Synthetic, structural mimetics of the β-hairpin flap of HIV-1 protease inhibit enzyme function

Accepted Manuscript

Synthetic, structural mimetics of the β-hairpin flap of HIV-1 protease inhibit

enzyme function

Jay Chauhan, Shen-En Chen, Katherine J. Fenstermacher, Aurash Naser-

Tavakolian, Tali Reingewertz, Rosene Salmo, Christian Lee, Emori Williams,

Mithun Raje, Eric Sundberg, Jeffrey J. DeStefano, Ernesto Freire, Steven

Fletcher

PII:

S0968-0896(15)30027-4

DOI:

http://dx.doi.org/10.1016/j.bmc.2015.09.002

BMC 12549

Reference:

Bioorganic & Medicinal Chemistry

To appear in:

Received Date:

Revised Date:

Accepted Date:

9 July 2015

20 August 2015

3 September 2015

Please cite this article as: Chauhan, J., Chen, S-E., Fenstermacher, K.J., Naser-Tavakolian, A., Reingewertz, T.,

Salmo, R., Lee, C., Williams, E., Raje, M., Sundberg, E., DeStefano, J.J., Freire, E., Fletcher, S., Synthetic, structural

mimetics of the β-hairpin flap of HIV-1 protease inhibit enzyme function, Bioorganic & Medicinal Chemistry

(2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.09.002

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Synthetic, structural mimetics of the -

hairpin flap of HIV-1 protease inhibit

enzyme function

Jay Chauhan,^aShen-En Chen,^bKatherine J. Fenstermacher,^cAurash Naser-Tavakolian,^bTali

Reingewertz,^dRosene Salmo,^aChristian Lee,^eEmori Williams,^fMithun Raje,^aEric Sundberg,^dJeffrey

J. DeStefano,^cErnesto Freire^band Steven Fletcher^a,g*

^aDepartment of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N. Pine St.,

Baltimore, MD 21201, USA

^bDepartment of Biology, Johns Hopkins University, 3400 N. Charles St, Baltimore, MD 21218, USA

^cDepartment of Cell Biology & Molecular Genetics, University of Maryland, College Park, MD 20742, USA

^dDepartment of Microbiology and Immunology, University of Maryland School of Medicine, 725 West Lombard

St., Baltimore, MD 21201, USA

^ePharmD Program, University of Maryland School of Pharmacy, 20 N. Pine St., Baltimore, MD 21201, USA

^fVivien T Thomas Medical Arts Academy, 100 N Calhoun St., Baltimore, MD 21223, USA

^gUniversity of Maryland Greenebaum Cancer Center, 22 S. Greene St., Baltimore, MD 21201, USA

Bioorganic & Medicinal Chemistry

Synthetic, structural mimetics of the -hairpin flap of HIV-1 protease inhibit enzyme

function

Jay Chauhan,^aShen-En Chen,^bKatherine J. Fenstermacher,^cAurash Naser-Tavakolian,^bTali Reingewertz,^d

Rosene Salmo,^aChristian Lee,^eEmori Williams,^fMithun Raje,^aEric Sundberg,^dJeffrey J. DeStefano,^c

Ernesto Freire^band Steven Fletcher^a,g*

^aDepartment of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N. Pine St., Baltimore, MD 21201, USA

^bDepartment of Biology, Johns Hopkins University, 3400 N. Charles St, Baltimore, MD 21218, USA

^cDepartment of Cell Biology & Molecular Genetics, University of Maryland, College Park, MD 20742, USA

^dDepartment of Microbiology and Immunology, University of Maryland School of Medicine, 725 West Lombard St., Baltimore, MD 21201, USA

^ePharmD Program, University of Maryland School of Pharmacy, 20 N. Pine St., Baltimore, MD 21201, USA

^fVivien T Thomas Medical Arts Academy, 100 N Calhoun St., Baltimore, MD 21223, USA

^gUniversity of Maryland Greenebaum Cancer Center, 22 S. Greene St., Baltimore, MD 21201, USA

*Tel.: +1-410-706-6361; Fax: +1-410-706-5017; e-mail: sfletcher@rx.umaryland.edu

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

Small-molecule mimetics of the -hairpin flap of HIV-1 protease (HIV-1 PR) were designed

based on a 1,4-benzodiazepine scaffold as a strategy to interfere with the flap–flap protein–

protein interaction at the “mouth” of the active site. Michaelis-Menten kinetics suggest our

small-molecules are competitive inhibitors, which indicates the mode of inhibition is through

binding the active site or sterically blocking access to the active site, as designed. More

generally, a new bioactive scaffold for HIV-1PR inhibition has been discovered, with the most

potent compound inhibiting the protease with a modest K_iof 10 M.

Available online

Keywords:

HIV-1 protease

-hairpin

1,4-benzodiazepine

protein–protein interaction

proteomimetic

1. Introduction

are not thermodynamically stable⁶and so inhibitors designed to

recognize the folded -strands would need to organize its target

HIV-1 protease (HIV-1 PR) is a retroviral enzyme that is

essential for the life-cycle of HIV, and is, therefore, an important

target for AIDS therapy.^1-3Indeed, there are now eleven FDA-

approved HIV-1PR inhibitors. HIV-1 PR cleaves newly

synthesized gag and gag-pol polyproteins at the appropriate

places to create mature protein components of an infectious

virion that proceeds to further infect normal host cells.^1-3Owing

to the high mutation rate within the active site, however, the

enzyme becomes resistant to current drugs that target this region

peptide sequence(s) into -strand(s). Nevertheless, Schramm,

Chimelewksi and others have made some progress in this area

with “interfacial peptides”,^8-17although new drugs with this

mechanism of action are yet to be identified.

Extensive X-ray crystal structures have revealed that HIV-1

PR is a C2-symmetric homodimer where the enzyme’s active site

is “gated” by two glycine-rich, anti-parallel -hairpin flaps

(residues 43-58) that recognize each other predominantly through

hydrophobic interactions.¹⁸These flaps “open” and “close” to

allow peptide substrates to enter, and products to exit, the active

site of HIV-1 PR; more importantly, they are essential for proper

placement of the substrate within the active site. Accordingly,

interfering with this PPI in some manner might be expected to

compromise enzymatic activity. In fact, Král and Konvalinka

have demonstrated that metallacarboranes are capable of

competitively inhibiting HIV-1 PR through an unconventional

binding mode wherein the metallacarboranes bind the flap-

proximal region of the active, approximately the S3 and S3’

and so novel modes of inhibition are urgently required.^4,5

A

complementary strategy to targeting the active site is to disrupt

the protein–protein interaction (PPI) in the “whiskers” region of

the dimeric complex that is comprised of four interdigitating -

strands, two from each monomer (the N-terminal residues 1-4

and the C-terminal residues 96-99). This is a particularly

appealing target since this PPI accounts for 75% of the

dimerization stabilization energy,⁶and the mutation rate here is

lower than in the active site, suggesting inhibitors targeting this

region may be more refractory to resistance.⁷However, there is a

considerable entropic challenge here since the isolated monomers

hypothesized that bulky,

hydrophobic R²groups

would putatively mimic I47,

and tert-butyl esters at the R³

position were designed to

mimic I54. We proposed to

also include carboxylic acids

here to assist in compound

solubility. Accordingly, -

hairpin flap mimetics based

on the generic structure in

Figure 1D were prepared

according to Scheme 1.

Regioselective nitration of 1 followed by benzylic oxidation

with Jones’s reagent afforded ketone 2. Esterification with tert-

butanol and N,N-dicyclohexylcarbodiimide (DCC) then delivered

tert-butyl ester 3. Attempted reduction of the nitro group in 3

with stannous chloride (SnCl₂.2H₂O) yielded a mixture of the

desired product 4 along with the corresponding 3-methyl-2,1-

benzisoxazole (anthranil) that was presumably generated through

heterocyclization of the intermediate hydroxylamine with the

ketone. We investigated this more fully, and have published our

findings elsewhere.²⁵Complete transformation of 3 into 4 was

instead accomplished via catalytic hydrogenation, which may

have occurred through direct reduction of the aryl nitro group to

the respective aniline and/or reduction of the weak N-O bond

within the 2,1-benzisoxazole generated in situ or a combination

thereof. Fmoc-protected amino acids (R¹group) were pre-

activated with isobutyl chloroformate then coupled to aniline 4 to

deliver amides 5, which were not isolated; subsequent base-

mediated cyclization delivered the key BZD nucleus, as in

compounds 6. Deprotection of the tert-butyl esters in 6 with TFA

furnished acids 7. Alternatively, the anilide nitrogen of 6 was

alkylated under standard conditions with R²bromides to yield

target compounds 8, which, upon treatment with TFA, provided

the additional target compounds 9. We also prepared two

congeners of 8 and 9 in which the methyl imine was replaced by

a phenyl imine, as shown in Scheme 2, using similar chemistry to

that described previously.

Figure 1.: The semi-open and closed forms of HIV-1 PR, with

the active site indicated, including the catalytic aspartate residues

Asp25 and Asp25’), and the -hairpin flap regions highlighted

(residues 43-58 and 43’-58’). Green and cyan represent the two

HIV-1 PR monomers.

substrate binding sites, trapping the enzyme in the open

conformation.¹⁹

It is noteworthy that this binding mode was unprecedented as

all reported crystal structures of ligands bound to HIV-1 PR had

shown the enzyme in the closed conformation. It has been

speculated that the metallacarboranes inhibit HIV-1 PR through

preventing flap closure, and hence proper formation of the active

site, as this would still deliver competitive kinetics. Klebe and

co-workers have also targeted the open-flap conformation of

HIV-1 PR with pyrrolidine-based inhibitors.²⁰Thus, there exists

precedent for trapping the enzyme in the (semi-)open, inactive

conformation. We herein describe our efforts to disrupt the

enzymatic activity of HIV-1 PR through abrogating the PPI

between the -hairpin flaps with rationally designed synthetic

flap mimetics.

2. Results and Discussion

As a novel strategy to inhibit HIV-1 PR, we reasoned that the

equilibrium between the “semi-open” and “closed” states of the

enzyme could be intercepted by small-molecules that mimic one

of the -hairpin flaps found at the flap–flap PPI. Interaction of

one -hairpin flap with a flap surrogate would result in the

incomplete formation of the enzyme’s activity site, providing a

novel mechanism for the inhibition of HIV-1 PR. As mimetics of

-turns, -strands and -helices, 2-amido-2’-carboxamido-

diphenylacetylene derivatives exhibit a privileged role in

biomimicry, but their syntheses can be lengthy.^21-23In addition,

1,4-benzodiazepines (BZD) have also been utilized to imitate -

turns.²⁴Given their simpler synthesis, we selected the BZD

scaffold to reproduce the epitope of one of the -hairpin turns

located at the dynamic PPI between the two HIV-1PR monomers.

As shown in Table 1, a small batch of 1,4-benzodiazepines

lacking the R²group was initially prepared to gauge if this novel

scaffold could elicit inhibition of HIV-1 PR. Protocols measuring

HIV-1 PR activities using chromogenic and fluorogenic

substrates have been well-established.^26-29The HIV-1 PR

activities were measured spectroscopically allowing inhibition

constants, K_i, of the newly synthesized compounds to be

determined. Although most of the compounds in Table 1 did not

inhibit the enzyme, two of the most hydrophobic compounds,

tert-butyl esters 6f and 6g, exhibited appreciable inhibitory

activity (K_i= 64 M and 88 M, respectively), and so a second

set of compounds was prepared in which the anilide nitrogen

atom was alkylated with 4-cyclohexylbenzyl (R²) bromide to

afford the series of compounds illustrated in Table 2 that were

now proposed to be capable of mimicking I47. In general, the

bulkier and more hydrophobic the I50-mimicking R¹group, the

more potent was the inhibitor. Interestingly, the L- and D-leucine

derivatives 9c (K_i= 27 M) and 9d (K_i= 19 M) were more

potent than the L-isoleucine derivative 9e (K_i= 53 M). Whilst

there appeared little impact of the stereogenic centre on the

activity of the leucine derivatives 8c–9d, the L-phenylalanine

derivatives 8f and 9f were more potent than their D-counterparts.

In contrast to most instances in Table 1, the free acids (R³= H)

were more potent than their corresponding tert-butyl esters, the

latter of which were designed to approximate to I54. We surmise

this finding may be related to compound solubility. In order to

Residues 47-54 of the -hairpin flap are shown in Figures 2A

and 2B. A corresponding BZD proteomimetic of the flap tip is

given in Figure 2C. In order to simplify the synthetic chemistry

as well as attempt the mimicry of I47 and I54, the BZD was

inverted and then elaborated as shown in Figure 2D. [I50] of the

mimetic was intended to interact with I47/I54 of the target -

hairpin flap, whilst [I47] and [I54] were designed to recognize

I50 of the flap. R¹represents the side chain of the I50 mimetic

and may be readily varied by invoking different amino acids in

the construction of the BZD ring. To capture additional

hydrophobic interactions with the flap, the side-chain of G51 was

emulated by a methyl group, instead of a hydrogen atom. We

E.

B.

C.

A.

D.

Figure 2: A. Cartoon form of the -hairpin flap (residues 47-54) from a monomer of HIV-1PR; B. The peptide sequence of HIV-

1PR_47-54; C. A 1,4-benzodiazepine (BZD)-based mimetic of the four residues at the tip of the -hairpin flap; D. MM2 energy

minimized conformation of C (yellow, coloured by atom type) overlaid on -hairpin flap tip; E. Flipped BZD from C to simplify

the synthetic chemistry, and elaboration towards the additional mimicry of I47 and I54.

gauge if enhancing the hydrophobicity at the tip of the flap

mimetic would confer improved inhibition, the 5-methyl R⁴

group was replaced with a phenyl ring, although this resulted in

reduced inhibition (compare 9d with 16), which is consistent

with this group required to imitate only a small glycine side

chain. To expand our library further, we selected the most potent

compound from Table 2, 9d, and replaced the 4-

cyclohexylbenzyl group with a variety of alternative hydrophobic

groups as possible mimetics of I47; these compounds are shown

in Table 3. Once more, acids were more potent than their

corresponding tert-butyl esters. The larger, more hydrophobic R²

groups translated into increasingly improved inhibitors of HIV-1

PR. For example, compare 9h (R²= Bn) with 9i (R²= 4-tert-

butylbenzyl) and 9j (R²= 4-phenylbenzyl). For the series of alkyl

amides, the larger derivatives 9o and 9p, both with K_is of 11 M,

were more potent than the smaller derivative 9q (K_i= 75 M),

which may be due to better mimicry of I47. Others have also

observed improved inhibition of HIV-1 PR through the addition

of long chain alkyl groups to moderately active inhibitors.¹⁶An

MM2 energy-minimized conformation of 9d overlaid with one of

the -hairpin flaps of HIV-1PR is shown in Figure 3, which

reveals good mimicry of I47 and I50.

designed, and as reported elsewhere.^19,20Attempts to acquire a

co-crystal structure of 9o with HIV-1 PR failed, possibly due to

the modest binding affinity of the compound coupled with its

poor solubility. We are presently developing some second-

generation compounds that might address these drawbacks.

To examine if our compounds could inhibit HIV virus

replication within cells, we first studied the general cytotoxicity

of four of our most potent compounds: 9d, 9j, 9o and 9p. For this

purpose, MT-4 cells were utilized, which are a lymphocyte-

derived cell line used for HIV tissue culture, particularly for

assessing anti-retroviral cytotoxicity. The data is shown in Figure

5. All but 9p were tolerated up to 50 M, but all proved

particularly cytotoxic at 100 M. We then tested the ability of the

compounds to inhibit HIV replication in TZM-bl cells, a HeLa-

derived cell line expressing CCR5, CXCR4, and CD4 that

contains a luciferase gene under the control of a HIV tat-driven

promoter. As shown in Figure 5, which is presented as relative

luminescence units (RLUs) of a control (HIV infection without

any inhibitor), all compounds appeared to inhibit HIV replication

within cells at doses of 50 M or higher. However, according to

Figure 5, the HIV infectivity data for 9j and 9p may be attributed

O

a, b

c

d

e

Me

NO₂

Me

NO₂

Me

NH₂

HO₂C

tBuO₂C

1

2

3

4

Me

O

Me

N

Me

N

f

h

R¹

tBuO₂C

NH

R³O₂C

N

R²

R³O₂C

N

O

NHFmoc

O

H

O

R¹

6 (R³= tBu)

7 (R³= H)

8 (R³= tBu)

9 (R³= H)

5

g

in part, or completely, to the general cytotoxicities exhibited by

these compounds. Two of our more promising compounds, 9d

and 9o, will be subjected to further structure–activity refinement

to enhance the inhibition of HIV-1 PR in vitro towards the

development of more potent, and less cytotoxic, inhibitors of

HIV replication within cells.

Scheme 1: (a) HNO₃, H₂SO₄; (b) CrO₃, H₂SO₄; (c) tBuOH,

DCC, DMAP, CH₂Cl₂; (d) 10% Pd/C, H₂, MeOH; (e) 1. isobutyl

chloroformate, NMM, THF; 2. FmocHN(R¹)CO₂H, NMM; (f)

HNEt₂, CH₃CN; (g) TFA, CH₂Cl₂; (h) R²CH₂Br, K₂CO₃, DMF.

To investigate the mode of inhibition of our BZD-based

compounds, 9o was studied further. According to Figure 4, 9o is

a competitive inhibitor of HIV-1 PR. While these findings

indicate that 9o possibly binds the active site of HIV-1 PR, it

does not rule out a potential mechanism by which the small-

molecule sterically blocks access to the active site, as originally

indicates that our compounds function as competitive inhibitors

either through (a) binding in the active site, or (b) sterically

blocking access to the active site. Efforts towards the

determination of the specific binding mode of these new HIV-1

PR inhibitors is underway, and second-generation inhibitors

based on lead compounds 9d and 9o are presently a focus of our

laboratory

In conclusion, a new series of HIV-1 PR inhibitors was

developed based on BZD-inspired small-molecule mimicry of

one of the -hairpin turns found at the flap–flap PPI, which is the

gated access to the enzyme’s active site. Our molecules possibly

mimic I47, I50 and G51. Although the exact binding mode

remains unknown at this time, Michaelis-Menten analysis

Me

a

b

HO₂C

NO₂

tBuO₂C

NO₂

10

11

O

c, d

e

H

tBuO₂C

NO₂

tBuO₂C

NO₂

12

13

O

N

f-h

R³O₂C

N

tBuO₂C

NH₂

O

14

15 (R³= tBu)

16 (R³= H)

i

Scheme 2: (a) tBuOH, DCC, DMAP, CH₂Cl₂; (b) 1. DMF-dimethyl acetal, 5 h, 140 °C; 2. NaIO₄; (c) PhMgBr, 0 °C; (d) CrO₃,

H₂SO₄; (e) 10% Pd/C, H₂, MeOH; (f) 1. isobutyl chloroformate, NMM, CH₂Cl₂; 2. FmocHN(R¹)CO₂H, NMM, THF; (g) HNEt₂,

CH₃CN; (h) 4-cyclohexylbenzyl bromide, K₂CO₃, DMF; (i) TFA, CH₂Cl₂.

Table 1. Inhibition of HIV-1 PR by BZDs unfunctionalized at the anilide nitrogen. K_iis the inhibition constant, the concentration

of inhibitor that produces half-maximal inhibition.

O

R¹

HN

N

O

Me

₃O

R

Compound

R¹

R³

K_i( M)

Compound

6e

R¹

R³

K_i( M)

6a

7a

6b

7b

> 200

H

199 ± 16

H

7e

6f

158 ± 5

64 ± 9

> 200

Me

> 200

H

7f

> 200

H

6c

7c

6d

7d

193 ± 32

>200

6g

7g

88 ± 14

H

> 200

>200

Table 2. Inhibition of HIV-1 PR by BZDs alkylated at the anilide nitrogen with a 4-cyclohexylbenzyl moiety. K_iis the inhibition

constant, the concentration of inhibitor that produces half-maximal inhibition.

O

R¹

N

O

R⁴

₃O

R

Compound

R¹

R³

R⁴

K_i( M)

169 ± 27

85 ± 24

88 ± 6

Compound

16

R¹

R³

R⁴

K_i( M)

68 ± 13

H

Ph

8a

9a

8b

9b

H

Me

H

Me

8e

108 ± 12

Me

H

9e

8f

53 ± 7

54 ± 7

56 ± 9

H

8c

9c

8d

9d

76 ± 7

27 ± 4

65 ± 12

9f

47 ± 12

H

Me

8g

9g

131 ± 8

72 ± 11

19 ± 4

88 ± 5

H

15

Ph

Table 3. SAR of the anilide R²group of the BZD compounds. K_iis the inhibition constant, the concentration of inhibitor that

produces half-maximal inhibition.

O

R²

N

O

Me

₃O

R

Compound

8h

R²

R³

K_i( M)

Compound

9n

R²

R³

K_i( M)

75 ± 7

> 200

H

NH

O

9h

8i

H

> 200

8o

9o

> 200

NH

O

9

147 ± 19

11 ± 3

H

8p

9p

8q

9q

165 ± 11

11 ± 3

9i

H

46 ± 7

NH

O

15

H

8j

9j

62 ± 5

21 ± 2

15

> 200

5

H

75 ± 7

H

5

8k

89 ± 7

8r

9r

8s

143 ± 9

N

NBoc

NH

O

9k

8l

> 200

H

> 200

H

119 ± 40

O

> 200

NCbz

9l

> 200

OH

O

9s

8t

9t

81 ± 28

H

8m

N

NCbz

O

N

> 200

9m

8n

> 200

H

NFmoc

N

49 ± 15

H

95 ± 9

NH

O

Figure 3. An MM2 energy minimized conformation of 9d (yellow, coloured by atom type) overlaid with one of the -hairpin flaps

from HIV-1PR (cyan).

Figure 4. Compound 9o is a competitive inhibitor of HIV-1 PR.

Figure 5. Cytotoxicities of select HIV-1PR inhibitors to MT-4 cells.

Figure 6. HIV-1 virus replication inhibition by select HIV-1PR inhibitors in HeLa CD4+ TZM-bl cells. RLU = relative luminescence

units, relative to untreated control.

3. Experimental

3.1 Chemistry

General

= 7.5, 1 H, Ar), 2.89 (q, 2 H, CH₂), 1.23 (t, 3 H, CH₃). To a

round bottom flask equipped with a magnetic stirrer bar was

charged with NaIO₆(4.54 g, 20.00 mmol) in MeCN (50 mL). 0.1

M CrO₃in MeCN (5 mL, 0.50 mmol) and 4-ethyl-3-nitrobenzoic

acid (1.95 g, 10.0 mmol) were added to the reaction mixture,

which was allowed to stir at room temperature overnight. The

reaction mixture was decanted, washed with EtOAc and reduced

in vacuo. The residue was partitioned between EtOAc (100 mL)

and H₂O (50 mL), washed with 5% Na₂S₂O₃(50 mL), saturated

aqueous NaCl (50 mL), dried over MgSO₄, filtered and reduced

in vacuo. The residue was purified by column chromatography

(CH₂Cl₂/MeOH) to provide the title compound as a cream solid

(1.56 g): δ_H(DMSO-d₆, 500 MHz) 13.90 (br s, 1 H, CO₂H), 8.50

(s, 1 H, Ar), 8.34 (d, J = 8.0, 1 H, Ar), 7.88 (d, J = 8.0, 1 H, Ar),

2.60 (s, 3 H, CH₃).

Unless otherwise stated, all reaction were performed

under an inert atmosphere (N₂). Reagents and solvents were ACS

grade, and purchased from Sigma-Aldrich, Alfa Aesar, Oakwood

and TCI America. Anhydrous solvents were used as provided

from Sigma-Aldrich. Reactions were monitored by thin-layer

chromatography (TLC), visualizing with a UV lamp and/or

KMnO₄stain. Flash column chromatography was performed with

silica gel 60 Å (70-230 mesh, Merck). H and ¹³C NMR spectra

1

were recorded on a Varian INOVA 400 MHz NMR spectrometer

at 25 °C. Chemical shifts are reported in parts per million (ppm).

Data for H NMR are reported thus: chemical shift (δ ppm)

1

(multiplicity, coupling constant (Hz), integration), where

multiplicities are: s = singlet, d = doublet, t = triplet, m =

multiplet. The residual solvent peak was used as an internal

reference: CDCl₃(δ_H7.26; δ_C77.21) and d₆-DMSO (δ_H2.50; δ_C

39.51). Mass spectra were obtained on an Electrospray TOF

(ESI-TOF) mass spectrometer (Bruker AmaZon X). All final

molecules were deemed to be >90% pure by reversed-phased

HPLC using a Waters 1525 analytical/preparative HPLC fitted

with a C18 reversed-phase column (Atlantis T3 (T3) or

Symmetry (S): 4.6 mm x 150 mm) according to the following

conditions with solvents (A) H₂O/0.1% TFA, (B) CH₃CN–H₂O,

9:1 with 0.1% TFA at 1 ml min-1: (I) a gradient of 100% A to

100% B over 22 min (T3); (II) a gradient of 100% A to 100% B

over 22 min, then maintained for 13 min at 100% B (T3); (III) a

gradient of 100% A to 100% B over 10 min (S); (IV) a gradient

of 50% A to 100% B over 22 min (S); (V) an isocratic gradient of

100% B over 22 min (S). HPLC data are presented as retention

time (t_R(min)), purity (%), condition (I, II, II, IV or V).

tert-Butyl 4-acetyl-3-nitrobenzoate (3). To a round bottom flask

equipped with a magnetic stirrer bar was charged with 2 (209 mg,

1.00 mmol), tBuOH (115 L, 1.20 mmol) and DMAP (12 mg,

0.10 mmol) in CH₂Cl₂(6 mL). N,N-Dicyclohexylcarbodiimide

(DCC; 218 mg, 1.20 mmol) was added, and the reaction mixture

was stirred at room temperature for 3 h. The reaction mixture was

reduced in vacuo, and the residue was purified by column

chromatography (hexane/EtOAc) to provide the title compound

as a yellow oil (217 mg, 88%): δ_H(DMSO-d₆, 500 MHz) 8.46 (s,

1 H, Ar), 8.31 (d, J = 8.0, 1 H, Ar), 7.89 (d, J = 8.0, 1 H, Ar),

2.60 (s, 3 H, CH₃), 1.59 (s, 9 H, 3 x CH₃).

tert-Butyl 4-acetyl-3-aminobenzoate (4). To a round bottom flask

equipped with a magnetic stirrer bar was charged with nitro

acetophenone (300 mg, 1.44 mmol) in MeOH (10 mL). Pd/C (60

mg, 20 mol%) was added and the reaction put under H₂gas for 1

h. The reaction mixture was filtered through Celite, reduced in

vacuo and the residue was purified by silica gel column

chromatography (hexane/EtOAc) to provide the title compound

as a yellow solid (150 mg): δ_H(DMSO-d₆, 500 MHz) 7.82 (d, J =

8.0, 1 H, Ar), 7.35 (br s, 3 H, Ar, NH₂), 6.99 (d, J = 8.0, 1 H, Ar),

2.53 (s, 3 H, CH₃), 1.54 (s, 9 H, 3 x CH₃).

4-Acetyl-3-nitrobenzoic acid (2).²⁵To a round bottom flask

equipped with a magnetic stirrer bar was charged with HNO₃(25

mL) and 4-ethylbenzoic acid (5 g, 33.3 mmol). The reaction was

cooled to -10 °C and H₂SO₄(20 mL) was added and the reaction

was allowed to stir at room temperature for 1 h. The reaction

was poured over ice and the precipitate filtered to give 4-ethyl-3-

nitrobenzoic acid as a cream solid (5.9 g, 92 %): δ_H(DMSO-d₆,

500 MHz) 8.36 (s, 1 H, Ar), 8.15 (d, J = 7.5, 1 H, Ar), 7.67 (d, J

General Procedure A: Preparation of 1,4-Benzodiazepines 6.

To a round bottom flask equipped with a magnetic stirrer bar was

6.4, 3 H, CH₃); δ_C(DMSO-d₆, 100 MHz) 171.0, 166.4, 163.9,

137.5, 133.3, 131.6, 128.8, 123.0, 121.7, 81.4, 57.7, 27.7, 25.4,

16.9; LRMS: m/z C₁₆H₂₀N₂O₃requires: 288.1, found: 289.2

[M+H]; t_R= 1.57 (90.8%, III).

charged with N-Fmoc amino acid

4 (1.50 mmol), N-

methylmorpholine (1.60 mmol) and THF (10 mL). The reaction

was cooled to 0 °C and isobutyl chloroformate (1.60 mmol) was

added dropwise. The reaction was stirred at 0 °C for 30 min,

aniline (1.00 mmol) was added in one portion and the reaction

refluxed for 20 h. The reaction was cooled, quenched with

saturated aqueous NH₄Cl (1 mL), partitioned between EtOAc (60

mL) and H₂O (30 mL). The aqueous phase was extracted with

EtOAc (3 × 10 mL) and the organic portions combined, washed

with H₂O (10 mL), brine (10 mL), dried over MgSO₄, filtered and

reduced in vacuo. The residue was dissolved in MeCN (10 mL),

Et₂NH (2 mL) was added at room temperature and stirred for 3 h.

The reaction was reduced in vacuo and purified by silica gel

column chromatography (hexane/EtOAc) to provide the title

compound.

(S)-3,5-Dimethyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepine-

8-carboxylic acid (7b). Prepared according to General Procedure

C, to give the title compound as a cream solid (34 mg, 98 %): mp

58-60 °C; δ_H(DMSO-d₆, 400 MHz) 11.25 (s, 1 H, NH), 8.05 (d, J

= 8.0, 1 H, Ar), 7.84 (d, J = 1.2, 1 H, Ar), 7.81 (dd, J = 8.0, 1.2, 1

H, Ar), 3.96 (q, J = 6.4, 1 H, CHCO), 2.74 (s, 3H, CH₃CN), 1.50

(d, J = 6.4, 3 H, CH₃); δ_C(DMSO-d₆, 100 MHz) 169.4, 166.3,

139.3, 136.4, 131.6, 124.2, 123.1, 56.0, 24.3, 14.1; LRMS: m/z

C₁₂H₁₂N₂O₃requires: 232.1, found: 233.2 [M+H]; t_R= 1.25

(97.6%, III).

(S)-tert-Butyl

3-isobutyl-5-methyl-2-oxo-2,3-dihydro-1H-

benzo[e][1,4]diazepine-8-carboxylate (6c). Prepared according

to General Procedure A, to give the title compound as a cream

solid (280 mg, 85%): mp 84-86 °C; δ_H(DMSO-d₆, 400 MHz)

10.55 (s, 1 H, NH), 7.83 (d, J = 8.4, 1 H, Ar), 7.70 (s, 1 H, Ar),

7.65 (d, J = 8.4, 1 H, Ar), 3.32-3.25 (m, 1 H, CHCO), 2.83 (s,

3H, CH₃CN), 1.88-1.81 (m, 1 H, CHMe₂), 1.78-1.68 (m, 2 H,

CH₂iPr), 1.55 (s, 9 H, 3 x CH₃), 0.85 (d, J = 5.6, 3 H, CH₃), 0.71

(d, J = 5.6, 3 H, CH₃); δ_C(DMSO-d₆, 100 MHz) 170.2, 166.7,

163.9, 137.5, 133.3, 131.5, 128.8, 123.1, 121.7, 81.4, 60.5, 27.7,

25.4, 23.9, 23.2, 21.8; LRMS: m/z C₁₉H₂₆N₂O₃requires: 330.2,

found: 331.3 [M+H]; t_R= 5.61 (97.0%, I).

General Procedure B: Preparation of N-alkylated-1,4-

benzodiazepines 8. To a round bottom flask equipped with a

magnetic stirrer bar was charged with 1,4-benzodiazepine (0.15

mmol), K₂CO₃(0.45 mmol) and DMF (3 mL). The reaction was

cooled to 0 °C and alkyl halide (0.30 mmol) in DMF (1 mL) was

added. The reaction was stirred at room temperature for 3 h. The

reaction was partitioned between EtOAc (30 mL) and H₂O (10

mL). The organic phase was washed with H₂O (3 × 10 mL), brine

(10 mL), dried over MgSO₄, filtered and reduced in vacuo. The

residue was purified by column chromatography (hexane/EtOAc)

to provide the title compound.

(S)-3-Isobutyl-5-methyl-2-oxo-2,3-dihydro-1H-

General Procedure C: Deprotection of 1,4-Benzodiazepines 6

and 8. To a round bottom flask equipped with a magnetic stirrer

bar was charged with 1,4-benzodiazepines 6 or 8 (0.10 mmol)

and CH₂Cl₂(1 mL). TFA (1 mL) was added at room temperature

and stirred for 4 h. The reaction was reduced in vacuo, co-

evaporated with chloroform and lyophilized from MeCN/H₂O

(1:1) to provide the title compound 7 or 9 as its TFA salt.

benzo[e][1,4]diazepine-8-carboxylic acid (7c). Prepared

according to General Procedure C, to give the title compound as

a cream solid (38 mg, 99 %): mp 113-115 °C; δ_H(DMSO-d₆, 400

MHz) 11.09 (s, 1 H, NH), 7.99 (d, J = 8.4, 1 H, Ar), 7.81 (d, J =

1.2, 1 H, Ar), 7.78 (dd, J = 8.4, 1.2, 1 H, Ar), 3.66 (t, J = 6.4, 1 H,

CHCO), 2.66 (s, 3H, CH₃CN), 1.92-1.71 (m, 3 H, CHMe₂,

CH₂iPr), 0.89 (d, J = 6.0, 3 H, CH₃), 0.77 (d, J = 6.0, 3 H, CH₃);

δ_C(DMSO-d₆, 100 MHz) 169.2, 166.5, 138.9, 135.6, 131.1,

128.7, 124.1, 122.9, 114.8, 59.2, 37.6, 24.9, 24.3, 23.4, 22.1;

LRMS: m/z C₁₅H₁₈N₂O₃requires: 274.1, found: 275.2 [M+H]; t_R

= 3.63 (92.6%, I).

tert-Butyl

5-methyl-2-oxo-2,3-dihydro-1H-

benzo[e][1,4]diazepine-8-carboxylate (6a). Prepared according

to General Procedure A, to give the title compound as a white

solid (246 mg, 90%): mp 186-188 °C; δ_H(DMSO-d₆, 400 MHz)

10.53 (s, 1 H, NH), 7.80 (d, J = 8.4, 1 H, Ar), 7.70 (s, 1 H, Ar),

7.64 (d, J = 8.4, 1 H, Ar), 3.90 (s, 2 H, CH₂CO), 2.40 (s, 3H,

CH₃CN), 1.55 (s, 9 H, 3 x CH₃); δ_C(DMSO-d₆, 100 MHz) 170.1,

168.5, 163.9, 137.9, 133.3, 131.3, 129.1, 123.1, 121.7, 81.4, 56.2,

27.7, 25.6; LRMS: m/z C₁₅H₁₈N₂O₃requires: 274.1, found: 275.3

[M+H]; t_R= 1.59 (98.7%, III).

(R)-tert-Butyl

3-isobutyl-5-methyl-2-oxo-2,3-dihydro-1H-

benzo[e][1,4]diazepine-8-carboxylate (6d). Prepared according

to General Procedure A, to give the title compound as a yellow

solid (273 mg, 83%): mp 85-87 °C; δ_H(DMSO-d₆, 400 MHz)

10.55 (s, 1 H, NH), 7.83 (d, J = 8.4, 1 H, Ar), 7.70 (s, 1 H, Ar),

7.65 (d, J = 8.4, 1 H, Ar), 3.32-3.25 (m, 1 H, CHCO), 2.83 (s, 3

H, CH₃CN), 1.88-1.81 (m, 1 H, CHMe₂), 1.78-1.68 (m, 2 H,

CH₂iPr), 1.55 (s, 9 H, 3 x CH₃), 0.85 (d, J = 5.6, 3 H, CH₃), 0.71

(d, J = 5.6, 3 H, CH₃); δ_C(DMSO-d₆, 100 MHz) 170.2, 166.7,

163.9, 137.5, 133.3, 131.5, 128.8, 123.1, 121.7, 81.4, 60.5, 27.7,

25.4, 23.9, 23.2, 21.8; LRMS: m/z C₁₉H₂₆N₂O₃requires: 330.2,

found: 331.2 [M+H]; t_R= 4.48 (99.1%, I).

5-Methyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepine-8-

carboxylic acid (7a). Prepared according to General Procedure C,

to give the title compound as a cream solid (33 mg, 99 %): mp

>250 °C; δ_H(DMSO-d₆, 400 MHz) 11.11 (s, 1 H, NH), 8.00 (d, J

= 8.0, 1 H, Ar), 7.82 (d, J = 1.6, 1 H, Ar), 7.78 (dd, J = 8.0, 1.6, 1

H, Ar), 4.13 (s, 2 H, CH₂CO), 2.69 (s, 3H, CH₃CN); δ_C(DMSO-

d₆, 100 MHz) 168.3, 166.0, 138.9, 135.5, 131.1 127.6, 123.8,

122.6, 52.6, 24.4; LRMS: m/z C₁₁H₁₀N₂O₃requires: 218.1, found:

219.3 [M+H]; t_R= 1.27 (98.1%, III).

(R)-3-Isobutyl-5-methyl-2-oxo-2,3-dihydro-1H-

benzo[e][1,4]diazepine-8-carboxylic acid (7d). Prepared

according to General Procedure C, to give the title compound as

a yellow solid (39 mg, 99%): mp 121-123 °C; δ_H(DMSO-d₆, 400

MHz) 11.09 (s, 1 H, NH), 7.99 (d, J = 8.4, 1 H, Ar), 7.81 (d, J =

1.2, 1 H, Ar), 7.78 (dd, J = 8.4, 1.2, 1 H, Ar), 3.66 (t, J = 6.4, 1 H,

CHCO), 2.66 (s, 3 H, CH₃CN), 1.92-1.71 (m, 3 H, CHMe₂,

CH₂iPr), 0.89 (d, J = 6.0, 3 H, CH₃), 0.77 (d, J = 6.0, 3 H, CH₃);

δ_C(DMSO-d₆, 100 MHz) 169.2, 166.5, 138.9, 135.6, 131.1,

128.7, 124.1, 122.9, 114.8, 59.2, 37.6, 24.9, 24.3, 23.4, 22.1;

(S)-tert-Butyl

3,5-dimethyl-2-oxo-2,3-dihydro-1H-

benzo[e][1,4]diazepine-8-carboxylate (6b). Prepared according

to General Procedure A, to give the title compound as a white

solid (267 mg, 93%): mp 80-82 °C; δ_H(DMSO-d₆, 400 MHz)

10.53 (s, 1 H, NH), 7.80 (d, J = 8.4, 1 H, Ar), 7.69 (d, J = 1.2, 1

H, Ar), 7.65 (dd, J = 8.4, 1.2, 1 H, Ar), 3.44 (q, J = 6.4, 1 H,

CHCO), 2.38 (s, 3H, CH₃CN), 1.55 (s, 9 H, 3 x CH₃), 1.54 (d, J =

LRMS: m/z C₁₅H₁₈N₂O₃requires: 274.1, found: 275.2 [M+H]; t_R

= 3.68 (92.1%, I).

166.8, 163.9, 139.2, 137.3, 133.4, 131.6, 129.4, 128.9, 128.0,

125.9, 123.1, 121.7, 81.4, 64.3, 36.9, 27.7, 25.4; LRMS: m/z

C₂₂H₂₄N₂O₃requires: 364.2, found: 365.2 [M+H]; t_R= 3.53

(96.1%, III).

(S)-tert-Butyl 3-((R)-sec-butyl)-5-methyl-2-oxo-2,3-dihydro-1H-

benzo[e][1,4]diazepine-8-carboxylate (6e). Prepared according

to General Procedure A, to give the title compound as a white

solid (277 mg, 84 %): mp 81-83 °C; δ_H(DMSO-d₆, 400 MHz)

10.51 (s, 1 H, NH), 7.82 (d, J = 8.4, 1 H, Ar), 7.69 (s, 1 H, Ar),

7.65 (d, J = 8.0, 1 H, Ar), 2.89 (d, J = 9.2, 1 H, CHCO), 2.39 (s,

3H, CH₃CN), 2.18-2.12 (m, 1 H, CH), 1.79-1.73 (m, 1 H, CH),

1.55 (s, 9 H, 3 x CH₃), 1.09-1.02 (m, 1 H, CH), 0.88 (d, J = 6.5, 3

H, CHCH₃), 0.82 (t, J = 7.6, 3 H, CH₂CH₃); δ_C(DMSO-d₆, 100

MHz) 168.9, 166.3, 163.9, 137.4, 133.3, 131.5, 128.8, 123.1,

121.7, 81.4, 67.2, 34.4, 27.7, 25.4, 24.4, 15.8, 10.8; LRMS: m/z

C₁₉H₂₆N₂O₃requires: 330.2, found: 331.2 [M+H]; t_R= 3.98

(91.2%, III).

(R)-3-benzyl-5-methyl-2-oxo-2,3-dihydro-1H-

benzo[e][1,4]diazepine-8-carboxylic acid (7g). Prepared

according to General Procedure C, to give the title compound as

a cream solid (41 mg, 88%): mp 83-85 °C; δ_H(DMSO-d₆, 400

MHz) 11.00 (s, 1 H, NH), 7.91 (d, J = 8.4, 1 H, Ar), 7.76 (d, J =

1.6, 1 H, Ar), 7.74 (dd, J = 8.0, 1.6, 1 H, Ar), 7.29-7.21 (m, 4 H,

Ar), 7.18-7.13 (m, 1 H, Ar), 3.85 (t, J = 6.4, 1 H, CHCO), 3.40

(dd, J = 14.0, 6.4, 1 H, CH_aH_bAr), 3.22 (dd, J = 14.0, 6.4, 1 H,

CH_aH_bAr), 2.56 (s, 3H, CH₃CN); δ_C(DMSO-d₆, 100 MHz)

169.0, 166.5, 138.5, 138.4, 135.0, 130.6, 129.9, 128.6,

126.7,124.1, 122.7, 63.1, 35.6, 25.2; LRMS: m/z C₁₈H₁₆N₂O₃

requires: 308.1, found: 309.2 [M+H]; t_R= 3.91 (90.0%, I).

(S)-3-((R)-sec-Butyl)-5-methyl-2-oxo-2,3-dihydro-1H-

benzo[e][1,4]diazepine-8-carboxylic acid (7e). Prepared

according to General Procedure C, to give the title compound as

a cream solid (38 mg, 99%): mp 73-75 °C; δ_H(DMSO-d₆, 400

MHz) 11.0 (s, 1 H, NH), 7.96 (d, J = 8.0, 1 H, Ar), 7.78-7.75 (m,

2 H, Ar), 3.40-3.23 (m, 1 H, CHCO), 2.61 (s, 3H, CH₃CN), 2.22-

2.12 (m, 1 H, CH), 1.80-1.66 (m, 1 H, CH), 1.14-1.06 (m, 1 H,

CH), 0.94 (d, J = 6.8, 3 H, CHCH₃), 0.84 (t, J = 7.6, 3 H,

CH₂CH₃); δ_C(DMSO-d₆, 100 MHz) 168.0, 166.5, 138.6, 130.6,

124.1, 122.7, 114.5, 66.0, 33.7, 25.1, 24.8, 16.0, 11.1; LRMS:

m/z C₁₅H₁₈N₂O₃requires: 274.1, found: 275.2 [M+H]; t_R= 3.70

(92.2%, I).

tert-Butyl

1-(4-cyclohexylbenzyl)-5-methyl-2-oxo-2,3-dihydro-

(8a). Prepared

1H-benzo[e][1,4]diazepine-8-carboxylate

according to General Procedure B, to give the title compound as

a white solid (52 mg, 78%): mp 171-173 °C; δ_H(DMSO-d₆, 400

MHz) 7.85 (s, 1 H, Ar), 7.75 (d, J = 8.4, 1 H, Ar), 7.68 (d, J =

8.4, 1 H, Ar), 7.12 (d, J = 8.0, 2 H, Ar), 7.00 (d, J = 8.0, 2 H, Ar),

5.05 (d, J = 16.0, 1 H, CH_aH_bAr), 4.98 (d, J = 16.0, 1 H,

CH_aH_bAr), 4.40 (d, J = 11.2, 1 H, CH_aH_bCO), 3.69 (d, J = 11.2, 1

H, CH_aH_bCO), 2.46-2.40 (m, 1 H, Cy), 2.38 (s, 3H, CH₃CN),

1.77-1.66 (m, 5 H, Cy), 1.46 (s, 9 H, 3 x CH₃), 1.39-1.17 (m, 5 H,

Cy); δ_C(DMSO-d₆, 100 MHz) 168.6, 168.2, 163.6, 146.3, 140.8,

134.5, 133.8, 133.4, 128.1, 126.7, 126.4, 124.8, 122.6, 81.3, 79.2,

78.9, 78.6, 55.9, 49.6, 43.3, 33.9, 33.8, 27.6, 26.3, 25.5, 24.9;

LRMS: m/z C₂₈H₃₄N₂O₃requires: 446.3, found: 447.2 [M+H]; t_R

= 27.74 (100%, II).

(S)-tert-Butyl

3-benzyl-5-methyl-2-oxo-2,3-dihydro-1H-

benzo[e][1,4]diazepine-8-carboxylate (6f). Prepared according to

General Procedure A, to give the title compound as a white solid

(316 mg, 87%): mp 85-87 °C; δ_H(DMSO-d₆, 400 MHz) 10.59 (s,

1 H, NH), 7.78 (d, J = 8.0, 1 H, Ar), 7.68 (s, 1 H, Ar), 7.63 (d, J =

8.0, 1 H, Ar), 7.24-7.18 (m, 4 H, Ar), 7.14-7.10 (m, 1 H, Ar),

3.54 (t, J = 6.0, 1 H, CHCO), 3.36 (dd, J = 13.6, 6.0, 1 H,

CH_aH_bAr), 3.16 (dd, J = 13.6, 6.0, 1 H, CH_aH_bAr), 2.37 (s, 3H,

CH₃CN), 1.53 (s, 9 H, 3 x CH₃); δ_C(DMSO-d₆, 100 MHz) 169.6,

166.8, 163.9, 139.2, 137.3, 133.4, 131.6, 129.4, 128.9, 128.0,

125.9, 123.1, 121.7, 81.4, 64.3, 36.9, 27.7, 25.4; LRMS: m/z

C₂₂H₂₄N₂O₃requires: 364.2, found: 365.2 [M+H]; t_R= 5.38

(100%, III).

1-(4-Cyclohexylbenzyl)-5-methyl-2-oxo-2,3-dihydro-1H-

benzo[e][1,4]diazepine-8-carboxylic acid (9a). Prepared

according to General Procedure C, to gie the title compound as a

cream solid (49 mg, 98%): mp 103-105 °C; δ_H(DMSO-d₆, 400

MHz) 7.97 (s, 1 H, Ar), 7.88 (d, J = 8.0, 1 H, Ar), 7.80 (d, J =

8.0, 1 H, Ar), 7.12 (d, J = 8.0, 2 H, Ar), 6.99 (d, J = 8.0, 2 H, Ar),

5.14 (d, J = 16.0, 1 H, CH_aH_bAr), 4.98 (d, J = 16.0, 1 H,

CH_aH_bAr), 4.42 (d, J = 11.2, 1 H, CH_aH_bCO), 3.90 (d, J = 11.2, 1

H, CH_aH_bCO), 2.52 (s, 3H, CH₃CN), 2.46-2.38 (m, 1 H, Cy),

1.76-1.65 (m, 5 H, Cy), 1.38-1.16 (m, 5 H, Cy); δ_C(DMSO-d₆,

100 MHz) 167.3, 165.9, 146.4, 141.1, 134.2, 134.1, 129.1, 126.7,

126.4, 125.3, 123.3, 54.1, 49.8, 33.8, 26.3, 25.5, 24.4; LRMS:

m/z C₂₄H₂₆N₂O₃requires: 390.2, found: 391.2 [M+H]; t_R= 4.66

(100%, I).

(S)-3-Benzyl-5-methyl-2-oxo-2,3-dihydro-1H-

benzo[e][1,4]diazepine-8-carboxylic acid (7f). Prepared

according to General Procedure C, to give the title compound as

a cream solid (41 mg, 99 %): mp 78-80 °C; δ_H(DMSO-d₆, 400

MHz) 11.00 (s, 1 H, NH), 7.91 (d, J = 8.4, 1 H, Ar), 7.76 (d, J =

1.6, 1 H, Ar), 7.74 (dd, J = 8.0, 1.6, 1 H, Ar), 7.29-7.21 (m, 4 H,

Ar), 7.18-7.13 (m, 1 H, Ar), 3.85 (t, J = 6.4, 1 H, CHCO), 3.40

(dd, J = 14.0, 6.4, 1 H, CH_aH_bAr), 3.22 (dd, J = 14.0, 6.4, 1 H,

CH_aH_bAr), 2.56 (s, 3H, CH₃CN); δ_C(DMSO-d₆, 100 MHz)

169.0, 166.5, 138.5, 138.4, 135.0, 130.6, 129.9, 128.6,

126.7,124.1, 122.7, 63.1, 35.6, 25.2; LRMS: m/z C₁₈H₁₆N₂O₃

requires: 308.1, found: 309.2 [M+H]; t_R= 4.61 (95.5%, I).

(S)-tert-Butyl

1-(4-cyclohexylbenzyl)-3,5-dimethyl-2-oxo-2,3-

dihydro-1H-benzo[e][1,4]diazepine-8-carboxylate (8b). Prepared

according to General Procedure B, to give the title compound as

a white solid (55 mg, 81%): mp 66-68 °C; δ_H(DMSO-d₆, 400

MHz) 7.88 (s, 1 H, Ar), 7.78 (d, J = 8.0, 1 H, Ar), 7.70 (d, J =

8.0, 1 H, Ar), 7.12 (d, J = 7.6, 2 H, Ar), 6.97 (d, J = 7.6, 2 H, Ar),

5.04 (s, 2 H, CH₂Ar), 3.65 (q, J = 6.4, 1 H, CHCO), 2.46-2.37 (m,

1 H, Cy), 2.35 (s, 3H, CH₃CN), 1.77-1.65 (m, 5 H, Cy), 1.48-1.40

(m, 12 H, 4 x CH₃), 1.39-1.17 (m, 5 H, Cy); δ_C(DMSO-d₆, 100

MHz) 169.7, 166.4, 163.7, 146.3, 140.5, 134.7, 134.3, 133.4,

127.9, 126.7, 126.5, 124.7, 122.8; LRMS: m/z C₂₉H₃₆N₂O₃

requires: 460.3, found: 461.3 [M+H]; t_R= 5.97 (96.3%, VI).

(R)-tert-Butyl

3-benzyl-5-methyl-2-oxo-2,3-dihydro-1H-

benzo[e][1,4]diazepine-8-carboxylate (6g). Prepared according

to General Procedure A, to give the title compound as a white

solid (291 mg, 80%): mp 90-92 °C; δ_H(DMSO-d₆, 400 MHz)

10.59 (s, 1 H, NH), 7.78 (d, J = 8.0, 1 H, Ar), 7.68 (s, 1 H, Ar),

7.63 (d, J = 8.0, 1 H, Ar), 7.24-7.18 (m, 4 H, Ar), 7.14-7.10 (m, 1

H, Ar), 3.54 (t, J = 6.0, 1 H, CHCO), 3.36 (dd, J = 13.6, 6.0, 1 H,

CH_aH_bAr), 3.16 (dd, J = 13.6, 6.0, 1 H, CH_aH_bAr), 2.37 (s, 3H,

CH₃CN), 1.53 (s, 9 H, 3 x CH₃); δ_C(DMSO-d₆, 100 MHz) 169.6,

(S)-1-(4-cyclohexylbenzyl)-3,5-dimethyl-2-oxo-2,3-dihydro-1H-

benzo[e][1,4]diazepine-8-carboxylic acid (9b). Prepared

according to General Procedure C, to give the title compound as

a cream solid (51 mg, 99%): mp 70-72 °C; δ_H(DMSO-d₆, 400

MHz) 8.00 (s, 1 H, Ar), 7.89 (d, J = 8.0, 1 H, Ar), 7.81 (d, J =

8.0, 1 H, Ar), 7.11 (d, J = 8.0, 2 H, Ar), 6.95 (d, J = 8.0, 2 H, Ar),

5.20 (d, J = 16.0, 1 H, CH_aH_bAr), 4.99 (d, J = 16.0, 1 H,

CH_aH_bAr), 3.89 (q, J = 6.8, 1 H, CHCO), 2.49 (s, 3H, CH₃CN),

2.46-2.38 (m, 1 H, Cy), 1.75-1.65 (m, 5 H, Cy), 1.48 (d, J = 6.8,

3 H, CH3), 1.35-1.16 (m, 5 H, Cy); δ_C(DMSO-d₆, 100 MHz)

168.7, 166.0, 146.4, 140.8, 134.4, 134.2, 129.0, 126.7, 126.5,

125.3, 123.6, 56.3, 50.1, 33.9, 33.8, 26.2, 25.5, 24.0, 15.7;

LRMS: m/z C₂₅H₂₈N₂O₃requires: 404.2, found: 405.2 [M+H]; t_R

= 4.59 (90.2%, V).

4.98 (d, J = 16.0, 1 H, CH_aH_bAr), 3.65 (t, J = 7.2, 1 H, CHCO),

2.45 (s, 3 H, CH₃CN), 2.44-2.37 (m, 1 H, Cy), 2.01-1.62 (m, 8 H,

CHMe₂, CH₂iPr, Cy), 1.38-1.11 (m, 5 H, Cy), 0.87 (d, J = 6.4, 3

H, CH₃), 0.74 (d, J = 6.4, 3 H, CH₃); δ_C(DMSO-d₆, 100 MHz)

168.2, 166.1, 146.4, 140.7, 134.3, 134.1, 133.1, 128.6, 126.7,

126.5, 125.7, 123.7, 59.6, 50.0, 43.3, 33.9, 33.8, 26.3, 25.5, 24.4,

23.9, 23.0, 21.8; LRMS: m/z C₂₈H₃₄N₂O₃requires: 446.3, found:

447.3 [M+H]; t_R= 6.57 (100%, I).

(S)-tert-Butyl 3-((R)-sec-butyl)-1-(4-cyclohexylbenzyl)-5-methyl-

2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepine-8-carboxylate

(8e). Prepared according to General Procedure B, to give the title

compound as a white solid (70 mg, 93%): mp 75-77 °C; δ_H

(DMSO-d₆, 400 MHz) 7.89 (s, 1 H, Ar), 7.79 (d, J = 8.0, 1 H,

Ar), 7.70 (d, J = 8.0, 1 H, Ar), 7.12 (d, J = 7.6, 2 H, Ar), 6.95 (d,

J = 7.6, 2 H, Ar), 5.07 (d, J = 16.0, 1 H, CH_aH_bAr), 5.00 (d, J =

16.0, 1 H, CH_aH_bAr), 3.07 (d, J = 9.6, 1 H, CHCO), 2.46-2.37

(m, 1 H, Cy), 2.34 (s, 3H, CH₃CN), 2.31-2.65 (m, 1 H, CH),

1.81-1.62 (m, 6 H, CH, Cy), 1.46 (s, 9 H, 3 x CH₃), 1.38-1.13 (m,

5 H, Cy), 1.04-0.96 (m, 1 H, CH), 0.89 (d, J = 6.4, 3 H, CHCH₃),

0.81 (t, J = 7.6, 3 H, CH₂CH₃); δ_C(DMSO-d₆, 100 MHz) 167.8,

166.4, 163.6, 146.4, 140.5, 134.7, 134.0, 133.4, 127.8, 126.8,

126.4, 124.8, 122.7, 81.4, 67.3, 49.7, 43.3, 34.6, 33.9, 33.8, 27.6,

26.3, 25.5, 24.8, 24.3, 15.6, 10.7; LRMS: m/z C₃₂H₄₂N₂O₃

requires: 502.3, found: 503.3 [M+H]; t_R= 25.48 (95.6%, II).

(S)-tert-Butyl 1-(4-cyclohexylbenzyl)-3-isobutyl-5-methyl-2-oxo-

2,3-dihydro-1H-benzo[e][1,4]diazepine-8-carboxylate

(8c).

Prepared according to General Procedure B, to give the title

compound as a white solid (63 mg, 84%): mp 77-79 °C; δ_H

(DMSO-d₆, 400 MHz) 7.90 (s, 1 H, Ar), 7.79 (d, J = 8.4, 1 H,

Ar), 7.71 (d, J = 8.4, 1 H, Ar), 7.12 (d, J = 8.0, 2 H, Ar), 6.96 (d,

J = 8.0, 2 H, Ar), 5.04 (s, 2 H, CH₂Ar), 3.49 (t, J = 6.8, 1 H,

CHCO), 2.47-2.38 (m, 1 H, Cy), 2.35 (s, 3H, CH₃CN), 2.00-1.62

(m, 8 H, CHMe₂, CH₂iPr, Cy), 1.47 (s, 9 H, 3 x CH₃), 1.41-1.10

(m, 5 H, Cy), 0.86 (d, J = 6.4, 3 H, CH₃), 0.72 (d, J = 6.4, 3 H,

CH₃); δ_C(DMSO-d₆, 100 MHz) 168.9, 166.8, 163.7, 146.4,

140.5, 134.7, 134.2, 133.5, 127.9, 126.8, 126.5, 124.9, 122.8,

81.4, 60.4, 49.8, 43.3, 33.9, 33.8, 27.6, 26.3, 25.5, 24.8, 24.0,

23.2, 21.9; LRMS: m/z C₃₂H₄₂N₂O₃requires: 502.3, found: 503.3

[M+H]; t_R= 29.28 (90.5%, II).

(S)-3-((R)-sec-Butyl)-1-(4-cyclohexylbenzyl)-5-methyl-2-oxo-2,3-

dihydro-1H-benzo[e][1,4]diazepine-8-carboxylic acid (9e).

Prepared according to General Procedure C, to give the title

compound as a yellow solid (55 mg, 98%): mp 94-96 °C; δ_H

(DMSO-d₆, 400 MHz) 7.99 (s, 1 H, Ar), 7.83 (d, J = 8.4, 1 H,

Ar), 7.78 (d, J = 8.4, 1 H, Ar), 7.10 (d, J = 8.4, 2 H, Ar), 6.90 (d,

J = 8.4, 2 H, Ar), 5.20 (d, J = 16.0, 1 H, CH_aH_bAr), 4.97 (d, J =

16.0, 1 H, CH_aH_bAr), 3.21 (d, J = 10.0, 1 H, CHCO), 2.59-2.54

(m, 1 H, Cy), 2.40 (s, 3H, CH₃CN), 2.29-2.25 (m, 1 H, CH),

1.82-1.62 (m, 6 H, CH, Cy), 1.43-1.15 (m, 5 H, Cy), 1.07-0.95

(m, 1 H, CH), 0.90 (d, J = 6.4, 3 H, CHCH₃), 0.82 (t, J = 7.2, 3 H,

CH₂CH₃); δ_C(DMSO-d₆, 100 MHz) 167.2,166.1, 146.4, 140.5,

134.3, 133.7, 133.5, 128.3, 126.7, 126.4, 125.3, 123.4, 66.6, 49.8,

34.2, 33.9, 26.3, 25.5, 24.4, 24.3, 15.5, 10.6; LRMS: m/z

C₂₈H₃₄N₂O₃requires: 446.3, found: 447.2 [M+H]; t_R= 20.77

(100%, I).

(S)-1-(4-cyclohexylbenzyl)-3-isobutyl-5-methyl-2-oxo-2,3-

dihydro-1H-benzo[e][1,4]diazepine-8-carboxylic acid (9c).

Prepared according to the general procedure B, to give the title

compound as a cream solid (55 mg, 99%): mp 63-65 °C; δ_H

(DMSO-d₆, 400 MHz) 8.01 (d, J = 1.2, 1 H, Ar), 7.86 (d, J = 8.0,

1 H, Ar), 7.80 (dd, J = 8.4, 1.2, 1 H, Ar), 7.10 (d, J = 8.4, 2 H,

Ar), 6.93 (d, J = 8.4, 2 H, Ar), 5.21 (d, J = 16.0, 1 H, CH_aH_bAr),

4.98 (d, J = 16.0, 1 H, CH_aH_bAr), 3.65 (t, J = 7.2, 1 H, CHCO),

2.45 (s, 3H, CH₃CN), 2.44-2.37 (m, 1 H, Cy), 2.01-1.62 (m, 8 H,

CHMe₂, CH₂iPr, Cy), 1.38-1.11 (m, 5 H, Cy), 0.87 (d, J = 6.4, 3

H, CH₃), 0.74 (d, J = 6.4, 3 H, CH₃); δ_C(DMSO-d₆, 100 MHz)

168.2, 166.1, 146.4, 140.7, 134.3, 134.1, 133.1, 128.6, 126.7,

126.5, 125.7, 123.7, 59.6, 50.0, 43.3, 33.9, 33.8, 26.3, 25.5, 24.4,

23.9, 23.0, 21.8; LRMS: m/z C₂₈H₃₄N₂O₃requires: 446.3, found:

447.3 [M+H]; t_R= 4.72 (90.2%, V).

(R)-tert-Butyl 1-(4-cyclohexylbenzyl)-3-isobutyl-5-methyl-2-oxo-

(S)-tert-Butyl 3-benzyl-1-(4-cyclohexylbenzyl)-5-methyl-2-oxo-

2,3-dihydro-1H-benzo[e][1,4]diazepine-8-carboxylate

(8d).

2,3-dihydro-1H-benzo[e][1,4]diazepine-8-carboxylate

(8f).

Prepared according to General Procedure B, to give the title

compound as a white solid (64 mg, 85%): mp 75-77 °C; δ_H

(DMSO-d₆, 400 MHz) 7.90 (s, 1 H, Ar), 7.79 (d, J = 8.4, 1 H,

Ar), 7.71 (d, J = 8.4, 1 H, Ar), 7.12 (d, J = 8.0, 2 H, Ar), 6.96 (d,

J = 8.0, 2 H, Ar), 5.04 (s, 2 H, CH₂Ar), 3.49 (t, J = 6.8, 1 H,

CHCO), 2.47-2.38 (m, 1 H, Cy), 2.35 (s, 3 H, CH₃CN), 2.00-1.62

(m, 8 H, CHMe₂, CH₂iPr, Cy), 1.47 (s, 9 H, 3 x CH₃), 1.41-1.10

(m, 5 H, Cy), 0.86 (d, J = 6.4, 3 H, CH₃), 0.72 (d, J = 6.4, 3 H,

CH₃); δ_C(DMSO-d₆, 100 MHz) 168.9, 166.8, 163.7, 146.4,

140.5, 134.7, 134.2, 133.5, 127.9, 126.8, 126.5, 124.9, 122.8,

81.4, 60.4, 49.8, 43.3, 33.9, 33.8, 27.6, 26.3, 25.5, 24.8, 24.0,

23.2, 21.9; LRMS: m/z C₃₂H₄₂N₂O₃requires: 502.3, found: 503.3

[M+H]; t_R= 28.86 (96.5%, II).

Prepared according to General Procedure B, to give the title

compound as a white solid (62 mg, 78%): mp 81-83 °C; δ_H

(DMSO-d₆, 400 MHz) 7.85 (s, 1 H, Ar), 7.75 (d, J = 8.4, 1 H,

Ar), 7.67 (d, J = 8.4, 1 H, Ar), 7.25-7.19 (m, 4 H, Ar), 7.16-7.13

(m, 1 H, Ar), 7.08 (d, J = 7.6, 2 H, Ar), 6.90 (d, J = 7.6, 2 H, Ar),

5.03 (s, 2 H, CH₂Ar), 3.74 (t, J = 6.8, 1 H, CHCO), 3.43 (dd, J =

13.2, 6.8, 1 H, CH_aH_bAr), 3.22 (dd, J = 13.2, 6.8, 1 H, CH_aH_bAr),

2.47-2.36 (m, 1 H, Cy), 2.33 (s, 3H, CH₃CN), 1.81-1.61 (m, 5 H,

Cy), 1.45 (s, 9 H, 3 x CH₃), 1.34-1.16 (m, 5 H, Cy); δ_C(DMSO-

d₆, 100 MHz) 168.3, 166.9, 163.6, 146.4, 140.3, 138.9, 134.5,

134.1, 133.5, 129.5, 128.0, 127.9, 126.7, 126.5, 126.0, 124.8,

122.7, 81.4, 64.2, 49.8, 43.3, 37.2, 33.9, 33.8, 27.6, 26.2, 25.5,

24.8. LRMS: m/z C₃₅H₄₀N₂O₃requires: 536.3, found: 537.3

[M+H]; t_R= 27.86 (95.8%, II).

(R)-1-(4-Cyclohexylbenzyl)-3-isobutyl-5-methyl-2-oxo-2,3-

dihydro-1H-benzo[e][1,4]diazepine-8-carboxylic acid (9d).

Prepared according to General Procedure C, to give the title

compound as a yellow solid (55 mg, 99%): mp 87-89 °C; δ_H

(DMSO-d₆, 400 MHz) 8.01 (d, J = 1.2, 1 H, Ar), 7.86 (d, J = 8.0,

1 H, Ar), 7.80 (dd, J = 8.4, 1.2, 1 H, Ar), 7.10 (d, J = 8.4, 2 H,

Ar), 6.93 (d, J = 8.4, 2 H, Ar), 5.21 (d, J = 16.0, 1 H, CH_aH_bAr),

(S)-3-Benzyl-1-(4-cyclohexylbenzyl)-5-methyl-2-oxo-2,3-dihydro-

1H-benzo[e][1,4]diazepine-8-carboxylic acid (9f). Prepared

according to General Procedure C, to give the title compound as

a cream solid (58 mg, 98%): mp 94-96 °C; δ_H(DMSO-d₆, 400

MHz) 7.97 (s, 1 H, Ar), 7.78 (d, J = 8.4, 1 H, Ar), 7.74 (d, J =

8.4, 1 H, Ar), 7.25-7.19 (m, 4 H, Ar), 7.17-7.13 (m, 1 H, Ar),

7.07 (d, J = 8.0, 2 H, Ar), 6.87 (d, J = 8.0, 2 H, Ar), 5.24 (d, J =

16.0, 1 H, CH_aH_bAr), 4.94 (d, J = 16.0, 1 H, CH_aH_bAr), 3.83 (t, J

= 6.4, 1 H, CHCO), 3.42 (dd, J = 14.0, 6.4, 1 H, CH_aH_bAr), 3.24

(dd, J = 14.0, 6.4, 1 H, CH_aH_bAr), 2.44-2.36 (m, 1 H, Cy), 2.37

(s, 3H, CH₃CN), 1.76-1.63 (m, 5 H, Cy), 1.37-1.15 (m, 5 H, Cy);

δ_C(DMSO-d₆, 100 MHz) 168.0, 166.0, 146.4, 140.2, 138.5,

134.2, 133.8, 133.6, 129.4, 128.2, 128.0, 126.7, 126.5,

126.0,125.3, 123.4, 63.6, 49.7, 43.2, 36.6, 33.9, 33.8, 26.2, 25.5,

24.6; LRMS: m/z C₃₁H₃₂N₂O₃requires: 480.2, found: 481.2

[M+H]; t_R= 23.88 (99.0%, II).

(dd, J = 8.4, 1.2, 1 H, Ar), 7.26 (t, J = 7.6, 2 H, Ar), 7.19 (t, J =

7.6, 1 H, Ar), 7.02 (d, J = 7.6, 2 H, Ar), 5.26 (d, J = 16.0, 1 H,

CH_aH_bAr), 5.02 (d, J = 16.0, 1 H, CH_aH_bAr), 3.61 (t, J = 6.8, 1 H,

CHCO), 2.43 (s, 3 H, CH₃CN), 1.99-1.91 (m, 1 H, CHMe₂),

1.88-1.68 (m, 2 H, CH₂iPr), 0.88 (d, J = 6.8, 3 H, CH₃), 0.73 (d, J

= 6.8, 3 H, CH₃); δ_C(DMSO-d₆, 100 MHz) 168.5, 166.1, 140.4,

137.0, 133.7, 128.5, 128.4, 127.1, 126.6, 125.3, 123.4, 59.9, 49.9,

24.6, 24.0, 23.1, 22.2, 21.9; LRMS: m/z C₂₂H₂₄N₂O₃requires:

364.2, found: 365.2 [M+H]; t_R= 4.63 (98.1%, I).

(R)-tert-Butyl 1-(4-(tert-butyl)benzyl)-3-isobutyl-5-methyl-2-oxo-

(R)-tert-Butyl 3-benzyl-1-(4-cyclohexylbenzyl)-5-methyl-2-oxo-

2,3-dihydro-1H-benzo[e][1,4]diazepine-8-carboxylate

(8i).

2,3-dihydro-1H-benzo[e][1,4]diazepine-8-carboxylate

(8g).

Prepared according to General Procedure B, to give the title

compound as a white solid (65 mg, 92%): mp 82-83 °C; δ_H

(DMSO-d₆, 400 MHz) 7.88 (s, 1 H, Ar), 7.80 (d, J = 8.0, 1 H,

Ar), 7.71 (d, J = 8.0, 1 H, Ar), 7.30 (d, J = 8.0, 2 H, Ar), 7.00 (d,

J = 8.0, 2 H, Ar), 5.08 (d, J = 16.0, 1 H, CH_aH_bAr), 4.99 (d, J =

16.0, 1 H, CH_aH_bAr), 3.50 (t, J = 6.8, 1 H, CHCO), 2.36 (s, 3 H,

CH3CN), 1.98-1.89 (m, 1 H, CHMe₂), 1.86-1.68 (m, 2 H,

CH₂iPr), 1.46 (s, 9 H, 3 x CH₃), 1.23 (s, 9 H, 3 x CH₃), 0.86 (d, J

= 6.4, 3 H, CH₃), 0.72 (d, J = 6.4, 3 H, CH₃); δ_C(DMSO-d₆, 100

MHz) 169.0, 166.8, 163.7, 149.4, 140.6, 134.9, 134.1, 133.5,

127.9, 126.2, 125.3, 124.9, 122.7, 81.5, 60.4, 49.8, 34.1, 27.6,

24.9, 24.0, 23.2, 21.9; LRMS: m/z C₃₀H₄₀N₂O₃requires: 476.3,

found: 477.3 [M+H]; t_R= 6.45 (95.6%, V).

Prepared according to General Procedure B, to give the title

compound as a white solid (73 mg, 91%): mp 78-80 °C; δ_H