Design, synthesis and evaluation of novel 2-hydroxypyrrolobenzodiazepine-5, 11-dione analogues as potent angiotensin converting enzyme (ACE) inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.05.031

A series of novel 10-substituted 2-hydroxypyrrolobenzodiazepine-5,11-diones designed through structure based rational hybridization approach, synthesized by the cyclodehydration of isotonic anhydride with (2S,4R)-4-hydroxypyrrolidine- 2-carboxylic acid followed by N-substitution, were evaluated as angiotensin converting enzyme (ACE) inhibitors. Among all the new compounds screened (2R,11aS)-10-((4-bromothiophen-2-yl)methyl)-2-hydroxy-2,3-dihydro-1H-benzo[e] pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)dione, 5v (IC₅₀: 0.272 μM) emerged as most active non-carboxylic acid ACE inhibitor with minimal toxicity comparable to clinical drugs Lisinopril, Benazepril and Ramipril. Favorable binding characteristics in docking studies also supported the experimental results.

Full text of DOI:10.1016/j.bmc.2013.05.031

Bioorganic & Medicinal Chemistry 21 (2013) 4485–4493
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of novel
2-hydroxypyrrolobenzodiazepine-5,11-dione analogues as potent
angiotensin converting enzyme (ACE) inhibitors
Dinesh Addla a, Anvesh Jallapally a,b, Abhinav Kanwal b, Balasubramanian Sridhar c,
Sanjay K. Banerjee b, , Srinivas Kantevari a,
⇑
⇑
a CPC Division (Organic Chemistry Division-II), CSIR-Indian Institute of Chemical Technology, Hyderabad 500607, India
b Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500607, India
c Laboratory of X-ray Crystallography, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 10-substituted 2-hydroxypyrrolobenzodiazepine-5,11-diones designed through struc-
ture based rational hybridization approach, synthesized by the cyclodehydration of isotonic anhydride
with (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid followed by N-substitution, were evaluated as
angiotensin converting enzyme (ACE) inhibitors. Among all the new compounds screened (2R,11aS)-
10-((4-bromothiophen-2-yl)methyl)-2-hydroxy-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11
Received 23 April 2013
Revised 20 May 2013
Accepted 21 May 2013
Available online 31 May 2013
(10H,11aH)dione, 5v (IC50: 0.272 lM) emerged as most active non-carboxylic acid ACE inhibitor with
Keywords:
minimal toxicity comparable to clinical drugs Lisinopril, Benazepril and Ramipril. Favorable binding char-
acteristics in docking studies also supported the experimental results.
Ó 2013 Elsevier Ltd. All rights reserved.
ACE Inhibitors
Benzodiazepines
Hypertension
Molecular hybridization
Proline
1. Introduction
hyperkalaemia, dry cough, skin rashes and are not tolerated in pa-
tients with long-term treatment.9 We are now in a unique position
Despite of considerable therapeutic advances, cardiovascular
disease (CVD) remained responsible for ꢀ27% deaths worldwide,
with 80% occurring in developing countries.1,2 Recent statistics re-
vealed that seven out of ten are suffering from multi-factorial CVD
and the numbers are likely to go even higher in coming years.2
Hypertension (a common quantitative trait under polygenic vascu-
lar disorder) is one of the most important treatable factors of CVD.3
Angiotensin-I converting enzyme (ACE) is a key regulator of blood
pressure, due to its critical role in renin–angiotensin–aldosterone
system (RAAS)4 the inhibitors of ACE have emerged as first-line
therapy for hypertension in younger patients and second line in
geriatric patients.5,6 Driven by clinical evidence which demon-
strates the morbidity and mortality benefits, ACE has become an
important target for drug development in treating hypertension.
Discovery of Captopril,7 the first orally active ACE inhibitor,
encouraged researchers’ worldwide to design and develop ACE
inhibitors(using the structure of carboxy peptidase) and were
clinically tested.8 However, these synthetic drugs (Fig. 1) are
reported plethora of side effects to say few, renal impairment,
to capitalize proven information (the real target molecule) for the
design of new and more effective next generation ACE inhibitors10
with the potential for greater efficacy in controlling hypertension.
The natural products generally derived from plants or microbes are
essential source for new chemical entities.11
They offer promising and amazing chemical diversity, there by
inspiring the development of structurally diverse new molecules
to play a vital role in drug discovery.12
Among the known naturally occurring alkaloids, pyrrolo[2,1-
c][1,4]benzodiazepines (PBDs)13 isolated from various Streptomy-
ces species (a class of biologically active compounds with good
pharmacological profile). These natural and synthetic PBDs
(Fig. 2) exert anticancer activity through their interaction towards
protein sequences to interfere and are currently in clinical develop-
ment.14 On the other hand, clinical ACE drugs (Fig. 1) found to pos-
sess proline or modified proline type architecture as a critical
pharmacophore responsible for exhibiting ACE inhibitor activ-
ity.15–17 With growing interest in use of pyrrolo[2,1-c][1,4]benzo-
diazepine (PBD) ring system as
a potential pharmacophoric
fragment.16
We envisaged that manoeuvring PBD with structural features of
ACE inhibitors in one molecular frame could deliver a new scaffold
for biological evaluation. Herein, we present an efficient synthesis
⇑
Corresponding authors. Tel.: +91 4027191438.
E-mail addresses: skbanerjee@iict.res.in (S.K. Banerjee), kantevari@yahoo.com
(S. Kantevari).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.031

4486
D. Addla et al. / Bioorg. Med. Chem. 21 (2013) 4485–4493
(A) Proline
H3C
H3C
O
N
SH
N
COOH
N
H
HOOC
Enalapril
O
HOOC
Captopril
H2N
-O
P
O
N
N
N
H
COOH
HOOC
O
HOOC
O
Lisinopril
Fosinopril
(B) Modified proline as scaffold
HO
CH3
H
H
CH3
S
N
CH3
H
N
COOEt
N
H
SH
H
N
COOEt
N
H
O
HOOC
O
HOOC
O
HOOC
Perindopril
Rentiapril
Tandolapril
O
S
N
N
HOOC
O
O
CH3
H
H
HOOC
HN
O
N
N
S
O
COOEt
O
H
O
Ramipril
HOOC
Imidapril
Zofenopril
Figure 1. ACE inhibitor drugs having (A) proline; (B) modified proline as scaffold.
Benazepril and Ramipril. In vitro cytotoxicity evaluations and
molecular docking studies of lead compounds are also described.
A more holistic approach for design strategy would be through,
(i) correlation of the structural features of widely used drugs
(Fig. 1); (ii) identification of common pharmacophoric unit(s) and
(iii) incorporate them to generate newer scaffolds with potential
activity profile.17 With the fact that, proline is a key pharmaco-
phoric fragment in drugs such as Captopril, Enalapril and Lisinopril,
we18 vision to use this information in designing a newer scaffold. A
more recent ACE inhibitor, Benazepril with fused seven member
cyclic ring has structural similarity to basic benzodiazepine archi-
tecture. This rekindled our curiousness to inculcate a design strat-
egy15 integrating proline fragment in benzodiazepine architecture
with appended liphophilic functionality. The explicit design of new
molecules for the present study is depicted in Figure 3.
N
R6
R5
H
N
O
H
H
CH3
HO
N
R
H3C
N
O
O
OH
Tomamycin (R5 = OCH3; R6= OH;R=CH3)
Prothracarcin (R5 = R6= H;R= CH3)
O
Chicamycin A
H3C
O
H
N
H
N
OH
H
HO
HO
O
H
H3C
N
O
O
N
CH3
R'
O
R
CH3
O
HO
Neothramycin A(R' = H; R= OH)
Neothramycin B (R' = OH; R = H)
DC-81(R'=R=H)
CH3
HO
Sibiromycin
NH
CH3
2. Results and discussion
2.1. Synthesis
Figure 2. Natural pyrrolo[2,1-c][1,4]benzodiazepines.
and bio-evaluation of pyrrolo[2,1-c][1,4]benzodiazepine derived
proline embodied hybrid heterocycles, designed through molecu-
lar hybridization approach. The inclusion of proline unit is due to
the architectural similarity with pyrrolo portion in benzodiazepine
(Fig. 3). All the new hybrid heterocycles 3 and 5a–w were evalu-
ated for in vitro ACE inhibitor activity using recently developed
high-throughput screening method.18 The ACE inhibition of new
analogues was correlated with known reference drugs Lisinopril,
Synthetic approach for the preparation of hydroxy pyrrolo[2,1-
c][1,4]benzodiazepine-5,11-dione 5a–w was presented in Scheme
1.16 Commercially available isotonic anhydride 1 was taken as
starting material. Cyclodehydration of 1 with readily available
(2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid 2 in DMSO re-
sulted (2R,11aS)-2-hydroxy-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-
a][1,4]diazepine-5,11(10H,11aH)dione 3 in excellent yield. This

D. Addla et al. / Bioorg. Med. Chem. 21 (2013) 4485–4493
4487
OEt
O
NH
N
O
O
OH
H2N
OH
N
O
Benazepril
N
N
N
H
COOH
O
HOOC
O
R
N
H
Lisinopril
New Analogues
N
O
OH
Pyrrolobenzodiazepine
Figure 3. Structure based rational design strategy.
O
N
O
O
O
OH
OH
R
Br
OH
N
DMSO
120 o
4a-w
O
HO
NH
N
C
K2CO3, DMF
o C - RT
N
H
O
N
H
O
O
0
2
3
R
5
1
O
R =
O
H
H3C
5c
O
X
5d
(3.5h, 87%)
5a
(4h, 80%) 5b
(4h, 79%)
(4.5h, 81%)
Y
F
5e:
X= H, Y= Cl (4.5h, 81%)
5f: X= H, Y= Br (4h,87%)
5g:
F
F
F
F
X= Br,Y= H (3.5h, 86%)
5h: X= H, Y= OMe (4h,90%)
MeO
OMe
Cl
F
5i:
5j:
F
X= OMe, Y= H (4h, 89%)
X= NO2, Y=H (4h, 90%)
OMe
F
5p (4.5h, 86%)
F
5q (4h, 86%)
5r (4.5h, 85%)
5s (4.5h, 84%)
5k: X= NH2, Y=H (4h, 92%)
5l:
X= COOMe, Y= H (5h, 89%)
O
O
Br
5m: X= COOH, Y=H (5h, 91%)
5n:
5o:
X= Cl, Y= F (4h, 89%)
X= NO2, Y=Cl (6h, 93%)
O
S
Br
Cl
5w (5h, 85%)
5v (4.5h, 89%)
5t
(5h, 87%)
5u (4h, 92%)
Scheme 1. Synthesis of new 10-substituted hydroxypyrrolo[2,1-c][1,4] benzodiazepine-5,11-dione analogues 5a–w.
hydroxy pyrrolobenzodiazepine diones frame unit 3, was then
alkylated with series of alkyl or benzyl bromides 4b–w (methyl
iodide in case of 4a), in the presence of K2CO3 in DMF to give 10-
substituted (2R,11aS)-2-hydroxy-2,3-dihydro-1H-benzo[e]pyrrol-
o[1,2-a][1,4]diazepine-5,11(10H,11aH)diones 5a–w in excellent
yields (81–93%). All of the compounds 5a–w were purified through
silica gel column chromatography (HPLC purity >96%) and were
fully characterized by IR, 1H NMR, 13C NMR, electron spray ioniza-
tion (ESI), and high-resolution mass spectral (HRMS) analysis (Sup-
plementary data). Further single crystal X-ray diffraction analysis
of 5g unambiguously confirmed the structure and stereochemistry
(Fig. 4).19
2.2. Pharmacology
In vitro angiotensin converting enzyme (ACE) inhibition of ‘24’
new analogues 3 and 5a–w were examined using recently devel-
oped high-throughput colorimetric screening method.20 Most of
these anti-hypertensive peptides have been characterized by the
rabbit lung ACE inhibitor assay, based on the hydrolysis of the syn-
thetic peptide hippuryl–histidyl–leucine (HHL). Angiotensin con-
verting enzyme (ACE) hydrolyses HHL to hippuric acid (HA) and
Figure 4. ORTEP representation of 5g with thermal displacement ellipsoids drawn
at the 30% probability.
histidyl–leucine (HL). HA released is directly proportional to the
ACE activity. In this screening method, the released hippuric acid

4488
D. Addla et al. / Bioorg. Med. Chem. 21 (2013) 4485–4493
Figure 5. In vitro angiotensin converting enzyme (ACE) inhibition of new analogues 3, 5a–w and standard drugs.
Table 1
MTT assay inhibitory activities (IC50) in
lM
Compound
HEK 293a
A549b
Lisinopril
Ramipril
Benazepril
5f
5g
5r
5v
443
414
301
398
496
331
281
307
392
358
400.4
486.7
418.7
361
a
HEK 293: Human Embryonic Kidney 293.
A549: Human alveolar basal epithelial cells.
b
Figure 6. Dose–response curves of synthetic hydroxy pyrrolobenzodiazepine dione
derivatives 5f, 5g, 5r, 5v and standard drugs.
IC50 values of all the three reference drugs, Lisinopril (0.262
Ramipril (0.253 M) and Benazepril (0.212 M).
lM),
l
l
from the substrate hippuryl–histidyl–leucine (HHL) was trans-
formed into yellow colour by mixing with pyridine and benzene
sulfonyl chloride. The resulted yellow colour was determined col-
orimetrically at absorbance 410 nm. The average ACE inhibitor
activity was measured in triplicate for new analogues 5a–w and
the standard drugs Lisinopril, Benazepril and Ramipril (Fig. 5).
The toxicity evaluation of four most potent ACE inhibitors 5f–g,
5r and 5v was assessed by (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay21 against HEK 293 (nor-
mal human embryonic kidney) and A549 (adeno carcinomic hu-
man alveolar basal epithelial) cell lines. The inhibitory activities
(IC50) in
lM are summarized in Table 1. The compounds 5f–g, 5r
The experiment carried out at 1.0 lM concentration of test com-
and 5v evaluated, exhibit higher inhibitory concentration (IC50
)
pounds 3 and 5a–w revealed 28–91% of ACE inhibition activity.
Among all the PBD derived analogues, four compounds 5f–g, 5r
and 5v posses better ACE inhibitory activity (>75%) compared with
the other substituted analogues. Among all the substituted PBDs,
the trimethoxy derivative 5r revealed highest ACE inhibition and
5l has least ACE inhibitory activity. Relating to the reference drugs
Lisinopril, Benazepril and Ramipril these four analogues 5f–g, 5r
and 5v were comparably active with 86%, 75%, 91% and 80% ACE
inhibition, respectively. Pyrrolobenzodizepine-5,11-dione 3, show-
ing 57% ACE inhibition, suggested the importance of PBD as a phar-
macophoric fragment in the development of new ACE inhibitors.
Also to note that, these three active analogues, 5f–g and 5v has bro-
mo functionality on phenyl and thiophenyl rings. In case of 5e, 5p–
q and 5s–t having fluoro/chloro groups was resulted in lower ACE
inhibition compared to the respective benzyl analogue 5d. Further,
the inhibitory concentration at 50% (IC50) ACE activity for 5f–g, 5r
and 5v along with reference drugs was calculated from dose–re-
sponse curves (Fig. 6) obtained by plotting the percentage inhibi-
tion verses the concentration.
at a magnitude of ꢀ1000 times more than the concentration
(IC50) of ACE inhibition activity and are considered nontoxic. Under
similar conditions, toxicity of new analogues 5f–g, 5r and 5v were
comparable with the evaluated reference drugs Lisinopril, Bena-
zepril and Ramipril.
2.3. Molecular modelling
To gain insight into the ACE-ligand binding mode, the most po-
tent analogues 5f–g, 5r and 5v were docked on to tACE (PDB code:
1O86) using Molegro Virtual Docker (MVD) software.22 The inter-
molecular interactions between tACE binding site residues and
predicted poses for lead analogue 5v are shown in Figure 7. Since
the first report from Acharya and co-workers on crystal structure
of Lisinopril bound–ACE complex,17,23 a greater understanding of
binding site was emerged for the development of new ACE inhibi-
tors. To validate the Molegro Virtual Docker (MVD) software, ini-
tially Lisinopril was docked and the results obtained were
correlated with reported molecular interactions.19 Similar to Lisin-
opril, the new potent analogue 5v also share interactions at S1, S2,
S10 and S20 binding subsites at the active site of enzyme. Lisinopril
and potent 5v ligand as the same interactions at Ala 356, His 513
Evaluation of IC50 data for 5f (0.418
(0.376 M) and 5v (0.272 M), revealed 5v as the best lead com-
pound with lowest IC50 (0.272 M). This result also correlate with
lM), 5g (0.658 lM), 5r
l
l
l

D. Addla et al. / Bioorg. Med. Chem. 21 (2013) 4485–4493
4489
neat liquids or KBr pellets and absorptions are reported in cmꢁ1
.
NMR spectra were recorded on 300 (Bruker) and 500 MHz (Varian)
spectrometers in appropriate solvents using TMS as internal stan-
dard or the solvent signals as secondary standards and the chemi-
cal shifts are shown in d scales. Coupling constants J are expressed
in Hertz. 13C NMR spectra were recorded on 75 and 125 MHz spec-
trometers. High-resolution mass spectra were obtained by using
ESI-QTOF mass spectrometry. Reagents and all solvents were ana-
lytically pure and were used without further purification. All the
experiments were monitored by analytical thin layer chromatogra-
phy (TLC) performed on silica gel GF254 pre-coated plates. After
elution, plate was visualized under UV illumination at 254 nm
for UV active materials. Further visualization was achieved by
staining with PMA and charring on a hot plate. Silica gel finer than
200 mesh was used for column chromatography. Appropriate
names for all the new compounds were given with the help of
ChemBioOffice 2010.
4.1. General procedure for preparation of (2R,11aS)-2-hydroxy-
2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4] diazepine-5,11
(10H,11aH)-dione (3)
Figure 7. Best predicted binding mode of ligand 5v at the tACE binding site.
and His 353. Hence, the subsite S20 plays a major role in tACE
binding.
A
mixture of 1H-benzo[d][1,3]oxazine-2,4-dione 1 (5.0 g,
30.6 mmol) and (2S,4R)-4-hydroxy pyrrolidine-2-carboxylic acid
2 (4.42 g, 33.7 mmol) in DMSO was refluxed for 4 h. After comple-
tion (by TLC), the reaction mixture was cooled at room tempera-
ture, water (50 mL) was added, solid precipitate thus formed was
filtered, washed with water and dried to give the pure (2R,11aS)-
2-hydroxy-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-
Docking results suggest that the hydroxyl group on pyrrolo res-
idue of 5v is positioned by strong hydrogen bonds from His513
(2.74 Å), Tyr523 (2.35 Å) and His353(2.69 Å) in S1 and S20 subsites.
The central carbonyl group of phenyl ring also interacts with
Ala356 (2.99 Å). These interactions in 5v bound ACE complex were
in close relation with reported ACE-drug complexes of Lisinopril
and Captopril.17,23,25 Also to observe that Arg522 (3.43 Å) in S2
subsite make interaction with ‘N’ attached to phenyl group in ben-
zodiazepine architecture. The tilted bromo thiophene residue
makes additional electrostatic interaction with residues of
Glu411 (3.33 Å). Such interaction of new molecules at the subsites
depends on the nature of substituent attached and their staking in
hydrophobic cavity between Phe512 and Val518 residues in S1 and
S1/S2 subsites of ligand bound ACE complex. Such staking of aryl or
thiophene substituent was well corroborating with the binding site
information reported for ACE bound other drugs, Enalapril and Fos-
inopril.24,25 At this point of investigation, it was remarkable to note
that almost all the functional groups of 5v were occupied with
either hydrogen bonding or other non-covalent lipophilic interac-
tions and were important in exerting greater affinity towards tACE
enzyme and then ACE inhibition activity.
5,11(10H,11aH)-dione
3
as brown solid. Mp: 223–225 °C
½
a 2D5
ꢂ
þ 448 (c 1.0, CH3OH). 1H NMR (300 MHz, DMSO-d6) d 10.29
(s, 1H), 7.89 (d, J = 7.74 Hz, 1H), 7.45 (t, J = 7.54 Hz, 1H), 7.21 (t,
J = 7.17 Hz, 1H), 7.13 (d, J = 7.93 Hz, 1H), 5.09 (s, 1H), 4.49 (br s,
1H), 4.21 (t, J = 6.23 Hz, 1H), 3.82–3.86 (m, 1H), 3.59 (dd,
J = 4.34 Hz, J = 7.93 Hz, 1H), 2.78–2.86 (m, 1H), 2.04–2.17 (m, 1H).
HR-MS (ESI) Calcd for C12H12N2O3Na [M+H]+: 255.07401. Found
255.07408.
4.2. General procedure for preparation of N-substituted
pyrrolobenzodiazepine derivatives 5a–w
(2R,11aS)-2-Hydroxy-2,3-dihydro-1H-benzo[e]pyrrole[1,2-
a][1,4]diazepine-5,11(10H,11aH)-dione 3 (0.4 g, 1.71 mmol) and
K2CO3 (0.473 g, 3.43 mmol) in DMF at 0 °C for 30 min was added
substituted alkyl bromides 4a–w (2.06 mmol) to the reaction and
stirred for 4 h under inert conditions. After completion (by TLC),
the reaction mixture was filtered through Buchner funnel and
washed with EtOAc. The product was extracted with EtOAc
(3 ꢃ 30 mL) and cold water (20 mL). Combined organic phase
was dried over Na2SO4 and evaporated under vacuum. The
crude residue thus obtained was further purified by silica gel col-
umn chromatography eluted with mixture of ethyl acetate and
hexane.
3. Conclusion
In this present study, we report a new series of 10-substituted
2-hydroxy-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaze pine-
5,11(10H,11aH) diones as angiotensin converting enzyme inhibi-
tors. Among them (2R,11aS)-10-((4-bromothiophen-2-yl)methyl)-
2-hydroxy-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-
5,11(10H,11aH)dione, 5v (IC50: 0.272 lM) emerged as most active
non-carboxylic acid ACE inhibitor with minimal toxicity compara-
ble to clinical drugs Lisinopril, Benazepril and Ramipril. The struc-
ture activity correlation of lead analogue 5v was also made using
molecular docking studies. Docking study revealed that bulky
and hydrophobic group substituents affected in vitro activities.
The results presented here can lead to generate potential new
PBD based non-carboxylic acid ACE inhibitors.
4.2.1. (2R,11aS)-2-Hydroxy-10-methyl-2,3-dihydro-1H-
benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione 5a
Brown syrup. ½a 2D5
ꢂ
þ 186:3 (c 1.0, CH3OH). 1H NMR (300 MHz,
CD3OD)
d 7.82 (dd, J = 1.51 Hz, J = 6.23 Hz, 1H), 7.62 (t,
J = 6.98 Hz, 1H), 7.43 (d, J = 8.12 Hz, 1H), 7.35 (t, J = 7.93 Hz, 1H),
4.48–4.54 (m, 1H), 4.25 (dd, J = 5.66 Hz, J = 2.26 Hz, 1H), 3.57–
3.72 (m, 2H), 3.39 (s, 3H), 2.81–2.90 (m, 2H), 2.00–2.09 (m, 1H).
13C NMR (75 MHz, CD3OD) d 171.1, 167.9, 142.1, 133.7, 130.8,
129.8, 126.8, 123.5, 69.7, 57.4, 54.7, 36.5, 35.8. IR (Neat) 3428,
2923, 2853, 1675, 1629, 1467, 1423, 1384,769 cmꢁ1. HR-MS (ESI)
Calcd for C13H15N2O3 [M+H]+: 247.10772. Found 247.10773.
4. Experimental section
Melting points were measured with a Fischer-Johns melting
point apparatus and are uncorrected. IR spectra were recorded as

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D. Addla et al. / Bioorg. Med. Chem. 21 (2013) 4485–4493
4.2.2. (2R,11aS)-2-Hydroxy-10-(2-oxopropyl)-2,3-dihydro-1H-
benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione 5b
(d, J = 7.24 Hz, 1H), 5.04–5.15 (m, 3H), 4.48–4.50 (m, 1H), 4.34 (t,
J = 6.20 Hz, 1H), 3.70 (dd, J = 3.10 Hz, J = 9.31 Hz, 1H), 3.58 (dd,
J = 4.13 Hz, J = 7.24 Hz, 1H), 2.82–2.87 (m, 1H), 2.04–2.09 (m, 1H).
13C NMR (75 MHz, CDCl3 + DMSO-d6) d 167.5, 163.5, 138.2, 137.6,
130.6, 128.8, 128.6, 128.5, 128.3, 128.1, 124.4, 123.8, 120.9, 66.6,
54.3, 52.2, 49.6, 33.5. IR (KBr) 3418, 2962, 1677, 1602, 1471,
1384, 1218, 1149, 1086, 950, 867, 764, 669 cmꢁ1. HR-MS (ESI)
Calcd for C19H18N2O3Br [M+H]+: 401.0500. Found 401.0497.
Light yellow syrup. ½a D25
ꢂ
þ 267:3 (c 1.0, CH3OH). 1H NMR
(300 MHz, CDCl3) d 7.87 (d, J = 3.30 Hz, 1H), 7.47 (t, J = 8.30 Hz,
1H), 7.29 (t, J = 7.55 Hz, 1H), 7.03 (d, J = 8.30 Hz, 1H), 4.86 (d,
J = 18.1 Hz, 1H), 4.59–4.63 (m, 1H), 4.30–4.34 (m, 1H), 4.19 (d,
J = 18.1 Hz, 1H), 3.84–3.89 (m, 1H), 3.61–3.67 (m, 1H), 2.83–2.95
(m, 2H), 2.28 (s, 3H), 1.97–2.16 (m, 1H). 13C NMR (75 MHz, CDCl3)
d 202.0, 168.9, 165.8,132.4, 130.3, 129.0,126.2, 121.8, 69.0,59.4,
55.7, 54.0, 34.9, 29.6. IR (KBr) 3414, 2923, 2854, 1728, 1629,
1464, 1417, 1214, 766 cmꢁ1. HR-MS (ESI) Calcd for C15H17N2O4
[M+H]+: 289.1188. Found 289.1193.
4.2.7. (2R,11aS)-10-(4-Bromobenzyl)-2-hydroxy-2,3-dihydro-
1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione
5g
Mp: 150–152 °C
½
a 2D5
ꢂ
þ 556:0 (c 1.0, CH3OH). 1H NMR
4.2.3. Ethyl 2-((2R,11aS)-2-hydroxy-5,11-dioxo-2,3,11,11a-tetra
hydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-10-(5H)-yl)
acetate 5c
(300 MHz, DMSO-d6) d 7.85 (d, J = 7.74 Hz, 1H), 7.46 (t,
J = 8.12 Hz, 1H), 7.39 (d, J = 8.30 Hz, 2H), 7.27 (q, J = 7.74 Hz,
J = 10.1 Hz, 2H), 7.03 (d, J = 8.30 Hz, 2H), 4.99–5.07 (m, 2H), 4.55
(br s, 1H), 4.33 (t, J = 7.55 Hz, 1H), 3.81 (d, J = 12.46 Hz, 1H), 3.60
(dd, J = 4.72 Hz, J = 7.55 Hz, 1H), 2.82–2.96 (m, 2H), 1.99–2.16 (m,
1H). 13C NMR (75 MHz, CDCl3 + DMSO-d6) d 168.4, 164.7, 138.6,
135.6, 131.5, 131.0, 129.5, 129.3, 128.1, 125.4, 121.8, 120.2, 67.7,
55.4, 53.3, 50.7, 34.5. IR(KBr) 3399, 3007, 2939, 1678, 1632,
1572, 1464, 1391, 1214, 986, 872, 757 cmꢁ1. HR-MS (ESI) Calcd
for C19H18N2O3Br [M+H]+: 401.04953. Found 401.04961.
Mp: 195–197 °C
(500 MHz, DMSO-d6)
½
a 2D5
ꢂ
þ 360:0 (c 1.0, CH3OH). 1H NMR
d 7.86 (d, J = 7.99 Hz, 1H), 7.55 (t,
J = 6.99 Hz, 1H), 7.34 (t, J = 6.99 Hz, 1H), 7.26 (d, J = 7.99 Hz, 1H),
4.57 (d, J = 17.9 Hz, 1H), 4.49 (br s, 1H), 4.41 (d, J = 17.9 Hz, 1H),
4.29 (t, J = 6.99 Hz, 1H), 4.19 (q, J = 7.99 Hz, J = 7.99 Hz, 2H), 3.74
(d, J = 12.9 Hz, 1H), 3.57 (dd, J = 4.99 Hz, J = 6.99 Hz, 1H), 2.80–
2.84 (m, 2H), 2.00–2.09 (m, 1H), 1.26 (t, J = 7.99 Hz, 3H). 13C NMR
(75 MHz, CDCl3 + DMSO-d6) d 167.9, 167.3, 164.0, 138.1, 131.0,
128.8, 128.3, 124.8, 121.0, 66.9, 60.1, 54.4, 52.6, 49.9, 33.7, 12.8.
IR (KBr): 3349, 2990, 1752, 1681, 1571, 1466, 1375, 1202, 1080,
4.2.8. (2R,11aS)-2-hydroxy-10-(3-methoxybenzyl)-2,3-dihydro-
1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione
761, 590 cmꢁ1
.
HR-MS (ESI) Calcd for C16H19N2O5 [M+H]+:
5h
319.12885. Found 319.12873.
Mp: 102–105 °C
½
a 2D5
ꢂ
þ 355:3 (c 1.0, CH3OH). 1H NMR
(300 MHz, CDCl3) d 7.85 (d, J = 6.23 Hz, 1H), 7.41 (t, J = 6.61 Hz,
1H), 7.17–7.28 (m, 3H), 6.67–6.77 (m, 3H), 5.14 (d, J = 15.8 Hz,
1H), 4.98 (d, J = 15.8 Hz, 1H), 4.63–4.68 (m, 1H), 4.35 (t,
J = 6.04 Hz, 1H), 3.90 (d, J = 11.3 Hz, 1H), 3.74 (s, 3H), 3.67 (dd,
J = 4.53 Hz, J = 8.12 Hz, 1H), 2.95–3.05 (m, 2H), 2.11–2.19 (m, 1H).
13C NMR (75 MHz, CDCl3) d 168.9, 165.7, 159.8, 139.6, 138.3,
132.1, 130.3, 129.7, 126.1, 122.2, 118.9, 112.7, 112.2, 69.2, 56.0,
55.1, 53.9, 52.5, 35.1. IR 3401, 2929, 1675, 1463, 1385, 1260,
1219, 1158, 1049, 771 cmꢁ1. HR-MS (ESI) Calcd for C20H21N2O4
[M+H]+: 353.14958. Found 353.14977.
4.2.4. (2R,11aS)-10-Benzyl-2-hydroxy-2,3-dihydro-1H-
benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione 5d
Mp: 180–182 °C
½
a 2D5
ꢂ
þ 324:3 (c 1.0, CH3OH). 1H NMR
(300 MHz, CDCl3) d 7.86 (d, J = 6.42 Hz, 1H), 7.42 (t, J = 6.42 Hz,
1H), 7.13–7.31 (m, 7H), 5.17 (d, J = 15.67 Hz, 1H), 5.00 (d,
J = 15.67 Hz, 1H), 4.65–4.68 (m, 1H), 4.36 (t, J = 6.23 Hz, 1H),
3.89–3.95 (m, 1H), 3.67 (dd, J = 4.53 Hz, J = 4.53 Hz, 1H), 2.96–
3.06 (m, 2H), 2.12–2.20 (m, 1H). 13C NMR (75 MHz,
CDCl3 + DMSO-d6)
d 168.0, 164.4, 138.4, 135.9, 131.0, 129.1,
128.8, 127.6, 126.2, 125.6, 124.8, 121.3, 67.4, 55.0, 52.9, 50.9,
34.1. IR (KBr) 3401, 2925, 1676, 1630, 1464, 1385, 1219, 1092,
771 cmꢁ1. HR-MS (ESI) Calcd for C19H19N2O3 [M+H]+: 323.1395.
Found 323.1398.
4.2.9. (2R,11aS)-2-hydroxy-10-(4-methoxybenzyl)-2,3-dihydro-
1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione
5i
Mp: 124–126 °C
½
a 2D5
ꢂ
þ 433:3 (c 1.0, CH3OH). 1H NMR
4.2.5. (2R,11aS)-10-(3-Chlorobenzyl)-2-hydroxy-2,3-dihydro-
1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione
(500 MHz, CDCl3) d 7.78 (br s, 1H), 7.34 (t, J = 6.99 Hz, 1H), 7.15–
7.18 (m, 2H), 6.98 (d, J = 7.99 Hz, 2H), 6.72 (d, J = 8.99 Hz, 2H),
4.95 (s, 2H), 4.58 (br s, 1H), 4.24 (t, J = 6.99 Hz, 1H), 3.80 (d,
J = 12.9 Hz, 1H), 3.67 (s, 3H), 3.60 (d, J = 12.9 Hz, 1H), 2.87–2.96
(m, 2H), 2.03–2.08 (m, 1H). 13C NMR (75 MHz, CDCl3) d 168.8,
165.8, 158.8, 139.6, 132.1, 130.3, 129.7, 128.7, 128.2, 126.1,
122.4, 114.1, 69.3, 56.1, 55.1, 53.9, 52.0, 35.0. IR (KBr) 3402,
2933, 1676, 1572, 1464, 1295, 1246, 1092, 956, 769 cmꢁ1. HR-MS
(ESI) Calcd for C20H21N2O4 [M+H]+: 353.1501. Found 353.1517.
5e
Mp: 110 °C ½a 2D5
ꢂ
þ 387:8 (c 1.0, CH3OH). 1H NMR (300 MHz,
CDCl3 + DMSO-d6) d 7.86 (d, J = 7.74 Hz, 1H), 7.47 (t, J = 7.17 Hz,
1H), 7.16–7.32 (m, 5H), 7.03 (d, J = 6.98 Hz, 1H), 5.12 (d,
J = 15.8 Hz, 1H), 5.04 (d, J = 15.8 Hz, 1H), 4.55 (br s, 1H), 4.35 (t,
J = 6.04 Hz, 1H), 3.79–3.83 (m, 1H), 3.62 (dd, J = 4.91 Hz
J = 7.55 Hz, 1H), 2.87–2.96 (m, 2H), 2.08–2.16 (m, 1H). 13C NMR
(75 MHz, CD3OD) d 170.7, 167.8, 140.6, 140.6, 135.5, 133.8,
131.2, 131.1, 130.9, 128.5, 128.3, 127.4, 126.4, 124.3, 69.3, 57.6,
54.7, 52.0, 35.8. IR (KBr) 3418, 2930, 2096, 1644, 1601, 1463,
1384, 1218, 1164, 1078, 959, 856, 771 cmꢁ1. HR-MS Calcd for
4.2.10. (2R,11aS)-2-Hydroxy-10-(4-nitrobenzyl)-2,3-dihydro-
1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione
5j
C
19H18N2ClO3 [M+H]+: 357.1005. Found 357.1017.
Mp: 80–82 °C ½a 2D5
ꢂ
þ 425:3 (c 1.0, CH3OH). 1H NMR (300 MHz,
CDCl3 + DMSO-d6) d 8.17 (d, J = 9.06 Hz, 2H), 7.85 (d, J = 7.55 Hz,
1H), 7.46 (t, J = 6.79 Hz, 1H), 7.27–7.33 (m, 3H), 7.16 (d,
J = 8.30 Hz, 1H), 5.18 (s, 2H), 4.63–4.68 (m, 1H), 4.39 (t,
J = 7.55 Hz, 1H), 3.66 (dd, J = 4.53 Hz, J = 7.55 Hz, 1H), 3.90–3.95
(m, 1H), 2.93–3.01 (m, 2H), 2.14–2.22 (m, 1H). 13C NMR (75 MHz,
CDCl3) d 169.0, 165.6, 147.2, 144.1, 139.1, 132.4, 130.6, 129.5,
127.5, 126.5, 124.0, 121.9, 69.0, 56.0, 54.1, 52.1, 34.9. IR (KBr)
4.2.6. (2R,11aS)-10-(3-Bromobenzyl)-2-hydroxy-2,3-dihydro-
1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione
5f
Mp: 171–173 °C
½
a 2D5
ꢂ
þ 268:0 (c 1.0, CH3OH). 1H NMR
(500 MHz, DMSO-d6)
d 7.80 (d, J = 8.27 Hz, 1H), 7.49 (t,
J = 8.27 Hz, 1H), 7.28–7.35 (m, 4H), 7.17 (t, J = 8.27 Hz, 1H), 7.07

D. Addla et al. / Bioorg. Med. Chem. 21 (2013) 4485–4493
4491
3401, 1679, 1632, 1519, 1463, 1385, 1346, 1219, 1092, 771 cmꢁ1
.
HR-MS (ESI) Calcd for C19H18N3O5 [M+H]+: 368.1246. Found
368.1258.
4.2.15. (2R,11aS)-10-(3-Chloro-4-nitrobenzyl)-2-hydroxy-2,3-
dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-
5,11(10H,11aH)-dione 5o
Mp: 81–83 °C ½a 2D5
ꢂ
þ 321:3 (c 1.0, CH3OH). 1H NMR (300 MHz,
CDCl3) d 7.87 (dd, J = 2.26 Hz, J = 6.04 Hz, 1H), 7.72 (d, J = 1.51 Hz,
1H), 7.47–7.52 (m, 2H), 7.27–7.34 (m, 2H), 7.16 (d, J = 7.55 Hz,
1H), 5.10 (s, 2H), 4.62–4.68 (m, 1H), 4.37 (dd, J = 6.04 Hz,
J = 2.26 Hz, 1H), 3.90 (d, J = 13.5 Hz, 1H), 3.65 (dd, J = 4.53 Hz,
J = 8.30 Hz, 1H), 2.87–3.01 (m, 2H), 2.11–2.22 (m, 1H). 13C NMR
(75 MHz, CDCl3) d 169.0, 165.6, 159.7, 156.4, 139.3, 137.7, 132.3,
130.9, 130.5, 129.6, 126.4, 123.1, 121.9, 115.4, 69.1, 56.0, 54.0,
51.9, 35.0. IR (KBr) 3390, 2929, 1679, 1631, 1535, 1463, 1389,
1250, 1049, 756 cmꢁ1. HR-MS (ESI) Calcd for C19H16N3O5ClNa
[M+Na]+: 424.06707. Found 424.06598.
4.2.11. (2R,11aS)-10-(4-Aminobenzyl)-2-hydroxy-2,3-dihydro-
1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione
5k
Mp: 95–97 °C ½a 2D5
ꢂ
þ 480:0 (c 1.0, CH3OH). 1H NMR (300 MHz,
CD3OD) d 7.74 (dd, J = 1.51 Hz, J = 6.60 Hz, 1H), 7.48–7.57 (m,
2H), 7.27–7.32 (m, 1H), 6.81 (d, J = 8.49 Hz, 2H), 6.54 (d,
J = 8.49 Hz, 2H), 5.34 (d, J = 15.1 Hz, 2H), 4.79 (d, J = 15.1 Hz, 1H),
4.52–4.58 (m, 1H), 4.33 (dd, J = 5.47 Hz, J = 2.64 Hz, 1H), 3.57–
3.72 (m, 2H), 2.87–2.96 (m, 1H). 13C NMR (75 MHz,
CDCl3 + DMSO-d6)
d 168.2, 164.8, 145.5, 138.7, 131.1, 129.2,
127.3, 125.0, 121.9, 114.3, 67.7, 55.3, 53.0, 50.8, 34.3. IR (KBr)
3357, 2933, 1671, 1628, 1465, 1396, 1249, 1052, 956, 715,
620 cmꢁ1. HR-MS (ESI) Calcd for C19H20N3O3 [M+H]+: 338.1504
found: 338.1514.
4.2.16. (2R,11aS)-10-(3,5-Difluorobenzyl)-2-hydroxy-2,3-
dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-
5,11(10H,11aH)-dione 5p
½
a 2D5
ꢂ
þ 269:6 (c 1.0, CH3OH). 1H NMR (300 MHz, CD3OD) d.7.81
(dd, J = 1.70 Hz, J = 6.23 Hz, 1H), 7.55–7.60 (m, 1H), 7.43 (d,
J = 7.55 Hz, 1H), 7.32–7.38 (m, 1H), 6.70–6.81 (m, 3H), 5.33 (d,
J = 16.05 Hz, 1H), 5.03 (d, J = 16.05 Hz, 1H), 4.52–4.58 (m, 1H),
4.41 (dd, J = 5.66 Hz, J = 2.45 Hz, 1H), 3.70–3.76 (m, 1H), 3.63 (dd,
J = 4.72 Hz, J = 7.74 Hz, 1H), 2.84–2.98 (m, 2H), 2.05–2.14 (m, 1H).
13C NMR (75 MHz, CD3OD) d 170.8, 167.8,166.3, 166.1, 143.0,
140.5, 133.9, 131.0, 127.5, 124.1, 111.2, 110.9, 103.5, 69.8, 57.6,
54.8, 51.9, 35.8. IR (KBr) 3392, 2932, 1678, 1628, 1463, 1391,
4.2.12. Methyl 4-(((2R,11aS)-2-hydroxy-5,11-dioxo-2,3,11,11a-
tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-10(5H)-
yl)methyl)benzoate 5l
½
a 2D5
ꢂ
þ 262:2 (c 1.0, CH3OH). 1H NMR (300 MHz, CD3OD) d 7.84
(d, J = 8.30 Hz, 2H), 7.77 (dd, J = 1.88, J = 6.42 Hz, 1H), 7.48–7.54
(m, 1H), 7.40 (d, J = 8.30 Hz, 1H), 7.26–7.31 (m, 1H), 7.19 (d,
J = 8.30 Hz, 2H), 5.37 (d, J = 16.24 Hz, 1H), 5.04 (d, J = 16.2 Hz,
1H), 4.51–4.57 (m, 1H), 4.37 (dd, J = 5.6 Hz, J = 2.45 Hz, 1H), 3.82
(s, 3H), 3.59–3.75 (m, 2H), 2.83–2.96 (m, 2H), 2.04–2.13 (m, 1H).
13C NMR (75 MHz, CDCl3) d 168.9, 166.6, 165.7, 141.9, 139.4,
132.2, 130.4, 130.0, 129.4, 129.2, 126.6, 126.2, 122.0, 69.0, 56.0,
54.0, 52.5, 52.0, 34.9. IR (KBr) 3413, 2952, 1719, 1680, 1463,
1389, 1281, 1218, 1019, 770, 714 cmꢁ1. HR-MS (ESI) Calcd for
C21H21N2O5 [M+H]+: 381.14450. Found 381.14462.
1118, 992, 760 cmꢁ1
. HR-MS (ESI) Calcd for C19H17O3N2F2
[M+H]+: 359.12018. Found 359.12025.
4.2.17. (2R,11aS)-10-(2-Chloro-3,5-difluorobenzyl)-2-hydroxy-
2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-
5,11(10H,11aH)-dione 5q
Mp: 161–163 °C
½
a 2D5
ꢂ
þ 248:4 (c 1.0, CH3OH). 1H NMR
(300 MHz, CDCl3) d 7.75 (dd, J = 1.54 Hz, J = 6.04 Hz, 1H), 7.42–
7.48 (m, 1H), 7.34 (d, J = 8.30 Hz, 1H), 7.22 (t, J = 7.55 Hz, 1H),
6.90–6.97 (m, 1H), 6.78–6.88 (m, 1H), 5.80 (d, J = 15.10 Hz, 1H),
4.94 (d, J = 15.10 Hz, 1H), 4.62–4.68 (m, 1H), 4.26 (dd, J = 5.28 Hz,
J = 3.02 Hz, 1H), 3.85–3.90 (m, 1H), 3.65 (dd, J = 4.53 Hz,
J = 7.55 Hz, 2H), 2.94–3.04 (m, 1H), 2.09–2.16 (m, 1H). 13C NMR
(75 MHz, CDCl3) d 169.2, 165.5, 158.7, 156.1, 137.5, 131.6, 130.1,
126.3, 122.5, 116.6, 166.3, 114.6, 114.3, 69.0, 56.2, 42.4, 34.9. IR
(KBr) 3393, 2928,1681,1635,1471, 1421, 1388, 1237, 1095,
761 cmꢁ1. HR-MS (ESI) Calcd for C19H15O3N2ClF2Na [M+Na]+:
415.06315. Found 415.06210.
4.2.13. 4-(((2R,11aS)-2-Hydroxy-5,11-dioxo-2,3,11,11a-
tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-10(5H)-
yl)methyl)benzoic acid 5m
½
a 2D5
ꢂ
þ 442:0 (c 1.0, CH3OH). 1H NMR (300 MHz,
CDCl3 + DMSO-d6)
d
7.96 (d, J = 8.12 Hz, 2H), 7.89 (d,
J = 6.98 Hz, 1H), 7.42 (t, J = 9.8 Hz, 1H), 7.17–7.31 (m, 4H), 5.22
(d, J = 16.0 Hz, 1H), 5.05 (d, J = 16.0 Hz, 1H), 4.56–4.59 (m, 1H),
4.37 (t, J = 7.55 Hz, 1H), 3.83–3.88 (m, 1H), 3.58–3.71 (m, 2H),
2.91–2.99 (m, 1H), 2.10–2.18 (m, 1H). 13C NMR (75 MHz,CD3OD)
d 170.7, 167.9, 163.9, 143.5, 140.6, 133.8, 131.1, 130.9, 128.1,
127.4, 124.2, 69.8, 57.6, 54.8, 52.3, 35.8. IR (KBr) 3403, 2954,
4.2.18. (2R,11aS)-2-Hydroxy-10-(3,4,5-trimethoxybenzyl)-2,3-
dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-
5,11(10H,11aH)-dione 5r
1683, 1466, 1421, 1393, 1251, 1108, 1053, 987, 843, 764 cmꢁ1
.
HR-MS (ESI) Calcd for C20H18N2O5Na [M+Na]+; 389.1113. Found
389.1108.
½
a 2D5
ꢂ
þ 264:3 (c 1.0, CH3OH). 1H NMR (300 MHz, CDCl3) d 7.84 (d,
J = 7.74 Hz, 1H), 7.46 (t, J = 7.74 Hz, 1H), 7.25–7.29 (m, 2H), 6.30 (s,
2H), 5.17 (d, J = 15.6 Hz, 1H), 4.97 (d, J = 15.6 Hz, 1H), 4.66 (br s,
1H), 4.36 (t, J = 7.7 Hz, 1H), 3.92 (d, J = 12.8 Hz, 1H), 3.78 (s, 3H),
3.76 (s, 6H), 3.62–3.71 (m, 1H), 2.96–3.05 (m, 2H), 2.11–2.19 (m,
1H). 13C NMR (75 MHz, CDCl3) d 169.0, 165.7, 153.3, 139.4, 132.3,
132.1, 130.3, 129.9, 126.2, 122.5, 103.5, 69.1, 60.7, 56.0, 53.9,
52.1, 35.0. IR (KBr) 3430, 2936, 1675, 1634, 1462, 1423, 1330,
1219, 1125, 772 cmꢁ1. HR-MS (ESI) Calcd for C22H25N2O6 [M+H]+:
413.1712 found: 413.1729.
4.2.14. (2R,11aS)-10-(4-Chloro-3-fluorobenzyl)-2-hydroxy-2,3-
dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-
5,11(10H,11aH)-dione 5n
½
a 2D5
ꢂ
þ 249:7 (c 1.0, CH3OH). 1H NMR (300 MHz, CDCl3) d 7.88
(dd, J = 1.70 Hz, J = 6.04 Hz, 1H), 7.43–7.34 (m, 1H), 7.27–7.34 (m,
2H), 7.17 (d, J = 8.30 Hz, 1H), 6.98 (dd, J = 2.07 Hz, J = 7.74 Hz,
1H), 6.88 (dd, J = 1.32 Hz, J = 6.98 Hz, 1H), 5.11 (d, J = 15.6 Hz,
1H), 4.95 (d, J = 15.6 Hz, 1H), 4.64–4.69 (m, 1H), 4.36 (dd,
J = 6.04 Hz, J = 1.88 Hz, 1H), 3.88–3.94 (m, 1H), 3.67 (dd,,
J = 4.53 Hz, J = 8.12 Hz, 1H), 2.96–3.04 (m, 2H), 2.11–2.21 (m, 1H).
13C NMR (75 MHz, CDCl3) d 169.0, 165.6, 159.7, 139.3, 137.8,
132.3, 130.9, 130.5, 129.6, 126.4, 123.0, 121.9, 115.4, 115.1, 69.1,
56.0, 54.0, 51.9. IR (KBr) 3384, 2927, 1628, 1490, 1464, 1423,
1060, 764 cmꢁ1. HR-MS (ESI) Calcd. for C19H17N2O3ClF [M+H]+:
375.09062. Found 375.09062.
4.2.19. (2R,11aS)-2-Hydroxy-10-(perfluorobenzyl)-2,3-dihydro-
1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione
5s
½
a 2D5
ꢂ
þ 294:2 (c 1.0, CH3OH). 1H NMR (300 MHz, CDCl3) d 7.80
(m, 1H), 7.50–7.56 (m, 1H), 7.30 (dd, J = 7.55 Hz, J = 6.04 Hz, 2H),
5.75 (d, J = 15.1 Hz, 1H), 4.84 (d, J = 15.1 Hz, 1H), 4.61–4.67 (m,

4492
D. Addla et al. / Bioorg. Med. Chem. 21 (2013) 4485–4493
1H), 4.25 (dd, J = 5.28 Hz, J = 2.26 Hz, 1H), 3.85–3.90 (m, 1H), 3.63
(dd, J = 4.53 Hz, J = 8.30 Hz, 1H), 2.87–3.00 (m, 2H), 2.08–2.17 (m,
1H). 13C NMR (75 MHz, CDCl3) d 169.0, 165.3, 137.6, 132.1, 130.5,
130.2, 126.7, 122.1, 109.5, 69.0, 56.0, 53.8, 39.6, 34.8. IR (KBr)
3392, 2927, 1698, 1631, 1505,1463, 1249, 1124, 1040, 997, 955,
764, 713, 609 cmꢁ1. HR-MS (ESI) Calcd for C19H14N2O3F5 [M+H]+:
413.09191. Found 413.09193.
5. X-ray crystallography
X-ray data for the compounds were collected at room tempera-
ture using a Bruker Smart Apex CCD diffractometer with graphite
monochromatic MoKa radiation (k = 0.71073 Å) with x-scan
method. Preliminary lattice parameters and orientation matrices
were obtained from four sets of frames.
Integration and scaling of intensity data were accomplished
using SAINT program. The structure was solved by direct methods
using SHELXS97 and refinement was carried out by full-matrix
least-squares technique using SHELXL97. Anisotropic displace-
ment parameters were included for all non-hydrogen atoms. The
atom C12 of 5 g is disordered over two sites (C12/C121) and the
site-occupation factors refined to 0.708(7) and 0.292(7). EADP
and DFIX constraints were applied to the disordered atoms. The
O bound H atoms in 5 g were located in a difference density
map and refined isotropically. All other H atoms were located in
difference Fourier maps and subsequently geometrically opti-
mized and allowed for as riding atoms, with C–H = 0.93–0.97 Å,
with Uiso(H) = 1.5 Ueq(C) for methyl H or 1.2 Ueq(C). The methyl
groups were allowed to rotate but not to tip. The absolute config-
uration of the procured material was known in advance and was
confirmed by unambiguous refinement of the absolute structure
parameter.
4.2.20. (2R,11aS)-10-(2-(4-Chlorophenyl)-2-oxoethyl)-2-
hydroxy-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4] diazepine-
5,11(10H,11aH)-dione 5t
Mp: 141–143 °C
½
a 2D5
ꢂ
þ 374:7 (c 1.0, CH3OH). 1H NMR
(500 MHz, CDCl3) d 7.87–7.91 (m, 3H), 7.43–7.47 (m, 3H), 7.28 (t,
J = 6.9 Hz, 1H), 7.09 (d, J = 7.9 Hz, 1H), 5.47 (d, J = 17.9 Hz, 1H),
4.77 (d, J = 17.9 Hz, 1H), 4.59 (br s, 1H), 4.34 (t, J = 6.99 Hz, 1H),
3.87 (d, J = 13.99 Hz, 1H), 3.63 (dd, J = 3.99 Hz, J = 3.99 Hz, 1H),
2.87–2.95 (m, 2H), 2.09–2.16 (m, 1H). 13C NMR (75 MHz, CDCl3)
d 192.0, 169.0, 165.8, 140.5, 139.6, 132.8, 132.4, 130.3, 129.4,
129.2, 126.2, 121.9, 69.0, 56.2, 55.8, 54.0, 34.9. IR (KBr) 3388,
3012, 2934, 1699, 1466, 1420, 1222, 1095, 984, 764 cmꢁ1. HR-MS
(ESI) Calcd for C20H18N2O4Cl [M+H]+: 385.0955. Found 385.0950.
4.2.21. (2R,11aS)-10-(2-(4-Bromophenyl)-2-oxoethyl)-2-
hydroxy-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaz epine-
5,11(10H,11aH)-dione 5u
Crystal data for 5g: C19H17BrN2O3, M = 401.26, colorless plate,
0.12 ꢃ 0.06 ꢃ 0.06 mm3, monoclinic, space group P21 (No. 4),
a = 5.7253 (6), b = 12.3763 (13), c = 11.9108 (13) Å, b = 94.641
(2)°, V = 841.21(15) Å3, Z = 2, Dc = 1.584 g/cm3, F0 0 0 = 408, CCD
Mp: 159–161 °C
½
a 2D5
ꢂ
þ 421:3 (c 1.0, CH3OH). 1H NMR
(300 MHz, CDCl3) d 7.80–7.86 (m, 3H), 7.61 (d, J = 8.68 Hz, 2H),
7.40–7.46 (m, 1H), 7.24–7.29 (m, 1H), 7.08 (d, J = 7.74 Hz, 1H),
5.47 (d, J = 17.5 Hz, 1H), 4.75 (d, J = 17.5 Hz, 1H), 4.53–4.62 (m,
1H), 4.27–4.32 (m, 1H), 3.85–3.90 (m, 1H), 3.60 (dd, J = 4.53 Hz,
J = 7.93 Hz, 1H), 2.83–2.91 (m, 2H), 2.04–2.13 (m, 1H). 13C NMR
(75 MHz, CDCl3 + DMSO-d6) d 191.2, 167.7, 164.0, 138.2, 132.2,
130.9, 130.6, 128.6, 128.4, 128.1, 127.3, 124.5, 120.9, 66.8, 54.6,
54.3, 52.4, 33.6. IR (KBr) 3337, 3086, 2935, 1707, 1677, 1620,
1461, 1418, 1383, 1302, 1117, 1095, 988, 765 cmꢁ1. HR-MS (ESI)
Calcd for C20H17N2O4BrNa [M+Na]+: 451.02639. Found 451.02524.
Area Detector, MoKa radiation, k = 0.71073 Å, T = 294(2) K,
2hmax = 50.0°, 7355 reflections collected, 2836 unique
(Rint = 0.0246). Final GooF = 1.046, R1 = 0.0252, wR2 = 0.0647, R
indices based on 2683 reflections with I >2
r
(I) (refinement on
F2), 230 parameters, 1 restraint.
l
= 2.465 mmꢁ1. Absolute struc-
ture parameter = 0.048(8).
6. ACE inhibition assay
ACE inhibition assay was performed using the colorimetric
method. 1 g of rabbit lung acetone powder (Sigma–Aldrich,
USA, Cat. No. L0756) was homogenized with 10 mL of 0.05 M so-
dium borate buffer (pH 8.2) containing 0.3 M NaCl and 0.5% Tri-
ton X-100 at 4 °C followed by centrifugation at 15,000 rpm for
60 min at 4 °C. Supernatant was used as a source of ACE enzyme
for this assay. ACE activity was assayed by monitoring the release
of Hippuric acid (HA) from the hydrolysis of hippuryl–histidyl–
leucine (HHL) (Sigma–Aldrich, USA, Cat No. H1635). The enzyme
4.2.22. (2R,11aS)-10-((4-Bromothiophen-2-yl)methyl)-2-
hydroxy-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-
5,11(10H,11aH)-dione 5v
½
a 2D5
ꢂ
þ 261:6 (c 1.0, CH3OH). 1H NMR (500 MHz, CDCl3) d 7.86
(dd, J = 1.51 Hz, J = 6.04 Hz, 1H), 7.48–7.54 (m, 1H), 7.28–7.35 (m,
2H), 7.11 (d, J = 1.51 Hz, 1H), 6.88 (s, 1H), 5.20 (d, J = 15.8 Hz,
1H), 5.05 (d, J = 15.8 Hz, 1H), 4.63–4.69 (m, 1H), 4.29 (dd,
J = 6.04 Hz, J = 2.26 Hz, 1H), 3.86–3.91 (m, 1H), 3.66 (dd,
J = 4.53 Hz, J = 8.30 Hz, 1H), 2.96–3.04 (m, 2H), 2.10–2.20 (m, 1H).
13C NMR (75 MHz, CDCl3) d 168.8, 165.5, 141.0, 139.1, 132.3,
130.5, 129.7, 128.8, 126.5, 123.1, 122.1, 109.0, 69.2, 56.0, 54.0,
reaction was started by adding 7
0.05 M sodium borate buffer (pH 8.2) containing 0.3 M NaCl
and 50 l of 5 mM substrate (HHL) followed by incubation at
ll of ACE enzyme, 12.5 ll of
l
48.1,
34.9.
IR
(KBr)
3355,3101,2926,1642,1462,1247,1093,983,762 cmꢁ1. HR-MS (ESI)
Calcd for C17H16N2O3BrS [M+H]+: 407.00595. Found 407.00578.
37 °C for 30 min. The reaction was arrested by the addition of
0.1 mL of 1 M HCl. After stopping the reaction, 0.2 mL of pyridine
(SD Fine chemical, India) was added followed by 0.1 mL of ben-
zene sulphonyl chloride (SD Fine chemical, India). The solution
was mixed by inversion for 1 min and cooled on ice. The yellow
color developed was measured at 410 nm. The decreased concen-
tration of HA in the test reaction compared with the control reac-
tion was expressed as percentage inhibition and calculated from
the equation: Inhibition% = 100 ꢁ [ATest/AControl] ꢃ 100, where
ATest = absorbance of test reaction and AControl = absorbance of
control reaction. The therapeutic drug Lisinopril, Benazepril and
Ramipril was used as standard. Depending on the inhibition assay
of all synthesized derivatives, we selected effective derivatives 5f,
5g, 5r and 5v for measuring IC50, concentration which inhibit 50%
of enzyme activity. IC50 value was measured by plotting the per-
centage inhibition versus the serial concentrations of the chosen
derivatives.
4.2.23. (2R,11aS)-10-(Dibenzo[b,d]furan-4-ylmethyl)-2-
hydroxy-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-
5,11(10H,11aH)-dione 5w
½
a 2D5
ꢂ
þ 192:0 (c 1.0, CH3OH). 1H NMR (300 MHz, CD3OD) d 7.90
(d, J = 7.74 Hz, 1H), 7.80 (dd, J = 2.07, J = 4.53 Hz, 1H), 7.65 (dd,
J = 1.32 Hz, J = 6,42 Hz, 1H), 7.41–7.57 (m, 4H), 7.14–7.32 (m, 4H),
5.90 (d, J = 15.6 Hz, 1H), 5.20 (d, J = 15.6 Hz, 1H), 4.56–4.63 (m,
1H), 4.37 (dd, J = 5.28 Hz, J = 2.83 Hz, 1H), 3.68 (d, J = 4.53 Hz,
2H), 2.92–3.00 (m, 2H), 2.05–2.14 (m, 1H). 13C NMR (75 MHz,
CD3OD) d 170.6, 167.7, 157.3, 154.9, 140.3, 133.4, 131.1, 130.6,
128.4, 127.7, 127.1, 125.6, 125.0, 124.5, 124.1, 123.9, 121.7,
121.0, 112.8, 69.8, 57.7, 54.7, 47.6, 35.8. IR (KBr) 3403, 2925,
2854, 1677, 1633, 1463, 1186, 755 cmꢁ1. HR-MS (ESI) Calcd for
C
25H21O4N2 [M+H]+: 413.14958. Found 413.14947.

D. Addla et al. / Bioorg. Med. Chem. 21 (2013) 4485–4493
4493
port, encouragement and assistance through Network (NWP 0054,
NWP 0055, SMiLE CSC 0111, ORIGIN CSC 0108) & MLP 0002 pro-
jects. S.K.B. is thankful to Department of Biotechnology (DBT) for
providing Ramalingaswami Fellowship. D.A. & A.K. (SRF) is thank-
ful to CSIR for financial assistance. We also thank Rene Thomsen,
Molegro, Denmark for providing evaluation copy of Molegro Vir-
tual Docker Software.
7. In vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide assay
Cytotoxicity activity of active compounds 5f, 5g, 5r and 5v were
studied by means of a colorimetric microculture assay (MTT).6 It
was measured by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl tetrazolium bromide (MTT) by mitochondrial succinate
dehydrogenase to insoluble coloured formazan product, using
Dulbecco’s modified Eagle’s medium (DMEM, Sigma, USA) for nor-
mal HEK 293 cells and A549 cancer cells. 1 ꢃ 104 cells/well were
Supplementary data
seeded in 100 ll DMEM, supplemented with 10% FBS in each well
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.05.031.
of 96-well micro culture plates and incubated for 24 h at 37 °C in a
CO2 incubator. The desired concentrations of the compounds/com-
plexes were made and added to the wells with respective vehicle
References and notes
control. After 24 h of incubation, add 10 ll MTT (3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (5 mg/mL) was
added to each well and the plates were further incubated for 4 h.
Then the supernatant from each well was carefully removed, form-
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Molegro Virtual Docker (MVD, Ver: 2012.5.0.0) is a program for
determining the most conformations of how a ligand will bind to
an enzyme site. The identification of ligand binding modes is done
iteratively by evaluating a number of candidate solutions (ligand
conformations) and estimating the energy of their interactions
with the enzyme. The highest scoring solutions are returned for
further analysis. MVD performs flexible ligand docking, so the opti-
mal geometry of the ligand will be determined during the docking.
Docking studies of active ligands 5f, 5g, 5r and 5v were performed
using this software. Crystal structure of t-ACE (PDB ID: 1O86) along
with the co-factor (Zinc) was downloaded from RCSB Protein Data
Bank. Structure of tACE (residues 37-625) adopts an overall ellip-
soid shape with central groove that extends for about 30 Å into
molecules and divides the protein into two sub domains. Zinc is
an important catalytic component of ACE, bound at the active site.
The boundaries of the groove are provided by helices
a and b
strands. Structure of tACE—lisinopril complex used to locate the ac-
tive site of the molecules. tACE contains 27 helices. Structure of
tACE revealed the location of two buried chloride ions seperated
by 20.3 Å. All the structures of new compounds 5f, 5g, 5r and 5v
were prepared by using Chem Draw and saved to mol file format.
These structures were further converted into mol2 file format by
using Marvin Sketch. MVD was used to perform computational
studies, cavity prediction, assigning bond orders, structure refine-
ment, defining the active binding sites of the t-ACE and structure
preparation. Residues involved in hydrogen bonding interaction
with ligands are as follows: Glu 162, Gln 281, Ala 354, His 353,
Tyr 520, Ala 356, Tyr 523, Arg 522, His 513, Lys 511. As most of
the ligands are surrounded by the above residues, it can be con-
cluded that these residues are highly conserved and plays an
important functional roles. Interaction of the ligands with these
residues might be changing the active site structure, which leads
to inhibition of enzyme. These ligands bind with high affinity
against tACE and show low free energy.
24. (a) Natesh, R.; Schwager, S. L. U.; Evans, H. R.; Sturrock, E. D.; Acharya, K. R.
Biochemistry 2004, 43, 8718; (b) Hooper, N. M.; Tuner, A. J. Nat. Struct. Mol. Biol.
2003, 10, 155.
Acknowledgements
25. (a) Bhuyan, B. J. Curr. Sci. 2011, 101, 881; (b) Brew, K. Trends Pharmacol. Sci.
2003, 24, 391.
Authors are thankful to Dr. M. Lakshmikantam, Director, Dr. V. J.
Rao, Head, C P C Division, IICT, Hyderabad for their continuous sup-