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

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, structureactivity 

relationship revealed that electron-withdrawing substituents at the paraposition 

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 

ii

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. 

iii

Table of Contents 

i. Title Page. 

ii-iii. 

iv. 

Abstract. 

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. 

iv

List of Tables 

Table I. 

Table II. 

Initial lead optimization/IC50 values for Acyclic analogs. 

Van der Waals radii distances and Conformations between 

groups. 

Table III. 

Table IV. 

Table V. 

IC50 values for Cyclic analogs. 

Anti-cryptosporidial/toxoplasmodial activities. 

Anti-parasitic activities against potential biowarfare agents. 

v

List of Illustrations/Figures 

Figure I. 

Figure II: 

IMPDH reaction scheme. 

CpIMPDH inhibitor identified by HTS. 

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

trifluoromethyl- series. 

Figure IV. 

Figure V. 

Scheme I. 

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

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

General Procedure for the Synthesis of Amide Derivatives. 

vi

Introduction 

Outbreaks worldwide have instigated drug discovery programs against the 

waterborne protozoan parasite Cryptosporidium parvum 1 . A calf is capable of 

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

bioterrorism threat 2 . 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 metabolism 3 . 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. 

1

Figure I. IMPDH reaction scheme 

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

nicotinamide adenosine dinucleotide (NAD + ) site 4 . This NAD + site has been exploited 

in high throughput screens as a potential drug target for C. parvum 5 . These 

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

inhibitors which exhibit in vitro cell culture model of infection 6 . 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. 

2

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). 

3

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 

4

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* 

5

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 

6

D35 51 Me 

thiazolyl) 

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

thiazolyl) 

>5000 ND 

The D series shares the same general trend with the other two series 6 ; 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 halogen 6,11 . 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 halogen 8,9 ; 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 halogen 9 . For example, the difference in 

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

7

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 pipi 

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 electronwithdrawal 

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). 

8

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 

9

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 CpIMPDH 6 . 

ChemBio3D Ultra MM2 minimizations of compounds 30 and 34. 

10

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. 

11

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

groups. 

Cmpd 

Å 

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. 

34 

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. 

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 

12

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 acceptors 9 . 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 

13

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 

14

enzofuran 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 enzymes 12 ; 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 intermediate 12 . 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/mol 13 . The inactivity 

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

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 

15

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 

Tree 10 ; Topliss Tree suggests next compound be the extremely electronwithdrawing 

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. 

16

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 1 H 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 

17

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. 

18

Conclusion 

We have hypothesized, applied, and tested a new phenomenon of pseudoring 

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 

19

oom 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 1 H 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 

20

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) 1 H 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. 

21

(3) Ethyl 2-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)acetate. 1 H 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. 1 H 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. 1 H NMR (DMSOd6, 

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. 1 H 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). 

22

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)acetamide. 

1 H 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). 


1 H 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). 



23

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. 1 H 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)acetamide. 

1 H 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. 1 H 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)acetamide. 


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)acetamide. 


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). 

24

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



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

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

1 H 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)acetamide. 

1 H 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. 1 H 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. 1 H 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). 



25

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. 1 H 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. 1 H 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. 1 H 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-dihydrophthalazin-1-yl)acetamide. 

1 H 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-dihydrophthalazin-1-yl)acetamide. 


26

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-dihydrophthalazin-1-yl)acetamide. 


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-dihydrophthal-azin-1-yl)acetamide. 


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-dihydrophthalazin-1-yl)acetamide. 

1 H 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-dihydrophthalazin-1-yl)acetamide. 


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). 

27

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

1-yl)acetamide. 1 H 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. 1 H 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- 


28

(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-dihydrophthalazin-1-yl)acetamide. 

1 H 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-dihydrophthalazin-1-yl)acetamide. 


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-dihydrophthalazin-1-yl)acetamide. 


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). 

29

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

1 H 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-dihydrophthalazin-1-yl)acetamide. 

1 H 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)acetamide. 

1 H 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. 1 H 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. 1 H 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, 

30

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-dihydrophthalazin-1-yl)acetamide. 

1 H 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. 1 H 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. 1 H 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), 

31

(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)acetamide. 

1 H 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. 1 H 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). 

32

Bibliography 

1. Huang, D.R.; White, A.C. Gastroenterol. Clin . North Am. 2006, 35, 291-314; 

Corso, P.S; Kramer, M.H.; Blair, K.A.; Addiss, D.G.; Davis, J.P.; Haddix, A.C. 

Emerg. Infect. Dis. 2003, 9, 426-31. 

2. Dupont, H.L.; Chappell, C.L.; Sterling, C.R.; Okhuysen, P.C.; Rose, J.B.; 

Kakubowski, W. N. Engl. J. Med. 1995, 332, 855-59. 

3. Abrahamsen, M.S.; Templeton, T.J.; Enomoto, S.; Abrahante, J.E.; Zhu, G.; 

Lancto, C.A.; Deng, M.; Liu, C.; Widmer, G.; Tzipori, S. et al. Science 2004, 304, 

441-45; Striepen, B.; Pruijssers, A.J.; Huang, J.; Li, C.; Gubbels, M.J.; Umejiego, 

N.N.; Hedstrom, L.; Kissinger, J.C. Proc. Natl. Acad. Sci. USA 2004, 101, 3154- 

59; Xu, P.; Widmer, G.; Wang, Y.; Ozaki, L.S.; Alves, J.M.; Serrano, M.G.; Puiu, D.; 

Manque, P.; Akiyoshi, D.; Mackey, A.J. et al. Nature 2004, 431, 1107-12. 

4. Ratcliffe, A.J. Drug Discov. Dev. 2006, 9, 595-605. 

5. Umejiego, N.N.; Gollapalli, D.; Sharling, L.; Volftsun, A.; Lu, J.; Benjamin, N.N.; 

Stroupe, A.H.; Riera, T.V.; Striepen, B.; Hedstrom, L. Chemistry and Biology 

2008, 15, 70-77. 

6. Maurya, S. K.; Gollapalli, D. R.; Kirubakaran, S.; Zhang, M.; Johnson, C. R.; 

Benjamin, N. N.; Hedstrom, L.; Cuny, G. D. J Med Chem 2009, 52, 4623; 

MacPherson, I.S.; Kirubakaran, S.; Gorla, S.K.; Riera, T.V.; Alejandro D’Aquino, 

J.; Zhang, M.; Cuny, G.D.; Hedstrom, L. J. Am. Chem. Soc. 2010, 132, 1230-31. 

7. Mylari, B.L., Larson, E.R., Beyer, T.A., Zembrowski, W.J., Aldinger, C.E., Dee, 

M.F., Siegel, T.W., Singleton, D.H. J. Med. Chem. 1991, 34, 108-122; Niebel, C., 

Lokshin, V., Sigalov, M., Krief, P., Khodorkovsky, V. Eur J. Org. Chem. 2008, 

3689-3699. 

8. Metrangolo, P.; Neukirch, H.; Pilati, T.; Resnati, G. Acc. Chem. Res. 2005, 38, 

386-395. 

9. Brammer, L.; Bruton, E.A.; Sherwood, P. Crystal Growth & Design. 2001, 1, 

277-90; Schwӧbel, J.; Ebert, R.; Kȕhne, R.; Schȕȕrmann, G. J. Chem. Inf. Model. 

2009, 49, 956-62. 

10. Topliss, J.G. J. Med. Chem. 1972, 15, 1007-1011. 

11. Bondi, A. J. Phys. Chem. 1964, 68, 441-451. 

12. Ravtndranath, V.; Burka, T., Boyd, M. R., Pharmacologist, 1983, 25, 171 

;Ravindranath, V.; Boyd, M. R., Toxicol. Appl. Pharmacol., 1985, 78, 370-376; 

Adam, W.; Hadjiarapoglou, L.; Peters, K.; Sauter, M. J. Am. Chem. Soc., 1993, 

115, 8603–8608. 

13. Kistiakowsky, G. B.; Ruhoffh, J. R.; Smith, A.; Vaughan, W. E. J. Am. Chem. Soc., 

1936, 58, 146–153. 

33

Synthesis and Biological Evaluation of Phthalazinone Inhibitors of ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?