Synthesis and Biological Evaluation of Phthalazinone Inhibitors of

Synthesis and Biological Evaluation of Phthalazinone Inhibitors of Cryptosporidium

parvum Inosine-5’-Monophosphate Dehydrogenase

Master’s Thesis

Presented to

The Faculty of the Graduate School of Arts and Sciences

Brandeis University

Department of Chemistry

Lizbeth Hedstrom, Advisor

In Partial Fulfillment

of the Requirements for

Master’s Degree

By

Corey R. Johnson

August 2011

ii

ABSTRACT

Phthalazinone Inhibitors of Cryptosporidium parvum Inosine-5’-Monophosphate Dehydrogenase

A thesis presented to the Chemistry Department

Graduate School of Arts and Sciences

Brandeis University

Waltham, Massachusetts

By Corey R. Johnson

Cryptosporidium parvum has a salvaged guanine nucleotide biosynthetic pathway

for metabolism in which inosine-5’-monophosphate dehydrogenase (IMPDH) plays

a key role. The characterization of C. parvum IMPDH has instigated a drug discovery

program exploiting a nicotinamide adenine dinucleotide (NAD+) active site for

selective inhibition over its human counterpart. A series of N-aryl-4-oxophthalazine

acetamide inhibitors are described. For acyclic substituted anilines, structure-

activity relationship revealed that electron-withdrawing substituents at the para-

position are required. An additional substituent in the meta-position improved

potency up to 10-fold. It was proposed that pseudo-ring formation due to π-π

interactions of substituents at the 3- and 4- positions of the aniline ring may be

iii

inducing enzyme inhibition. Further optimization utilizing the pseudo-ring

hypothesis has resulted in the discovery of new benzofuranamide analogs exhibiting

low nanomolar enymatic inhibition against CpIMPDH, and low nanomolar minimum

inhibitory concentration against the fatal disease-causing biowarfare bacteria

Franciscella tularensis.

iv

Table of Contents

i. Title Page.

ii-iii. Abstract.

iv. Table of Contents.

v. List of Tables.

vi. List of Illustrations/Figures.

1-3. Introduction.

4-18. Results and Discussion.

19. Conclusion.

19-20. Methods.

20-32. Experimental.

33. Bibliography.

v

List of Tables

Table I. Initial lead optimization/IC50 values for Acyclic analogs.

Table II. Van der Waals radii distances and Conformations between

groups.

Table III. IC50 values for Cyclic analogs.

Table IV. Anti-cryptosporidial/toxoplasmodial activities.

Table V. Anti-parasitic activities against potential biowarfare agents.

vi

List of Illustrations/Figures

Figure I. IMPDH reaction scheme.

Figure II: CpIMPDH inhibitor identified by HTS.

Figure III. Intramolecular interactions of 3-halo-, 3-methyl- and 3-

trifluoromethyl- series.

Figure IV. Ball-and-stick model of Cmpd 30. (ChemBio3D Ultra)

Figure V. Ball-and-stick model of Cmpd 34. (ChemBio3D Ultra)

Scheme I. General Procedure for the Synthesis of Amide Derivatives.

1

Introduction

Outbreaks worldwide have instigated drug discovery programs against the

waterborne protozoan parasite Cryptosporidium parvum1. A calf is capable of

producing enough oocysts to infect millions of people rendering C. parvum a

bioterrorism threat2. Drug discovery for C. parvum is challenging due to absence of

continuous cell culture. Moreover, drugs currently on the market for C. parvum are

ineffective.

Genomic analysis of C. parvum has revealed a guanine nucleotide

biosynthetic pathway which implies that inosine-5’-monophosphate dehydrogenase

(IMPDH) could serve a key role in parasite metabolism3. Guanine nucleotide

pathways generally abide by the following sequence: AMP is converted to IMP by

adenine deaminase (ADA); IMP is converted to XMP by IMPDH; XMP is converted to

GMP by GMP synthase. The absence of guanine/xanthine

phosphoribosyltransferases or guanosine/xanthosine kinases, or collectively

salvage enzymes in this scheme, indicates that IMPDH controls the guanine

nucleotide metabolism sequence for this parasite.

IMPDH oxidizes IMP to XMP via a redox reaction that produces NADH and a

Cysteine-linked covalent enzyme intermediate E-XMP*, and a hydrolysis reaction

which releases XMP. Since NADH absorbs light at wavelength 340 nm, its

production via concentration can be detected by UV-VIS spectrometer, a decrease in

its concentration was used as the indicator of C. parvum IMPDH inhibition.

2

Figure I. IMPDH reaction scheme

C. parvum’s IMPDH differs from human IMPDH due to a diverged

nicotinamide adenosine dinucleotide (NAD+) site4. This NAD+ site has been exploited

in high throughput screens as a potential drug target for C. parvum5. These

discoveries have resulted in two previously published series of C. parvum IMPDH

inhibitors which exhibit in vitro cell culture model of infection6. In this work, we

have established a structure-activity relationship of phthalazine-based inhibitors

derived from another hit from the same high throughput screening which exhibit

potency in cell culture model of infection.

Figure II: CpIMPDH inhibitor identified by HTS

The lead compound (7) exhibited an IC50 of 945 nM upon resynthesis.

3

Compound 7 and its analogs were prepared following a 4-step sequence (Scheme 1)

beginning with the Wittig olefination of phthalic anhydride (1) with

carbethoxymethylidenetriphenylphosphorane in chloroform to produce (E)-ethyl-

2-(3-oxoisobenzofuran-1(3H)-ylidene)acetate (2)7. Then, a Gabriel synthesis in

which a hydrazine was added to the phthalide in refluxing ethanol to produce a

phthalazine acetoester(3 & 4)7. The acetoester was then hydrolyzed to produce a

phthalazine acetic acid(5 & 6)7. and the acetic acid was amidated with various

aromatic amines in DMSO to afford the final compounds (7-60) which were tested

for in vitro enzyme assay (Minjia Zhang, Hedstrom Laboratory) and anti-parasitic

activity (Striepen Laboratory, Univ. of Georgia).

4

Results and Discussion

Scheme I. General Procedure for the Synthesis of Amide Derivatives.

Reagents and Conditions : a) Ph3PCHCO2Et, CHCl3, reflux, 16 h. b) NH2-NH2 or

NH2-NHMe, EtOH, reflux, 3 h. c) 3M NaOH/THF, reflux, 2 h followed by

acidification. d) R’-NH2, EDC, HOBt, DIPEA, DMSO, 3-5 h.

Table I. Initial lead optimization/IC50 values for Acyclic analogs.

Cmpd R R’ (-)-BSA (nM) (+)-BSA (nM)

D1 7 Me 4-OMePh 1000 ± 250 970 ± 250

D20 8 Me 3-OMePh >5000 ND

5

D21 9 Me 2-OMePh >5000 ND

D23 10 Me -CH2(4-OMePh) >5000 ND

D22 11 Me 4-OEtPh >5000 ND

D40 12 Me 4-OCF3Ph >5000 ND

D30 13 Me 4-FPh >5000 ND

D24 14 Me 4-ClPh 240 ± 58 240 ± 54

D29 15 Me 4-BrPh 120 ± 11 120 ± 20

D75 16 Me 4-CNPh 950* 940*

D34 17 Me 4-MePh >5000 ND

D39 18 Me 4-CF3Ph >5000 ND

D6 19 Me 4-iPrPh >5000 ND

D14 20 Me tBu >5000 ND

D8 21 Me NH2 >5000 ND

D19 22 Me 4-SO2MePh >5000 ND

D27 23 Me 3,4-ClPh 60 ± 6.5 100 ± 2.5

D32 24 Me 2,4-ClPh >5000 ND

D43 25 Me 3,5-ClPh >5000 ND

D42 26 Me 3,4,5-ClPh >5000 ND

D46 27 Me 3,4,5-FPh >5000 ND

D31 28 Me 3-Cl-4-BrPh 42 ± 0 110 ± 1.5

D45 29 Me 3-CF3-4-BrPh 23* 41*

6

D48 30 Me 3-CF3-4-ClPh 13 ± 0.82 25 ± 7.1

D50 31 Me 3-CF3-4-CNPh 81 ± 32 130 ± 100

D49 32 Me 3-Cl-4-CNPh 290 ± 51 470 ± 170

D60 33 Me 3-CF3-4-FPh 150 ± 32 150 ± 41

D53 34 Me 3-CF3-4-OMePh >5000 ND

D54 35 Me 3-CF3-4-NH2Ph >5000 ND

D51 36 Me 3-CF3Ph >5000 ND

D58 37 H 3-CF3-4-ClPh 40 ± 1.7 61 ± 1.7

D59 38 H 3-CF3-4-BrPh 17* 42*

D52 39 Me 3-F-4-ClPh 200 ± 41 140 ± 45

D68 40 Me 3-Me-4-ClPh 66 ± 12 100 ± 26

D74 41 Me 3-Me-4-BrPh 82* 150*

D76 42 Me 3-Me-4-CNPh 650* 640*

D69 43 Me 3-OMe-4-ClPh 31* 31*

D57 44 Me 2-CF3-4-ClPh >5000 ND

D56 45 Me 2-CF3-4-BrPh >5000 ND

D33 46 Me 2-F-4-BrPh 1500* 1700*

D38 47 Me 2,4-BrPh >5000 ND

D41 48 Me 2-naphthyl 61* 134*

D77 49 Me 3,4-CNPh 2500* 2500*

D28 50 Me 2-(1,3,4- >5000 ND

7

thiazolyl)

D35 51 Me 2-(5-Me(1,3,4-

thiazolyl) >5000 ND

The D series shares the same general trend with the other two series6; that is,

halogen groups in the para-position larger than fluorine (Compounds 14 and 15) are

preferred for activity and potency increases with the van der Waals area/volume of

the halogen6,11. For ether groups in the para position, this trend is not present as the

bigger ethoxy group (Cmpd 11) is inactive. The 3,4- combination of halogens

(Cmpds 23 and 28) exhibits greater potency but does not increase with van der

Waals area of the functional groups. From this discovery came the idea that the

substituents were forming pseudo-rings to suit a conformation preferred by

CpIMPDH. We hypothesized that these interactions to be either halogen bonding or

π-π interactions, and designed experiments to test either phenomena correlating to

CpIMPDH inhibition.

Halogen bonding is the non-covalent interaction between an electron-rich

functional group and a halogen8,9; it is analogous to hydrogen bonding. Halogen

bonding improves with increased differences in lewis acidity of the acceptor

halogen group (lewis acid) and the donor group (lewis base)9. Acidity increases

down the periodic table. Fluorine is the least acidic halogen and the most basic, and

astatine is the most acidic and least basic halogen9. For example, the difference in

substituent lewis acidities of compound 27 (3,4-ClPh) is significantly less than

8

compound 31 (3-Cl-4-BrPh) because bromine is more acidic than chlorine. The

increase in potency from compound 27 to compound 31 correlates to the increased

differences in substituent lewis acidities for these compounds which insists halogen

bonding induces enzyme inhibition. Also, the difference in substituent lewis

acidities for compound 29 (3-CF3-4-BrPh) is significantly less than that of

compound 30 (3-CF3-4-ClPh). However, there was no increase in potency from

compound 30 to compound 29. Compounds 29 and 30 did not follow the expected

potency differences correlating to the differences in substituent lewis acidities

which insists that enzyme inhibition is not induced by halogen bonding. This

discovery lead to designed experiments for which the presence of halogen-pi or pi-

pi interactions could be determined vital to induction of enzyme inhibition.

Compounds 33 (3-CF3-4-FPh) and 31 (3-CF3-4-CNPh) with the more

electron-withdrawing fluorine and cyano moieties in the para-position did not

exhibit greater potency than 30 (3-CF3-4-ClPh). We believe this is due to the

electron-withdrawing of the para-positioned functional group. Bromine is not very

electron-withdrawing, but cyano and fluorine groups are. The cyano and fluorine

groups are more competitive with the trifluoromethyl group for electron-

withdrawal from the aromatic ring which perturbs the basicity of the fluorine

groups of the trifluoromethyl substituent and disrupt the interactive relationship.

The inactivity of 13 (4-FPh) suggests presence of pseudo-ring formation in 33 (3-

CF3-4-FPh).

9

Figure III. Intramolecular interactions of 3-halo-, 3-methyl- and 3-

trifluoromethyl- series.

X= -Cl, -CH3, -OMe, -CN, -F, -Br.

Chlorine is a moderate competitor to the trifluoromethyl group and is more

electron-demanding than bromine, which makes it more suitable for this interaction

as observed with 30. The inactivity of compounds 24-27 (2,4-ClPh; 3,5-ClPh; 3,4,5-

ClPh; 3,4,5-FPh) confirm that this relationship is exclusive to 3,4-substitutions.

Compound 39 (3-F-4-ClPh) suggests that fluorine directly connected to the

aromatic ring may be too strong a donor for the chlorine group, and may actually

promote repulsion between the adjacent chlorine substituent. Fluorines are

typically strongly basic due to their electron-withdrawal from their counterparts in

organic compounds, but in this series the carbon of the trifluoromethyl group limits

the availability of electrons to be withdrawn by the fluorines because of its lower

electronegativity and its incapability to expand its octet. The oxygens of a nitro

group are more basic/electron-rich than fluorines of the trifluoromethyl group

because of the higher electronegativity and its capability to expand its octet via its

10

lone pair. Electron-withdrawing para-substitutions will compete with the

trifluoromethyl group for electrons withdrawn from the aromatic ring.

Compounds 40 and 41 (3-Me-4-Cl, 4-Br) instigated the idea of resonance

hybrid double-bond interactions with the electron-donating methyl group in the

meta-position which enhanced inhibitory activity from 14 (4-ClPh) and is

equipotent with its 3-chloro counterparts (Cmpds 23 and 28). Compound 42 (3-Me-

4-CNPh) confirms that the existing trend is due exclusively to π-π interactions of

resonance hybrids with no halogen substituents on the aniline ring. Therefore, we

conclude that one or more of the fluorines of the trifluoromethyl group or the

methyl group in the meta-position is forming pseudo-rings via π-π interactions with

the para-substituent via resonance hybridization. Outstandingly, compounds 23

(3,4-ClPh) and 40 (3-Me-4ClPh) have similar potencies against CpIMPDH. Other

factors concerning the 3-trifluoromethyl SAR were discussed earlier in this work.

Compound 37 (3-CF3-4-ClPh) suggests that removal of the methyl at N-2 is

detrimental to inhibitory activity when 3-trifluoromethyl-4-chloro moiety is

installed on aromatic ring. The enhanced inhibitory activity from 37 (3-CF3-4-ClPh)

to 38 (3-CF3-4-BrPh) shows that this subseries of compounds follows the size trend

for the other series (A, C) of inhibitors against CpIMPDH6.

ChemBio3D Ultra MM2 minimizations of compounds 30 and 34.

11

Figure IV. Ball-and-stick model of Cmpd 30. (ChemBio3D Ultra)

Figure V. Ball-and-stick model of Cmpd 34. (ChemBio3D Ultra)

Parameters: 500 iterations, Step Interval = 1.0 femtoseconds (fs), Frame Interval =

10 fs, Heating/Cooling Rate = 1 kcal/atom/picosecond, Target Temperature = 310 K.

12

Table II. Van der Waals radii distances and Conformations between functional

groups.

Å Cmpd

Cl, F(25) Cl, F(26) Cl, F(27) vdW Sum Conf.

30 3.03 4.41 3.06 3.22 Gauche - Cl, F(25),

F(27) O, F(26) O, F(27) O, F(28) vdW Sum Conf.

4.09 2.71 2.91 2.99 Gauche -

F(28), F(27), O

C, F(26) C, F(27) C, F(28) vdW Sum Conf. 34

5.48 4.03 4.12 3.17 Out of plane -

C

ChemBio3D Ultra was used to energy minimize 30 (3-CF3-4-ClPh) and 34 (3-

CF3-4-OMePh) for the van der Waals radii distances, molecular conformations, and

pseudo-ring formations. The distances between F(25) and F(27) of the

trifluoromethyl group and the chlorine of 30 are significantly less than the sum of

the van der Waals radii (3.22 Å)11 and indicates presence of an interaction between

the elements.

In compound 34 (3-CF3-4-OMePh), the distance between two of the fluorines

of the trifluoromethyl group and the ether are less than the sum of their Van der

Waals radii (2.99 Å)11; similarity in distance (2.74 Å, 2.87 Å) suggests gauche

conformation. As predicted, the distance between the fluorines of the

trifluoromethyl group and the methyl of the methoxy group are greater than van der

Waals radii (3.17 Å). We believe the lack of inhibitory activity of 34 and similar

compounds is due to the out-of-plane conformation of groups like the methyl of

13

methoxy group. We believe that this consequential out-of-plane group is responsible

for the inactivity of compound 10 (4-OEtPh) in comparison with its active

counterpart compound 6 (4-OMePh) also.

The potency differences between 30 (3-CF3-4-ClPh) and 23 (3,4-ClPh) are

assumed due to a preference for the 5-membered pseudo-ring over the 4-membered

pseudo-ring by the enzyme. This hypothesis instigated the synthesis of 2-

benzofuranamides to further investigate this phenomenon. Halogens and olefins are

sometimes classified as weak hydrogen-bond acceptors9. We proposed that the

hydrogen-bond accepting ether oxygen of the furan would replace the role of the

chlorine as a hydrogen bond acceptor, and the ethylene group would both occupy

the space of and replace the role of the trifluoromethyl group as a hydrogen bond

acceptor.

Table III. IC50 values for Cyclic analogs.

Cmpd R (-)-BSA (nM) (+)-BSA (nM)

D61 52

200 ± 76 180 ± 80

14

D64 53

>5000 ND

D62 54

70 ± 22 100 ± 45

D67 55

20 ± 6.6 150 ± 25

D73 56

4.0 ± 1.8 33 ± 14

D72 57

>5000 ND

D78 58

1500* 1900*

D70 59

>5000 ND

D71 60

>5000 ND

The benzofuran analog (52) exhibited moderate inhibitory activity as

expected. A methyl was added at the 2-position to investigate generality of the

15

benzofuran series of inhibitors. However, the “naked” double bond of the

benzofuran moiety is very prone to oxidation which would deem it metabolically

labile to liver enzymes12; with acyclic substituents in the 2,3-positions of the furan

ring as large as a phenyl group, the double bond is still metabolized to the highly

reactive epoxide intermediate12. Therefore, fused rings were added to the 2,3-

positions of the benzofuran (Cmpds 55, 56, and 57) in an effort to divert oxidation

from this site to that of the fused ring moieties should in vivo testing with these

compounds be attempted. Fused ring analogs were more active than its methylated

and non-methylated counterparts. Aromatization of the fused-ring in Compound 55

(6,7,8,9-tetrahydrodibenzofuran-3-yl) to 56 (dibenzofuran-3-yl) resulted in ~3-fold

increase of inhibitory activity. Aromatic rings are significantly higher in energy than

their saturated counterparts with the exception of cyclohexadiene; the heats of

hydrogenation of benzene and cyclohexene are 208 and 102 kJ/mol13. The inactivity

of 57 (dibenzofuran-2-yl) verifies the requirement of the ether oxygen in the para-

position of the aniline ring and intolerance in the meta-position. The carbazoles

(Cmpds 59 and 60) were completely inactive, which implies that the amines are not

tolerated by the enzyme. This suggests that functional groups only capable of very

weak hydrogen-bond acceptor capabilities such as olefins, halogens, ether oxygens,

and resonating small alkyl groups are permitted by the enzyme. Moreover, the fused

furan substitutions are strongly electron-donating in opposition to the 3,4-halogen

and pseudo-halogen counterparts which confirms that increasing inhibition is not

due to the electron-deficiency of the aniline ring. Moreover, the fused furan

16

substitutions are strongly electron-donating in opposition to the 3,4-halogen and

pseudo-halogen counterparts which confirms that increasing inhibition is not due to

the electron-deficiency of the aniline ring which one may interpret from the Topliss

Tree10; Topliss Tree suggests next compound be the extremely electron-

withdrawing 3-CF3-4-NO2Ph from 3-CF3-4-ClPh for enhanced inhibition; in this work

we have achieved higher inhibition with the extremely electron-donating

dibenzofuran from 3-CF3-4-ClPh.

Cell-culture model of infection

Table IV. Anti-cryptosporidial/toxoplasmodial activities.

Performed by the Striepen Laboratory at Univ. of Georgia.

Cmpd Toxo WT

(µM)

Toxo HX

(µM)

Toxo WT/

CpIMPDH

(µM)

Selectivity

WT/

CpIMPDH

C. parvum

(µM)

LIVE/DEAD

assay

(µM)

29 5 ± 3 12 ± 11 0.24 ± 0.2 40 - -

30 23 ± 4 17 ± 12 0.10 ± 0.2 83 3.1 -

31 - - - - - >10

55 - - - - - 7.7

56 3 ± 3 3 ± 2 0.40 ± 0.08 7 >10 -

*minimum concentration where toxicity observed.

17

Trends in potency seemed to increase with added energy in the benzofuran

series. However, compound 56 is inactive in the cell-culture model of infection of the

parasite, and compound 30 is active. The acidities of the amide hydrogen in both

compounds differ. It was hypothesized that the decreasing the acidity of the amide

enhances the solubility of 56 over 30 in aqueous solvents. However, there is no

correlation between solubility and potency in this series. Furthermore, the amide

hydrogen is strongly acidic in the presence of an electron-deficient aromatic ring

(30) and less acidic in presence of an electron-rich aromatic ring (56). Often, acidic

protons will stabilize hydrogen-bond donation to nearby acceptors such as N-3 of

the phthalazine ring. In the case of 56, the acetamide linker should be non-planar to

the phthalazine ring whereas 30 should be planar due to pseudo-ring formation. The

parasite in cell-culture is selective of the conformation of 30 but not 56. This is more

apparent in the cell-culture model data of compound 55 which is still a high-energy

molecule compared to the initial series, but maintains potency against the parasite.

Compound 55 is not as electron-donating as 56 due to resonance of the aromatic

versus non-aromatic ring. The amide proton is more acidic in 55 than in 56 which

would enhance formation of the pseudo-ring with N-3 of the phthalazine ring.

Moreover, the 1H NMR spectra of 56 shows 2 peaks for the amide nitrogen

indicating free rotation, different conformations about the amide bond.

Compound 30 exhibited insignificant activity in a mouse model of infection

(250 mg/kg in corn oil with 10% DMSO), which we accredit to its insolubility

correlating to pseudo-ring conformation due to the strong H-bond between N-3 and

18

the very acidic amide hydrogen. Compound 30 is only soluble in DMSO at low

concentrations; the same phenomena exists for the precursor acid. Compound 56,

however, dissolves in a variety of solvents which is why it was used for the

screening against other bacteria.

Table V. Anti-parasitic activities against potential biowarfare agents.

Performed by the New England Regional Center of Excellenece/ Biodefense and

Emerging Infectious Diseases (NERCE/BEID).

Minimum inhibitory concentrations [MIC] (µM)

Cmpd E. coli

A.

baumannii

S.

aureus

S.

pneumoniae

B.

anthracis

F.

tularensis

Y.

pestis

56 ≥20 10 20 20 20 0.039 20

The bacteria used in this screening were selected based on a structural motif

of their IMPDHs. Compound 56 because of its solubility in the prescribed buffer for

this screen over compound 30 was selected and tested for its minimum

concentration necessary for complete inhibition of bacterial growth. Compound 56

exhibited significant activity against Franciscella tularensis with a MIC in the

nanomolar range.

19

Conclusion

We have hypothesized, applied, and tested a new phenomenon of pseudo-

ring formations due to π-π interactions between functional groups to the synthesis

of very potent furan-based inhibitors which exploited the structural-activity

relationships of trending CpIMPDH inhibition. We conclude that inhibition of

CpIMPDH is exclusive to hydrogen or halogen-bond acceptor groups in either the 4

or 3,4-positions of the aniline ring. Then, we submitted our best compounds from

the enzyme assay for cell-culture model assays attaining low micromolar inhibition.

Future endeavors include x-ray diffraction of compounds 29 30, 34, and 42 for

further analysis of the CpIMPDH inhibition trend discussed in this article, and

optimization and screening of new dibenzofuranamide derivatives against F.

tularensis.

Methods

Biology

Determination of IC50 values. (performed by Minjia Zhang of Hedstrom Laboratory)

Inhibition of recombinant CpIMPDH, purified from E. coli, was assessed by

monitoring the production of NADH by fluorescence at varying inhibitor

concentrations (25 pM - 5 μM). IMPDH was incubated with inhibitor for 5 min at

20

room temperature prior to addition of substrates. The following conditions were

used: 50 mM Tris-HCl, pH 8.0, 100 mM KCl, 3 mM EDTA, 1 mM dithiothreitol (DTT)

(assay buffer) at 25 °C, 10 nM CpIMPDH, 300 μM NAD and 150 μM IMP. To

characterize the non-specific binding of inhibitors, assays were also carried out in

the presence of 0.05% BSA (fatty acid free). IC50 values were calculated for each

inhibitor according to Equation 1 using the SigmaPlot program (SPSS, Inc.):

υi = υ0/(1+[I]/IC50) (Eq. 1)

where υi is initial velocity in the presence of inhibitor (I) and υo is the initial velocity

in the absence of inhibitor. Inhibition at each inhibitor concentration was measured

in quadruplicate and averaged; this value was used as υi. The IC50 values were

determined three times except those marked by a *; the averages are reported in

Tables 1, 2, and 3.

Experimental

Unless otherwise noted, all reagents and solvents were purchased from

commercial sources and used without further purification. All reactions were

performed under nitrogen atmosphere unless otherwise noted. The NMR spectra

were obtained using a 400 or 500 MHz spectrometer. All 1H NMR spectra are

reported in δ units ppm and are referenced to tetramethylsilane (TMS) if conducted

in CDCl3 or to the central line of the quintet at 2.49 ppm for samples in d6-DMSO. All

21

chemical shift values are also reported with multiplicity, coupling constants and

proton count. Coupling constants (J values) are reported in hertz. Column

chromatography was carried out on SILICYCLE SiliaFlash silica gel F60 (40–63 μm,

mesh 230–400).

Synthesis of (E)-ethyl-2-(3-oxoisobenzofuran-1(3H)-ylidene)acetate (2).

A solution of phthalic anhydride (10 g, 67.48 mmol) and

(carbethoxymethylene)triphenylphosphorane (23.5 g, 67.48 mmol) in CHCl3 under

nitrogen atmosphere was refluxed for 36 h. Evaporation of CHCl3 and purification of

the residue by column chromatography yielded 12 g (81%) of the product.

(2) 1H NMR (DMSO-d6, 400 MHz): (8.87, d, 1H, J= 8 Hz), (8.01, d, 1H, J= 8 Hz), (7.96, t,

1H, J= 8 Hz), (7.92, t, 1H, J= 8 Hz), (6.24, s, 1H), (4.23, q, 2H, J= 8 Hz), (1.26, t, 3H, J= 8

Hz).

General Procedure for the synthesis of 4-oxophthalizinyl-1-acetic ethyl esters.

Compound 2 (10.0 g, 46 mmol) was refluxed in ethanol for 1 h under

nitrogen atmosphere, then 1.2 eq (2.5 g, 55 mmol) of the appropiate hydrazine was

added to the solution, and the solution heated at reflux for 3 hr. The reaction

mixture was neutralized with 1 eq of 1 M HCl in water (50 mL), and extracted with

CHCl3, which was dried with anhydrous MgSO4. Evaporation of CHCl3 yielded 9.8 g

(87%) of the desired product.

22

(3) Ethyl 2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)acetate. 1H NMR

(DMSO-d6, 400 MHz): (8.21, d, 1H, J= 8 Hz), (7.96, t, 1H, J= 8 Hz), (7.90, 2H), (4.14, t,

2H, J= 8 Hz), (4.10, s, 3H), (1.19, t, 3H, J= 10 Hz).

(4) Ethyl 2-(4-oxo-3,4-dihydrophthalazin-1-yl)acetate. 1H NMR (DMSO-d6, 400

MHz): (8.12, d, 1H, J= 8 Hz), (7.80, t, 1H, J= 7 Hz), (7.71, 2H), (3.96, q, 2H, J= 6-8 Hz),

(3.90, s, 2H), (3.19, s, 1H), (1.02, t, 3H, J= 8 Hz).

General procedure for the synthesis of 4-oxopthalazinyl-1-acetic acids.

A solution of ester 3 or 4 (9.0 g, 37 mmol) and 10 eq. 3M NaOH (122 mL) in

THF was refluxed for 3 hr under nitrogen atmosphere. The reaction mixture was

acidified with 1.2 eq of 10 M HCl (47 mL), diluted with water, and extracted with

CHCl3, which was dried with anhydrous MgSO4. Evaporation of CHCl3 yielded 7.9 g

(5) or 7.8 g (6) (99%) of the desired product.

(5) 2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)acetic acid. 1H NMR (DMSO-

d6, 400 MHz): (9.20, d, 1H, J= 8 Hz), (7.94, t, 1H, J= 8 Hz), (7.88, 2H), (3.99, s, 2H),

(3.71, s, 3H).

(6) 2-(4-oxo-3,4-dihydrophthalazin-1-yl)acetic acid. 1H NMR (DMSO-d6, 400

MHz): (8.14, d, 1H, J= 8 Hz), (7.83, t, 1H, J= 8 Hz), (7.74, m, 2H, J= 8 Hz), (3.94, s, 2H),

(2.39, s, 1H).

23

General procedure for the synthesis of 4-oxopthalazinyl-1-N-arylacetamides.

1.2 eq of triethylamine (TEA) (56mg, 0.55 mmol) was added to a solution of

acid (4) or (5) (100mg, 0.46 mmol), 1.2 eq 1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide hydrochloride (EDCI-HCl) (105mg, 0.55

mmol), 1.2 eq hydroxybenzotriazole (HOBt) (74mg, 0.55 mmol) and 1.2 eq of aniline

in 3mL DMSO under nitrogen atmosphere for 3 h. The reaction mixture was washed

with 1.2 mL HCl and water, extracted with CHCl3, then washed again with NaHCO3,

and dried with anhydrous MgSO4. Evaporation of CHCl3 and purification of the

residues by column chromatography yielded the desired products in yields ranging

from 5-95%.

(7) N-(4-methoxyphenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)acet-

amide. 1H NMR (DMSO-d6, 400 MHz): (10.22, 1H, s), (8.30, d, 1H, J= 8 Hz), (7.95, m,

2H, J= 8 Hz), (7.86, t, 1H, J= 7 Hz), (7.48, d, 2H, J= 8 Hz), (6.88, d, 2H, J= 8 Hz), (4.06, s,

2H), (3.72, s, 3H), (3.71, s, 3H).


amide. 1H NMR (DMSO-d6, 400 MHz): (10.27, s, 1H), (8.30, d, 1H, J= 8 Hz), (7.95, m,

2H, J= 7-8 Hz), (7.87, t, 1H, J= 8 Hz), (7.30, s, 1H), (7.21, t, 1H, J= 8 Hz), (7.11, d, 1H, J=

8 Hz), (6.64, d, 1H, J= 8 Hz), (4.09, s, 2H), (2.73, s, 3H), (2.70, s, 3H).



24

1H, J= 6-8 Hz), (7.86, t, 1H, J= 8 Hz), (7.29, s, 2H), (7.20, t, 1H, J= 8 Hz), (7.10, d, 1H, J=

9 Hz), (6.69, d, 1H, J= 8 Hz), (4.09, s, 2H), (2.72, s, 3H), (2.69, s, 3H).

(10) N-(4-methoxybenzyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)-

acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.31, 1H, s), (9.20, d, 1H, J= 8 Hz), (7.95,

q, 2H, J= 8 Hz), (7.87, m, 2H, J= 7 Hz), (7.46, d, 1H, J= 8 Hz), (6.96, d, 2H, J= 8 Hz),

(4.05, s, 3H), (3.97, q, 2H, J= 7-8 Hz), (3.72, s, 3H), (1.29, S, 3H).

(11) N-(4-ethoxyphenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)acet-

amide. 1H NMR (DMSO-d6, 400 MHz): (10.22, 1H, s), (8.31, d, 1H, J= 7 Hz), (7.96, q,

2H, J= 8 Hz), (7.87, t, 1H, J= 7 Hz), (7.48, d, 2H, J= 8 Hz), (6.86, d, 2H, J= 8 Hz), (4.06, s,

3H), (3.97, q, 2H, J= 8-10 Hz), (3.73, s, 3H), (1.20, t, 3H, J= 8 Hz).

(12) 2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)-N-(4-(trifluoromethoxy)-

phenyl)acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.70, S, 1H), (8.26, d, 1H, J= 8

Hz), (7.91, q, 2H, J= 8 Hz), (7.83, t, 1H, J= 8 Hz), (7.75, d, 2H, J= 8 Hz), (7.64, d, 2H, J= 8

Hz), (4.11, s, 2H), (3.68, s, 3H).

(13) N-(4-fluorophenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)acet-


2H, J= 8 Hz), (7.85, t, 1H, J= 8 Hz) (7.59, q, 2H, J= 7 Hz), (7.15, t, 2H, J= 8 Hz), (4.09, s,

2H), (3.72, s, 3H).

(14) N-(4-chlorophenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)acet-


2H, J= 7-8 Hz), (7.87, t, 1H, J= 8 Hz), (7.62, d, 2H, J= 7 Hz), (7.37, d, 2H, J= 8 Hz), (4.11,

s, 2H), (3.73, s, 3H).

25

(15) N-(4-bromophenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)acet-



s, 2H), (3.72, s, 3H).

(16) N-(4-cyanophenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)acet-

amide. 1H NMR (DMSO-d6, 400 MHz): (10.91, s, 1H), (8.31, d, 1H, J= 8 Hz), (7.96, d,

2H, 7 Hz), (7.93, d, 1H, J= 8 Hz), (7.87, t, 1H, J= 8 Hz), (7.78, 3H), (4.16, s, 2H), (3.72, s,

3H).

(17) N-(4-methylphenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)acet-

amide. 1H NMR (DMSO-d6, 400 MHz): (10.27, s, 1H), (8.30, d, 1H, J= 8 Hz), (7.95, d,

1H, J= 8 Hz), (7.93, d, 1H, J= 8 Hz), (7.87, t, 1H, J= 8 Hz), (7.46, d, 2H, J= 8 Hz), (7.10, d,

2H, J= 8 Hz), (4.06, s, 2H), (3.72, s, 3H).

(18) 2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)-N-(4-(trifluoromethyl)-

phenyl)acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.54, s, 1H), (8.26, d, 1H, J= 8

Hz), (7.91, d, 2H, J= 8 Hz), (7.80, t, 1H, J= 8 Hz), (7.65, d, 2H, J= 8 Hz), (7.28, d, 2H, J= 8

Hz), (4.07, s, 2H), (3.65, s, 3H).

(23) N-(3,4-dichlorophenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)-

acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.68, s, 1H), (8.30, d, 1H, J= 8 Hz), (7.95,

d, 3H, J= 8 Hz), (7.88, t, 1H, J= 8 Hz), (7.58, d, 1H, J= 8 Hz), (7.49, d, 1H, J= 8 Hz), (4.12,

s, 2H), (3.72, s, 3H).



26

d, 1H, J= 8 Hz), (7.94, d, 1H, J= 8 Hz), (7.88, t, 1H, J= 8 Hz), (7.71, t, 2H, J= 7-8 Hz),

(7.41, d, 1H, J= 8 Hz), (4.19, s, 2H), (3.72, s, 3H).



d, 2H, J= 7 Hz), (7.94, 1H), (7.71, s, 2H), (7.36, s, 1H), (4.19, s, 2H), (3.78, s, 3H).

(26) N-(3,4,5-trichlorophenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-

yl)acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.96, s, 1H), (8.27, d, 1H, J= 8 Hz),

(8.02, 2H), (7.94, 2H), (4.20, s, 2H), (3.79, s, 3H).

(27) N-(3,4,5-trifluorophenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-

yl)aceta-mide. 1H NMR (DMSO-d6, 400 MHz): (10.71, s, 1H), (8.26, d, 1H, J= 8 Hz),

(7.90, s, 2H), (7.83, 1H), (7.45, 2H), (4.07, s, 2H), (3.67, s, 3H).

(28) N-(4-bromo-3-chlorophenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-

1-yl)-acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.67, s, 1H), (8.20, d, 1H, J= 8 Hz),

(7.97, d, 1H, J= 7 Hz), (7.94, d, 2H, J= 8 Hz), (7.87, t, 1H, J= 7 Hz), (7.70, d, 1H, J= 8 Hz),

(7.41, d, 1H, J= 8 Hz), (4.12, s, 2H), (3.72, s, 3H).

(29) N-(4-bromo-3-(trifluoromethyl)phenyl)-2-(3-methyl-4-oxo-3,4-dihydro-

phthalazin-1-yl)acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.83, s, 1H), (8.21, d,

1H, J= 8 Hz), (8.19, d, 1H, J= 7 Hz), (7.96, q, 2H, J= 8 Hz), (7.88, t, 1H, J= 7-8 Hz), (7.84,

d, 1H, J= 8 Hz), (7.75, d, 1H, J= 12 Hz), (4.15, s, 2H), (2.74, s, 3H).

(30) N-(4-chloro-3-(trifluoromethyl)phenyl)-2-(3-methyl-4-oxo-3,4-dihydro-


27

1H, J= 7 Hz), (8.17, d, 1H, J= 9 Hz), (7.95, q, 2H, J= 8 Hz), (7.88, t, 1H, J= 7 Hz), (7.82, d,

1H, J= 8 Hz), (7.68, d, 1H, J= 8 Hz), (4.06, s, 2H), (3.72, s, 3H).

(31) N-(4-cyano-3-(trifluoromethyl)phenyl)-2-(3-methyl-4-oxo-3,4-dihydro-


1H, J= 8 Hz), (8.27, s, 1H), (8.11, d, 1H, J= 8 Hz), (7.95, q, 3H, J= 7-8 Hz), (7.88, t, 1H, J=

8 Hz), (4.20, s, 2H), (3.72, s, 3H).

(32) N-(3-chloro-4-cyanophenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-


(8.14, s, 1H), (7.90, q, 2H, J= 8 Hz), (7.82, t, 1H, J= 8 Hz), (7.77, d, 1H, J= 8 Hz), (7.63,

d, 1H, J= 8 Hz), (4.09, s, 2H), (3.67, s, 3H).

(33) N-(4-fluoro-3-(trifluoromethyl)phenyl)-2-(3-methyl-4-oxo-3,4-dihydro-

phthal-azin-1-yl)acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.74, s, 1H), (8.30, d,

1H, J= 8 Hz), (8.09, d, 1H, J= 8 Hz), (7.97, d, 1H, J= 8 Hz), (7.93, d, 1H, J= 8 Hz), (7.87, t,

1H, J= 8 Hz), (7.82, t, 1H, J= 7-8 Hz), (7.49, t, 1H, J= 10 Hz), (4.13, s, 2H), (3.72, s, 3H).

(34) N-(4-methoxy-3-(trifluoromethyl)phenyl)-2-(3-methyl-4-oxo-3,4-dihyd-

rophthalazin-1-yl)acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.57, s, 1H), (8.34,

d, 1H, J= 8 Hz), (8.00, q, 3H, J= 7-8 Hz), (7.91, t, 1H, J= 7-8 Hz), (7.81, d, 1H, J= 8 Hz),

(7.28, d, 1H, J= 8 Hz), (4.14, s, 2H), (3.89, s, 3H), (3.77, s, 3H).

(35) N-(4-amino-3-(trifluoromethyl)phenyl)-2-(3-methyl-4-oxo-3,4-dihydro-


1H, J= 8 Hz), (7.95, q, 2H, J= 7-8 Hz), (7.86, t, 1H, J= 7-8 Hz), (7.69, s, 1H), (7.39, d, 1H,

J= 8 Hz), (6.78, d, 1H, J= 8 Hz), (5.40, s, 2H), (4.09, s, 2H), (3.71, s, 3H).

28

(36) N-(3-(trifluoromethyl)phenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-

1-yl)acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.72, s, 1H), (8.31, d, 1H, J= 8 Hz),

(8.09, s, 1H), (7.97, d, 1H, J= 8 Hz), (7.93, d, 1H, J= 8 Hz), (7.87, t, 1H, J= 8 Hz), (7.77,

d, 1H, J= 8 Hz), (7.56, t, 1H, J= 8 Hz), (7.42, d, 1H, J= 8 Hz), (4.14, s, 2H), (3.72, s, 3H).

(37) N-(4-chloro-3-(trifluoromethyl)phenyl)-2-(4-oxo-3,4-dihydrophthalazin-


(8.19, s, 1H), (7.94, d, 2H, J= 8 Hz), ( 7.86, q, 1H, J= 7-8 Hz), (7.82, d, 1H, J= 7 Hz),

(7.67, d, 1H, J= 8 Hz), (4.10, s, 2H), (3.32, s, 3H).

(38) N-(4-bromo-3-(trifluoromethyl)phenyl)-2-(4-oxo-3,4-dihydrophthalazin-


(8.16, 1H), (7.93, 2H), (7.84, 1H), (7.80, d, 1H, J= 8 Hz), (7.72, d, 1H, J= 8 Hz), (4.08, s,

2H).

(39) N-(4-chloro-3-fluorophenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-

yl)a-cetamide. 1H NMR (DMSO-d6, 400 MHz): (10.70, s, 1H), (8.30, d, 1H, J= 8 Hz),

(7.94, m, 2H, J=7-8 Hz), (7.87, t, 1H, J= 7 Hz), (7.75, d, 1H, J= 8 Hz), (7.53, t, 1H, J= 8

Hz), (7.34, d, 1H, J= 8 Hz), (4.12, s, 2H), (3.71, s, 3H).

(40) N-(4-chloro-3-methylphenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-


(7.95, m, 2H, J= 8 Hz), (7.87, t, 1H, J= 8 Hz), (7.59, s, 1H), (7.40, d, 1H, J= 8 Hz), (7.34,

d, 1H, J= 8 Hz), (4.10, s, 2H), (3.73, s, 3H), (2.26, s, 3H).

(41) N-(4-bromo-3-methylphenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-


29

(7.95, q, 2H, J= 7-8 Hz), (7.87, t, 1H, J= 8 Hz), (7.60, s, 1H), (7.49, d, 1H, J= 8 Hz), (7.36,

d, 1H, J= 8 Hz), (4.10, s, 2H), (3.73, s, 3H), (2.26, s, 3H).

(42) N-(4-cyano-3-methylphenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-


(7.95, m, 2H, J= 8 Hz), (7.88, t, 1H, J= 7 Hz), (7.70, d, 2H, J= 8 Hz), (7.58, d, 1H, J= 8

Hz), (4.15, s, 2H), (3.71, s, 3H).

(43) N-(4-chloro-3-methoxyphenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalaz-

in-1-yl)acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.51, s 1H), (8.28, d, 1H, J= 8

Hz), (7.92, q, 2H, J= 7-8 Hz), (7.85, t, 1H, J= 8 Hz), (7.51, s, 1H), (7.31, d, 1H, J= 8 Hz),

(7.09, d, 1H, J= 8 Hz), (4.09, s, 2H), (3.78, s, 3H), (3.70, s, 3H).

(44) N-(4-chloro-2-(trifluoromethyl)phenyl)-2-(3-methyl-4-oxo-3,4-dihydro-


1H, J= 8 Hz), (7.94, q, 2H, J= 7-8 Hz), (7.88, t, 1H, J= 7-8 Hz), (7.83, s, 1H), (7.77, d, 1H,

J= 8 Hz), (7.56, d, 1H, J= 8 Hz), (4.13, s, 2H), (3.73, s, 3H).

(45) N-(4-bromo-2-(trifluoromethyl)phenyl)-2-(3-methyl-4-oxo-3,4-dihydro-


1H, J= 8 Hz), (7.94, q, 2H, J= 8 Hz), (7.87, t, 1H, J= 8 Hz), (7.82, s, 1H), (7.77, d, 1H, J= 8

Hz), (7.56, d, 1H, J= 8 Hz), (4.12, s, 2H), (3.72, s, 3H).

(46) N-(4-bromo-2-fluorophenyl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-


(7.95, 2H), (7.85, q, 2H, J= 8 Hz), (7.63, d, 1H, J= 8 Hz), (7.26, d, 1H, J= 8 Hz), (4.19, s,

2H), (3.72, s, 3H).

30

(48) 2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)-N-(naphthalen-2-yl)acet-

amide. 1H NMR (DMSO-d6, 400 MHz): (10.61, s, 1H), (8.32, d, 1H, J= 8 Hz), (8.29, s,

1H), (8.04, d, 1H, J= 8 Hz), (7.97, t, 1H, J= 8 Hz), (7.89, q, 2H, J= 7 Hz), (7.85, d, 1H, J=

8 Hz), (7.79, d, 1H, J= 8 Hz), (4.19, s, 2H), (3.75, s, 3H).

(50) 2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)-N-(1,3,4-thiadiazol-2-yl)-


d, 2H, J= 7 Hz), (7.89, m, 1H), (4.27, s, 2H), (3.71, s, 3H).

(51) N-(5-methyl-1,3,4-thiadiazol-2-yl)-2-(3-methyl-4-oxo-3,4-dihydrophtha-

lazin-1-yl)acetamide. 1H NMR (DMSO-d6, 400 MHz): (8.30, d, 1H, J= 8 Hz), (7.93, s,

2H), (7.86, 1H), (4.25, s, 2H), (3.70, s, 3H), (2.60, s, 3H).

(52) N-(benzofuran-5-yl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)acet-

amide. 1H NMR (DMSO-d6, 400 MHz): (10.39, s, 1H), (8.30, d, 1H, J= 8 Hz), (7.96, q,


d, 1H, J= 7 Hz), (4.11, s, 2H), (3.72, s, 3H).

(53) N-(benzo[d]oxazol-5-yl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)-

acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.69, s, 1H), (8.68, s, 1H), (8.21, d, 1H, J=

8 Hz), (8.19, s, 1H), (7.96, m, 2H, J= 8 Hz), (7.87, t, 1H, J= 8 Hz), (7.74, d, 1H, J= 8 Hz),

(7.43, d, 1H, J= 8 Hz), (4.15, s, 2H), (3.73, s, 3H).

(54) N-(2-methylbenzofuran-5-yl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-

1-yl)acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.23, s, 1H), (8.30, d, 1H, J= 8 Hz),

(7.99, d, 1H, J= 8 Hz), (7.94, t, 1H, J= 8 Hz), (7.87, t, 1H, J= 8 Hz), (7.82, s, 1H), (7.41, d,

31

1H, J= 8 Hz), (7.31, d, 1H, J= 8 Hz), (6.52, s, 1H), (4.09, s, 2H), (3.72, s, 3H), (2.40, s,

3H).

(55) N-(6,7,8,9-tetrahydrodibenzo[b,d]furan-5-yl)-2-(3-methyl-4-oxo-3,4-di-

hydrophthalazin-1-yl)acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.21, s, 1H),

(8.26, d, 1H, J= 8 Hz), (7.96, d, 1H, J= 8 Hz), (7.90, t, 1H, J= 8 Hz), (7.83, t, 1H, J= 8 Hz),

(7.76, s, 1H), (7.35, d, 1H, J= 8 Hz), 7.24, d, 1H, J= 8 Hz), (4.06, s, 2H), (3.68, s, 3H),

(1.91, d, 4H, J= 8 Hz), (1.72, q, 4H, J= 8 Hz).

(56) N-(dibenzo[b,d]furan-2-yl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-


(8.13, s, 1H), (8.07, d, 1H, J= 8 Hz), (8.01, d, 1H, J= 8 Hz), (7.96, t, 1H, J= 8 Hz), (7.88, t,

1H, J= 8 Hz), (7.66, d, 1H, J= 8 Hz), (7.49, d, 1H, J= 8 Hz), (7.46, d, 1H, J= 8 Hz), (7.37, t,

1H, J= 8 Hz), (4.18, s, 2H), (3.74, s, 3H).

(57) N-(dibenzo[b,d]furan-3-yl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-

yl)ace-tamide. 1H NMR (DMSO-d6, 400 MHz): (10.57, s, 1H), (8.44, s, 1H), (8.32, d,

1H, J= 8 Hz), (8.06, d, 1H, J= 8 Hz), (8.02, d, 1H, J= 8 Hz), (7.96, t, 1H, J= 8 Hz), (7.88, t,

1H, J= 8 Hz), (7.88, t, 1H, J= 8 Hz), (7.69, d, 1H, J= 8 Hz), (7.66, d, 1H, J= 8 Hz), (7.59, d,

1H, J= 9 Hz), (7.51, t, 1H, J= 8 Hz), (7.46, t, 1H, J= 8 Hz), (7.38, t, 1H, J= 8 Hz), (4.17, s,

2H), (3.74, s, 3H).

(58) 2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)-N-(1,2,4-trimethyl-

1,2,3,4-tetrahydrobenzofuro[3,2-c]pyridin-8-yl)acetamide. 1H NMR (DMSO-d6,

400 MHz): (10.46, s, 1H), (8.36, d, 1H, J= 8 Hz), (8.06, d, 1H, J= 8 Hz), (8.00, t, 2H, J= 8

Hz), (7.93, t, 1H, J= 7-8 Hz), (7.51, d, 1H, J= 8 Hz), (7.39, d, 1H, J= 8 Hz), (4.17, s, 2H),

32

(3.79, s, 3H), (3.40, s, 2H, J= 10 Hz), (3.14, s, 2H), (2.56, s, 3H), (2.46, s, 3H), (2.31, s,

1H), (1.40, s, 3H), (1.28, s, 1H), (1.24, d, 3H, J= 10 Hz).

(59) N-(9H-carbazol-3-yl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)acet-

amide. 1H NMR (DMSO-d6, 400 MHz): (11.19, s, 1H), (10.26, s, 1H), (8.38, s, 1H),

(8.31, d, 1H, J= 8 Hz), (8.04, d, 1H, J= 8 Hz), (7.97, q, 2H, J= 8 Hz), (7.88, t, 1H, J= 8 Hz),

(7.49, t, 1H, J= 8 Hz), (7.43, m, 2H, J= 7-8 Hz), (7.36, t, 1H, J= 8 Hz), (7.11, t, 1H, J= 6

Hz), (4.13, s, 2H), (3.75, s, 3H).

(60) N-(9-ethyl-9H-carbazol-3-yl)-2-(3-methyl-4-oxo-3,4-dihydrophthalazin-

1-yl)acetamide. 1H NMR (DMSO-d6, 400 MHz): (10.39, s, 1H), (8.41, s, 1H), (8.31, d,

1H, J= 8 Hz), (8.03, t, 2H, J= 8 Hz), (7.96, t, 1H, J= 8 Hz), (7.97, t, 1H, J= 8 Hz), (7.56, d,

2H, J= 10 Hz), 7.43, t, 1H, J= 8 Hz), (7.15, t, 1H, J= 8 Hz), (4.40, q, 2H, J= 8 Hz), (4.14, s,

2H), (3.74, s, 3H), (1.29, t, 3H, J= 8 Hz).

33

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