Novel inhibitors of bacterial virulence: Development of 5,6-dihydrobenzo[h]quinazolin-4(3H)-ones for the inhibition of group A streptococcal streptokinase expression

Bioorganic & Medicinal Chemistry 21 (2013) 1880–1897

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Bioorganic & Medicinal Chemistry

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Novel inhibitors of bacterial virulence: Development

of 5,6-dihydrobenzo[h]quinazolin-4(3H)-ones for the inhibition

of group A streptococcal streptokinase expression

Bryan D. Yestrepsky ^a,, Yuanxi Xu ^c,, Meghan E. Breen ^a, Xiaoqin Li ^b, Walajapet G. Rajeswaran ^a,

Jenny G. Ryu ^a, Roderick J. Sorenson ^a, Yasuhiro Tsume ^b, Michael W. Wilson ^a, Wenpeng Zhang ^b,

a,

Duxin Sun ^b, Hongmin Sun ^c,, Scott D. Larsen

⇑

^aVahlteich Medicinal Chemistry Core, Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, United States

^bDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, United States

^cSchool of Medicine, Department of Internal Medicine, University of Missouri, Columbia, MO 65212, United States

a r t i c l e i n f o

a b s t r a c t

Article history:

Resistance to antibiotics is an increasingly dire threat to human health that warrants the development of

new modes of treating infection. We recently identiﬁed 1 (CCG-2979) as an inhibitor of the expression of

streptokinase, a critical virulence factor in Group A Streptococcus that endows blood-borne bacteria with

ﬁbrinolytic capabilities. In this report, we describe the synthesis and biological evaluation of a series of

novel 5,6-dihydrobenzo[h]quinazolin-4(3H)-one analogs of 1 undertaken with the goal of improving

the modest potency of the lead. In addition to achieving an over 35-fold increase in potency, we identiﬁed

structural modiﬁcations that improve the solubility and metabolic stability of the scaffold. The efﬁcacy of

two new compounds 12c (CCG-203592) and 12k (CCG-205363) against bioﬁlm formation in Staphylococ-

cus aureus represents a promising additional mode of action for this novel class of compounds.

Received 30 November 2012

Revised 8 January 2013

Accepted 15 January 2013

Available online 1 February 2013

Keywords:

Group A Streptococcus

Virulence inhibitor

Antibiotic

Streptokinase

Antibiotic resistance

Metabolic oxidation

Bioﬁlm

Antivirulence

1. Introduction

infection (‘strep throat’) and superﬁcial skin infections, systemic

infections with GAS such as streptococcal toxic shock syndrome,

Bacterial infection remains one of the most critical threats to

human health, especially in third world countries and in children.

Streptococcus pyogenes, or Group A Streptococcus (GAS), represents

one aspect of this global threat. GAS is a ubiquitous human patho-

gen responsible for up to 700 million infections per year world-

wide.¹While most commonly associated with upper respiratory

rheumatoid fever, and necrotizing fasciitis are very often fatal.

Cases of systemic GAS infection are thought to approach 500,000

cases annually, with a mortality rate of 15–35%.^2,3

The rapid emergence of bacterial resistance to antibiotic treat-

ment is an alarming aspect of systemic infection, which can rapidly

progress in the absence of effective treatment. As dangerous resis-

tant strains such as methicillin-resistant Staphylococcus aureus

(MRSA) and vancomycin-resistant Enterococcus (VRE) become

increasingly prevalent, especially in the hospital setting,^4,5the

necessity for antibiotics with novel mechanisms of action grows.

Virulence factors are among the targets currently being evaluated

for antibiotic activity.⁶By inhibiting the mechanisms bacteria use

to spread and thrive in a host organism, virulence inhibitors are in-

tended to retain highly infective pathogenic bacteria in a relatively

benign state, allowing additional time for the host’s immune sys-

tem or a course of antibiotics to clear the infection. Since inhibitors

of virulence do not necessarily affect the reproduction of bacterial

cells, they may represent an avenue of antibiotic treatment that

Abbreviations: GAS, Group A Streptococcus (Streptococcus pyogenes); SK, strep-

tokinase; MRSA, methicillin-resistant Staphylococcus aureus; VRE, vancomycin-

resistant Enterococcus; HTS, high-throughput screening; SAR, Structure–activity

relationship; MLM, mouse liver microsomal extract; CID, collision-induced disso-

ciation; ESI, electrospray ionization; EPI, enhanced product ion scan; MRM, multiple

reaction monitoring; EMS, enhanced mass spectrometry; DMEM, Dulbecco’s

modiﬁed Eagle’s medium; THY, Todd-Hewitt media with 0.2% yeast extract.

⇑

Corresponding authors. Tel.: +1 573 882 2296 (H.S.); tel.: +1 734 615 0454

(S.D.L.).

E-mail addresses: sunh@health.missouri.edu (H. Sun), sdlarsen@umich.edu (S.D.

Larsen).

These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.bmc.2013.01.046

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

1881

does not encourage the development of resistance. A number of

virulence mechanisms have been targeted for inhibition by small

molecules in the literature, including bacterial toxins,⁷adhesion

factors,⁸quorum-sensing systems,^9,10and virulence-inducing sig-

naling pathways.¹¹Though no anti-virulence drugs have yet pro-

gressed to use in the clinic, inhibitors of anthrax toxin cell

permeability,¹²N-acyl homoserine lactone-mediated quorum

sensing,¹³and QseC histidine sensor kinase activation¹¹have dem-

onstrated efﬁcacy in murine models of infection.

Sun et al. previously identiﬁed the protein streptokinase (SK) as

a potent virulence factor in GAS.¹⁴SK is an activator of human plas-

minogen, which generates the ﬁbrinolytic plasmin and allows the

bacteria to circumvent the host’s defense mechanism of clotting

around foci of infection. We recently reported a high-throughput

screening (HTS) campaign that identiﬁed lead compound 1 (CCG-

2979, Fig. 1) and analog 6b (CCG-102487) as capable of reducing

the expression of streptokinase at the transcriptional level, con-

ﬁrmed by Western blot and RNA microarray analysis. This com-

pound was also found to elicit a signiﬁcant protective effect in a

transgenic mouse model of GAS infection.¹⁵These ﬁndings

prompted the initiation of a structure-activity relationship (SAR)

study based on 1 with the intent of increasing its modest potency

potency and to identify areas on the molecule amenable to substi-

tution without loss of activity.

2. Chemistry

Access to the core structure of 1 was accomplished with a

known 5-step synthetic pathway (Scheme 1). b-aminoester 4 was

synthesized from cyclohexanone via a Knoevenagel condensation

with ethyl cyanoacetate followed by Michael addition of benzyl-

magnesium chloride, and ﬁnally acid-mediated cyclization.¹⁶The

2-thioxo-dihydropyrimidinone ring could then be installed via

nucleophilic addition of 4 to allyl isothiocyanate, followed by

base-catalyzed cyclization.¹⁷Finally, sulfur alkylation under basic

conditions gave access to 1 and S-substituted analogs 6a–f. Addi-

tional analogs that replace the sulﬁde with ether (7a) and amine

(7b) linkages were accessed via the S-methyl derivative 6f, either

by direct nucleophilic substitution with sodium ethoxide, or fur-

ther functionalization to the methyl sulfone and displacement with

ethylamine under basic conditions.¹⁸

Given the relative ease with which the original spirocyclohexyl-

substituted analogs were synthesized, we were surprised to ﬁnd

that we were not able to generate gem-dimethyl analogs (e.g.,

12, Scheme 2) using this scheme. While Michael addition of ben-

zylmagnesium chloride into ethyl 2-cyano-3-methylbut-2-enoate

proceeded as desired, no set of conditions was successful in gener-

ating the gem-dimethyl analog of 4. It was ﬁnally determined that

the b-aminoesters 10a–d could be accessed in one step beginning

from the corresponding o-tolunitriles 9a–d, available commercially

or by SN_Araddition of cyanide into the corresponding chlorotolu-

enes 8a,b¹⁹(Scheme 2). In the presence of LDA, smooth addition

of 9a–d to the b-position of ethyl 3,3-dimethyl acrylate afforded

an enol ester intermediate that readily cyclized in the presence

of ZnI₂, providing amino esters 10a–d.²⁰Modiﬁed conditions for

condensation with alkyl isothiocyanates were required to efﬁ-

ciently produce the 2-thioxo-dihydropyrimidinone intermediates

11a–f. Subsequent alkylation at sulfur ultimately allowed the syn-

thesis of gem-dimethyl analogs 12a–n in a total of 3 to 4 steps

(Scheme 2). Further elaboration of these compounds to generate

ether and amino derivatives 13a,b was achieved in the same man-

ner as for 7a,b. Chemoselective sulfur oxidation of 12a via treat-

ment with 1.1 eq of Oxone^Ò21or an excess of mCPBA delivered

(% of plasmin activity at 50 lM = 48 28%) and improving the pre-

dicted pharmacokinetic properties of the scaffold. In particular, we

were concerned about its high lipophilicity (cLogP = 7.1), which

can be associated with poor solubility and metabolic stability. Ulti-

mately we would also like to identify the macromolecular target

for 1, which is currently unknown due to the phenotypic nature

of the HTS and biological assay. To design afﬁnity probes for this

purpose, we needed to perform SAR in order to both improve

N

O

S

N

1

Figure 1. Structure of HTS lead compound 1.

H

NC

CO₂Et

O

NH₂

N

S

a

b,c

d,e

N

CO₂Et

O

5

2

3

4

f

N

O

X

N

O

S

R

g or h

N

6f

from

7a, X = O

7b, X = NH

1 6a-f

,

Scheme 1. Preparation of spirocyclohexyl analogs of 1. Reagents and conditions: (a) ethyl cyanoacetate, AcOH, NH₄OAc, toluene, 150 °C, 5 h; (b) BnMgCl, Et₂O, RT, 72 h; (c)

H₂SO₄, 0 °C to rt, 7 h; (d) allyl isothiocyanate, EtOH, reﬂux, 10 h; (e) KOH, EtOH/H₂O (1:1), reﬂux, 10 h; (f) R-X, base, EtOH or MEK, 0–70 °C, 10 min–16 h; (g) NaOEt, EtOH,

40 °C, 48 h; (h) (i) mCPBA, DCM, rt, 16 h, (ii) EtNH₂, K₂CO₃, THF/DMF (1:1), rt, 5 h.

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B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

R¹

R³

R²

R³

R²

a

b

NH₂

R²

Cl

CN

Me

CO₂Et

9a: R¹= OMe, R², R³= H

9b: R¹= R³= H, R²= OMe

9c: R¹, R²= H, R³= OMe

10a-d

8a:

R

¹= OMe, R²= H

8b: R¹= H, R²= OMe

R , R , R³= H

1

2

9d:

c

R¹

R³

R²

R³

R²

N

O

X

H

e or f

d

N

S

N

S

R⁵

N

from 12n

N

R⁴

O

13a

: X = O

13b: X = NH

11a-d: R⁴= allyl

11e

12a-n

: R⁴= Et, R¹= R²= R³= H

h

g

11f:

R

⁴= Me, R¹= OMe, R²= R³= H

12a

from

O

S

O

N

O

S

N

O

14

15

Scheme 2. Synthesis of gem-dimethyl substituted analogs of 1. Reagents and conditions: (a) NiBr₂, Zn(dust), PPh₃, then KCN, THF, 50–60 °C, 24 h; (b) LDA, diglyme, ꢀ78 °C,

1 h, then ethyl 3,3-dimethyl acrylate, ZnI₂, ꢀ78 °C to rt, 2 h; (c) R⁴-NCS, AcOH, EtOH, 70 °C, 16 h; (d) R⁵-X, Cs₂CO₃, MEK or DMF, 70 °C, 16 h; (e) NaOEt, EtOH, 40 °C, 48 h; f) (i)

mCPBA, DCM, 0 °C to RT, 16 h, (ii) EtNH₂, K₂CO₃, THF/DMF (1:1), rt, 5 h; (g) 1.1 equiv Oxone, THF/H₂O (1:1), 0 °C to rt, 16 h; (h) mCPBA, DCM, rt, 12 h.

sulfoxide and sulfone-modiﬁed analogs 14 and 15, respectively. No

epoxidation of the allyl group was observed under these mild oxi-

dation conditions.

Small changes or additions to the core procedure allowed access

to a number of other substitution patterns. Beginning from the

methoxy-substituted aryl ethers 12a–c, treatment with BBr₃re-

vealed phenols 16a–c, which could be elaborated into a diverse

set of aryl ethers 17a–k (Scheme 3). Exploring diversity at the N-

and O-positions of the pyrimidinone required assembling the

unsubstituted thioxo-dihydropyrimidinone ring via treatment of

10a or 10d with benzoyl isothiocyanate, followed by ring closure

and hydrolysis of the resulting N-benzoyl group with aqueous

KOH to generate 18a and 18d²²(Scheme 4). Selective alkylation

R¹

H

N

a

S

NH₂

NH

9

O

HO

CO₂Et

8

O

18a,d

N

O

N

S

10a, R₁= OMe

7

a

10d

, R₁= H

N

b,c

O

16a-c

12a-c

R¹

b

N

S

R²

N

S

R

O

R²

+

N

R³

N

S

O

R³

20a-c

O

N

19a-b

O

17a-k

Scheme 4. Generation of N- and O-alkylated substitution pairs 19a–b and 20a–c.

Reagents and conditions: (a) (i) benzoyl isothiocyanate, EtOH, 75 °C, 5 h, (ii) KOH,

EtOH/H₂O (2:1) 70 °C, 2 h; (b) R²-X, NaHCO₃, DMF, rt ꢀ70 °C, 30 min–16 h; (c) R³-X,

base, EtOH or DMF, 70–80 °C, 16–24 h.

Scheme 3. Synthesis of aryl ethers 17a–k. .Reagents and conditions: (a) BBr₃, DCM,

0 °C to rt, 16 h; (b) R-X, Cs₂CO₃, MEK or DMF, rt ꢀ70 °C, 16 h.

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

1883

of the sulfur was achieved under neutral conditions, while subse-

quent base-catalyzed alkylation of the amide generally gave some

mixture of N- and O-alkylated analogs 19a–b and 20a–c.

Finally, carboxylic acid derivatives at the 8-position (Scheme 5)

were accessed via lithiation and carboxylation of 4-bromo-2-meth-

ylbenzonitrile 21, followed by cyclization and esteriﬁcation to gen-

erate 23. Sensitivity to the standard acid-catalyzed cyclization with

allyl isothiocyanate necessitated the use of the stepwise procedure

seen for the conversion of 10 to 18 (Scheme 4). Subsequent alkyla-

tions at sulfur and nitrogen delivered the 8-methylcarboxylate

analog 25, which could be saponiﬁed to acid 26. Amides 27a and

27b were accessed via amide coupling conditions using HOBt and

EDC.

sponse titrations to determine approximate IC₅₀values using the

same assay.

During the course of evaluating the new analogs, it was observed

that there was a relatively high level of inter-assay variability in the

activity assay. There are a number of likely contributing factors,

most notably the variability inherent to working in a live bacterial

system. For example, it has been noted that minimum inhibitory

concentration (MIC) values for determining bacteriotoxicity tend

to vary by up to three-fold from assay to assay.²³Furthermore,

the currently unknown macromolecular target of these compounds

likely affects a pathway including a transcription factor, which are

known to be low-abundance proteins²⁴that exhibit signiﬁcant

temporal expression changes, potentially contributing to the vari-

ability. It was noted early into the SAR effort that 12h was both

moderately active and particularly consistent from assay to assay.

In an effort to reduce the number of anomalous data points, 12h

was included in each assay as a positive control. Assays in which

12h exhibited activity deviating from the mean activity at 5 and

3. Results and discussion

3.1. SK expression assay

50 lM by more than one standard deviation (calculated from a total

of 35 assays) were discarded.

All new compounds were evaluated for their ability to suppress

SK expression via a chromogenic assay of plasmin activity. Two

identical cultures of GAS were grown in the presence of the desired

concentration of test compound or DMSO and allowed to grow to

OD₆₀₀ꢁ1.0. After centrifugation, an aliquot of supernatant was

combined with human plasma and synthetic plasmin substrate

S-2403 (see Section 5.1.1). Decreased expression of SK induced

by the test compound lowers the amount of activated plasmin, in

turn reducing the rate of cleavage of S-2403 to colored product

p-nitroaniline. The concentration of p-nitroaniline was measured

by absorbance at 405 nm (A₄₀₅) and compared to the DMSO con-

trol. Activity data is reported as the ratio of A₄₀₅(test) divided by

A₄₀₅(control) (T/C). To provide an approximate readout of potency,

3.2. SAR development

We ﬁrst examined a series of spirocycloalkyl substituted com-

pounds with varying substitution patterns at the 2-position of

the pyrimidinone ring system (Table 1). Aromatic substitution at

R¹(6a–c) did not appreciably increase potency over 1, but methyl

substitution (6f) conferred

a

modest increase in activity

(IC₅₀= 19 M vs >50

l

lM). Interestingly, polar groups were toler-

ated as part of smaller alkyl groups at –XR¹(e.g., 6d and 7a). The

replacement of sulfur with nitrogen (7b) led to an unacceptable de-

crease in growth. Similarly, basic amine functionality appended to

sulfur (6e) led to growth inhibition in some trials. We deﬁned

the assay was run initially at both 5 and 50 lM of test compound.

An optical density reading at 600 nm (OD₆₀₀) gave an estimate of

cell density and thus overall bacterial growth inhibition; this data

acceptable growth inhibition to be no greater than 15% at 50 lM

of test compound. The favorable potency of commercially available

spirocyclopentyl analog 28 relative to closely related spiro-

cyclohexyl analog 6b led us to hypothesize that the size of the

geminal substitution on the central ring could be reduced, allowing

is also reported for the higher concentration of compound (50 lM)

as a ratio of the OD₆₀₀of the test culture divided by the OD₆₀₀of

the DMSO control culture. Selected compounds that inhibited SK

activity by more than 50% at 50 lM were subjected to full dose–re-

O

R

O

H

N

c,d

b

H₂N

EtO

S

CN

HN

21 R = Br

22

O

a

24

R = CO₂H

23

e,f

O

R

NHR

N

O

S

h

S

N

O

N

25 R = CO₂Me

26

27a, b

g

R = CO₂H

Scheme 5. Synthesis of carboxylic acid derivatives 24–26. Reagents and conditions: (a) nBuLi, then CO₂, THF, ꢀ78 °C to rt, 1 h; (b) (i) LDA, diglyme, ꢀ78 °C, 20 min, then ethyl

3,3-dimethyl acrylate, ZnI₂, ꢀ78 °C to rt, 3 h, (ii) TMS-CHN₂, MeOH/toluene (1:5), rt, 30 min; (c) benzoyl isothiocyanate, EtOH, reﬂux, 2.5 h; (d) NaOMe, MeOH, 70 °C, 2 h; (e)

2-methoxyethyl-4-methylbenzenesulfonate, Cs₂CO₃, DMF, 40 °C to rt, 16 h; (f) allyl bromide, NaOMe, MeOH, 65 °C, 2 h; (g) NaOH, MeOH, rt, 16 h; (h) NH₂R, HOBt, EDC, THF,

rt, 16 h.

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B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

Table 1

Inhibition of SK activity by spirocycloalkyl analogs

X

N

O

R¹

N

R²

n

a

c

No.

R¹

R²

X

n

5

l

M SK T/C

50

l

M SK T/C

50

l

M Growth T/C^b

IC₅₀

>50

(

l

M)

MLM t_1/2(min)

Aq Sol.^d

(lM)

1

n-Bu

4-MeOPhCH₂

PhCH₂

4-AcNHPhCH₂

MeOCH₂CH₂

Me₂NCH₂CH₂

Me

Et

PhCH₂

Allyl

Et

S

O

NH

S

1

0

0.84 0.14

1.00 0.33

0.74 0.16

0.96 0.46

0.47 0.28

0.72 0.12

0.60 0.19

0.53 0.23

0.78 0.47

0.44 0.06

0.48 0.28

0.82 0.44

0.57 0.21

0.74 0.28

0.29 0.15

0.52 0.37

0.42 0.03

0.14 0.05

0.56 0.70

0.23 0.16

0.95 0.08

1.05 0.06

0.96 0.06

0.96 0.14

0.99 0.10

0.88 0.20

0.98 0.03

1.02 0.09

0.32 0.18

1.07 0.04

37.2

0.6

9

2

6a

6b

6c

6d

6e

6f

7a

7b

28

>50

9

6.4

9

19 6.0

13.5

22

5

14.5 3.5

9

2

a

Ratio of A₄₀₅of SK-cleaved substrate in GAS culture treated with the indicated concentration of test compound divided by A₄₀₅of DMSO control (see Section 5.1.1). Values

are the mean of at least three experiments standard deviation.

b

Ratio of OD (600 nm) for growth of GAS in the presence of test compound divided by DMSO control. Values are the mean of at least three experiments standard

deviation.

c

Half-life of parent compound during incubation with mouse liver microsomes.

Kinetic solubility of compound in aqueous Todd Hewitt bacterial media.

d

Table 2

Inhibition of SK activity by gem-dimethyl analogs

X

N

O

R¹

N

R²

a

c

No.

R¹

R²

X

5

l

M SK T/C

50

l

M SK T/C^a

50

l

M Growth T/C^b

IC₅₀

(l

M)

MLM t_1/2(min)

Aq Sol.^d

(lM)

12d

12e

12f

12g

12h

12i

13a

13b

19a

29

H₂NCOCH₂

1-pyridylCH₂

HOCH₂CH₂

NCCH₂

MeOCH₂CH₂

Et

Allyl

Et

Allyl

S

O

NH

S

0.79 0.17

0.40 0.09

0.48 0.17

0.37 0.17

0.35 0.11

0.41 0.23

0.34 0.08

0.48 0.19

0.71 0.40

0.65 0.25

0.24 0.07

0.54 0.10

0.64 0.16

0.13 0.07

—

0.23 0.06

0.13 0.07

—

0.23 0.10

—

0.61 0.32

0.35 0.14

0.17 0.08

0.27 0.12

1.00 0.02

1.05 0.02

0.06 0.03

1.00 0.03

0.86 0.10

0.39 0.15

0.50 0.23

0.06 0.03

0.97 0.07

1.03 0.03

0.89 0.05

1.01 0.02

<2

20.6

1.7

0.8

0.9

1.1

9.4

34.5 7.5

22.5 4.5

3.2 0.6

Et

MeOCH₂CH₂

PhCH₂

Et

4-MeOPhCH₂CH₂

Allyl

S

10.8 3.0

2.0 0.5

9

2

30

31

EtO₂CCH₂

a

Ratio of A₄₀₅of SK-cleaved substrate in GAS culture treated with the indicated concentration of test compound divided by A₄₀₅of DMSO control (see Section 5.1.1). Values

are the mean of at least three experiments standard deviation.

b

Ratio of OD (600 nm) for growth of GAS in the presence of test compound divided by DMSO control. Values are the mean of at least three experiments standard

deviation.

c

Half-life of parent compound during incubation with mouse liver microsomes.

Kinetic solubility of compound in aqueous Todd Hewitt bacterial media.

d

reduction of both the lipophilicity and the molecular weight of fu-

ture analogs.

substitution of the N-position of the pyrimidinone was found to

lead to bacterial toxicity (12i) or attenuation of activity (19a) com-

pared to N-allyl (12h). Overall, the S-Et and S-CH₂CH₂OMe analogs

30 and 12h were the optimum compounds from this series

(Table 2).

Our attention then turned to exploring substitution of the aro-

matic ring (Table 3) at 3 different positions. Assessment of methyl

ether analogs 12a–c revealed that substitution at the 8- and 9- posi-

tions conferred a moderate increase in potency over 30, while sub-

stitution of the 7-position did not. The installation of larger and/or

more polar ethers (17a–d, 17h–k) generally led to the attenuation

of these gains, though a few compounds retained activity similar

Exploration of analogs with gem-dimethyl substitution of the

central ring (Table 2) began with the assessment of commercially

available analogs 29, 30, and 31, all of which showed an increase

in potency in comparison to 1. Polar substitution at the R¹position

was generally well-tolerated (12e, 12g, 12h, 31) except for primary

amide 12d. Introducing an alcohol on the sidechain (12f) to im-

prove solubility led to unacceptable bacteriotoxicity. This toxicity

was also observed with heteroatomic analogs 13a and 13b versus

sulfur analog 30, leading us to conclude that retention of the sulfur

was necessary to avoid inhibition of bacterial growth. Altering the

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

1885

Table 3

Inhibition of SK activity by analogs with 7-, 8-, and 9-position substitution

R³

9

8

7

N

O

S

R¹

N

c

No.

R¹

R³

5

l

M SK T/C^a

50

l

M SK T/C^a

50

l

M Growth T/C^b

IC₅₀

(lM)

MLM t_1/2(min)

Aq Sol.^d

(lM)

12a

12b

12c

12j

12k

17a

17b

17c

17d

Et

9-OMe

7-OMe

8-OMe

0.10 0.06

0.26 0.07

0.18 0.09

0.47 0.25

0.33 0.15

0.58 0.24

0.84 0.23

0.46 0.35

0.64 0.67

0.07 0.02

0.29 0.18

0.07 0.01

0.25 0.18

0.28 0.16

0.43 0.15

0.40 0.21

0.36 0.41

0.47 0.48

0.73 0.07

0.28 0.26

0.87 0.19

0.89 0.16

0.92 0.16

1.02 0.10

0.94 0.08

0.85 0.22

1.02 0.10

1.3 0.6

3.1 0.7

6.9 0.3

2.3

0.8

0.6

9

2

14.5 3.5

MeOCH₂CH₂

Allyl

Et

8-OMe

9-MeOCH₂CH₂O

7-MeOCH₂CH₂O

8-MeOCH₂CH₂O

8-EtO₂CCH₂

Et

O

0.53^e

0.08^e

0.41^e

8-

17e

Et

O

N

17f

17g

17h

17i

17j

17k

25

Et

8-O-iPr

8-NCCH₂O

8-n-BuO

8-PhCH₂CH₂O

8-CH₃(CH₂)₆O

8-HO₂CCH₂O

8-CO₂Me

0.29 0.19

0.45 0.25

0.48 0.26

0.68 0.34

1.03 0.18

0.96 0.21

0.71 0.30

0.75 0.36

1.14 0.24

0.13 0.12

0.20 0.17

0.35 0.29

0.59 0.55

0.87 0.12

0.44 0.12

0.58 0.19

0.70 0.52

0.91 0.18

0.87 0.23

0.94 0.15

0.87 0.10

1.01 0.03

1.05 0.03

0.86 0.07

1.01 0.04

0.86 0.11

1.07 0.02

4.1 0.7

14.5 3.5

MeOCH₂CH₂

26

27a

8-CO₂H

8-CONHBn

O

8-

27b

MeOCH₂CH₂

0.84 0.10

0.77 0.27

0.99 0.02

N

NH

H

N

a

Ratio of A₄₀₅of SK-cleaved substrate in GAS culture treated with the indicated concentration of test compound divided by A₄₀₅of DMSO control (see Section 5.1.1). Values

are the mean of at least three experiments standard deviation.

b

Ratio of OD (600 nm) for growth of GAS in the presence of test compound divided by DMSO control. Values are the mean of at least three experiments standard

deviation.

c

Half-life of parent compound during incubation with mouse liver microsomes.

Kinetic solubility of compound in aqueous Todd Hewitt bacterial media.

Values derived from only 1 experiment due to toxicity.

d

e

Table 4

Effect of N- versus O-alkylation on GAS-SK inhibition and metabolic stability

R³

N

S

N

S

R¹

N

R²

O

R²

O

19

20

No.

R¹

R²

R³

5

l

M SK T/C^a

50

l

M SK T/C^a

50

l

M Growth T/C^b

IC₅₀

(lM)

MLM t_1/2(min)^c

19a

20a

19b

20b

12a

20c

MeOCH₂CH₂

Et

4-MeOPhCH₂CH₂

CF₃CH₂

Allyl

H

MeO

0.71 0.40

0.46 0.37

0.78 0.36

0.51 0.20

0.10 0.06

0.89 0.33

0.61 0.32

0.24 0.15

0.49 0.17

0.38 0.23

0.07 0.02

0.68 0.39

0.97 0.07

1.00 0.09

1.01 0.04

0.89 0.02

0.73 0.07

0.96 0.02

9.4

45.1

3.0

45.5

2.3

5.5 0.7

1.3 0.6

Allyl

8.3

a

Ratio of A₄₀₅of SK-cleaved substrate in GAS culture treated with the indicated concentration of test compound divided by A₄₀₅of DMSO control (see Section 5.1.1). Values

are the mean of at least three experiments standard deviation.

b

Ratio of OD (600 nm) for growth of GAS in the presence of test compound divided by DMSO control. Values are the mean of at least three experiments standard

deviation.

c

Half-life of parent compound during incubation with mouse liver microsomes.

to 30 (17f, g). Incorporation of a basic amine (17e) introduced sig-

niﬁcant growth inhibition. Benzoic acid-derived analogs 25–27 also

displayed reduced potency compared to 30. Ultimately, 9-methoxy

analog 12a was found to be the optimal compound from this series,

exhibiting a 90% reduction in streptokinase activity at 5 M and an

IC₅₀of 1.3 M, representing a greater than 35-fold improvement

l

1886

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

3.4. Solubility

oxidation to sulfone

S

In addition to microsomal stability, the aqueous solubility of

N

O

small molecules has been shown to be a critical physicochemical

property for predicting the efﬁcacy of compounds in living sys-

tems. Lipinski suggests that to achieve oral bioavailability at a dose

of 1 mg/kg, a compound minimally needs to achieve an aqueous

demethylation

O

N

dihydroxylation

solubility of 52

ꢂ150 M for small analogs such as 12a. Therefore, several key

l a solubility of

g/mL,²⁵corresponding to

l

compounds from the SAR library were assessed for kinetic solubil-

Figure 2. Potential routes of metabolism identiﬁed by MLM incubation and MS

analysis. No oxidation of the left-hand side of the molecule was observed.

ity in Todd Hewitt (THY) bacterial media. The solubility of all

compounds tested (Tables 1–3) was equal to or less than 25

lM,

with the exception of nitrile 12g (aq sol. = 34.5 7.5 M). It was

l

over 1, although some modest inhibition of bacterial growth at high

concentrations was introduced.

found that the solubility of these compounds was considerably

lower in phosphate-buffered saline (PBS) than in THY media (e.g.,

The ﬁnal analogs for the SAR effort were designed to assess the

effect of alkylation at the pyrimidinone oxygen in comparison to

identical substitution at nitrogen (Table 4). No general efﬁcacy

advantage for N- or O-alkylation was observed; O-alkylated 20a

was somewhat more potent than 19a, while 12a remained much

more potent than its O-alkylated counterpart 20c. Compounds

19b and 20b displayed similarly weak activity.

2

1 vs 9

2 lM [PBS:THY] for 1, 3 1 vs 34.5 7.5 lM [PBS:THY]

for 12g), suggesting that protein binding may increase the effective

aqueous solubility of this compound class. Preliminary activity

studies that included human serum albumin (HSA) in the reaction

mixture also suggested some compounds in this series may bind to

protein.^àThe measured solubility has a weak negative correlation to

the cLogP (R²= 0.533), but the most soluble compound 12g does in-

deed have the lowest cLogP (3.55). Further decreases in cLogP

should logically result in greater aqueous solubility; however, nearly

all efforts to signiﬁcantly decrease lipophilicity, including replace-

ment of the fused phenyl ring with pyridyl and replacement of the

central ring with lactones or lactams (unpublished data), have thus

far led to the reduction or loss of activity.

3.3. Microsomal stability and metabolite identiﬁcation

In our previous publication identifying 1 and 6b as inhibitors of

SK expression, it was determined that 1 displayed efﬁcacy in mouse

models of GAS infection while 6b did not, despite having similar

activity in bacterial assays.¹⁵We hypothesized that a difference in

susceptibility to oxidative metabolism might be playing a role in

the differential activity in mammalian systems. Incubation of each

of these compounds in mouse liver microsomal extract (MLM) sup-

ported this hypothesis, with 1 displaying more than 60-fold higher

stability to oxidation than 6b (t_1/2= 37.2 min vs 0.6 min, respec-

tively). A survey of several more compounds in the series (Tables

1–3) revealed that most were highly unstable (t_1/2<5 min), despite

several structural modiﬁcations that would potentially reduce their

propensity to oxidation, including lowered lipophilicity, fewer

unsubstituted aliphatic/oleﬁnic/aryl carbons, and replacement of

sulfur with oxygen or nitrogen. Interestingly, we also noted that

the spirocyclohexyl compounds were generally more stable than

the corresponding gem-dimethyl compounds (e.g., 6d t_1/

₂= 6.4 min vs 12h t_1/2= 1.7 min), despite their higher lipophilicity.

A metabolite ID study was performed on compound 6d to eluci-

date the most metabolically labile sites of the scaffold. Possible

metabolites were identiﬁed by LC–MS analysis, then fragmented

via collision-induced dissociation (CID). Subsequent MS analysis of

the resulting metabolite fragments allowed us to deduce the struc-

ture of each metabolite. This study indicated that the most metabol-

ically labile portions of the molecule were the substitutions on the

pyrimidinone ring (Fig. 2). No oxidation was indicated on the spiro-

cyclohexane ring or any of the four unsubstituted aromatic carbons.

Based on the ﬁndings of the metabolite ID study, we prepared ﬁve

analogs of our optimum compound 12a that deactivated this portion

of the scaffold to oxidation (Table 5); however, these were also found

to be quickly metabolized. Interestingly, metabolic assessment of

the O-alkylated compounds 20a–c (Table 4) showed that they were

consistently more stable (4 to 15-fold) than their N-alkylated coun-

terparts. This observation, paired with the generally greater stability

of the hindered spirocyclohexyl analogs versus the corresponding

gem-dimethyl analogs, suggests that hydrolysis of the pyrimidinone

amide may play a key role in the metabolic breakdown of these com-

pounds. Compound 20a, despite being four-fold less potent than 12a

3.5. Mammalian cytotoxicity

A representative subset of compounds (1, 12a, 12c, 12h, 19a,

20a, 29) was assessed for toxic effect in HeLa cells using a standard

tetrazolium-formazan assay of cell viability. It was found that none

of the compounds inhibited growth by more than 35% up to

100 lM, the highest concentration tested.

3.6. Bioﬁlm inhibition

As reported previously, a gene expression microarray assay of

GAS mRNA levels after incubation with 6b conﬁrmed the down-

regulation of several virulence genes, including streptokinase,

antiphagocytic factors, and cytolytic toxins. Reduced transcrip-

tion of genes related to cell adhesion and bioﬁlm formation, such

as laminin- and ﬁbronectin-binding proteins and collagen-like

surface protein, was also observed.¹⁵Bioﬁlm formation is critical

for bacterial colonization of solid substrates in the body and

serves to mechanically sequester bacteria from the effects of

the immune system and antibiotics.²⁶Diminishing the ability

of bacteria to form bioﬁlms represents a new mode of action

by which compounds in this series can exert anti-virulence

activity, and more importantly, expands the range of susceptible

bacterial species to clinically important strains such as S. aureus.

The compounds produced for the SK effort were assessed for

their ability to inhibit bioﬁlm formation in this strain using a

previously developed plate-based OD assay.²⁷These assays iden-

tiﬁed compounds 12c (CCG-203592) and 12k (CCG-205363) as

potent S. aureus bioﬁlm inhibitors; these results are reported in

greater detail elsewhere.²⁸

(IC₅₀= 5.5 lM vs 1.3 lM), demonstrates the potential viability of O-

alkylated analogs as more metabolically stable alternatives to the N-

à

Compound 1 showed attenuated efﬁcacy in the presence of 10% HSA compared to

the standard assay conditions. This effect was less pronounced for the more

alkylated compounds.

hydrophilic compound 12g (data not shown).

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

1887

Table 5

Analogs of 12a with potentially reduced metabolic liability

O

N

O

R¹

R²

N

R¹

R²

5

l

M SK T/C^a

50

l

M SK T/C^a

50 l

M Growth T/C^b

MLM t_1/2(min)

c

No.

12a

12l

12m

14

15

19b

S-Et

S-CH₂CF₃

S-Et

(SO)Et

(SO₂)Et

S-Et

Allyl

Me

Allyl

CF₃CH₂

0.10 0.06

0.45 0.19

0.66 0.20

1.00 0.13

0.83 0.31

0.78 0.36

0.07 0.02

0.22 0.08

0.20 0.14

0.51 0.31

0.70 0.13

0.49 0.17

0.73 0.07

0.86 0.07

0.83 0.15

1.01 0.05

1.03 0.03

1.01 0.04

2.3

4.8

1.7

2.8

3.8

3.0

a

Ratio of A₄₀₅of SK-cleaved substrate in GAS culture treated with the indicated concentration of test compound divided by A₄₀₅of DMSO control (see Section 5.1.1). Values

are the mean of at least three experiments standard deviation.

b

Ratio of OD (600 nm) for growth of GAS in the presence of test compound divided by DMSO control. Values are the mean of at least three experiments standard

deviation.

c

Half-life of parent compound during incubation with mouse liver microsomes.

H₃N

O

Cl

MeO

O

H

N

H

N

NO₂

O

N

H

O

N

H

N

H

O

Ph

S-2403

32

Figure 3. Structure of chromogenic plasmin substrate S-2403 and microsomal stability assay internal standard 32.

system to generate increased expression of SK and other virulence

factors.³⁰

4. Conclusions

Extracellular GAS-SK activity was measured by a previously de-

We report the preparation and biological evaluation of 45 ana-

logs of 1, resulting in the achievement of a greater than 35-fold

improvement in activity (IC₅₀>50 lM to 1.3 lM) over compound

scribed chromogenic assay.¹⁵Brieﬂy, a single colony of UMAA2616

was inoculated into Todd-Hewitt broth containing 0.2% yeast ex-

tract (THY) (Difco, Detroit, MI) supplemented with 100 lg/mL

streptomycin and grown overnight at 37 °C. Vials of THY medium

(4 mL) containing different concentrations of test compound in a

ﬁnal concentration of 0.1% DMSO were inoculated with 4 ll of

1 with optimum analog 12a. During the optimization process, we

observed a general instability of new analogs to oxidative metabo-

lism. Through careful structure metabolism relationship analysis,

we have developed a hypothesis that a primary route of metabo-

lism for this class of compounds is through hydrolysis of the pyri-

midinone amide, which should guide the design of new analogs

with improved potential for activity in vivo. Murine infection stud-

ies are in fact currently planned for the most stable active analogs

from this work (6f, 12g, 20a, 20b) and will be reported in due

course. Concurrently, a more focused effort toward optimizing

the bioﬁlm inhibition activity of this class of compounds is

underway.

the UMAA2616 overnight culture. The cultures were grown in trip-

licate at 37 °C to an OD₆₀₀ꢁ1.0. Each sample was then centrifuged

at 11,000g for 8 min. An aliquot (20 lL) of supernatant was mixed

with 100

ll PBS, 10

ll human plasma (Innovative Research, Novi,

MI), and 10

ll of 1 mg/ml S-2403 solution (Fig. 3; Diapharma

Group Inc., West Chester, OH), then incubated at 37 °C for 2 h.

Activity was reported as the ratio of SK activity as measured by

absorbance at 405 nm of the test cultures compared to that of a

culture treated with only DMSO. A GAS strain deﬁcient in SK

activity, UMAA2641,³¹was used as a blank. The SK activity was

calculated based on a standard curve made with serial dilutions

of control UMAA2616 cultures grown under the same conditions

with 0.1% DMSO. The numbers were then corrected for inhibition

of growth by each test compound versus DMSO control, as mea-

sured by OD₆₀₀. The experiments were performed in triplicate to

obtain mean and standard deviation values for each test

concentration.

5. Experimental section

5.1. Biological/physicochemical characterization

5.1.1. SK expression assay

The GAS strain UMAA2616 used in this study was derived from

the GAS M type 1 strain MGAS166.²⁹UMAA2616 was originally

designated as UMAA2392 and contains mutations in the CovR/S

1888

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

5.1.2. Aqueous solubility assay

(EMS) full scan, and precursor scanning detection modes. Only the

ions detected in the test sample and absent in both control samples

were regarded as possible metabolites. Based on the MS²and MS³

spectra of the possible metabolites and the proposed fragmentation

pathways of 6d, the likely structures of the metabolites were deduced.

Compounds were dissolved in DMSO to a stock concentration

of 10 mM. 100 L of each stock was diluted with DMSO through

a series of twelve 35% dilutions, resulting in working solutions

with concentrations ranging from 10 mM to 0.0875 mM. 2 L of

each working solution was added to one well of a clear plastic,

round-bottom 96-well plate containing 200 of

l

L

5.1.5. Mammalian cytotoxicity assay

standard-strength Todd-Hewitt broth or phosphate-buffered sal-

ine solution warmed to 37 °C, resulting in ﬁnal testing concentra-

tions of 0.87 to 99 lM. After addition of compound to all test

HeLa cells (ATCC, Manassas, VA) were cultured in Dulbecco’s

modiﬁed Eagle’s medium (DMEM) (Life Technologies, Grand Island,

NY) supplemented with 10% fetal bovine serum, 1% penicillin-

wells, the plate was loaded into a Molecular Devices SpectraMax

Plus UV/Vis spectrophotometer, shaken for 15 seconds, and incu-

bated for 5 min at 37 °C. Measurement of the OD₆₀₀resulted in a

series of curves. The concentration at which the average (n = 3 for

each concentration and test compound) OD₆₀₀reading rose above

background (OD₆₀₀P0.005 AU) was noted, and the aqueous sol-

ubility is reported as the mean concentration between this point

and the previous point ( the concentration difference between

the two points ^⁄0.5).

streptomycin and 1%

sphere. Cell suspensions (200

ent concentrations (0.39

DMSO were plated in 96-well plates and cultured for 24 h in tripli-

cate. CellTiter 96 Aqueous One Solution reagent (Promega, Madison,

L

-glutamine at 37 °C under 5% CO₂atmo-

l

L, 2.5 ꢃ 10⁴cells/well) with differ-

lM to 100 lM) of test compounds or

WI) (20 lL) was added to each sample and incubated for 2 h. Viabil-

ity was reported as the ratio of absorbance at 490 nm of the test

compound wells in comparison to DMSO control. The experiment

was repeated three times to obtain mean and standard error values.

5.1.3. Microsomal stability assay

5.2. Chemistry procedures

Stock solutions of test compounds were generated via the dilu-

tion of 100 mM DMSO stocks 1000-fold with phosphate-buffered

General information: Chemical names follow CAS nomenclature.

Starting materials were purchased from Fisher, Sigma–Aldrich Lan-

caster, Fluka or TCI-America and were used as supplied unless

otherwise indicated. All reaction solvents were purchased from

Fisher and used as received. Reactions were monitored by TLC

using precoated silica gel 60 F254 plates. All anhydrous reactions

were run under an atmosphere of dry nitrogen. Silica gel chroma-

tography was performed with silica gel (220–240 mesh) obtained

from Silicycle. Solvent abbreviations used: CDCl₃, deutero-chloro-

form; DCM, dichloromethane; DMSO, dimethyl sulfoxide; EtOH,

ethanol; MEK, methyl ethyl ketone; THF, tetrahydrofuran; DMF,

N,N-dimethylformamide; EtOAc, ethyl acetate; hex, hexanes. Re-

agent abbreviations used: Cs₂CO₃, cesium carbonate; Na₂SO₄, so-

dium sulfate; MgSO₄, magnesium sulfate; mCPBA, meta-chloro

peroxybenzoic acid; KOH, potassium hydroxide; LDA, lithium

diisopropylamide; NaHCO₃, sodium bicarbonate; NaOMe, sodium

methoxide; TMS, trimethylsilyl; HOBt 1-hydroxybenzotriazole;

EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; ZnI₂, zinc

iodide.

NMR spectra were recorded on a Bruker 400 MHz, Bruker

500 MHz, Varian 400 MHz, or Varian 500 MHz spectrometer.

Chemical shifts are reported in d (parts per million), by reference

to the hydrogen residues of deuterated solvent as internal standard

CDCl₃: d = 7.28 (¹H NMR), or in reference to the hydrogen peaks of

tetramethylsilane, d = 0.00 (¹H NMR). Mass spectra were recorded

on a Micromass LCT time-of-ﬂight instrument utilizing electro-

spray ionization operating in positive-ion (ESI+) or negative-ion

(ESIꢀ) modes where indicated. Melting points were measured on

a MEL-TEMP melting point apparatus and are uncorrected. The

purity of the compounds was assessed via analytical rpHPLC with

one of three gradient methods. ‘Method A’: 10% B to 90% B over

6 min, hold at 90% B for 7 additional minutes; ‘Method B’: 50% B

to 90% B, hold at 90% B for 7 additional minutes; ‘Method C’: 90%

B over 12 min (solvent A H₂O, solvent B acetonitrile, C18 column,

saline containing up to 10% MeOH as a co-solvent. To 366

100 mM phosphate buffer containing 3.3 mM MgCl₂was added

10 L of 20 mg/mL mouse liver microsomal extract (XenoTech,

Lenexa, KS) and 4 L of test compound solution (100 M). Enzy-

matic oxidation was initiated by adding 15 L of 16.7 mg/mL

lL of

l

NADPH in 100 mM phosphate buffer containing 3.3 mM MgCl₂

(ﬁnal concentrations of mouse liver microsomes and test com-

pound = 0.5 mg/mL and 1

carried out at 37 °C for 60 min. At each time point (0, 1, 3, 5,

10, 30, and 60 min), an aliquot of the reaction mixture (30 L)

was removed and added to 90 L of cold acetonitrile containing

lM, respectively). The reactions were

l

a known concentration of internal standard compound (32, Fig. 3)

to quench the reaction. The quenched sample mixtures were cen-

trifuged at 16,000g for 10 min. The supernatant was then analyzed

via an LC–MS/MS system equipped with a reverse-phase column.

LC–MS/MS analysis of test compounds was performed on an

LC–MS/MS-3200 system (AB Sciex, Framingham, MA) equipped

with an electrospray ionization (ESI) source. The AB Sciex LC-

MS/MS 2800 system consisting of Agilent 1200 series (Agilent,

Santa Clara, CA) with a Zorbax Extend C-18 column (5 lm,

50 ꢃ 2.1 mm) was used for the separation and the efﬂuent from

the column was directly fed into the ionization source. The sys-

tem was controlled by Analyst software (version 1.4.2) to collect

and process data. The mobile phase consisted of water containing

0.1% formic acid (Solvent A) and acetonitrile containing 0.1% for-

mic acid (Solvent B) for all test compounds with the solvent B

gradient changing from 10–95% during a 15 min run. The LC–

MS/MS was operated at a ﬂow rate of 0.4 mL/min. Metabolic

half-lives were derived from the rate of disappearance of the par-

ent compound MS/MS readout as determined by comparison to

the internal standard.

5.1.4. Metabolite identiﬁcation study

Metabolic oxidation and HPLC separation were performed in

the same manner as in the metabolic half-life determination as-

says. Samples from the metabolic oxidation of 6d were injected

into the HPLC and subjected to enhanced product ion (EPI), MS/

MS (MS²), and MS³scanning to obtain MS²and MS³fragmentation

spectra of each potential metabolite derived from the M+H ion of

6d. Samples of 6d incubated with inactivated (boiled) MLM or

omitting the addition of NADPH to the reaction mixture served

as negative controls. Potential metabolites were identiﬁed using

multiple reaction monitoring (MRM), enhanced mass spectrometry

3.5 um, 4.6 ꢃ 100 mm, 254 nm

l).

5.2.1. Ethyl 2-cyano-2-cyclohexylideneacetate (3)

To a solution of cyclohexanone (1.50 ml, 14.47 mmol) in tolu-

ene (24.12 ml) was added ethyl cyanoacetate (1.556 ml,

14.62 mmol), acetic acid (0.166 ml, 2.89 mmol), and ammonium

acetate (0.112 g, 1.447 mmol). The mixture was heated to reﬂux

at 150 °C in a Dean-Stark apparatus. After 5 h, the reaction was

cooled and washed with water and saturated NaHCO₃solution.

The organics were dried over Na₂SO₄, ﬁltered, and concentrated.

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

1889

The resulting residue (2.48 g, 89% yield) was used without further

5.2.6. 3-Allyl-2-((4-methoxybenzyl)thio)-3H-

spiro[benzo[h]quinazoline-5,1’-cyclohexan]-4(6H)-one (6a)

puriﬁcation. ¹H NMR (500 MHz, CDCl₃) d (ppm) 4.29 (q, J = 7.2 Hz,

2H), 3.00 (t, J = 6.0 Hz, 2H), 2.68 (t, J = 6.1 Hz, 2H), 1.82 (p,

J = 6.0 Hz, 2H), 1.75 (p, J = 6.1 Hz, 2H), 1.70–1.65 (m, 2H), 1.37 (t,

J = 7.2 Hz, 3H).

Prepared from 5 according to General Method A, using 4-

methoxybenzyl bromide as the alkylating agent, KOH as the base,

and ethanol as the solvent. Isolated as a clear oil (97 mg, 72% yield).

¹H NMR (500 MHz, CDCl₃) d (ppm) 8.17 (d, J = 7.6 Hz, 1H), 7.41–

7.36 (m, 3H), 7.34 (t, J = 6.8 Hz, 1H), 7.24 (d, J = 7.2 Hz, 1H), 6.89

(d, J = 8.6 Hz, 2H), 5.94 (ddt, J = 17.1, 10.7, 5.6 Hz, 1H), 5.37–5.18

(m, 2H), 4.67 (d, J = 5.6 Hz, 2H), 4.57 (s, 2H), 3.82 (s, 3H), 3.07 (s,

2H), 2.72–2.51 (m, 2H), 1.74 (d, J = 12.8 Hz, 1H), 1.66–1.50 (m,

4H), 1.49–1.32 (m, 3H). ESI+MS m/z = 481.2 (M+Na⁺). HPLC (Meth-

od C, t_R= 5.11 min), purity >95%.

5.2.2. Ethyl 4⁰-amino-1⁰H-spiro[cyclohexane-1,2-naphthalene]-

3⁰-carboxylate (4)

1 M benzylmagnesium chloride in diethyl ether (18.92 ml) was

added dropwise to a solution of 3 (1.828 g, 9.46 mmol) in diethyl

ether (6.31 ml) at room temperature. The mixture was stirred at

room temperature for 3 days, then 10% hydrochloric acid

(7.83 ml, 255 mmol) was added dropwise at 0 °C while stirring.

The organic layer was separated, washed with water, dried over

Na₂SO₄, ﬁltered, and concentrated to dryness in vacuo. Concen-

trated H₂SO₄(4.59 ml) was added dropwise to the crude solid at

0 °C. The mixture was stirred at room temperature for 7 h, then

ice water was added to the mixture resulting in a precipitate.

The precipitate was dissolved in Et₂O and washed with 28% NH₃

solution. The organic layer was washed with water, dried over

Na₂SO₄, ﬁltered, and concentrated to an orange oil (1.66 g, 68%

yield). ¹H NMR (500 MHz, CDCl₃) d (ppm) 7.42 (d, J = 6.8 Hz, 1H),

7.36–7.25 (m, 2H), 7.22 (d, J = 6.8 Hz, 1H), 6.04 (br s, 2H), 4.25 (q,

J = 7.0 Hz, 2H), 2.87 (s, 2H), 2.24–2.05 (m, 2H), 1.76–1.20 (m, 9H).

5.2.7. 3-Allyl-2-(benzylthio)-3H-spiro[benzo[h]quinazoline-5,1’-

cyclohexan]-4(6H)-one (CCG-102487) (6b)

Prepared from 5 according to General Method A, using benzyl

bromide as the alkylating agent, KOH as the base, and ethanol as

the solvent. Isolated as a clear oil (95 mg, 75% yield). ¹H NMR

(400 MHz, CDCl₃)

d (ppm) 8.11 (d, J = 7.4 Hz, 1H), 7.44 (d,

J = 7.2 Hz, 2H), 7.39–7.27 (m, 5H), 7.21 (d, J = 7.2 Hz, 1H), 5.92

(ddt, J = 15.8, 10.7, 5.5 Hz, 1H), 5.32–5.21 (m, 2H), 4.66 (d,

J = 5.5 Hz, 2H), 4.59 (s, 2H), 3.04 (s, 2H), 2.65–2.53 (m, 2H), 1.71

(d, J = 12.7 Hz, 1H), 1.61–1.52 (m, 4H), 1.43–1.32 (m, 3H). ESI+MS

m/z = 429.2 (M+H⁺), 451.2 (M+Na⁺). HPLC (Method C,

t_R= 4.68 min), purity >95%.

5.2.3. 3-Allyl-2-thioxo-2,3-dihydro-1H-

5.2.8. N-(4-(((3-Allyl-4-oxo-4,6-dihydro-3H-

spiro[benzo[h]quinazoline-5,1⁰-cyclohexan]-4(6H)-one (5)

Intermediate 4 (1.66 g, 5.82 mmol) and allyl isothiocyanate

(0.600 ml, 6.13 mmol) were dissolved in ethanol (9.69 ml) and re-

ﬂuxed at 85 °C. for 10 h. A solution of KOH (0.653 g, 11.63 mmol) in

water (9.69 ml) was then added and the reaction mixture was re-

ﬂuxed for 3 h. The cooled reaction mixture was acidiﬁed to pH

3.0–3.5 resulting in precipitation. The precipitate was collected

via vacuum ﬁltration, washed with water, and recrystallized from

butanol. Recovered as pale yellow crystals (587 mg, 29.8% yield).

TLC R_f= 0.25 (10% EtOAc/hex). ¹H NMR (500 MHz, CDCl₃) d (ppm)

9.28 (s, 1H), 7.50–7.35 (m, 3H), 7.32 (d, J = 7.4 Hz, 1H), 6.00 (ddt,

J = 16.2, 11.3, 5.8 Hz, 1H), 5.37 (dd, 1H), 5.27 (dd, 1H), 5.06 (d,

J = 5.8 Hz, 2H), 3.03 (s, 2H), 2.48 (td, J = 13.3, 4.4 Hz, 2H), 1.72 (d,

J = 13.3 Hz, 1H), 1.62–1.44 (m, 4H), 1.33 (d, J = 14.3 Hz, 3H).

spiro[benzo[h]quinazoline-5,1’-cyclohexan]-2-

yl)thio)methyl)phenyl)acetamide (6c)

Prepared from 5 according to General Method A, using N-(4-

(chloromethyl)phenyl)acetamide as the alkylating agent, Cs₂CO₃

as the base, and MEK as the solvent. Isolated as a white solid

(71 mg, 83% yield). ¹H NMR (500 MHz, CDCl₃) d (ppm) 8.11 (d,

J = 7.2 Hz, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.39 (d, J = 8.5 Hz, 2H),

7.35 (t, J = 7.3 Hz, 1H), 7.31 (t, J = 6.9 Hz, 1H), 7.22 (d, J = 7.3 Hz,

1H), 7.16 (s, 1H), 5.91 (ddt, J = 15.9, 10.5, 5.6 Hz, 1H), 5.35–5.11

(m, 2H), 4.65 (d, J = 5.6 Hz, 2H), 4.55 (s, 2H), 3.49 (d, J = 5.3 Hz,

1H), 3.04 (s, 2H), 2.65–2.51 (m, 2H), 2.18 (s, 3H), 1.71 (d,

J = 13.4 Hz, 1H), 1.60–1.51 (m, 4H), 1.45–1.32 (m, 3H). ESI+MS m/

z = 508.2 (M+Na⁺). HPLC (Method C, t_R= 2.75 min), purity >95%.

5.2.9. 3-Allyl-2-((2-methoxyethyl)thio)-3H-

5.2.4. General Method A for generating compounds 1, 6a–e

Compound 5 (100 mg, 0.295 mmol) was combined with base

(0.443 mmol) and alkylating agent (0.325 mmol) in ethanol or

MEK (1.75 mL). The suspension was warmed to 70 °C and allowed

to stir for 16 h. The suspension was subsequently diluted with

EtOAc and washed with water and brine. The isolated organic layer

was then dried over MgSO₄, ﬁltered, and concentrated in vacuo.

Further puriﬁcation via ﬂash chromatography (0% to 10% EtOAc/

hex) delivered the desired compounds in 72–83% yield.

spiro[benzo[h]quinazoline-5,1’-cyclohexan]-4(6H)-one (6d)

Prepared from 5 according to General Method A, using 2-

methoxyethyl p-toluenesulfonate as the alkylating agent, KOH as

the base, and ethanol as the solvent. Isolated as a yellow oil

(88 mg, 75% yield). ¹H NMR (500 MHz, CDCl₃) d (ppm) 8.08 (d,

J = 7.5 Hz, 1H), 7.38 (t, J = 7.3 Hz, 1H), 7.33 (t, J = 6.8 Hz, 1H), 7.24

(d, J = 7.2 Hz, 1H), 5.96 (ddt, J = 15.9, 11.0, 5.6 Hz, 1H), 5.35–5.26

(m, 2H), 4.71 (d, J = 5.6 Hz, 2H), 3.79 (t, J = 6.2 Hz, 2H), 3.56 (t,

J = 6.2 Hz, 2H), 3.44 (s, 3H), 3.06 (s, 2H), 2.66–2.56 (m, 2H), 1.73

(d, J = 13.0 Hz, 1H), 1.62–1.54 (m, 4H), 1.44–1.35 (m, 3H). ESI+MS

m/z = 397.2 (M+H⁺), 419.2 (M+Na⁺). HPLC (Method C,

t_R= 3.54 min), purity >95%.

5.2.5. 3-Allyl-2-(butylthio)-3H-spiro[benzo[h]quinazoline-5,1’-

cyclohexan]-4(6H)-one (CCG-2979) (1)

Prepared from 5 according to General Method A, using 1-iodo-

butane as the alkylating agent, Cs₂CO₃as the base, and MEK as

the solvent. Isolated after ﬂash chromatography (0–5% EtOAc/

hex) as white crystals (87 mg, 74% yield). ¹H NMR (400 MHz,

CDCl₃) d (ppm) 8.08 (d, J = 6.7 Hz, 1H), 7.38–7.28 (m, 2H), 7.20 (d,

J = 6.8 Hz, 1H), 5.99–5.87 (m, 1H), 5.33–5.22 (m, 2H), 4.68 (d,

J = 5.2 Hz, 2H), 3.31 (t, J = 7.3 Hz, 2H), 3.03 (s, 2H), 2.67–2.52 (m,

2H), 1.80 (p, J = 7.4 Hz, 2H), 1.71 (d, J = 11.3 Hz, 1H), 1.63–1.45

(m, 6H), 1.44–1.31 (m, 3H), 0.98 (t, J = 7.4 Hz, 3H). ESI+MS m/

z = 395.2 (M+H⁺), 417.2 (M+Na⁺). HPLC (Method C, t_R= 7.44 min),

purity >95%.

5.2.10. 3-Allyl-2-((2-(dimethylamino)ethyl)thio)-3H-

spiro[benzo[h]quinazoline-5,1’-cyclohexan]-4(6H)-one (6e)

Prepared from 5 according to General Method A, using b-

dimethylaminoethyl bromide hydrobromide as the alkylating

agent, KOH as the base, and MEK as the solvent. Isolated as white

crystals (88 mg, 75% yield). ¹H NMR (500 MHz, CDCl₃) d (ppm) 8.10

(d, J = 7.6 Hz, 1H), 7.35 (t, J = 7.3 Hz, 1H), 7.30 (t, J = 7.4 Hz, 1H),

7.21 (d, J = 7.1 Hz, 1H), 5.93 (ddt, J = 15.9, 10.4, 5.5 Hz, 1H), 5.32–

5.23 (m, 2H), 4.69 (d, J = 5.5 Hz, 2H), 3.46 (t, J = 7.0 Hz, 2H), 3.04

(s, 2H), 2.73 (t, J = 7.0 Hz, 2H), 2.64–2.53 (m, 2H), 2.34 (s, 6H),

1890

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

1.71 (d, J = 12.6 Hz, 1H), 1.59–1.51 (m, 4H), 1.42–1.33 (m, 3H).

ESI+MS m/z = 410.3 (M+H⁺). HPLC (Method A, t_R= 6.29 min), purity

>95%.

5.2.15. 3-Allyl-2-(ethylamino)-3H-spiro[benzo[h]quinazoline-

5,1⁰-cyclohexan]-4(6H)-one (7b)

Prepared from 6f according to General Method C. Isolated as a

white crystalline solid, 53% yield over 2 steps. ¹H NMR (500 MHz,

CDCl₃) d (ppm) 8.13 (d, J = 7.4 Hz, 1H), 7.34–7.27 (m, 2H), 7.18

(d, J = 6.6 Hz, 1H), 5.92 (ddt, J = 17.3, 10.5, 5.3 Hz, 1H), 5.36–5.26

(m, 2H), 4.66 (d, J = 5.2 Hz, 2H), 4.56 (d, J = 5.0 Hz, 1H), 3.57 (qd,

J = 7.2, 5.0 Hz, 2H), 3.01 (s, 2H), 2.64–2.54 (m, 2H), 1.70 (d,

J = 12.6 Hz, 1H), 1.62–1.48 (m, 5H), 1.38 (d, J = 12.8 Hz, 3H), 1.28

(t, J = 7.2 Hz, 3H). ESI+MS m/z = 350.2 (M+H⁺), 372.2 (M+Na⁺). HPLC

(Method B, t_R= 7.18 min), purity = 94%.

5.2.11. 3-Allyl-2-(methylthio)-3H-spiro[benzo[h]quinazoline-

5,1⁰-cyclohexan]-4(6H)-one (6f)

Compound 5 (150 mg, 0.443 mmol) was dissolved in absolute

EtOH at 0 °C (2.61 mL) to which KOH (37 mg, 0.665 mmol) and

methyl iodide (33 lL, 0.532 mmol) were added. The solution was

allowed to stir for 10 min, resulting in the precipitation of white

crystals. The suspension was diluted with H₂O and vacuum ﬁltered

to collect the precipitate. The precipitate was washed with water

and dried in vacuo. Recovered 150 mg (95% yield). ¹H NMR

5.2.16. General Method D for generating 9a and 9b

To anhydrous THF (6.4 mL) in a dry round-bottom ﬂask was

added nickel(II)bromide (279 mg, 1.28 mmol), zinc powder

(250 mg, 3.83 mmol), and triphenylphosphine (1.68 g, 6.39 mmol).

The mixture was heated to 50 °C and stirred for 30 min. The se-

lected o-chlorotoluene (8a or 8b, 12.78 mmol) was added, the tem-

perature raised to 60 °C, and the reaction was tightly capped and

allowed to stir 30 min, then potassium cyanide (1.66 g, 25.5 mmol)

was added over the course of 5 h in 2 equal portions. The mixture

was allowed to stir an additional 16 h. Water was added to quench

the reaction, and the suspension extracted 3ꢃ with ether. The

resulting organic layer was washed with H₂O and brine, dried over

MgSO₄, ﬁltered, and concentrated to a heterogeneous mixture of

white crystals and clear oil. The residue was diluted with 10 mL

of toluene, then methyl iodide (997 mg, 7.02 mmol) was added

and allowed to stir 16 h at room temperature. The resulting white

crystals were removed via vacuum ﬁltration. The ﬁltrate was con-

centrated in vacuo to a clear oil. Flash chromatography with 2%

EtOAc/hex delivered the pure product in 68–90% yield.

(500 MHz, CDCl₃)

d (ppm) 8.14 (d, J = 7.4 Hz, 1H), 7.35 (t,

J = 7.3 Hz, 1H), 7.31 (t, J = 6.8 Hz, 1H), 7.21 (d, J = 7.1 Hz, 1H), 5.94

(ddt, J = 15.9, 10.8, 5.6 Hz, 1H), 5.32–5.24 (m, 2H), 4.68 (d,

J = 5.6 Hz, 2H), 3.04 (s, 2H), 2.68 (s, 3H), 2.64–2.54 (m, 2H), 1.71

(d, J = 12.8 Hz, 1H), 1.60–1.51 (m, 4H), 1.42–1.33 (m, 3H). ESI+MS

m/z = 353.2 (M+H⁺), 375.2 (M+Na⁺). HPLC (Method C,

t_R= 4.19 min), purity >95%.

5.2.12. General Method B for generating 7a and 13a

Metallic sodium (78 mg, 3.40 mmol) was added to absolute eth-

anol (1.67 mL) and allowed to stir at room temperature for 30 min.

Once all solid had dissolved, the corresponding 2-methylthio 5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (6f or 12n, 0.284 mmol)

was added. The reaction was warmed to 40 °C and allowed to stir

for 48 h. After the completion of the reaction, the solution was di-

luted with H₂O and extracted with 3 portions of ethyl acetate. The

organic layers were combined, washed with water and brine, then

isolated, dried over MgSO₄, vacuum ﬁltered, and concentrated to a

yellow solid that was further puriﬁed via silica ﬂash chromatogra-

phy (15% EtOAc/hex), 69–73% yield.

5.2.17. 5-Methoxy-2-methylbenzonitrile (9a)

Synthesized from 8a according to General Method D. Isolated in

90% yield. ¹H NMR (400 MHz, CDCl₃) d (ppm) 7.21 (d, J = 8.5 Hz,

1H), 7.08 (d, J = 2.8 Hz, 1H), 7.03 (dd, J = 8.5, 2.8 Hz, 1H), 3.81 (s,

3H), 2.47 (s, 3H).

5.2.13. 3-Allyl-2-ethoxy-3H-spiro[benzo[h]quinazoline-5,1⁰-

cyclohexan]-4(6H)-one (7a)

Prepared according to General Method B from 6f. Isolated as a

light yellow crystalline solid (73 mg, 73% yield). ¹H NMR

(400 MHz, CDCl₃) d (ppm) 8.07 (d, J = 8.0 Hz, 1H), 7.38–7.25 (m,

2H), 7.20 (d, J = 6.8 Hz, 1H), 5.99–5.85 (m, 1H), 5.27–5.15 (m, 2H),

4.61 (d, J = 5.8 Hz, 3H), 4.56 (q, J = 7.2 Hz, 2H), 3.03 (s, 2H), 2.66–

2.53 (m, 2H), 1.70 (d, J = 12.1 Hz, 1H), 1.60–1.50 (m, 4H), 1.45 (t,

J = 7.2 Hz, 3H), 1.41–1.33 (m, 3H). ESI+MS m/z = 351.2 (M+H⁺),

373.2 (M+Na⁺) HPLC (Method B, t_R= 9.75 min), purity >95%.

5.2.18. 3-Methoxy-2-methylbenzonitrile (9b)

Synthesized from 8b according to General Method D. Isolated as

a clear oil, 68% yield. ¹H NMR (500 MHz, CDCl₃) d (ppm) 7.24 (t,

J = 7.9 Hz, 1H), 7.19 (d, J = 7.7 Hz, 1H), 7.03 (d, J = 8.1 Hz, 1H),

3.86 (s, 3H), 2.42 (s, 3H).

5.2.19. General Method E for synthesizing 10a–10d, 23

A dry round bottom ﬂask was charged with anhydrous diglyme

(24 mL) and diisopropylamine (2.91 mL, 20.4 mmol), then cooled

to ꢀ78 °C in a dry ice/acetone bath. To this solution was added

n-butyllithium (2.5 M in hexanes, 8.15 mL, 20.4 mmol). The reac-

tion was removed from the dry ice/acetone bath for 10 min, then

re-cooled to ꢀ78 °C. The desired o-tolunitrile 9a–d (6.79 mmol),

dissolved in anhydrous diglyme (2 mL), was then added slowly

dropwise then allowed to stir at -78 °C for 45 min.

A separate dry ﬂask was charged with anhydrous diglyme

(8.0 mL) and zinc powder (1.11 g, 17.0 mmol). Molecular iodine

(3.45 g, 13.6 mmol) was added portionwise over the course of

10 min. The suspension was subsequently heated via heat gun in

30-s intervals until a silver precipitate of ZnI₂had formed and all

iodine color had disappeared (caution: exothermic).

Ethyl-3,3-dimethyl acrylate (1.42 mL, 10.2 mmol) was added to

the ﬁrst ﬂask dropwise over 10 min. The ﬂask containing the ZnI₂

suspension was then added to the reaction vessel and the resulting

suspension allowed to stir for an additional 2 h, slowly warming to

room temperature. The reaction was quenched by the addition of

saturated ammonium chloride solution and the resulting biphasic

suspension was extracted with diethyl ether (3 ꢃ 30 mL), then

5.2.14. General Method C for generating 7b and 13b

Compound 6f or 12n (1.28 mmol) was dissolved in DCM

(19.3 mL), then mCPBA (70 wt%, 787 mg, 3.19 mmol) was added

and the reaction mixture allowed to stir over the course of 16 h

at room temperature. At this time the reaction mixture was di-

luted with DCM, washed with saturated aqueous NaHCO₃solu-

tion, water, and brine. The organic layer was dried over

MgSO₄, vacuum ﬁltered, and concentrated in vacuo. Puriﬁcation

via ﬂash chromatography isolated the sulfone intermediate

which was then dissolved in a 1:1 mixture of THF:DMF (2 mL).

Potassium carbonate (165 mg, 1.19 mmol) and 2 M ethylamine

solution in THF (1.00 mL, 2.00 mmol) were added, then the reac-

tion vessel was tightly capped and allowed to stir 5 h at RT. The

reaction mixture was diluted with diethyl ether, then washed

with 3 portions of H₂O followed by brine. The organic layer

was isolated, dried over MgSO₄, vacuum ﬁltered, and concen-

trated in vacuo. The residue was further puriﬁed by ﬂash chro-

matography (10–33% EtOAc/hex) and isolated in 41–53% yield

over 2 steps.

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

1891

washed with water (4 ꢃ 50 mL) and brine (1 ꢃ 50 mL). The organic

extract was dried over MgSO₄, vacuum ﬁltered, and concentrated

in vacuo. Further puriﬁcation via ﬂash chromatography (silica

gel, 5% EtOAc/hex) delivered the desired 10a–d in 33–59% yields.

7.08–7.01 (m, 2H), 6.00 (ddt, J = 16.8, 10.8, 5.8 Hz, 1H), 5.37 (d,

J = 16.8 Hz, 1H), 5.27 (d, J = 10.8 Hz, 1H), 5.06 (d, J = 5.8 Hz, 2H),

3.89 (s, 3H), 2.79 (s, 2H), 1.34 (s, 6H).

5.2.27. 3-alLyl-8-methoxy-5,5-dimethyl-2-thioxo-2,3,5,6-

tetrahydrobenzo[h]quinazolin-4(1H)-one (11c)

5.2.20. Ethyl 1-amino-7-methoxy-3,3-dimethyl-3,4-

dihydronaphthalene-2-carboxylate (10a)

Prepared according to General Method F from 10c and allyl iso-

thiocyanate. Puriﬁed via trituration of the crude organic isolate

with hexanes and diethyl ether; isolated as tan crystals (252 mg,

59% yield). TLC R_f= 0.11 (10% EtOAc/hex). ¹H NMR (400 MHz,

CDCl₃) d (ppm) 9.27 (s, 1H), 7.38 (d, J = 8.6 Hz, 1H), 6.88 (dd,

J = 8.6, 2.5 Hz, 1H), 6.80 (d, J = 2.5 Hz, 1H), 5.99 (ddt, J = 16.6,

10.4, 5.7 Hz, 1H), 5.36 (dd, J = 16.6, 1.3 Hz, 2H), 5.26 (dd, J = 10.4,

1.3 Hz, 1H), 5.06 (d, J = 5.7 Hz, 2H), 3.87 (s, 3H), 2.74 (s, 2H), 1.33

(s, 6H). ESI-MS m/z = 327 (MꢀH⁺).

Prepared according to General Method E from 9a. Isolated as a

pale yellow oil (623 mg, 33% yield). ¹H NMR (500 MHz, CDCl₃) d

(ppm) 7.11 (d, J = 8.1 Hz, 1H), 6.96 (s, 1H), 6.88 (d, J = 8.1 Hz, 1H),

6.27 (s, 2H), 4.27 (q, J = 7.1 Hz, 2H), 3.85 (s, 3H), 2.60 (s, 2H), 1.37

(t, J = 7.1 Hz, 3H), 1.20 (s, 6H).

5.2.21. Ethyl 1-amino-5-methoxy-3,3-dimethyl-3,4-

dihydronaphthalene-2-carboxylate (10b)

Prepared according to General Method E from 9b. Isolated as a

yellow oily solid (636 mg, 35% yield). ¹H NMR (500 MHz, CDCl₃) d

(ppm) 7.24 (t, J = 8.1 Hz, 2H), 7.04 (d, J = 8.1 Hz, 1H), 6.92 (d,

J = 8.1 Hz, 1H), 6.31 (s, 2H), 4.25 (q, J = 7.1 Hz, 2H), 3.85 (s, 3H),

2.66 (s, 2H), 1.34 (t, J = 7.1 Hz, 3H), 1.20 (s, 6H).

5.2.28. 3-Allyl-5,5-dimethyl-2-thioxo-2,3,5,6-

tetrahydrobenzo[h]quinazolin-4(1H)-one (11d)

Prepared according to General Method F from 10d and allyl iso-

thiocyanate. Puriﬁed via trituration of the crude organic isolate

with hexanes and diethyl ether; recovered in 24% yield. R_f= 0.34

(10% EtOAc/hex). ¹H NMR (500 MHz, CDCl₃) d (ppm) 9.28 (s, 1H),

7.50–7.37 (m, 3H), 7.29 (d, J = 7.4 Hz, 1H), 6.00 (ddt, J = 17.1,

10.3, 5.8 Hz, 1H), 5.37 (d, J = 17.1 Hz, 1H), 5.27 (d, J = 10.3 Hz,

1H), 5.07 (d, J = 5.8 Hz, 2H), 2.79 (s, 2H), 1.34 (s, 6H).

5.2.22. Ethyl 1-amino-6-methoxy-3,3-dimethyl-3,4-

dihydronaphthalene-2-carboxylate (10c)

Prepared according to General Method E from 9c. Isolated as

pale yellow crystals (1.108 g, 59% yield). TLC R_f= 0.14 (10%

EtOAc/hex). ¹H NMR (500 MHz, CDCl₃) d (ppm) 7.35 (d, J = 8.5 Hz,

1H), 6.81 (dd, J = 8.5, 2.4 Hz, 1H), 6.73 (d, J = 2.4 Hz, 1H), 6.37 (s,

1H), 4.26 (q, J = 7.1 Hz, 2H), 3.86 (s, 3H), 2.64 (s, 2H), 1.36 (t,

J = 7.1 Hz, 3H), 1.22 (s, 6H).

5.2.29. 3-Ethyl-5,5-dimethyl-2-thioxo-2,3,5,6-

tetrahydrobenzo[h]quinazolin-4(1H)-one (11e)

Prepared according to General Method F from 10d and ethyl

isothiocyanate. Puriﬁed via ﬂash chromatography (0% to 10%

EtOAc/hex); recovered in 31% yield. TLC R_f= 0.20 (10% EtOAc/

hex). ¹H NMR (500 MHz, CDCl₃) d (ppm) 9.24 (s, 1H), 7.49–7.35

(m, 3H), 7.29 (d, J = 7.4 Hz, 1H), 4.49 (q, J = 7.0 Hz, 2H), 2.79 (s,

2H), 1.35 (t, J = 7.0 Hz, 3H), 1.34 (s, 6H).

5.2.23. Ethyl 1-amino-3,3-dimethyl-3,4-dihydronaphthalene-2-

carboxylate (10d)

Prepared according to General Method E from 9d. Isolated as a

pale yellow oil (860 mg, 41% yield). TLC R_f= 0.30 (10% EtOAc/hex).

¹H NMR (500 MHz, CDCl₃) d (ppm) 7.42 (d, J = 8.6 Hz, 1H), 7.36–

7.29 (m, 2H), 7.20 (d, J = 7.3 Hz, 1H), 6.35 (s, 1H), 4.28 (q,

J = 7.1 Hz, 2H), 2.68 (s, 2H), 1.37 (t, J = 7.1 Hz, 3H), 1.22 (s, 6H).

5.2.30. 9-Methoxy-3,5,5-trimethyl-2-thioxo-2,3,5,6-

tetrahydrobenzo[h]quinazolin-4(1H)-one (11f)

Prepared according to General Method F from ethyl 10a and

methyl isothiocyanate. Puriﬁed via ﬂash chromatography (0–10%

EtOAc/hex) and recovered in 27% yield. ¹H NMR (400 MHz, CDCl₃)

d (ppm) 9.35 (s, 1H), 7.20 (d, J = 8.3 Hz, 1H), 6.99 (dd, J = 8.3, 2.3 Hz,

1H), 6.94 (d, J = 2.3 Hz, 1H), 3.87 (s, 3H), 3.74 (s, 3H), 2.71 (s, 2H),

1.33 (s, 6H).

5.2.24. General Method F for synthesizing compounds 11a–f

Glacial acetic acid (147 lL, 2.56 mmol) and an alkyl isothiocya-

nate (2.56 mmol) were combined with the selected intermediate

10a–d (1.28 mmol) in absolute ethanol (1.7 mL). The solution

was allowed to stir at reﬂux under nitrogen atmosphere for 1 h.

Additional allyl isothiocyanate (373 lL, 3.84 mmol) was added in

equal portions over the course of 3 h. The reaction was allowed

to stir at reﬂux 16 additional hours, then diluted with ethyl acetate.

The organic mixture was washed with water and brine, dried over

MgSO₄, ﬁltered, and concentrated in vacuo. Trituration or ﬂash

chromatography delivered compounds 11a–f in 21–59% yield.

5.2.31. General Method G for the generation of compounds 12a–

m

The selected intermediate 11a–f (0.101 mmol) was dissolved in

MEK (0.591 mL), to which Cs₂CO₃(66 mg, 0.201 mmol) and alkyl-

ating agent (0.151 mmol) were added. The reaction was heated

to 70 °C and allowed to stir 16 h. The reaction mixture was diluted

with H₂O and extracted 2ꢃ with EtOAc. The combined organic lay-

ers were washed with water and brine, then isolated, dried over

MgSO₄, vacuum ﬁltered, and concentrated in vacuo. Further puriﬁ-

cation via ﬂash chromatography in an appropriate solvent system

delivered the desired S-alkylated compounds in 24–88% yield.

5.2.25. 3-Allyl-9-methoxy-5,5-dimethyl-2-thioxo-2,3,5,6-

tetrahydrobenzo[h]quinazolin-4(1H)-one (11a)

Prepared according to General Method F from 10a and allyl iso-

thiocyanate. Puriﬁed via trituration with hexanes and diethyl ether

(125 mg, 21% yield). ¹H NMR (400 MHz, CDCl₃) d (ppm) 9.29 (s,

1H), 7.35 (d, J = 8.0 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 7.00 (s, 1H),

6.08 (ddt, J = 15.8, 10.9, 5.5 Hz, 1H), 5.45–5.30 (m, 2H), 5.18 (d,

J = 5.5 Hz, 2H), 3.85 (s, 3H), 2.73 (s, 2H), 1.34 (s, 6H).

5.2.32. 3-Allyl-2-(ethylthio)-9-methoxy-5,5-dimethyl-2,3,5,6-

tetrahydrobenzo[h]quinazolin-4(1H)-one (12a)

Prepared according to General Method G from intermediate

11a, using iodoethane as the alkylating agent. Isolated via ﬂash

chromatography as a white solid (74 mg, 71% yield). ¹H NMR

(400 MHz, CDCl₃) d (ppm) 7.70 (s, 1H), 7.10 (d, J = 8.2 Hz, 1H),

6.91 (d, J = 8.2 Hz, 1H), 5.93 (ddt, J = 15.8, 10.9, 5.5 Hz, 1H), 5.33–

5.21 (m, 2H), 4.68 (d, J = 5.5 Hz, 2H), 3.85 (s, 3H), 3.31 (q,

J = 7.3 Hz, 2H), 2.72 (s, 2H), 1.49 (t, J = 7.3 Hz, 3H), 1.37 (s, 6H).

5.2.26. 3-Allyl-7-methoxy-5,5-dimethyl-2-thioxo-2,3,5,6-

tetrahydrobenzo[h]quinazolin-4(1H)-one (11b)

Prepared according to General Method F from 10c and allyl iso-

thiocyanate. Puriﬁed via trituration of the crude organic isolate

with hexanes and diethyl ether (133 mg, 24% yield). ¹H NMR

(500 MHz, CDCl₃) d (ppm) 9.29 (s, 1H), 7.35 (t, J = 8.0 Hz, 1H),

1892

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

ESI+MS m/z = 357.2 (M+H⁺), 379.2 (M+Na⁺). HPLC (Method B,

t_R= 8.53 min) purity >95%.

5.2.38. 2-((3-Allyl-5,5-dimethyl-4-oxo-3,4,5,6-

tetrahydrobenzo[h]quinazolin-2-yl)thio)acetonitrile (12g)

Prepared according to General Method G from intermediate

5.2.33. 3-Allyl-2-(ethylthio)-7-methoxy-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (12b)

11d, using a-chloroacetonitrile as the alkylating agent. Isolated

via ﬂash chromatography (5% EtOAc/hex) as a white crystalline

solid (92 mg, 81% yield). ¹H NMR (500 MHz, CDCl₃) d (ppm)

8.16 (d, J = 7.0 Hz, 1H), 7.41–7.32 (m, 2H), 7.19 (d, J = 6.3 Hz,

1H), 5.91 (ddt, J = 16.1, 10.6, 5.6 Hz, 1H), 5.34–5.26 (m, 2H),

4.65 (d, J = 5.6 Hz, 2H), 4.06 (s, 2H), 2.80 (s, 2H), 1.39 (s, 6H).

ESI+MS m/z = 338.1 (M+H⁺), 360.1 (M+Na⁺). HPLC (Method B,

t_R= 5.57 min), purity >95%.

Prepared according to General Method G from intermediate

11b, using iodoethane as the alkylating agent. Isolated via ﬂash

chromatography (75 mg, 69% yield). ¹H NMR (500 MHz, CDCl₃) d

(ppm) 7.80 (d, J = 7.8 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 6.97 (d,

J = 8.1 Hz, 1H), 5.95 (ddt, J = 16.0, 10.6, 5.6 Hz, 1H), 5.34–5.25 (m,

2H), 4.70 (d, J = 5.5 Hz, 2H), 3.90 (s, 3H), 3.33 (q, J = 7.3 Hz, 2H),

2.82 (s, 2H), 1.49 (t, J = 7.3 Hz, 3H), 1.41 (s, 6H). ESI+MS m/

z = 357.2 (M+H⁺), 379.1 (M+Na⁺). HPLC (Method B, t_R= 8.52 min),

purity >95%.

5.2.39. 3-Allyl-2-((2-methoxyethyl)thio)-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (12h)

Prepared according to General Method G from intermediate

11d, using 2-methoxyethyl p-toluenesulfonate as the alkylating

agent. Isolated via ﬂash chromatography (5% EtOAc/hex) as a white

crystalline solid (86 mg, 72% yield). ¹H NMR (500 MHz, CDCl₃) d

(ppm) 8.08 (d, J = 7.4 Hz, 1H), 7.35 (t, J = 7.3 Hz, 1H), 7.31 (t,

J = 7.3 Hz, 1H), 7.19 (d, J = 7.1 Hz, 1H), 5.93 (ddt, J = 15.9, 10.8,

5.6 Hz, 1H), 5.32–5.24 (m, 2H), 4.70 (d, J = 5.6 Hz, 2H), 3.76 (t,

J = 6.2 Hz, 2H), 3.54 (t, J = 6.2 Hz, 2H), 3.42 (s, 3H), 2.79 (s, 2H),

1.38 (s, 6H). ESI+MS m/z = 357.1 (M+H⁺), 379.1 (M+Na⁺). HPLC

(Method B, t_R= 7.47 min), purity = 94%.

5.2.34. 3-Allyl-2-(ethylthio)-8-methoxy-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (CCG-203592) (12c)

Prepared according to General Method G from intermediate

11c, using iodoethane as the alkylating agent. Isolated as white

crystals via ﬂash chromatography (105 mg, 88% yield). ¹H NMR

(500 MHz, CDCl₃) d (ppm) 8.05 (d, J = 8.6 Hz, 1H), 6.84 (dd, J = 8.6,

2.4 Hz, 1H), 6.71 (d, J = 2.1 Hz, 1H), 5.93 (ddt, J = 15.8, 10.8,

5.5 Hz, 1H), 5.30–5.22 (m, 2H), 4.67 (d, J = 5.4 Hz, 2H), 3.86 (s,

3H), 3.30 (q, J = 7.3 Hz, 2H), 2.75 (s, 2H), 1.47 (t, J = 7.3 Hz, 3H),

1.38 (s, 6H). ESI+MS m/z = 357.2 (M+H⁺), 379.2 (M+Na⁺). HPLC

(Method B, t_R= 8.26 min), purity >95%.

5.2.40. 3-Ethyl-2-((2-methoxyethyl)thio)-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (12i)

5.2.35. 2-((3-Allyl-5,5-dimethyl-4-oxo-3,4,5,6-

Prepared according to General Method G from intermediate

11e, using 2-methoxyethyl p-toluenesulfonate as the alkylating

agent. Isolated as a clear oil after ﬂash chromatography (66 mg,

71% yield). ¹H NMR (500 MHz, CDCl₃) d (ppm) 8.09 (dd, J = 7.5,

1.3 Hz, 1H), 7.37 (td, J = 7.3, 1.6 Hz, 1H), 7.33 (td, J = 7.5, 1.4 Hz,

1H), 7.21 (d, J = 6.9 Hz, 1H), 4.14 (q, J = 7.1 Hz, 2H), 3.80 (t,

J = 6.2 Hz, 2H), 3.57 (t, J = 6.2 Hz, 2H), 3.46 (s, 3H), 2.81 (s, 2H),

1.43–1.36 (m, 9H). ESI+MS m/z = 345.2 (M+H⁺), 367.2 (M+Na⁺).

HPLC (Method B, t_R= 7.53 min), purity >95%.

tetrahydrobenzo[h]quinazolin-2-yl)thio)acetamide (12d)

Prepared according to General Method G from intermediate

11d, using 2-bromoacetamide as the alkylating agent. Further puri-

ﬁcation via ﬂash chromatography (2:1 EtOAc/hex) delivered the

desired product as a white crystalline solid (26 mg, 74% yield).

¹H NMR (500 MHz, CDCl₃) d (ppm) 8.07 (d, J = 7.1 Hz, 1H), 7.41–

7.31 (m, 2H), 7.20 (d, J = 7.9 Hz, 1H), 6.83 (s, 1H), 5.93 (ddt,

J = 17.3, 10.1, 5.6 Hz, 1H), 5.39 (s, 1H), 5.34–5.27 (m, 2H), 4.70 (d,

J = 5.6 Hz, 2H), 3.96 (s, 2H), 2.80 (s, 2H), 1.38 (s, 6H). ESI+MS m/

z = 378.1 (M+Na⁺). HPLC (Method B, t_R= 2.77 min), purity >95%.

5.2.41. 3-Allyl-8-methoxy-2-((2-methoxyethyl)thio)-5,5-

dimethyl-5,6-dihydrobenzo[h]quinazolin-4(3H)-one (12j)

Prepared according to General Method G from intermediate

11c, using 2-methoxyethyl p-toluenesulfonate as the alkylating

agent. Isolated as a crystalline white solid after ﬂash chromatog-

raphy (67 mg, 76% yield). ¹H NMR (500 MHz, CDCl₃) d (ppm)

8.01 (d, J = 8.6 Hz, 1H), 6.83 (d, J = 8.6 Hz, 1H), 6.71 (s, 1H),

5.92 (ddt, J = 16.1, 10.6, 5.5 Hz, 1H), 5.31–5.23 (m, 2H), 4.69 (d,

J = 5.5 Hz, 2H), 3.86 (s, 3H), 3.75 (t, J = 6.3 Hz, 2H), 3.52 (t,

J = 6.3 Hz, 2H), 3.42 (s, 3H), 2.75 (s, 2H), 1.38 (s, 6H). ESI+MS

m/z = 387.2 (M+H⁺), 409.2 (M+Na⁺). HPLC (Method B,

t_R= 7.05 min), purity >95%.

5.2.36. 3-Allyl-5,5-dimethyl-2-((pyridin-2-ylmethyl)thio)-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (12e)

Prepared according to General Method G from intermediate

11d, using 2-(bromomethyl)pyridine hydrobromide as the alkylat-

ing agent, as well as an additional equivalent of Cs₂CO₃to neutral-

ize the acid. Isolated via ﬂash chromatography (2:1 EtOAc/hex) as a

white crystalline solid (34 mg, 87% yield). ¹H NMR (500 MHz,

CDCl₃) d (ppm) 8.62 (d, J = 5.5 Hz, 1H), 8.11 (d, J = 7.7 Hz, 1H),

7.66 (t, J = 7.7 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.41–7.30 (m, 2H),

7.26–7.18 (m, 2H), 5.96 (ddt, J = 16.0, 10.5, 5.6 Hz, 1H), 5.35–5.26

(m, 2H), 4.76 (s, 2H), 4.73 (d, J = 5.6 Hz, 2H), 2.81 (s, 2H), 1.40 (s,

6H). ESI+MS m/z = 390.1 (M+H⁺), 412.1 (M+Na⁺). HPLC (Method

B, t_R= 2.36 min), purity >95%.

5.2.42. 3-Allyl-2-(allylthio)-8-methoxy-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (CCG-205363) (12k)

Prepared using a method similar to General Method G from

intermediate 11c, using allyl bromide as the alkylating agent,

DMF as the solvent, and stirring for 18 h at room temperature.

Flash chromatography (hexanes/diethyl ether 3:2) afforded the

product (22 mg, 78% yield) as a white solid; mp 75–78 °C. ¹H

NMR (400 MHz, dmso) d (ppm) 8.03 (d, J = 8.6, 1H), 6.92 (dd,

J = 8.6, 2.5, 1H), 6.86 (d, J = 2.3, 1H), 6.01 (dd, J = 16.9, 10.1, 1H),

5.94–5.78 (m, 1H), 5.39 (d, J = 17.0, 1H), 5.27–5.07 (m, 3H), 4.60

(d, J = 5.1,2H), 4.00 (d, J = 6.8, 2H), 3.82 (s, 3H), 2.75 (s, 2H), 1.29

(s, 3H), 1.24 (s, 3H). ESI+MS m/z = 369 (M+H⁺), 391 (M+Na⁺). HPLC

(Method A t_R= 9.40 min), purity = 96%.

5.2.37. 3-Allyl-2-((2-hydroxyethyl)thio)-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (12f)

Prepared according to General Method G from intermediate

11d, using 2-bromoethanol as the alkylating agent. Isolated via

ﬂash chromatography (5% EtOAc/hex) as a white crystalline solid

(27 mg, 78% yield). ¹H NMR (500 MHz, CDCl₃) d (ppm) 8.04 (d,

J = 7.2 Hz, 1H), 7.39–7.29 (m, 2H), 7.19 (d, J = 6.7 Hz, 1H), 5.94

(ddt, J = 15.9, 10.7, 5.6 Hz, 1H), 5.34–5.26 (m, 2H), 4.72 (d,

J = 5.6 Hz, 2H), 4.04 (t, J = 5.3 Hz, 2H), 3.55 (t, J = 5.6 Hz, 2H), 2.93

(s, 1H), 2.78 (s, 2H), 1.37 (s, 6H). ESI+MS m/z = 343.1 (M+H⁺),

365.1 (M+Na⁺). HPLC (Method B, t_R= 4.71 min), purity >95%.

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

1893

5.2.43. 3-Allyl-9-methoxy-5,5-dimethyl-2-((2,2,2-

5.2.48. 3-Allyl-2-(ethylsulﬁnyl)-9-methoxy-5,5-dimethyl-5,6-

triﬂuoroethyl)thio)-5,6-dihydrobenzo[h]quinazolin-4(3H)-one

(12l)

dihydrobenzo[h]quinazolin-4(3H)-one (14)

Compound 12a (20 mg, 0.056 mmol) was added to a 3.5:1 THF/

water mixture (0.617 mL), and the reaction then cooled to 0 °C. A

solution of Oxone (34 mg, 0.056 mmol) in water (0.411 mL) was

added slowly dropwise, and the reaction was allowed to stir

3.5 h at 0 °C, then allowed to warm to room temperature over

16 h. At this point the reaction was partitioned between EtOAc

and water. The organic layer was washed with water and brine,

then isolated, dried over MgSO₄, vacuum ﬁltered, and concentrated

under reduced pressure. The resulting residue was puriﬁed via

ﬂash chromatography (10–33% EtOAc/hex) to deliver the product

as light yellow crystals (13 mg, 62% yield). ¹H NMR (400 MHz,

CDCl₃) d (ppm) 7.66 (d, J = 2.7 Hz, 1H), 7.12 (d, J = 8.2 Hz, 1H),

6.93 (dd, J = 8.2, 2.7 Hz, 1H), 6.56–5.61 (m, 1H), 5.30 (d,

J = 10.3 Hz, 1H), 5.24 (d, J = 17.2 Hz, 1H), 5.13 (ab with 5.00; ddt,

J = 1.6, 5.0, 15.8 Hz, 1H), 5.00 (ab with 5.13; dd, J = 5.0, 15.8 Hz,

1H), 3.86 (s, 3H), 3.46–3.27 (m, 2H), 2.75 (s, 2H), 1.43 (t,

J = 7.5 Hz, 3H), 1.39 (s, 3H), 1.38 (s, 3H). ESI+MS m/z = 395.2

(M+H⁺). HPLC (Method A, t_R= 7.39 min), purity >95%.

Prepared according to General Method G from intermediate

11a, using 2,2,2-triﬂuoro-1-iodoethane as the alkylating agent.

Reaction was heated to 50 °C to avoid evaporation of the alkylating

agent. Flash chromatography (5–10% EtOAc/hex) delivered the

product (7 mg, 24% yield). ¹H NMR (400 MHz, CDCl₃) d (ppm)

7.61 (s, 1H), 7.10 (d, J = 8.1 Hz, 1H), 6.93 (d, J = 8.1 Hz, 1H), 5.93

(ddt, J = 16.5, 11.1, 5.6 Hz, 1H), 5.34–5.23 (m, 2H), 4.71 (d,

J = 5.6 Hz, 2H), 4.16 (q, J = 9.7 Hz, 2H), 3.84 (s, 3H), 2.73 (s, 2H),

1.37 (s, 6H). ESI+MS m/z = 411.2 (M+H⁺), HPLC (Method B,

t_R= 7.98 min), purity = 88%.

5.2.44. 2-(Ethylthio)-9-methoxy-3,5,5-trimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (12m)

Prepared according to General Method G from intermediate 11f,

using iodoethane as the alkylating agent. Isolated as a colorless oil

(20 mg, 55% yield) after ﬂash chromatography. ¹H NMR (400 MHz,

CDCl₃) d (ppm) 7.70 (s, 1H), 7.10 (d, J = 8.2 Hz, 1H), 6.90 (d,

J = 8.2 Hz, 1H), 3.85 (s, 3H), 3.50 (s, 3H), 3.32 (q, J = 7.3 Hz, 2H),

2.72 (s, 2H), 1.50 (t, J = 7.3 Hz, 3H), 1.38 (s, 6H). ESI+MS m/

z = 331.2 (M+H⁺), 353.2 (M+Na⁺). HPLC (Method A, t_R= 9.25 min),

purity >95%.

5.2.49. 3-Allyl-2-(ethylsulfonyl)-9-methoxy-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (15)

Compound 12a (28 mg, 0.79 mmol) and mCPBA (70 wt%, 48 mg,

0.196 mmol) were combined in DCM (1.2 mL) at 0 °C. The reaction

mixture was kept at 0 °C for 1 h, then allowed to warm to RT over

the course of 16 h. The reaction mixture was then diluted with

more DCM and washed with saturated sodium bicarbonate solu-

tion, water, and brine. The organic layer was isolated, dried over

MgSO₄, vacuum ﬁltered, and concentrated in vacuo. Further puriﬁ-

cation via ﬂash chromatography (5–10% EtOAc/hex) delivered the

desired product as a light yellow solid (19 mg, 62% yield). ¹H

NMR (400 MHz, CDCl₃) d (ppm) 7.43 (s, 1H), 7.14 (d, J = 8.2 Hz,

1H), 6.94 (d, J = 8.2 Hz, 1H), 6.04 (ddt, J = 16.8, 10.3, 5.5 Hz, 1H),

5.40 (d, J = 16.8 Hz, 1H), 5.30 (d, J = 10.3 Hz, 1H), 5.02 (d,

J = 5.5 Hz, 2H), 3.84 (s, 3H), 3.81 (q, J = 7.3 Hz, 2H), 2.75 (s, 2H),

1.62 (t, J = 7.3 Hz, 3H), 1.38 (s, 6H). ESI+MS m/z = 389.2 (M+H⁺),

411.2 (M+Na⁺). HPLC (Method A, t_R= 8.64 min), purity >95%.

5.2.45. 3-Allyl-5,5-dimethyl-2-(methylthio)-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (12n)

Intermediate 11d (116 mg, 0.39 mmol) was dissolved in EtOH

(2.3 mL), to which KOH (38 mg, 0.58 mmol) and methyl iodide

(27

lL, 0.43 mmol) were added. The solution was stirred for

30 min, precipitating a crystalline white solid. The solution was di-

luted with ethyl acetate and water. The aqueous layer was ex-

tracted with additional EtOAc, then the combined organic layers

were washed with water and brine. The organic layer was isolated,

dried over MgSO₄, vacuum ﬁltered, and concentrated in vacuo. The

recovered crystalline material (116 mg, 96% yield) was found to be

pure by NMR and used without further puriﬁcation. ¹H NMR

(400 MHz, CDCl₃) d 8.15 (d, J = 7.3, 1H), 7.38–7.28 (m, 2H), 7.18

(d, J = 6.9, 1H), 6.01–5.75 (m, 1H), 5.33–5.23 (m, 2H), 4.69 (d,

J = 5.5, 2H), 2.79 (s, 2H), 2.69 (s, 3H), 1.39 (s, 6H). HPLC (Method

B, t_R= 7.57 min), purity >95%.

5.2.50. General Method H for preparation of phenols 16a–c

Boron tribromide solution (1 M in dichloromethane, 4.42 mL)

was added gradually to a stirred solution of the selected interme-

diate 12a–c (2.1 mmol) in dichloromethane (14 mL) at room tem-

perature under N₂. The mixture was heated to reﬂux for 6 h, then

cooled to room temperature. Water (10 mL) was then added drop-

wise and the mixture was partitioned between DCM and water.

The layers were separated and the organic layer washed with

water, saturated aqueous NaHCO₃, and saturated brine, then dried

over MgSO₄. The solvent was removed under reduced pressure and

the residue was triturated in ethyl acetate, ﬁltered, and dried under

high vacuum. The resulting crystalline phenols 16a–c (43–62% iso-

lated yield) were used without further puriﬁcation.

5.2.46. 3-Allyl-2-ethoxy-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (13a)

Prepared according to General Method B from intermediate

12n. Isolated as a light yellow solid (22 mg, 69% yield). ¹H

NMR (500 MHz, CDCl₃) d (ppm) 8.09 (d, J = 7.3 Hz, 1H), 7.37–

7.27 (m, 2H), 7.18 (d, J = 7.5 Hz, 1H), 5.92 (ddt, J = 16.0, 10.3,

5.8 Hz, 1H), 5.25–5.16 (m, 2H), 4.62 (d, J = 5.8 Hz, 2H), 4.57 (q,

J = 7.1 Hz, 2H), 2.78 (s, 2H), 1.45 (t, J = 7.1 Hz, 3H), 1.37 (s, 6H).

ESI+MS m/z = 311.1 (M+H⁺), 333.1 (M+Na⁺). HPLC (Method B,

t_R= 7.94 min), purity >95%.

5.2.51. 3-Allyl-2-(ethylthio)-9-hydroxy-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (16a)

Prepared according to General Method H from compound 12a.

Isolated as a tan solid (60 mg, 62% yield). ¹H NMR (500 MHz, CDCl₃)

d (ppm) 7.91 (s, 1H), 7.05 (d, J = 8.2 Hz, 1H), 6.98 (d, J = 8.2 Hz, 1H),

5.90 (ddt, J = 16.3, 10.7, 5.6 Hz, 1H), 5.44–5.36 (m, 2H), 4.83 (d,

J = 5.6 Hz, 2H), 3.95 (q, J = 6.8 Hz, 2H), 2.75 (s, 2H), 1.52 (t,

J = 6.8 Hz, 3H), 1.36 (s, 6H).

5.2.47. 3-Allyl-2-(ethylamino)-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (13b)

Prepared according to General Method C from intermediate

12n. Isolated as a white crystalline solid (23 mg, 41% yield over 2

steps). ¹H NMR (500 MHz, CDCl₃) d (ppm) 8.15 (d, J = 7.3 Hz, 1H),

7.36–7.26 (m, 2H), 7.16 (d, J = 7.4 Hz, 1H), 5.92 (ddt, J = 17.3,

10.5, 5.3 Hz, 1H), 5.36–5.26 (m, 2H), 4.68 (d, J = 5.2 Hz, 2H), 4.58

(t, J = 4.9 Hz, 1H), 3.57 (qd, J = 7.2, 5.2 Hz, 2H), 2.77 (s, 2H), 1.37

(s, 6H), 1.28 (t, 3H). ESI+MS m/z = 310.1 (M+H⁺), 332.1 (M+Na⁺).

HPLC (Method B, t_R= 5.47 min), purity >95%.

5.2.52. 3-Allyl-2-(ethylthio)-7-hydroxy-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (16b)

Prepared according to General Method H from compound 12b.

Isolated as a tan solid (43 mg, 45% yield). ¹H NMR (500 MHz,

1894

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

CDCl₃) d (ppm) 7.76 (d, J = 7.8 Hz, 1H), 7.18 (t, J = 7.8 Hz, 1H), 6.89

(d, J = 7.8 Hz, 1H), 5.99–5.87 (m, 1H), 5.31–5.24 (m, 2H), 5.18 (s,

1H), 4.68 (d, J = 5.5 Hz, 2H), 3.31 (q, J = 7.4 Hz, 2H), 2.78 (s, 2H),

1.47 (t, J = 7.4 Hz, 3H), 1.41 (s, 6H).

J = 8.6 Hz, 1H), 6.86 (dd, J = 8.6, 2.4 Hz, 1H), 6.74 (d, J = 2.2 Hz,

1H), 5.92 (ddt, J = 15.9, 10.6, 5.4 Hz, 1H), 5.30–5.22 (m, 2H), 4.67

(d, J = 5.5 Hz, 2H), 4.18 (t, J = 5.0 Hz, 2H), 3.78 (t, J = 5.0 Hz, 2H),

3.47 (s, 3H), 3.30 (q, J = 7.4 Hz, 2H), 2.74 (s, 2H), 1.47 (t,

J = 7.3 Hz, 3H), 1.37 (s, 6H). ESI+MS m/z = 401.1 (M+H⁺), 423.1

(M+Na⁺). HPLC (Method B, t_R= 7.56 min), purity >95%.

5.2.53. 3-Allyl-2-(ethylthio)-8-hydroxy-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (16c)

Prepared according to General Method H from compound 12c.

Isolated as tan crystals (310 mg, 43% yield). ¹H NMR (500 MHz,

CDCl₃) d (ppm) 7.62 (d, J = 8.2 Hz, 1H), 6.85–6.79 (m, 2H), 5.88

(ddt, J = 16.3, 10.8, 5.9 Hz, 1H), 5.48–5.40 (m, 2H), 4.86 (d,

J = 5.9 Hz, 2H), 4.22 (q, J = 7.3 Hz, 2H), 2.74 (s, 2H), 1.59 (t,

J = 7.3 Hz, 3H), 1.31 (s, 6H).

5.2.58. Ethyl 2-((3-allyl-2-(ethylthio)-5,5-dimethyl-4-oxo-

3,4,5,6-tetrahydro-benzo[h]-quinazolin-8-yl)oxy)acetate (17d)

Prepared using General Method I from phenol intermediate 12c

and ethyl bromoacetate, stirred at room temperature for 2.5 h. The

mixture was poured into 40 mL of water, then the precipitate was

ﬁltered off, rinsed with water 3 times and dried to afford the prod-

uct (54 mg, 86% yield) as a white powder. ¹H NMR (400 MHz,

dmso) d (ppm) 8.01 (d, J = 8.6, 1H), 6.90 (dd, 1H), 6.86 (s, 1H),

5.94–5.83 (m, 1H), 5.22 (d, J = 10.2, 1H), 5.13 (d, J = 17.2, 1H),

4.85 (s, 2H), 4.59 (d, J = 4.3, 2H), 4.19 (q, 2H), 3.29 (q, 2H), 2.75

(s, 2H), 1.39 (t, J = 7.2, 3H), 1.29 (s, 6H), 1.23 (t, J = 7.1, 3H). ESI+MS

m/z = 429 (M+H⁺), 451 (M+Na⁺). HPLC (Method A, t_R= 10.21 min),

purity = 92%.

5.2.54. General Method I to generate compounds 17a–j

The selected phenol intermediate 12a–c (0.622 mmol) was dis-

solved in DMF (3.66 mL), to which Cs₂CO₃(304 mg, 0.933 mmol)

and an alkylating agent (0.716 mmol) were added. The resulting

suspension was stirred at 25 °C to 70 °C for 30 min to 16 h until

the completion of the reaction. Compounds were puriﬁed as indi-

cated in 35–92% yield.

5.2.59. 3-Allyl-2-(ethylthio)-5,5-dimethyl-8-(2-

morpholinoethoxy)-5,6-dihydrobenzo[h]-quinazolin-4(3H)-one

(17e)

5.2.55. 3-Allyl-2-(ethylthio)-9-(2-methoxyethoxy)-5,5-

dimethyl-5,6-dihydrobenzo[h]quinazolin-4(3H)-one (17a)

Prepared using General Method I from 12a and 2-methoxyethyl

p-toluenesulfonic ester, heated to 70 °C for 16 h. Reaction mixture

was partitioned between ethyl acetate and water, then the organic

layer washed with water and brine. The organic layer was isolated,

dried over MgSO₄, vacuum ﬁltered, and concentrated in vacuo. Fur-

ther puriﬁcation via ﬂash chromatography (0–10% EtOAc/hex)

delivered the desired product (38 mg, 65% yield). ¹H NMR

(500 MHz, CDCl₃) d (ppm) 7.71 (s, 1H), 7.09 (d, J = 8.2 Hz, 1H),

6.94 (d, J = 8.2 Hz, 1H), 5.93 (ddt, J = 16.0, 10.7, 5.6 Hz, 1H), 5.31–

5.23 (m, 2H), 4.68 (d, J = 5.5 Hz, 2H), 3.81–3.75 (m, 2H), 3.61–

3.55 (m, 2H), 3.47 (s, 3H), 3.31 (q, J = 7.3 Hz, 2H), 2.72 (s, 2H),

1.48 (t, J = 7.3 Hz, 3H), 1.37 (s, 6H). ESI+MS m/z = 401.2 (M+H⁺),

423.2 (M+Na⁺). HPLC (Method B, t_R= 7.83 min), purity >95%.

Prepared using General Method I from phenol intermediate 12c

and 4-(2-chloroethyl)-morpholine hydrochloride, stirred at 80 °C

for 4 h. The mixture was poured into water and extracted with

ethyl acetate three times, then the combined extracts were washed

with water and saturated brine, dried over MgSO₄, and concen-

trated in vacuo to a tan solid (0.035 g, 69% yield), mp 91–92 °C.

¹H NMR (400 MHz, dmso) d (ppm) 8.06 (d, 1H), 6.98 (dd, 1H),

6.92 (d, 1H), 5.93 (ddt, 1H), 5.23 (d, 1H), 5.19 (d, 1H), 4.64 (d,

2H), 4.21 (t, 2H), 3.65 (m, 4H), 3.34 (q, 2H), 2.80 (m, 4H), 2.53

(m, 2H), 1.44 (t, 3H), 1.35 (s, 6H). ESI+MS m/z = 456 (M+H⁺). HPLC

(Method A, t_R= 10.23 min), purity = 91%.

5.2.60. 3-Allyl-2-(ethylthio)-8-isopropoxy-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (17f)

5.2.56. 3-Allyl-2-(ethylthio)-7-(2-methoxyethoxy)-5,5-

Prepared using General Method I from phenol intermediate 12c,

using 2-bromopropane as the alkylating agent and stirring at room

temperature for 4 h. The reaction mixture was poured into water,

then extracted into ethyl acetate. The organic layer was washed with

water and brine, then isolated, dried over MgSO₄, vacuum ﬁltered,

and concentrated in vacuo delivering the ﬁnal product as a sticky

yellow oil (34 mg, 92% yield).. ¹H NMR (400 MHz, dmso) d (ppm)

8.04 (d, 1H), 6.94 (dd, 1H), 6.87 (d, 1H), 5.92 (m, 1H), 5.27 (dd, 1H),

5.18 (m, 1H), 4.82–4.69 (m, 1H), 4.64 (d, 2H), 3.31–3.39 (m, 2H),

2.79 (s, 2H), 1.44(t, 3H), 1.35 (m, 12H). ESI+MS m/z = 385 (M+H⁺),

407 M+Na⁺). HPLC (Method A, t_R= 9.25 min), purity = 93%.

dimethyl-5,6-dihydrobenzo[h]quinazolin-4(3H)-one (17b)

Prepared using General Method I from phenol intermediate 12b

and 2-methoxyethyl p-toluenesulfonic ester, heated to 70 °C for

16 h. Reaction mixture was partitioned between ethyl acetate

and water, then the organic layer washed with water and brine.

The organic layer was isolated, dried over MgSO₄, vacuum ﬁltered,

and concentrated in vacuo. Further puriﬁcation via ﬂash chroma-

tography (0–10% EtOAc/hex) delivered the desired product

(36 mg, 68% yield). ¹H NMR (500 MHz, CDCl₃) d (ppm) 7.78 (d,

J = 7.8 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H), 6.96 (d, J = 8.1 Hz, 1H),

5.99–5.87 (m, 1H), 5.31–5.23 (m, 2H), 4.68 (d, J = 5.5 Hz, 2H),

4.17 (dd, J = 5.5, 4.2 Hz, 2H), 3.80 (dd, J = 5.5, 4.2 Hz, 2H), 3.48 (s,

3H), 3.30 (q, J = 7.4 Hz, 2H), 2.83 (s, 2H), 1.47 (t, J = 7.4 Hz, 3H),

1.39 (s, 6H). ESI+MS m/z = 401.2 (M+H⁺), 423.2 (M+Na⁺). HPLC

(Method B, t_R= 7.91 min), purity >95%.

5.2.61. 2-((3-Allyl-2-(ethylthio)-5,5-dimethyl-4-oxo-3,4,5,6-

tetrahydrobenzo[h]quinazolin-8-yl)oxy)-acetonitrile (17g)

Prepared using General Method I from phenol intermediate 12c

using bromoacetonitrile as the alkylating agent and stirring at

room temperature for 4 h. The reaction mixture was partitioned

between ethyl acetate and water, then the organic layer was

washed with water and brine. After drying over MgSO₄, ﬁltration,

and concentration in vacuo, recrystallization of the residue from

isopropanol gave the pure product (13 mg, 39% yield); mp 130–

131 °C. ¹H NMR (400 MHz, dmso) d (ppm) 8.03 (d, 1H), 7.01 (dd,

1H),6.95 (d, 1H), 5.91–5.74 (m, 1H), 5.19 (m, 3H), 5.09 (d, 1H),

4.55 (d, 2H), 3.26 (q, 2H), 2.74 (s, 2H), 1.35 (t, 3H), 1.26 (s, 6H).

ESI+MS m/z = 382 (M+H⁺), 404 (M +Na⁺). HPLC (Method A,

t_R= 8.69 min), purity = 89%.

5.2.57. 3-Allyl-2-(ethylthio)-8-(2-methoxyethoxy)-5,5-

dimethyl-5,6-dihydrobenzo[h]quinazolin-4(3H)-one (17c)

Prepared using General Method I from phenol intermediate 12c

and 2-methoxyethyl p-toluenesulfonic ester, heated to 70 °C for

16 h. Reaction mixture was partitioned between ethyl acetate

and water, then the organic layer washed with water and brine.

The organic layer was isolated, dried over MgSO₄, vacuum ﬁltered,

and concentrated in vacuo. Further puriﬁcation via ﬂash chroma-

tography (0–10% EtOAc/hex) delivered the desired product

(39 mg, 71% yield). ¹H NMR (500 MHz, CDCl₃) d (ppm) 8.04 (d,

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

1895

5.2.62. 3-Allyl-8-butoxy-2-(ethylthio)-5,5-dimethyl-5,6-

5.2.67. 9-Methoxy-5,5-dimethyl-2-thioxo-2,3,5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (17h)

tetrahydrobenzo[h]quinazolin-4(1H)-one (18a)

Prepared according to General Method I from intermediate 12c

and 1-bromopentane. Isolated in 75% yield. ¹H NMR (500 MHz,

DMSO) d 8.00 (d, 1H), 6.90 (d, 1H), 6.84 (s, 1H), 5.94–5.77 (m,

1H), 5.21 (d, 1H), 5.13 (d, 1H), 4.59 (d, 2H), 4.03 (t, 2H), 3.28 (q,

2H), 2.74 (s, 2H), 1.71 (m, 2H), 1.48 (m, 2H), 1.41 (t, 3H), 1.29 (s,

6H), 0.95 (t, 3H). ESI+MS m/z = 399 (M+H⁺), 421 (M +Na⁺). HPLC

(Method C, t_R= 5.45 min), purity = 94%.

Generated using General Method J from b-aminoester 10a. Iso-

lated the desired product as a white solid (650 mg, 61% yield over 2

steps). ¹H NMR (400 MHz, CDCl₃) d (ppm) 9.32 (s, 2H), 7.20 (d,

J = 8.2 Hz, 1H), 7.00 (dd, J = 8.2, 2.4 Hz, 1H), 6.97 (d, J = 2.4 Hz,

1H), 3.87 (s, 3H), 2.71 (s, 2H), 1.32 (s, 6H).

5.2.68. 5,5-Dimethyl-2-thioxo-2,3,5,6-

tetrahydrobenzo[h]quinazolin-4(1H)-one (18d)

5.2.63. 3-Allyl-2-(ethylthio)-5,5-dimethyl-8-phenethoxy-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (17i)

Generated using General Method J from b-aminoester 10d. Iso-

lated the desired product as a white solid (2.7 g, 83% yield over 2

steps); R_f: 0.56 (2:3, EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃):

d (ppm) 9.95 (s, 1H, NH), 9.75 (s, 1H, NH), 7.57 (d, 1H, J = 7.7 Hz),

7.46 (t, 1H, J = 7.4 Hz), 7.39 (t, 1H, J = 7.5 Hz), 7.28 (d, 1H,

J = 7.5 Hz), 2.78 (s, 2H), 1.33 (s, 6H).

Prepared according to General Method I from intermediate 12c

and (2-bromoethyl)benzene. Isolated in 77% yield. ¹H NMR

(500 MHz, DMSO) d 8.05 (d, 1H), 7.5–7.29 (m, 5H), 6.97 (d, 1H),

6.91 (s, 1H), 6.09–5.81 (m, 1H), 5.27 (d, 1H), 5.18 (d, 1H), 4.64 (d,

2H), 4.31 (t, 2H), 3.33 (q, 2H), 3.12 (t, 2H), 2.79 (s, 2H), 1.45 (t,

3H), 1.33 (s, 6H). ESI+MS m/z = 447 (M+H⁺), 469 (M +Na⁺). HPLC

(Method A, t_R= 8.89 min), purity = 93%.

5.2.69. 2-((2-Methoxyethyl)thio)-3-(4-methoxyphenethyl)-5,5-

dimethyl-5,6-dihydrobenzo[h]quin-azolin-4(3H)-one, (19a) and

2-((2-Methoxyethyl)thio)-4-(4-methoxyphenethoxy)-5,5-

dimethyl-5,6-dihydrobenzo[h]quinazoline, (20a)

5.2.64. 3-Allyl-2-(ethylthio)-8-(heptyloxy)-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazolin-4(3H)-one (17j)

A solution of compound 18d (2.7 g, 10.45 mmole) and KOH

(590 mg, 10.45 mmole) in absolute EtOH (60 mL) was reﬂuxed

for 30 min. Then 2-methoxyethyl toluenesulfonic ester (2.41 g,

10.45 mmole) was added in EtOH (3 mL). The reaction was al-

lowed to reﬂux for 15 h, then allowed to cool to room tempera-

ture. The crystallized solid was ﬁltered, washed with EtOH

(3 mL) and water (75 mL), dried under suction and then under

high vacuum. A portion of the resulting S-alkylated compound

(25 mg, 0.079 mmol) in DMF (1 mL) was treated with lithium

carbonate (17 mg, 0.23 mmol) and 1-(2-bromoethyl)-4-methoxy-

benzene (18 mg, 0.084 mmol). The reaction was warmed to 80 °C

and allowed to stir 24 h, at which point additional lithium car-

bonate (17 mg, 0.23 mmol) and 1-(2-bromoethyl)-4-methoxy-

benzene (13 mg, 0.061 mmol) were added. After 24 additional h

at reﬂux, the solvent was removed in vacuo and the resulting

mixture of N and O-alkylated products were separated via ﬂash

chromatography (4% EtOAc/hex).

Prepared according to General Method I from intermediate 12c

and 1-bromoheptane. Isolated in 35% yield. ¹H NMR (500 MHz,

DMSO) d 7.95 (d, 1H), 6.85 (d, 1H), 6.80 (s, 1H), 5.89–5.78 (m,

1H), 5.18 (d, 1H), 5.09 (d, 1H), 4.54 (d, 2H), 3.98 (t, 2H), 3.23 (q,

2H), 2.70 (s, 2H), 1.69 (m, 2H), 1.45–1.12 (m, 17H), 0.84 (t, 3H).

ESI+MS m/z = 441 (M+H⁺), 463 (M +Na⁺). HPLC (Method C,

t_R= 11.67 min), purity >95%.

5.2.65. 2-((3-Allyl-2-(ethylthio)-5,5-dimethyl-4-oxo-3,4,5,6-

tetrahydrobenzo[h]quinazolin-8-yl)oxy)acetic acid (17k)

Compound 17d (40 mg, 0.93 mmol) was added to MeOH

(0.5 mL), and KOH solution (1 M, 0.75 mL) was added. The mix-

ture was stirred and heated at 50 °C for 90 min then cooled,

poured into 20 mL of water, stirred and acidiﬁed with 2 N HCl.

After 2 h the mixture was extracted twice with ethyl acetate.

The combined extracts were washed with water then saturated

brine and dried over MgSO₄. The solvent was removed under re-

duced pressure and the residue recrystallized from acetonitrile

to afford the product (10 mg, 27% yield) as a white solid; mp

167–168 °C. ¹H NMR (400 MHz, dmso) d 13.03 (s, 1H), 7.97 (d,

1H), 6.85 (dd, 1H), 6.82 (d, 1H), 5.91–5.74 (m, 1H), 5.19 (dd,

1H), 5.09 (d, 1H), 4.70 (d, 2H), 4.54 (d, 2H), 3.26–3.19 (m, 2H),

2.71 (s, 2H), 1.34 (t, 3H), 1.25 (s, 6H). ESI+MS m/z = 401

(M+H⁺), 423 (M+Na⁺); ESI-MS m/z = 399 (M - H⁺). HPLC (Method

A t_R= 7.69 min), purity = 93%.

5.2.70. 2-((2-Methoxyethyl)thio)-3-(4-methoxyphenethyl)-5,5-

dimethyl-5,6-dihydrobenzo[h]quin-azolin-4(3H)-one, (19a)

Yield: 5 mg (11% over 2 steps); R_f: 0.30 (1:9, EtOAc/hexanes); ¹H

NMR (400 MHz, CDCl₃) d (ppm) 8.08 (dd, 1H, J = 1.6 & 7.1 Hz),

7.26–7.37 (m, 4H), 7.19 (d, 1H, J = 7.2 Hz), 6.86 (d, 2H, J = 8.6 Hz),

4.19 (t, 2H, J = 8.3 Hz), 3.80 (s, 3H), 3.77 (t, 2H, J = 6.2 Hz), 3.56 (t,

2H, J = 6.2 Hz), 3.44 (s, 3H), 3.00 (t, 2H, J = 8.3 Hz), 2.79 (s, 2H) &

1.39 (s, 6H); ESI+MS m/z = 451 (M+H⁺), 473 (M+Na⁺). HPLC (Meth-

od B, t_R= 9.9 min), purity = 98.8%.

5.2.71. 2-((2-Methoxyethyl)thio)-4-(4-methoxyphenethoxy)-

5,5-dimethyl-5,6-dihydrobenzo[h]quinazoline, (20a)

5.2.66. General Method J for generating unsubstituted 2-

thioxopyrimidinone intermediates 18a, 18d, 24

Yield: 24 mg (54% over 2 steps); R_f: 0.38 (1:9, EtOAc/hex-

anes); ¹H NMR (400 MHz, CDCl₃) d (ppm) 8.20 (dd, 1H, J = 1.7

& 7.2 Hz), 7.29–7.36 (m, 2H), 7.14–7.21 (m, 3H), 6.85 (m, 2H),

4.60 (t, 2H, J = 7.0 Hz), 3.79 (s, 3H), 3.74 (t, 2H, J = 6.9 Hz), 3.41

(m, 5H), 3.06 (t, 2H, J = 7.0 Hz), 2.77 (s, 2H) & 1.25 (s, 6H);

ESI+MS m/z = 451 (M+H⁺), 473 (M+Na⁺). HPLC (Method B,

t_R= 10.1 min), purity = 97.3%.

To a solution of the selected b-aminoester intermediate (10a,

10d, 23)(12.64 mmol) in absolute EtOH (15 mL) was added ben-

zoyl isothiocyanate (1.7 mL, 12.64 mmol), and the reaction mix-

ture was warmed to reﬂux for 30 min. Additional benzoyl

isothiocyanate (400

lL, 2.7 mmol) was added in two equal por-

tions over the course of 1 hour, then the reaction was allowed to

cool. The thiourea adduct was isolated via crystallization from

ethanol or via ﬂash chromatography, then added to a solution

of KOH (1.21 g, 21.57 mmol) in ethanol/water (2:1, 30 mL). The

reaction mixture was warmed to reﬂux and allowed to stir for

1.5 h. The reaction mixture was cooled, then acidiﬁed to pH 5–

6 with 1 N HCl. The resulting precipitate was isolated via vac-

uum ﬁltration, washed with EtOH and water and dried under

high vacuum. Isolated in 61–83% yield.

5.2.72. 2-(Ethylthio)-9-methoxy-5,5-dimethyl-3-(2,2,2-

triﬂuoroethyl)-5,6-dihydrobenzo[h]quinazolin-4(3H)-one (19b)

and 2-(ethylthio)-9-methoxy-5,5-dimethyl-4-(2,2,2-

triﬂuoroethoxy)-5,6-dihydrobenzo[h]quinazoline (20b)

The 2-thioxo intermediate 18a (118 mg, 0.409 mmol) was dis-

solved in EtOH (2.4 mL), to which KOH (23 mg, 0.409 mmol) and

1896

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

iodoethane (64 mg, 0.409 mmol) were added. The reaction was

warmed to 75 °C and allowed to stir for 3 h. The reaction mix-

ture was diluted with 1 N HCl and the resulting precipitate

was collected via sintered glass funnel, washed with water,

and dried under high vacuum. A portion of the S-alkylated ad-

duct (27 mg, 0.085 mmol) was combined with 1,1,1-triﬂuoro-2-

30 min. Dry ice (solid CO2) was added and the solution was al-

lowed to stir 1 h while warming to room temperature. The reaction

was then concentrated in vacuo and the resulting residue was trit-

urated in ether and ﬁltered. The solid was dissolved in EtOAc and

washed with 2 M HCl and brine. The organic layer was isolated,

dried over MgSO4, concentrated in vacuo, then triturated in ether/

hexane and dried under high vacuum. Obtained 4-cyano-3-meth-

ylbenzoic acid 22 (1.5 g, 60.8% yield) as a white solid. ¹H NMR

(400 MHz, CDCl₃) d (ppm) 8.18 (s, 1H), 8.02–8.09 (m, 1H), 7.75–

7.81 (m, 1H), 2.61 (s, 3H).

iodoethane

(27 mg,

0.128 mmol)

and

Cs₂CO₃

(33 mg,

0.102 mmol) in DMF (500

lL). The reaction was tightly capped

and heated to 70 °C for 16 h. The reaction mixture was parti-

tioned between ethyl acetate and water, then the organic layer

was washed with water and brine. The organic layer was con-

centrated in vacuo and puriﬁed via ﬂash chromatography (0–

3% EtOAc/hex) to yield compounds 19b (6 mg, 13% yield over 2

steps) and 20b (17 mg, 36% yield over 2 steps) as crystalline

white solids.

5.2.77. 2-Ethyl 6-methyl 1-amino-3,3-dimethyl-3,4-

dihydronaphthalene-2,6-dicarboxylate (23)

Prepared according to General Method E from 4-cyano-3-meth-

ylbenzoic acid 22 (1.0 g, 6.2 mmol). The impure residue resulting

from concentrating the crude organic extract was dissolved in

20% methanolic toluene (20 ml) and treated with trimethylsilyldia-

zomethane (2.7 ml, 5.4 mmol). The mixture was stirred 30 min at

room temperature, then concentrated in vacuo. Puriﬁcation via

ﬂash chromatography (5% to 10% to 20% EtOAc/hex) yielded the de-

sired compound 23 as a yellow solid (0.78 g, 41.5% yield). ¹H NMR

(400 MHz, CDCl₃) d (ppm) 7.98–8.10 (m, 1H), 7.82–7.88 (m, 1H),

7.45–7.51 (m, 1H), 6.24 (br s, 2H), 4.31–4.39 (q, 2H), 3.93 (s, 3H),

2.68 (br s, 2H), 1.38–1.41 (t, 3H), 1.28 (s, 6H). HPLC (Method A,

t_R= 8.02 min), purity = 84%.

5.2.73. 2-(Ethylthio)-9-methoxy-5,5-dimethyl-3-(2,2,2-

triﬂuoroethyl)-5,6-dihydrobenzo[h]quinazolin-4(3H)-one (19b)

¹H NMR (400 MHz, CDCl₃) d (ppm) 7.68 (d, J = 2.8 Hz, 1H), 7.11

(d, J = 8.2 Hz, 1H), 6.93 (dd, J = 8.2, 2.8 Hz, 1H), 4.78 (q, J = 8.0 Hz,

2H), 3.85 (s, 3H), 3.35 (q, J = 7.3 Hz, 2H), 2.74 (s, 2H), 1.50 (t,

J = 7.3 Hz, 3H), 1.36 (s, 6H). ESI+MS m/z = 399.2 (M+H⁺), 421.2

(M+Na⁺). HPLC (Method B, t_R= 8.49 min), purity >95%.

5.2.74. 2-(Ethylthio)-9-methoxy-5,5-dimethyl-4-(2,2,2-

triﬂuoroethoxy)-5,6-dihydrobenzo[h]quinazoline (20b)

¹H NMR (400 MHz, CDCl₃) d (ppm) 7.81 (d, J = 2.7 Hz, 1H), 7.10

(d, J = 8.2 Hz, 1H), 6.94 (dd, J = 8.2, 2.7 Hz, 1H), 4.80 (q, J = 8.5 Hz,

2H), 3.87 (s, 3H), 3.21 (q, J = 7.3 Hz, 2H), 2.76 (s, 2H), 1.47 (t,

J = 7.3 Hz, 3H), 1.33 (s, 6H). ESI+MS m/z = 399.2 (M+H⁺). HPLC

(Method A, t_R= 9.84 min), purity >95%.

5.2.78. Methyl 5,5-dimethyl-4-oxo-2-thioxo-1,2,3,4,5,6-

hexahydrobenzo[h]quinazoline-8-carboxylate (24)

Prepared according to General Method J from b-aminoester 23.

Isolated as a white solid (550 mg, 61% yield over 2 steps) used

without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃) d 8.11–

8.15 (m, 1H), 7.87–7.89 (m, 1H), 7.78–7.81 (m, 1H), 3.89 (s, 3H),

2.82 (br s, 2H), 1.27 (s, 6H). HPLC (Method A, t_R= 6.26 min),

purity = 82%.

5.2.75. 4-(Allyloxy)-2-(ethylthio)-9-methoxy-5,5-dimethyl-5,6-

dihydrobenzo[h]quinazoline (20c)

The 2-thioxo intermediate 18a (118 mg, 0.409 mmol) was

dissolved in EtOH (2.4 mL), to which KOH (23 mg, 0.409 mmol)

and iodoethane (64 mg, 0.409 mmol) were added. The reaction

was warmed to 75 °C and allowed to stir for 3 h. The reaction

mixture was diluted with 1 N HCl, and the precipitate (92 mg,

71% yield) was collected via sintered glass funnel, washed with

water, and dried under high vacuum. A portion of the S-alkyl-

ated adduct (30 mg, 0.095 mmol) was combined with Cs₂CO₃

(37 mg, 0.114 mmol) and allyl bromide (17 mg, 0.142 mmol) in

5.2.79. Methyl 3-allyl-2-((2-methoxyethyl)thio)-5,5-dimethyl-4-

oxo-3,4,5,6-tetrahydrobenzo[h]quinazoline-8-carboxylate (25)

To a solution of methyl 5,5-dimethyl-4-oxo-2-thioxo-1,2,3,4,5,6-

hexahydrobenzo[h]quinazoline-8-carboxylate

24

(850 mg,

2.7 mmol) in DMF (10 mL) was added Cs₂CO₃(1.8 g, 5.4 mmol) fol-

lowed by 2-methoxyethyl 4-methylbenzenesulfonate (0.74 g,

3.2 mmol). The resulting mixture was stirred at 40 °C for 3 h, then al-

lowed to cool to room temperature and stir an additional 16 h. The

reaction mixture was diluted with EtOAc, then washed with water,

saturated NaHCO₃solution, and brine, then isolated and dried over

MgSO4. The organic extract was concentrated in vacuo and puriﬁed

by ﬂash chromatography, (5 to 30% EtOAc/hex) to obtain the S-2-

methoxyethyl adduct as a white solid. A portionof the resulting solid

(40 mg, 0.11 mmol) was dissolved in anhydrous methanol (1.5 mL)

along with sodium methoxide(10 mg, 0.21 mmol) and allyl bromide

(39 mg, 0.32 mmol). The reaction was heated to 65 °C and stirred for

30 min. Additional sodium methoxide (20 mg, 0.42 mmol) and allyl

bromide (78 mg, 0.64 mmol) were added in two equal portions over

the course of 1 hour and then allowed to stir an additional hour at

65 °C. The mixture was then cooled and concentrated in vacuo.

The resulting residue was partitioned between 2 M HCl and EtOAc.

The aqueous layer was extracted with additional EtOAc, then the or-

ganic extracts were combined, dried over MgSO₄, vacuum ﬁltered,

and concentrated in vacuo. Puriﬁcation by ﬂash chromatogaphy

(5–20% EtOAc/hex) provided methyl 3-allyl-2-((2-methoxy-

ethyl)thio)-5,5-dimethyl-4-oxo-3,4,5,6-tetrahydrobenzo[h]quinaz-

oline-8-carboxylate 25 (20 mg, 0.05 mmol, 30% yield over 2 steps).

¹H NMR (400 MHz, CDCl₃) d (ppm) 8.14–8.16 (m, 1H), 7.98–8.00

(m, 1H), 7.88 (s, 1H), 5.92–5.97 (m, 1H), 5.28–5.33(m, 2H), 3.82 (s,

3H), 3.78- 3.82 (t, 2H), 3.55–3.56 (m, 2H), 3.44 (s, 3H), 2.86 (s, 2H),

DMF (558

lL). The reaction mixture was tightly capped and

warmed to 75 °C, and allowed to stir for 16 h. The reaction mix-

ture was partitioned between ethyl acetate and water, then the

organic layer was washed with water and brine. The organic

layer was concentrated in vacuo, and the residue was puriﬁed

via ﬂash chromatography (0–5% EtOAc/hex) to yield 19e

(14 mg, 30% yield over 2 steps). ¹H NMR (400 MHz, CDCl₃) d

(ppm) 7.81 (d, J = 2.5 Hz, 1H), 7.08 (d, J = 8.2 Hz, 1H), 6.91 (dd,

J = 8.2, 2.5 Hz, 1H), 6.09 (ddt, J = 16.4, 10.6, 5.5 Hz, 1H), 5.40

(d, J = 16.4 Hz, 1H), 5.27 (d, J = 10.6 Hz, 1H), 4.94 (d, J = 5.5 Hz,

2H), 3.87 (s, 3H), 3.19 (q, J = 7.3 Hz, 2H), 2.74 (s, 2H), 1.47 (t,

J = 7.3 Hz, 3H), 1.33 (s, 6H). ESI+MS m/z = 357.2 (M+H⁺), 379.2

(M+Na⁺). HPLC (Method B, t_R= 10.41 min), purity >95%. N-allyl

compound 12a was also isolated (10 mg, 21% yield over

2

steps).

5.2.76. 4-Cyano-3-methylbenzoic acid (22)

A

solution of 2.5 M n-butyllithium in hexanes(6.7 mL,

16.8 mmol) was added to anhydrous THF (15 mL) at ꢀ78 °C. A

solution of 4-bromo-2-methylbenzonitrile 21 (3.0 g, 15.3 mmol)

in THF (15 mL) was then added dropwise and allowed to stir for

B. D. Yestrepsky et al. / Bioorg. Med. Chem. 21 (2013) 1880–1897

1897

1.46 (s, 6H). ESI+MS m/z = 415.1 (M+H ⁺). HPLC (Method A,

Acknowledgements

t_R= 9.06 min), purity = 95%.

This work was supported by NIH Grant P01HL573461 to HS, a

University of Michigan Life Sciences Institute Innovation Partner-

ship grant to HS and SDL, and NIH Pharmacological Sciences Train-

ing Program fellowships (Grant T32 GM007767) for BDY and MEB.

We thank Dr. David Ginsburg for his advice regarding optimization

of the SK activity assay.

5.2.80. 3-Allyl-2-((2-methoxyethyl)thio)-5,5-dimethyl-4-oxo-

3,4,5,6-tetrahydrobenzo[h]quinazoline-8-carboxylic acid (26)

To a solution of sodium hydroxide (3.1 mg, 0.055 mmol) in

methanol (0.5 mL) was added methyl 3-allyl-2-((2-methoxy-

ethyl)thio)-5,5-dimethyl-4-oxo-3,4,5,6-tetrahydrobenzo[h]quinaz-

oline-8-carboxylate 25 (20 mg, 0.06 mmol). The resulting mixture

was stirred overnight at room temperature, then concentrated in

vacuo and partitioned between 2 M HCl and EtOAc. The aqueous

phase was extracted with additional EtOAc, then the organic ex-

tracts were combined and washed with brine. The organic layer

was isolated, dried over MgSO₄, vacuum ﬁltered, and concentrated

in vacuo to a white powder (19 mg, 0.05 mmol, 86% yield). ¹H NMR

(400 MHz, CDCl₃) d (ppm) 8.18–8.19 (m, 1H), 8.01–8.05 (m, 1H),

7.95 (br s, 1H), 6.20–6.23 (m, 1H), 5.89–5.94 (m, 1H), 5.26–5.31

(m, 2H), 4.58–4.63 (m, 2H), 3.72–3.78 (m, 4H), 3.49–3.52 (m,

2H), 3.42 (s, 3H), 2.94–2.95 (m, 2H), 2.80 (s, 2H), 1.37 (s, 6H).

ESI+MS m/z = 400.49 (M+H⁺). HPLC (Method A, t_R= 7.5 min),

purity = 95%.

Supplementary data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.046.

References and notes

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concentrated in vacuo. Further puriﬁcation via ﬂash chromatogra-

phy (5–20% EtOAc/hex) delivered the desired compound (35 mg,

29% yield). ¹H NMR (400 MHz, CDCl₃) d (ppm) 8.12 (d, J = 8Hz,

1H), 7.70 (d,, J = 8Hz, 1H), 7.62 (br s, 1H), 7.32–7.38 (m, 4H),

6.39–4.41 (m, 1H), 5.89–5.93 (m, 1H), 5.26–5.31 (m, 2H), 4.67–

4.73 (4H, M), 3.75 (t, J = 4Hz, 2H), 3.53 (t, J = 4Hz, 2H), 3.42 (s,

3H), 2.82 (s, 2H), 1.56 (s, 6H). ESI+MS m/z = 490.3 (M+H⁺). HPLC

(Method A, t_R= 8.33 min), purity = 98.6%.

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5.2.82. N-(2-(1H-imidazol-4-yl)ethyl)-3-allyl-2-((2-

methoxyethyl)thio)-5,5-dimethyl-4-oxo-3,4,5,6-

tetrahydrobenzo[h]quinazoline-8-carboxamide (27b)

To a solution of 3-allyl-2-((2-methoxyethyl)thio)-5,5-dimethyl-

4-oxo-3,4,5,6-tetrahydrobenzo[h]quinazoline-8-carboxylic acid 26

(50 mg, 0.13 mmol), EDC (30 mg, 0.15 mmol), and HOBt (20 mg,

0.15 mmol) in dry THF (5 mL) was added 2-(1H-imidazol-4-yl)eth-

anamine hydrochloride (20 mg, 0.15 mmol). The reaction was al-

lowed to stir at room temperature for 16 h, then diluted with

diethyl ether and water. The organic layer was washed with satu-

rated Na₂CO₃solution, water, and brine, then isolated, dried over

MgSO₄, vacuum ﬁltered, and concentrated in vacuo. Further puriﬁ-

cation via ﬂash chromatography (5–30% EtOAc/hex) delivered the

desired compound (13 mg, 21% yield). ¹H NMR (400 MHz, CDCl₃)

d (ppm) 8.11 (d, J = 8Hz, 1H), 7.73 (d, J = 8Hz, 1H), 7.64 (br s, 2H),

7.22–7.24 (br s, 1H), 7.01 (br s, 1H), 5.87–5.95 (m, 1H), 5.26–5.28

(m, 2H), 4.69–4.70 (2H, M), 4.60–4.61 (2H, m), 3.73 (t, J = 4Hz,

2H), 3.2 (t, J = 4Hz, 2H), 3.41 (s, 3H), 2.82 (s, 2H), 1.38 (s, 6H).

(ESI+MS m/z = 494.2 (M+H⁺). HPLC (Method A, t_R= 5.29 min),

purity = 93%.

Article Doi

Novel inhibitors of bacterial virulence: Development of 5,6-dihydrobenzo[h]quinazolin-4(3H)-ones for the inhibition of group A streptococcal streptokinase expression

DOI: 10.1016/j.bmc.2013.01.046

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