.

Targeting the DNA repair defect in BRCA mutant cells as a therapeutic

strategy. Nature 2005, 434, 917−921.

(15) Kummar, S.; Chen, A.; Parchment, R. E.; Kinders, R. J.; Ji, J.;

Tomaszewski, J. E.; Doroshow, J. H. Advances in using PARP

inhibitors to treat cancer. BMC Med. 2012, 10, 25.

(16) Curtin, N. J.; Szabo, C. Therapeutic applications of PARP

inhibitors: anticancer therapy and beyond. Mol. Aspects Med. 2013, 34,

1217−1256.

(17) Papeo, G.; Casale, E.; Montagnoli, A.; Cirla, A. PARP inhibitors

in cancer therapy: an update. Expert Opin. Ther. Pat. 2013, 23, 503−

514.

(18) Underhill, C.; Toulmonde, M.; Bonnefoi, H. A review of PARP

inhibitors: from bench to bedside. Ann. Oncol. 2011, 22, 268−279.

(19) Turner, N.; Tutt, A.; Ashworth, A. Hallmarks of “BRCAness” in

sporadic cancers. Nat. Rev. Cancer 2004, 4, 814−819.

(20) Mendes-Pereira, A. M.; Martin, S. A.; Brough, R.; McCarthy, A.;

Taylor, J. R.; Kim, J. S.; Waldman, T.; Lord, C. J.; Ashworth, A.

Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors.

EMBO Mol. Med. 2009, 1, 315−322.

(21) Patel, M. R.; Pandya, K. G.; Lau-Cam, C. A.; Singh, S.; Pino, M.

A.; Billack, B.; Degenhardt, K.; Talele, T. T. Design and synthesis of

N-substituted indazole-3-carboxamides as poly(ADP-ribose)-

polymerase-1 (PARP-1) inhibitors. Chem. Biol. Drug Des. 2012, 79,

488−496.

(22) Kulkarni, S. S.; Singh, S.; Shah, J. R.; Low, W. K.; Talele, T. T.

Synthesis and SAR optimization of quinazoline-4(3H)-ones as

poly(ADP-ribose)polymerase-1 inhibitors. Eur. J. Med. Chem. 2012,

50, 264−273.

(34) Hogberg, T.; Paulis, T.; Johansson, L.; Kumar, Y.; Hall, H.;

Ogren, S. Potential antipsychotic agents. 7. Synthesis and

antidopaminergic properties of the atypical highly potent (S)-5-

bromo-2,3-dimethoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl]benzamide

and related compounds. A comparative study. J. Med. Chem. 1990, 33,

2305−2309.

(35) Meyers, A.; Reuman, M.; Gabel, R. A. Nucleophilic annulations

of aromatics. Novel route to benzo-fused ring systems via oxazoline

activation. J. Org. Chem. 1981, 46, 783−788.

(36) Meshram, H. M.; Premalatha, K.; Rameshbabu, K.; Eeshwaraiah,

B.; Yadav, Y. S. Zirconium(IV) chloride catalyzed cyclization of ortho-

allylphenols: synthesis of 2-methyl-2,3-dihydrobenzofurans. Synth.

Commun. 2004, 34, 3091−3097.

(23) Langelier, M. F.; Plank, J. L.; Roy, S.; Pascal, J. M. Structural

basis for DNA damage-dependent poly(ADP-ribosyl)ation by human

PARP-1. Science 2012, 336, 728−732.

(24) Ferraris, D. V. Evolution of poly(ADP-ribose) polymerase-1

(PARP-1) inhibitors. From concept to clinic. J. Med. Chem. 2010, 53,

4561−4584.

V

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(37) Rozenberg, V.; Dubrovina, N.; Sergeeva, E.; Antonov, D.;

Belokon, Y. An improved synthesis of (S)-(+)- and (R)-(−)-[2.2]-

paracyclophane-4-carboxylic acid. Tetrahedron: Asymmetry 1998, 9,

653−656.

(38) Irie, R.; Noda, K.; Ito, Y.; Matsumoto, N.; Katsuki, T. Catalytic

asymmetric epoxidation of unfunctionalized olefins using chiral

(salen)manganese(III) complexes. Tetrahedron: Asymmetry 1991, 2,

481−494.

(39) He, Q.; Gomma, H.; Rohani, S.; Zhu, J.; Jennings, M. Chiral

discrimination in diastereomeric salts of chlorine-substituted mandelic

acid and phenylethylamine. Chirality 2010, 22, 707−716.

(40) Stein, A. R.; Dawe, R. D.; Sweet, J. R. Preparation of chiral 1-

phenylethanols and bromides. Can. J. Chem. 1985, 63, 3442−3448.

(41) Riether, D.; Harcken, C.; Razavi, H.; Kuzmich, D.; Gilmore, T.;

Bentzien, J.; Pack, E. J. J.; Souza, D.; Nelson, R. M.; Kukulka, A.; Fadra,

T. N.; Zuvela-Jelaska, L.; Pelletier, J.; Dinallo, R.; Panzenbeck, M.;

Torcellini, C.; Nabozny, G. H.; Thomson, D. S. Nonsteroidal

dissociated glucocorticoid agonists containing azaindoles as steroid

A-ring mimetics. J. Med. Chem. 2010, 53, 6681−6698.

(42) Hadj-esfandiari, N.; Navidpour, L.; Shadnia, H.; Amini, M.;

Samadi, N.; Faramarzi, M. A.; Shafiee, A. Synthesis, antibacterial

activity, and quantitative structure−activity relationships of new (Z)-2-

(nitroimidazolylmethylene)-3(2H)-benzofuranone derivatives. Bioorg.

Med. Chem. Lett. 2007, 17, 6354−6363.

(43) Zlatoidsky, P.; Maliar, T. Synthesis and structure−activity

relationship study of the new set of trypsin-like proteinase inhibitors.

Eur. J. Med. Chem. 1999, 34, 1023−1034.

(44) Pielichowski, J.; Popielarz, R. Trichloroethylene in organic

synthesis: II. Reaction of trichloroethylene with secondary amines.

Tetrahedron 1984, 40, 2671−2675.

optimization of N³-phenylpyrazinones as corticotropin-releasing

factor-1 (CRF₁) receptor antagonists. J. Med. Chem. 2009, 52,

4161−4172.

(55) Rhoads, S. J.; Raulins, R.; Reynolds, R. D. The para-Claisen

rearrangement. I. The preparation and rearrangement of the α- and γ-

ethylallyl ethers of methyl ο-cresotinate. A reinvestigation. J. Am.

Chem. Soc. 1954, 76, 3456−3463.

(56) Nagarapu, L.; Aneesa; Satyender, A.; Chandana, G.; Bantu, R.

Synthesis and antimicrobial activity of novel analogs of trifenagrel. J.

Heterocycl. Chem. 2009, 46, 195−200.

(57) Katz, L.; Karger, L. S.; Schroeder, W.; Cohen, M. S. Hydrazine

derivatives. I. Benzalthio- and bisbenzaldithiosalicoylhydrazides. J. Org.

Chem. 1953, 18, 1380−1402.

(58) Ouach, A.; Gmouh, S.; Pucheault, M.; Vaultier, M. Onium salt

supported organic synthesis in water: application to Grieco’s

multicomponent reaction. Tetrahedron 2008, 64, 1962−1970.

(59) Ohno, T.; Ogawa, K.; Yano, S.; Fukushima, M.; Suzuki, N.;

Asao, T. Synthesis and structure−activity relationship of 4-substituted

benzoic acids and their inhibitory effect on the biosynthesis of fatty

acids and sterols. Arch. Pharm. 2005, 338, 147−158.

(60) Keelara, A.; Srinivasa, G. R.; Channe, G. D. Palladium-catalyzed

simple and efficient hydrogenative cleavage of azo compounds using

recyclable polymer-supported formate. Can. J. Chem. 2005, 83, 517−

520.

(61) Maede, Y.; Shimizu, H.; Fukushima, T.; Kogame, T.; Nakamura,

T.; Miki, T.; Takeda, S.; Pommier, Y.; Murai, J. Differential and

common DNA repair pathways for topoisomerase I- and II-targeted

drugs in a genetic DT40 repair cell screen panel. Mol. Cancer Ther.

2014, 13, 214−220.

(62) Steffen, J. D.; Tholey, R. M.; Langelier, M. F.; Planck, J. L.;

Schiewer, M. J.; Lal, S.; Bildzukewicz, M. A.; Yeo, C. J.; Knudsen, K. E.;

Brody, J. R.; Pascal, J. M. Targeting PARP-1 allosteric regulation offers

therapeutic potential against cancer. Cancer Res. 2014, 74, 31−37.

(63) Langelier, M. F.; Plank, J. L.; Servent, K. M.; Pascal, J. M.

Purification of human PARP-1 and PARP-1 domains from Escherichia

coli for structural and biochemical analysis. Methods Mol. Biol. 2011,

780, 209−226.

(64) Winter, G. Xia2: an expert system for macromolecular

crystallography data reduction. J. Appl. Crystallogr. 2010, 43, 186−190.

(65) Winn, M. D.; Isupov, M. N.; Murshudov, G. N. Use of TLS

parameters to model anisotropic displacements in macromolecular

refinement. Acta Crystallogr. 2001, D57, 122−133.

(66) Flack, H. D. On enantiomorph-polarity estimation. Acta

Crystallogr. 1983, A39, 876−881.

(45) Knoevenagel, E. Condensation of malonic acid with aromatic

aldehydes with ammonia and amines. J. Am. Chem. Soc. 1898, 31,

2596−2619.

(46) Scarpelli, R.; Boueres, J. K.; Cerretani, M.; Ferrigno, F.; Ontoria,

J. M.; Rowley, M.; Schultz-Fademrecht, C.; Toniatti, C.; Jones, P.

Synthesis and biological evaluation of substituted 2-phenyl-2H-

indazole-7-carboxamides as potent poly(ADP-ribose) polymerase

(PARP) inhibitors. Bioorg. Med. Chem. Lett. 2010, 20, 488−492.

(47) Ferraris, D.; Ficco, R. P.; Pahutski, T.; Lautar, S.; Huang, S.;

Zhang, J.; Kalish, V. Design and synthesis of poly(ADP-ribose)-

polymerase-1 (PARP-1) inhibitors. Part 3: In vitro evaluation of

1,3,4,5-tetrahydro-benzo[c][1,6]- and [c][1,7]-naphthyridin-6-ones.

Bioorg. Med. Chem. Lett. 2003, 13, 2513−2518.

(48) Varma, R.; Varma, M. Alumina-mediated condensation. A

simple synthesis of aurones. Tetrahedron Lett. 1992, 33, 5937−5940.

(49) Lea, T. V.; Suhb, J. H.; Kimb, N.; Park, H. In silico identification

of poly(ADP-ribose)polymerase-1 inhibitors and their chemosensitiz-

ing effects against cisplatin-resistant human gastric cancer cells. Bioorg.

Med. Chem. Lett. 2013, 23, 2642−2646.

(67) Hooft, R. W.; Straver, L. H.; Spek, A. L. Determination of

absolute structure using Bayesian statistics on Bijvoet differences. J.

Appl. Crystallogr. 2008, 41, 96−103.

(50) Murai, J.; Huang, S. N.; Das, B. B.; Renaud, A.; Zhang, Y.;

Doroshow, J. H.; Ji, J.; Takeda, S.; Pommier, Y. Trapping of PARP1

and PARP2 by clinical PARP inhibitors. Cancer Res. 2012, 72, 5588−

5599.

(51) Wahlberg, E.; Karlberg, T.; Kouznetsova, E.; Markova, N.;

̈

Macchiarulo, A.; Thorsell, A. G.; Pol, E.; Frostell, Å.; Ekblad, T.; Oncu,

̈

D.; Kull, B.; Robertson, G. M.; Pellicciari, R.; Schuler, H.; Weigelt, J.

Family-wide chemical profiling and structural analysis of PARP and

tankyrase inhibitors. Nat. Biotechnol. 2012, 30, 283−288.

̈

(52) Murai, J.; Huang, S. N.; Renaud, A.; Zhang, Y.; Ji, J.; Takeda, S.;

Morris, J.; Teicher, B.; Doroshow, J. H.; Pommier, Y. Stereospecific

PARP trapping by BMN 673 and comparison with olaparib and

rucaparib. Mol. Cancer Ther. 2014, 13, 433−443.

(53) Cohen, M. L.; Laceﬁeld, W. B. Preparation of 5-HT3 antagonist.

Eur. Pat. Appl. 307172 A2 19890315, 1989.

(54) Hartz, R. A.; Ahuja, V. T.; Rafalski, M.; Schmitz, W. D.; Brenner,

A. B.; Denhart, D. J.; Ditta, J. L.; Deskus, J. A.; Yue, E. W.; Arvanitis, A.

G.; Lelas, S.; Li, Y.; Molski, T. F.; Wong, H.; Grace, J. E.; Lentz, K. A.;

Li, J.; Lodge, N. J.; Zaczek, R.; Combs, A. P.; Olson, R. E.; Mattson, R.

J.; Bronson, J. J.; Macor, J. E. In vitro intrinsic clearance-based

W

dx.doi.org/10.1021/jm5002502 | J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery and structure-activity relationship of novel 2,3- dihydrobenzofuran-7-carboxamide and 2,3-dihydrobenzofuran-3(2 h)-one-7-carboxamide derivatives as poly(ADP-ribose)polymerase-1 Inhibitors

DOI: 10.1021/jm5002502

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm5002502

Full text of DOI:10.1021/jm5002502

Products guided by the article

R&D Labs maybe for 74727-56-3

Relevant to this article

Hot Product