Development of Selective CBP/P300 Benzoxazepine Bromodomain Inhibitors

Article

pubs.acs.org/jmc

Development of Selective CBP/P300 Benzoxazepine Bromodomain

Inhibitors

Tobias A. Popp,^†Cynthia Tallant,^‡,§Catherine Rogers,^‡,§Oleg Fedorov,^‡,§Paul E. Brennan,^‡,§

Susanne Muller,^‡,§Stefan Knapp,*^,‡,∥and Franz Bracher*^,†

̈

^†Department fur PharmazieZentrum fur Pharmaforschung, Ludwig-Maximilians-Universitat Munchen, Butenandtstrasse 5-13,

̈

D-81377 Munchen, Germany

̈

^‡Nuﬃeld Department of Clinical Medicine, University of Oxford, Structural Genomics Consortium, Old Road Campus Research

Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.

^§Nuﬃeld Department of Clinical Medicine, University of Oxford, Target Discovery Institute (TDI), Roosevelt Drive, Oxford OX3

7BN, U.K.

^∥Institute for Pharmaceutical Chemistry and Buchmann Institute for Life Sciences, Johann Wolfgang Goethe-University,

Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany

S

* Supporting Information

ABSTRACT: CBP (CREB (cAMP responsive element binding protein) binding protein (CREBBP)) and P300 (adenovirus

E1A-associated 300 kDa protein) are two closely related histone acetyltransferases (HATs) that play a key role in the regulation

of gene transcription. Both proteins contain a bromodomain ﬂanking the HAT catalytic domain that is important for the

targeting of CBP/P300 to chromatin and which oﬀeres an opportunity for the development of protein−protein interaction

inhibitors. Here we present the development of CBP/P300 bromodomain inhibitors with 2,3,4,5-tetrahydro-1,4-benzoxazepine

backbone, an N-acetyl-lysine mimetic scaﬀold that led to the recent development of the chemical probe I-CBP112. We present

comprehensive SAR of this inhibitor class as well as demonstration of cellular on target activity of the most potent and selective

inhibitor TPOP146, which showed 134 nM aﬃnity for CBP with excellent selectivity over other bromodomains.

heart development,³while heterozygous Cbp mice showed

defects in stem cell renewal. In adult mice, conditional deletion

INTRODUCTION

■

CBP/P300 are two evolutionary conserved and closely related

of Cbp in the hematopoetic system resulted in cell diﬀer-

entiation defects and impaired self-renewal of hematopoietic

stem cells (HSC).⁴Deregulation of CBP/P300 has been

reported in cancer and chromosomal rearrangement of the

CBP/P300 locus results in oncogenic fusions with either MOZ

acetyltransferase or the mixed linage leukemia (MLL) gene

product and CBP/MLL fusions have been detected in acute

myeloid leukemia (AML) or myelodysplastic syndromes

(MDS) after treatment with topoisomerase inhibitors.⁵

histone acetyl transferases (HATs) that function as transcrip-

tional regulators by acetylating histone tails and other nuclear

proteins and by acting as scaﬀolds for the assembly of

multiprotein complexes through protein interaction domains.¹

CBP/P300 activity is regulated on several levels: autoacetyla-

tion of a HAT domain adjacent loop inhibits CBP/P300

activity by competing with the binding of substrates, the RING

domain that regulates CBP/P300 turnover, and the PHD

bromodomain that is required for substrate recruitment.²

Studies in mice demonstrated that Cbp and p300 are essential

genes required for development. Homozygous deletion of Cbp

or p300 results in embryonic lethality early in development due

to impaired hematopoiesis, neurulation, cell proliferation, and

Received: May 24, 2016

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Because of the central function of CBP/P300 in disease

development and its critical role in many cellular processes, a

number of inhibitors have been developed that target domains

in CBP/P300. These inhibitors and chemical probes constitute

a rich resource that facilitates functional studies on CBP/P300

protein interaction and catalytic domains. The catalytic

function has been targeted by the pyrazolone C646, a

competitive HAT inhibitor.⁶However, C646 is reactive and

has multiple oﬀ-target activities and its thiol reactivity limits its

applications as a chemical probe in cellular systems.⁷

The KIX domain, an interaction domain mediating cAMP

response element binding protein (CREB) interaction, has

been targeted by several diﬀerent scaﬀolds.⁸The bromodomain

has been targeted by the inhibitor “ischemin”, an acetyl-lysine

competitive inhibitor with low μM activity. In addition, a

number of nonspeciﬁc fragments have been reported to target

the CBP bromodomain.⁹Bromodomains are acetyl-lysine

dependent protein interaction domains with predicted good

druggability,¹⁰which has been ﬁrst demonstrated by the

identiﬁcation of highly potent and selective inhibitors

developed for the BET (bromodomain and extraterminal

domain) family of transcriptional regulators.¹¹Despite their

conserved fold, the large sequence and structural diversity of

bromodomains oﬀers multiple opportunities for target or family

speciﬁc targeting using small molecules,¹²a property that has

been explored for the development of a large diversity of

inhibitors and chemical probes.¹³

inhibitor with good cellular activity.¹⁸Here we present the

optimization of the oxazepine series that led to the racemic

variant of 4 as well as the synthesis and a comprehensive SAR

analysis of 2,3,4,5-tetrahydro-1,4-benzoxazepines as selective

CBP/P300 bromodomain inhibitors.

RESULTS AND DISCUSSION

■

First CBP/P300 bromodomain inhibitors of this series were

identiﬁed by screening a commercial library of compounds

from Chembridge for CBP inhibition. This library was

extended to 48 compounds bearing a 2,3,4,5-tetrahydro-1,4-

benzoxazepine scaﬀold. Structures were screened by diﬀerential

scanning ﬂuorimetry (DSF) against CBP and BRD4(1) as an

example for a BET family bromodomain and an indicator for

inhibitor’s selectivity. Screened structures and their T_mshifts

with CBP and BRD4(1) are shown in Chart 1 (compounds 5−

52). Substances in this chart are sorted two dimensionally in

columns and rows, the ﬁrst row showing chlorophenyl moieties

at C-7 of the bicyclic scaﬀold and the second row electron

richer (methoxy/alkyl/thiomethyl)phenyl moieties at the same

position. Row 3 depicts heteroaromatic moieties at C-7, and

row 4 contains further and more advanced molecules with

several modiﬁcations. Substances with a cyclopropanecarbonyl

residue at N-4 are displayed in the ﬁrst column, then propionic

acid amides and acetamides in the next column, and in the last

column amides of bulkier carboxylic acids (row 1 and row 2

only). The best structure was 52, of which the (S)-enantiomer

is already established as the chemical probe 4.

The ﬁrst potent inhibitors for the CBP/P300 bromodomains

were based on 5- and 6-isoxazolylbenzimidazoles, acetyl-lysine

mimetic moieties that were initially developed for targeting

BET bromodomains¹⁴and that have been been later optimized

for CBP/P300 inhibition, such as compound 1 (CBP30)¹⁵

(Figure 1), as well as on a dihydroquinoxalinone based scaﬀold,

The active compounds of this commercial library did bind to

both CBP and BRD4(1), and an increase or decrease of

potency through exchange of structure elements mostly aﬀected

potency toward both bromodomains in equal measure. Among

the N-acyl moieties in the commercial compounds, cyclo-

propanecarbonyl and propanoyl groups appeared to be superior

to both smaller (acetyl in 14, 26, 27) and larger (e.g.,

compounds 15, 19, 28, 36) ones. It is noteworthy that activity

on CBP was found with compounds bearing a phenyl group at

C-7 with chloro and/or methoxy substituents (e.g., 9, 21). At

the same time, replacement of the methoxy group at C-9 with a

bulky, basic amine moiety resulted in enhanced binding (8),

whereas introduction of neutral alkyl ether moieties of similar

size at C-9 showed no clear tendency (6, 11). Introduction of a

thienylmethoxy residue at C-9 was detrimental (7, 12). Potency

was greatly reduced for compounds bearing basic, nitrogen

containing heterocycles at C-7 (37−39) and slightly reduced

for thiophene derivatives (40 vs 8). The introduction of

benzothiazole (41, 42) had no eﬀect compared to chlorophenyl

analogues (8, 11) but conﬁrmed the impression that the

combination of an electron-rich aromatic moiety at C-7 and a

bulky, basic amine moiety at C-9 might lead to potent

inhibitors. This hypothesis was strongly supported by very

strong CBP inhibition of the synthesized compounds 47, 51,

and especially 52. The (S)-enantiomer of 52 has meanwhile

been established as potent and to a good extent selective

chemical probe 4. Nevertheless, improvement of selectivity was

still a challenge because even the very potent CBP inhibitors

mentioned above still showed residual eﬀects on BRD4(1).

This prompted us to start a synthesis campaign in which all

edges of the 1,4-benzoxazepine scaﬀold were reinvestigated

systematically in order the gain further insight into the

inﬂuence of any single modiﬁcation on potency and selectivity

of these inhibitors.

Figure 1. Structures of potent CBP/P300 inhibitors.

compound 2.¹⁵The most selective inhibitor of the

isoxazolylbenzimidazole series, 1, has a K_Dof 21 nM for the

CBP bromodomain but maintains still considerable BET

bromodomain activity.¹⁶1 has strong anti-inﬂammatory

activity, reducing immune cell production of IL-17A and

other proinﬂammatory cytokines.¹⁶At a later stage, benzoic

acid derivatives, such as 3, were also proposed as CBP

inhibitors.¹⁷Inhibitors carrying a 1,4-benzoxazepine core led to

the development of 4 (I-CBP112), a selective CBP/P300 dual

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Chart 1. (This Page: Compounds 5−36) T_mShifts in °C, n = 3; (Next Page: Compounds 37−52) The (S)-Enantiomer of 52 Is

Inhibitor 4, T_mshifts in °C, n = 3

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Chart 1. continued

For investigation of variations at C-7, we used a readily

available 2,3,4,5-tetrahydro-1,4-benzoxazepine scaﬀold 53 bear-

ing a methoxy residue at C-9, and screening results are

displayed in Table 1. Most of the heteroaromatic variations at

C-7 (54−57) yielded only compounds with poor activity. Only

isoxazole 58 showed a signiﬁcant temperature shift (4.9 °C),

while in contrast to active benzothiazoles 41 and 42,

benzothiophene 59 interacted only weakly. Upon replacement

of the phenyl moiety at C-7 by an acetyl moiety (60), potency

was completely lost. While potency decreased according to our

expectations for some compounds bearing electron-deﬁcient

phenyl moieties at C-7 (61, 62), the nitrophenyl compound 63

showed modest activity (less than 4 °C). Surprisingly, the

electron-rich aminophenyl derivatives (64, 65) showed similar

temperature shifts, and modiﬁcations of the amino group

(sulfonamide 66, urea 67) led to increased potency. Regarding

the compounds with electron-rich phenyl moiety, 68 with a 3,5-

dimethoxyphenyl group showed the highest T_mshift in this

series, while related compounds 69, 70, 71, 72, and 73 still

showed acceptable ΔT_mon CBP.

Because the initial evaluation of purchased benzoxazepines

had already revealed that only small N-acyl residues on N-4 are

tolerated, we performed further modiﬁcations of the hitherto

most promising acyl groups without increasing the size of this

moiety too much (Table 2). These investigations were

performed on the basis of 77 (bearing a 3-chloro-4-

methoxyphenyl residue at C-7). We mainly aimed at the

introduction of small polar groups, enabling further polar

contacts, either to or mediated through the conserved water

molecules in the binding pocket. However, the compact and

polar urea derivatives 78 and 79 and the glycolic amide 80

showed no ΔT_m. The thiourea 81 and the thioamide 82

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Table 1. DSF Results for Compounds with Diﬀerent Moieties at C-7 (n = 3)

a

T_mshifts for lead structure 52: CBP 7.8 0.5 °C, BRD4(1) 2.1 0.4 °C.

showed reduced activity, too. Attempts to obtain more subtle

interactions via introduction of ﬂuorine atoms in the acyl

residue failed as well (83−85). Finally, the propionyl residue

was most favorable (86), and only the cyclopropanoyl (72) and

the methyl carbamate (87) were nearly as active. These data

suggest that water molecules in the CBP/P300 binding pocket

cannot be substituted by polar groups without losing some or

all binding activity of the inhibitor.

To investigate whether a 2,3,4,5-tetrahydro-1,4-benzothiaze-

pine can act as a bioisoster of the 2,3,4,5-tetrahydro-1,4-

benzoxazepine here, three benzothiazepine analogues (88−90)

bearing promising moieties at both C-7 and N-4 were

synthesized, but these compounds were found to be

signiﬁcantly less active than their benzoxazepine congeners

(compare 68 and 72) (Table 3). Consequently, further

investigation of this scaﬀold was not attractive. Likewise, a

newly developed approach to 2,3,4,5-tetrahydro-1H-1,4-benzo-

diazepines did not give a potent benzodiazepine analogue.¹⁹

The insights into the SAR based on purchased and

synthesized compounds were used to design a ﬁnal CBP

inhibitor with the following key features: 2,3,4,5-tetrahydro-1,4-

benzoxazepine scaﬀold, propionyl residue on N-4, dimethox-

yphenyl moiety at C-7, then combined with a polar substituent

bearing an amino group at C-9. This ﬁnal modiﬁcation was

inspired by the very potent but not absolutely selective CBP

inhibitors 47, 51, and 52 (racemate of 4). Starting from a newly

developed 2,3,4,5-tetrahydro-1,4-benzoxazepine-9-carboxylate

building block 91 (Scheme 5) and various diamines, a number

of N-aminoalkylamides were prepared and screened. Even the

intermediates 92−95 without amine function showed promis-

ing activity toward CBP combined with very high selectivity

(Table 4). While the N-piperidinoethyl amides 96 and 97

showed almost no activity toward BRD4(1) and good activity

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Table 2. DSF Results for Compounds with Diﬀerent N-Acyl Residues (n = 3)

a

T_mshifts for lead structure 52: CBP 7.8 0.5 °C, BRD4(1) 2.1 0.4 °C.

97). Finally, the 3-aminopiperidine-derived amides 100−103

showed very good potency toward CBP and very little potency

toward BRD4(1). Surprisingly, almost identical activities and

selectivities were obtained for the pairs of enantiomers and for

piperidine and N-methylpiperidine analogues.

Table 3. DSF Results for Benzothiazepine Compounds

(n = 3)

a

To assess the selectivity of 100 and 102 (named TPOP146)

over other bromodomains, these inhibitors were tested with 44

further bromodomains by DSF (Figure 2), and outstanding

selectivity was accomplished. It appears that the very low T_m

shifts with BRD4(1) result from the newly introduced amide

group at C-9. Noteworthy and strong activity was only

conﬁrmed for P300, which was not surprising due to the very

high sequence conservation of these two bromodomains: Out

of the 114 amino acids of the CBP bromodomain, P300 only

diﬀers by two amino acids in its α_Chelix (tyrosine for

phenylalanine and serine for alanine), and one remote amino

acid in each of the α_B- (isoleucine for valine) and α_AZhelices

(serine for asparagine). Conserved is the BC loop and the ZA

loop, lining the Kac binding site and inﬂuencing binding

speciﬁty.²⁰

We selected therefore 102 for further analysis. The K_dof 102

was determined in solution by isothermal titration calorimetry

(ITC). A constant of 134 10 nM was determined for CBP

(4: 151 6 nM) and 5.02 μM for BRD4(1), thus revealing

more than 30-fold selectivity (Figure 3).

A crystal structure of 102 with the CBP bromodomain

conﬁrmed the anticipated acetyl-lysine mimetic binding mode

and thus competitive inhibition mode. The structure was

a

T_mshifts for lead structure 52: CBP 7.8 0.5 °C, BRD4(1) 2.1 0.4

°C.

toward CBP, open chain compounds 98 and 99 were slightly

less potent but very selective, too. The compounds with 3,5-

dimethoxy phenyl substitution at C-7 (92, 94, 96) proved more

promising than those with 3,4-dimethoxy substitution (93, 95,

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Table 4. DSF Results for Compounds with Diﬀerent Moieties at C-9 (n = 3)

a

T_mshifts for lead structure 52: CBP 7.8 0.5 °C, BRD4(1) 2.1 0.4 °C.

reﬁned at atomic resolution (1.05 Å) with good data collection

and reﬁnement statistics (Supporting Information, pp S-4, S-5).

The ligand was well-deﬁned except for the distal ring atoms of

the 1-methylpiperidine ring that was oriented toward the

solvent (Figure 4A). The last amino acid of this bromodomain’s

α_Bhelix (N1168) and the ﬁrst amino acid of the α_Chelix

(R1173) greatly contribute to binding, while ZA loop’s Y1125

lines the binding site and binds via conserved water molecules

inside the pocket (Figure 4B,C). The electron-rich phenyl

moiety at C-7 interacts with R1173 by a π−cation interaction

and an induced binding mode (Figure 4D) as also described for

dihydroquinoxalinone based CBP inhibitors.²¹The newly

introduced carbonyl group at C-9 interacts through a water-

mediated hydrogen bond with N1168 (Figure 4E), and the acyl

group at N-4 acts as a Kac mimic moiety forming a hydrogen

bond to N1168 (Figure 4E) and a water-mediated hydrogen

bond to Y1125 (Figure 4F), as also conceivable in co-crystal

structures with 4.¹⁸

We next investigated whether 102 was able to inhibit

recruitment of CBP to chromatin in cells and studied the eﬀect

of this inhibitor using a ﬂuorescent recovery after photo

bleaching (FRAP) assay. Full-length CBP contains a large

number of protein and DNA interaction domains and is not

signiﬁcantly displaced by bromodomain inhibitors.¹⁸We used

therefore a construct harboring three CBP bromodomains

fused in frame by a linker region and a nuclear localization

signal fused to GFP (3xCBP^BRD/GFP) and increased aﬃnity of

this fusion protein by inhibiting HDAC activity using the pan-

HDAC inhibitor SAHA, which resulted in a suﬃcient FRAP

signal and residence time. Exposure to 1 μM 102 resulted in a

signiﬁcant decrease of recovery half-life that was comparable to

the construct that contained the bromodomain inactivating

mutation N1168F, demonstrating that 102 targets the CBP

bromodomain in the nucleus and is capable of competing with

acetyl-lysine mediated interactions of the CBP bromodomain in

cellular environments (Figure 5).

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aromatic moieties at C-7. Intermediate 53 was prepared

following an established approach to the 2,3,4,5-tetrahydro-

1,4-benzoxazepine backbone²²but starting with 5-bromo-2-

hydroxy-3-methoxybenzaldehyde (Scheme 1). Thus, reductive

amination with 2-aminoethanol and NaBH₄in THF/MeOH

gave intermediate 104 in 93% yield. After Boc-protection of the

amino group, ring closure to the benzoxazepine 106 was

performed in 91% yield using the Mitsunobu reagents DIAD

and triphenylphosphine. Acidic N-deprotection gave an almost

quantitative yield of the amine 107, which was reacted with

cyclopropanecarbonyl chloride to give the amide 53 with 71%

yield.

Suzuki cross-coupling of aryl bromide 53 with Pd(dppf)Cl₂

catalyst and various boronic acids and boronic acid pinacol

esters gave biaryls 54−55, 57−59, 61−64, 68−72, and 74−75

(Scheme 2, for details on the aryl residues, see Table 1).

Furthermore, aromatic aldehyde 61 was converted quantita-

tively with O-methylhydroxylamine into the O-methyloxime 76

and reduced to the benzyl alcohol 73 with NaBH₄with 97%

yield. The nitrophenyl compound 63 was reduced to the

corresponding aniline 65 using Raney nickel and hydrazine in

38% yield. This aniline was further functionalized with

mediocre yields into the sulfonamide 66 with ethanesulfonyl

chloride and the urea derivative 67 using ethyl isocyanate.

Finally, Stille cross-coupling of 53 with Pd(PPh₃)₂Cl₂catalyst

and tributyl(1-ethoxyvinyl)tin followed by acidic workup²³gave

the methyl ketone 60 in 67% yield, which was further converted

into the aminopyrimidine 56 in 57% using Bredereck’s reagent

and guanidinium carbonate.²⁴The same Stille coupling, but

with neutral aqueous workup, gave an enolether, which was

further converted to the aminothiazole 57 by treatment with

NBS and subsequent cyclization with thiourea²⁵(20% yield

over three steps).

Figure 2. Selectivity proﬁle of 102. Screened targets are indicated by a

colored dot. Temperature shifts are highlighted by red spheres

according to the magniture of the T_mshift as shown in the ﬁgure

capture.

The evaluation of the best moiety at the nitrogen at position

4 was performed with a benzoxazepine scaﬀold bearing a 3-

chloro-4-methoxyphenyl substituent at C-7, since at that time

this substitution pattern looked most promising. Intermediate

107 (Scheme 1) was subjected to a Suzuki cross-coupling

reaction with 3-chloro-4-methoxyphenylboronic acid to give

biaryl 77 in 48% yield (Scheme 3). Treatment of the secondary

amine 77 with acyl chlorides or carboxylic acid anhydrides gave

the urea 78, amides 83 and 86, and carbamate 87 (for details

on the acyl residues, see Table 2). EDC as coupling reagent and

appropriate carboxylic acids were used to obtain amides 84 and

85. (Thio)ureas 79 and 81 were accessible through iso(thio)-

cyanates, and glycolic acid amide 80 was obtained in acceptable

yield through reaction with neat ethyl glycolate. Finally,

Lawesson’s reagent was used to convert carboxamide 86 into

the corresponding thioamide 82.

To investigate the relevance of the heterocyclic backbone,

benzothiazepine analogues were prepared (Scheme 4). 2-

Amino-5-bromo-3-methoxybenzoic acid²⁶was converted into

the thiol 108 with 62% yield in a multistep reaction comprising

diazotation, reaction with potassium ethyl xanthate, and alkaline

hydrolysis.²⁷Acid-catalyzed esteriﬁcation^27bgave methyl ester

109, which underwent conversion into the benzothiazepinone

110 in a two-step reaction (thioetheriﬁcation with 2-

chloroethylamine,²⁸followed by base-mediated lactamiza-

tion²⁹) with 63% yield. Reduction of the lactam function with

BH₃−THF gave the crude amine, which was directly acylated

to give the amides 111 and 112 in moderate yields. Finally, a

standard Suzuki protocol gave good yields for the 7-aryl

derivatives 88, 89, and 90.

Figure 3. K_ddetermination of 102 with CBP by isothermal titration

calorimetry.

The in vitro results of compound 102 were particularly

promising because DSF results showed an excellent selectivity

of this compound for bromodomains of CBP and P300 sparing

BET bromodomains. Simultaneously to this report on the SAR,

the closely related 1,4-benzoxazepine 4 (S-isomer of 52)

demonstrated interesting activity in leukemia models, suppress-

ing clonogenic growth of primary blasts and cell diﬀerentiation.

An interesting aspect of this study was that 4 showed synergism

with BET inhibition, suggesting that design of dual BET/

CBP(P300) inhibitors may represent a new strategy for

leukemia treatment.¹⁸Because of the close structural similarity,

we did not repeat these experiments with 102. However, the

presented SAR of this compound series will provide interesting

insights for the further improvements of this scaﬀold for future

in vivo application of CBP/P300 bromodomain inhibitors.

CHEMISTRY

■

The 7-bromobenzoxazepine scaﬀold 53 served as a backbone

for the synthesis and assessment of compounds with diﬀerent

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Figure 4. Co-crystallization of CBP with 102. (A) 2FoFc OMIT map contoured at 2σ around the ligand. (B) Orientation of 102 with CBP and

details of the interactions with CBP’s N-acetyllysine binding site. The inhibitor and most relevant side chains are shown in ball and stick

representation. Water molecules are indicated by red spheres, and hydrogen bonds within the Kac binding pocket are shown as dotted lines. (C)

Overview on all interactions of 102 with CBP. (D) π−Cation interaction between R1173 of CBP (surface representation) and the electron-rich 3,5-

dimethoxyphenyl moiety of 102 (golden stick representation). (E) Water mediated hydrogen bond from the newly introduced amide group at C-9

to Y1168. (F) Conserved water molecules in the pocket binding to inhibitor and Y1125.

Having checked further scaﬀolds and having optimized the

N-acyl residue (N-propanoyl best) and the 7-aryl substituent

(dimethoxyphenyl best), we performed ﬁnal modiﬁcations at

C-9. Inspired by the most potent Chembridge substances

bearing a piperidinylmethoxy residue at this position, we

prepared the benzoxazepine scaﬀold 91, bearing an ester group

at C-9 as an attractive intermediate for the introduction of

aminoalkyl side chains. Starting point for the synthesis of 91

was 5-bromosalicylic acid, which underwent Duﬀ formylation

and subsequent esteriﬁcation in good yields.³⁰Then, following

the protocol described above for benzoxazepine scaﬀold 53, the

obtained methyl 5-bromo-3-formyl-2-hydroxybenzoate was

converted in four steps via intermediate 91 into benzoxazepine

115 (Scheme 5). Suzuki cross-coupling reactions and

subsequent alkaline ester hydrolyses gave the two isomeric 7-

(dimethoxyphenyl)-9-carboxy derivatives 94 and 95 in high

Figure 5. FRAP experiment demonstrating displacement of the CBP

yields. EDC-mediated amidation of these carboxylic acids with

bromodomain by 102 from chromatin. SAHA treated cells are

three diﬀerent diamines gave the amides 96−99 in moderate

indicated by †. Representative raw data traces are shown in the right

yields.

panel. The bromodomain inactivating mutant (N1168F) served as a

positive control.

Chiral compounds 100 and 101 were synthesized accord-

ingly from carboxylic acid 94 using both (S)- and (R)-

conﬁgured 3-amino-1-Boc-piperidine as building blocks and

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Scheme 1. Preparation of 1,4-Benzoxazepine Scaﬀold 53

a

Reagents and conditions: (a) 2-aminoethanol, NaBH₄, THF, MeOH, RT, 93%; (b) di-tert-butyl dicarbonate, NaHCO₃solution, EtOAc, RT, 69%;

(c) PPh₃, DIAD, CH₂Cl₂, RT, 91%; (d) HCl, 1,4-dioxane, MeOH, reﬂux, 94%; (e) cyclopropanecarbonyl chloride, DIPEA, CH₂Cl₂, 0 °C to RT,

71%.

TIKRKLDTGQYQEPWQYVDDVWLMFNNAWLYNRKTS-

RVYKFCSKLAEVFEQEIDPVMQSLG (140 amino acids).

subsequent, acidic Boc-deprotection. Finally these secondary

amines were N-methylated using formaldehyde and NaCNBH₃,

yielding N-methylpiperidines 102 and 103 (Scheme 6).

Likewise for BRD4(1) the Genbank ID was gi|19718731 and

expression cell line BL21(DE3)-R3-pRARE2 was used to express the

sequence MHHHHHHSSGVDLGTENLYFQSMNPPPPET-

SNPNKPKRQTNQLQYLLRVVLKTLWKHQFAWPFQQP-

VDAVKLNLPDYYKIIKTPMDMGTIKKRLENNYYWNAQECIQD-

FNTMFTNCYIYNKPGDDIVLMAEALEKLFLQKINELPTEE (148

amino acids).

Isothermal Titration Calorimetry (ITC). Experiments were

carried out on a VP-ITC microcalorimeter (MicroCal). All experi-

ments were performed at 15 °C in 20 mM HEPES, pH 7.5, 150 mM

NaCl. The titrations were conducted using an initial injection of 2 μL

followed by 34 identical injections of 8 μL. The dilution heats were

measured on separate experiments and were subtracted from the

titration data. Thermodynamic parameters were calculated using ΔG =

ΔH − TΔS = −RT ln K_B, where ΔG, ΔH, and ΔS are the changes in

free energy, enthalpy, and entropy of binding, respectively. In all cases,

a single binding site model was employed.

Thermal Shift Assay. Thermal melting experiments were carried

out using an Mx3005p real-time PCR machine (Stratagene). Proteins

were buﬀered in 10 mM HEPES, pH 7.5, 500 mM NaCl and assayed

in a 96-well plate at a ﬁnal concentration of 2 μM in 20 μL volume.

Compounds were added at a ﬁnal concentration of 10 μM. SYPRO

Orange (Molecular Probes) was added as a ﬂuorescence probe at a

dilution of 1:1000. Excitation and emission ﬁlters for the SYPRO

Orange dye were set to 465 and 590 nm, respectively. The

temperature was raised with a step of 3 °C per minute from 25 to

96 °C, and ﬂuorescence readings were taken at each interval. Data was

analyzed as previously described.^11a

Protein Crystallization. Aliquots of the puriﬁed proteins were set

up for crystallization using a mosquito crystallization robot (TTP

Labtech). Coarse screens were typically setup onto Greiner 3-well

plates using three diﬀerent drop ratios of precipitant to protein per

condition (200 + 100 nL, 150 + 150 nL, and 100 + 200 nL). All

crystallizations were carried out using the sitting drop vapor diﬀusion

method at 4 °C. CBP crystals with compound 102 (3 mM ﬁnal

concentration) were grown by mixing 100 nL of the protein (12.75

mg/mL) with 200 nL of reservoir solution containing 25% PEG 3350,

0.2 M MgCl₂, 0.1 M Bis Tris, pH 7.5.

CONCLUSION

■

For the ﬁrst time we herein present the systematic optimization

and the SAR of benzoxazepine-type compounds for the

inhibition of the closely related CBP/P300 bromodomains.

This study yielded 52 for CBP/P300 inhibition, which is the

racemate to the similarly potent, enantiomerically pure, and

meanwhile commercially available inhibitor 4. This selective

and potent compound has initially been developed as chemical

probe, but due to the involvement of the CBP/P300

bromodomains in several diseases (cancer, inﬂammatory

diseases, Rubinstein−Taybi syndrome) and promising results

in in vitro and in vivo experiments,¹⁸1,4-benzoxazepine-type

CBP/P300 inhibitors prove to be valuable candidates for

further preclinical studies. The preparation and characterization

of several series of novel 1,4-benzoxazepine compounds with

new substitution patterns and moieties is described in the next

section, allowing a comprehensive discussion of the SAR of the

1,4-benzoxazepines. The best, newly synthesized compound

102 exceeds 4 with regard to selectivity, and was well

characterized in biological experiments (K_d, FRAP, cocrystal-

lization). Its binding mode and orientation with CBP is

exemplarily shown and discussed in detail. The preparation of

novel 2,3,4,5-tetrahydro-1,4-benzothiazepines is also shown,

giving a total of 62 newly prepared and characterized

compounds. Because the 1,4-benzoxazepine element is reported

in various substances with anti-inﬂammatory,³¹antithrom-

botic,³²antitumor,^22,33and antiamyloid-β plaque activity,^34,35

this manuscript contains not only relevant information for

bromodomain researchers but also for scientists longing for

similar compounds for various applications.

EXPERIMENTAL SECTION

Data Collection and Structure Solution. Crystals were

cryoprotected using the well solution supplemented with additional

ethylene glycol and were ﬂash frozen in liquid nitrogen. Data were

collected at diamond beamline I04 at a wavelength of 1.0121 Å.

Indexing and integration was carried out using MOSFLM,³⁶and

scaling was performed with SCALA.³⁷Initial phases were calculated by

molecular replacement with PHASER³⁸using an ensemble of known

■

Protein Expression. cDNA encoding human bromodomains were

cloned, expressed, and puriﬁed as previously described:^11aTo obtain

CBP for screenings and co-crystallization, the Genbank ID gi|4758056

and expression cell line BL21(DE3)-R3 were used to express the

sequence MHHHHHHSSGVDLGTENLYFQSMRKKIFKPEELRQA-

LMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKNPMDLS-

J

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a

Scheme 2. Preparation of Benzoxazepine Inhibitors with Diﬀerent Moieties at C-7

a

Reagents and conditions: (a) various boronic acids/pinacol esters (see Experimental Section), Pd(dppf)Cl₂·CH₂Cl₂, DIPEA, H₂O/1,4-dioxane, 95

°C, 31−94%; (b) NaBH₄, CH₂Cl₂, MeOH, RT, 97%; (c) O-methylhydroxylamine, K₂CO₃, EtOH, RT, 99%; (d) Raney nickel, N₂H₄, MeOH, EtOH,

reﬂux, 38%; (e) ethanesulfonyl chloride, DMAP, pyridine, 0 °C to RT, 60%; (f) ethyl isocyanate, DIPEA, CH₂Cl₂, RT, 47%; (g) (i) Pd(PPh₃)₂Cl₂,

tributyl(1-ethoxyvinyl)tin, 1,4-dioxane, 140 °C, (ii) HCl, H₂O RT, 67%; (h) Bredereck’s reagent, DMF, 160 °C, then guanidinium carbonate,

K₂CO₃, DMF, 160 °C, 57%; (i) (i) Pd(PPh₃)₂Cl₂, tributyl(1-ethoxyvinyl)tin, 1,4-dioxane, 140 °C, (ii) NBS, THF, 0 °C to RT, (iii) thiourea, DMF,

RT, 20%.

bromodomain models (PDB IDs 3DWY, 2OSS, 2OUO, 2GRC,

2OO1, 3DAI, 3D7C). Initial models were built by ARP/wARP,³⁹and

building was completed manually with COOT.⁴⁰Reﬁnement was

carried out in REFMAC5.⁴¹Thermal motions were analyzed using

TLSMD,⁴²and hydrogen atoms were included in late reﬁnement

cycles. Data collection and reﬁnement statistics are compiled in the

Supporting Information, Table S5. The models and structure factors

have been deposited with PDB accession code 5J0D.

Fluorescence Recovery After Photo Bleaching (FRAP). FRAP

studies were performed essentially as described.⁴³In brief, U2OS cells

were transfected (Fugene HD; Roche) with mammalian over-

expression constructs a triplicated CBP bromodomain harboring a

nuclear localization sequence.¹⁸The imaging system consisted of a

Zeiss LSM 710 laser-scanning and control system (Zeiss) coupled to

an inverted Zeiss Axio Observer.Z1 microscope equipped with a high-

numerical-aperture (N.A. 1.3) 40× oil immersion objective (Zeiss).

Samples were placed in an incubator chamber in order to maintain

temperature and humidity. FRAP and GFP ﬂuorescence imaging were

both carried out with an argon-ion laser (488 nm) and with a PMT

detector set to detect ﬂuorescence between 500 and 550 nm. Once an

initial scan had been taken, a region of interest corresponding to

approximately 50% of the entire GFP positive nucleus was empirically

selected for bleaching. A time lapse series was then taken to record

GFP recovery using 1% of the power used for bleaching. The image

data sets and ﬂuorescence recovery data were exported from ZEN

2009, the microscope control software, into Origin to determine the

average half-time for full recovery for 10−20 cells per treatment point.

Data were analyzed using one-way ANOVA with Dunnetts’s multiple

comparisons test.

Compound Characterization. Melting points were determined

1

with a Buchi melting point B-540 and are uncorrected. H and ¹³C

̈

NMR spectra were recorded either with Avance III HD 400 MHz

Bruker BioSpin or Avance III HD 500 MHz Bruker BioSpin

spectrometers. Chemical shifts (δ) are given in ppm relative to TMS

K

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a

Scheme 3. Functionalizations at N-4

a

Reagents and conditions: (a) 3-chloro-4-methoxyphenylboronic acid, Pd(dppf)Cl₂·CH₂Cl₂, DIPEA, H₂O/1,4-dioxane, 95 °C, 48%; (b) propionyl

chloride, DIPEA, CH₂Cl₂, 0 °C to RT, 40%; (c) Lawesson’s reagent, THF, RT, 96%; (d) dimethylcarbamoyl chloride, DIPEA, CH₂Cl₂, 0 °C to RT,

74%; (e) (trimethylsilyl)isocyanate, CH₂Cl₂, then HCl in 1,4-dioxane, RT, 87%; (f) ethyl glycolate, 60 °C, 51%; (g) NaH, THF, then methyl

isothiocyanate, RT, 69%; (h) triﬂuoroacetic anhydride, DIPEA, DMAP, CH₂Cl₂, 0 °C to RT, 92%; (i) 3,3,3-triﬂuoropropionic acid, DMAP, EDC·

HCl, CH₂Cl₂, 0 °C to RT, 94%; (j) 2-ﬂuoropropionic acid, DMAP, EDC·HCl, 0 °C to RT, 81%; (k) methyl chloroformate, DIPEA, CH₂Cl₂, 0 °C to

RT, 94%.

or residual undeuterated solvent, and coupling constants (J) are given

in hertz (Hz). Splitting patterns are abbreviated as follows: s = singlet;

d = doublet; t = triplet; q = quartet; dd = doublet of doublet; m =

multiplet, br s = broad singlet. Some NMR spectra were recorded at

elevated temperature to suppress the appearance of rotameric double

peaks. Those occurred in the NMR spectra of almost all tested

substances and arise from the amide bond of the nitrogen of the seven-

membered ring. EI-Mass spectra were recorded at an ionization energy

of 70 eV either with a JMS GCmate II Jeol or a JEOL JMS-700

MStation. ESI-Mass spectra were recorded on a Thermo Finnigan

LTQ FT at 4 kV. Puriﬁcation by ﬂash column chromatography (FCC)

was performed using Silica Gel 60 from Merck KGaA. HPLC purity

analysis was individually performed on an Agilent 1100 series

apparatus with a G1311A QuatPump, a G1329A ALS autosampler,

and a G1316A ColComp column oven and Agilent ChemStation rev.

B04.02 as software. A G1315A DAD detector was set to 210 nm for

detection. Injection volume was 5 or 10 μL of a dilution of 100 μg/mL

(sample in mobile phase). Column temperature was 50 °C, ﬂow either

0.3 mL/min or 0.8 mL/min or 1.0 mL/min. Diﬀerent solvent mixtures

were used as mobile phase, from 50% to 25% water and from 50% to

75% acetonitrile, respectively. The water used for preparation of the

mobile phase contained 1% THF. The following columns were used:

Kinetex 2.6 u PFP, 100 A, (100 mm × 2.10 mm), Agilent Poroshell

120, PFP 2.7 μm, (3.0 mm × 100 mm), Varian Pursuit UPS 2.4

diphenyl (50 mm × 2.0 mm), and Agilent Poroshell 120, EC-C18 2.7

μm, (3.0 mm × 100 mm). All tested substances showed a purity

>95.0%. For microwave experiments, a CEM Discover was used with

power set to 300 W. Optical rotations were determined with a

PerkinElmer 241 polarimeter. Potent compounds were particularly

examined for assay interferences (PAINS), but the potent structures

oﬀered little chemical reactivity. Moreover, assays based on diﬀerent

principles were used to assess their potency. Finally, selectivity was

shown among similar targets (BRD4(1)). All this makes assay

interferences unlikely.

The combined organic layers were dried over MgSO₄, concentrated in

vacuo, and puriﬁed by FCC with EtOAc and hexanes.

Standard protocol 2 (Suzuki cross-coupling of 7-bromobenzox-

azepines and 7-bromobenzothiazepines with boronic acids and boronic

acid pinacol esters): Typically, to 0.30 mmol bromoarene, 0.36 mmol

boronic acid or boronic acid pinacol ester and 0.03 mmol [1.1′-

bis(diphenylphosphino)ferrocene]dichloropalladium(II) were added.

A mixture of 0.50 mL of water, 2.0 mL of 1,4-dioxane, and 1.2 mmol of

DIPEA was added, and the mixture was heated under nitrogen

atmosphere with vigorous stirring to 95 °C for 3.5 h. To this solution

was added either water or 0.5 M NaOH for compounds with basic

moieties or 0.5 M HCl for compounds with acidic moieties. The

mixture was extracted with CH₂Cl₂three times, and the combined

organic layers were dried over MgSO₄and concentrated in vacuo and

puriﬁed by FCC with EtOAc and hexanes.

Standard protocol 3 (conversion of the carboxylic acids 94 and 95

to carboxamides): Typically, 0.29 mmol of carboxylic acid and 0.35

mmol of EDC−HCl were dissolved in 3.0 mL of CH₂Cl₂and cooled

to 0 °C. Then 0.29 mmol of DIPEA, 0.35 mmol of the required

primary amine, and 2 mg of DMAP were added. The solution was

stirred at RT overnight. Then 1 M NaOH was added and the mixture

was extracted with EtOAc three times. The combined organic layers

were dried over MgSO₄, concentrated in vacuo, and puriﬁed by FCC.

1-(7-Bromo-9-methoxy-2,3-dihydro-1,4-benzoxazepin-4(5H)-yl)-

1-(cyclopropyl)methanone (53). Standard protocol 1 with 7.9 g (31

mmol) of 107 and 3.6 mL (40 mmol) of cyclopropanecarbonyl

chloride. FCC with EtOAc and hexanes (1:2, Rf 0.1) gave 7.4 g (22

1

mmol, 71%) of 53 as a white solid; mp 85−86 °C. H NMR (RT,

mixture of rotamers, 500 MHz, DMSO-d₆) δ = 7.29 (d, J = 2.1 Hz,

0.6H), 7.12 (d, J = 2.1 Hz, 0.6H), 7.08 (d, J = 2.1 Hz, 0.4H), 6.97 (d, J

= 2.1 Hz, 0.4H), 4.77 (s, 1.2H), 4.51 (s, 0.8H), 4.10−4.06 (m, 1.6H),

3.99−3.94 (m, 1.2H), 3.87−3.81 (m, 1.2H), 3.76 (s, 3H), 2.14−2.08

(m, 0.6H), 1.96−1.89 (m, 0.4H), 0.71−0.63 (m, 4H). ¹³C NMR (RT,

mixture of rotamers, 126 MHz, DMSO-d₆) δ = 173.1, 172.6, 153.1,

152.9, 148.3, 148.2, 134.9, 134.7, 124.9, 124.0, 116.1, 115.7, 115.5,

115.2, 73.2, 72.5, 57.0, 51.3, 49.8, 49.0, 48.2, 11.6, 8.4, 8.0. MS (EI+):

m/z calcd for (C₁₄H₁₆⁷⁹BrNO₃) 325.0314, found 325.0316.

Standard Synthetic Protocols. Standard protocol 1 (N-acylation

of benzoxazepines and benzothiazepines): Typically, 0.50 mmol of

educt was dissolved in 1.0 mL of CH₂Cl₂and 1.5 mmol of DIPEA was

added at 0 °C. Then 1.3 mmol acyl chloride was added, and the

mixture warmed to RT and stirred for 1.5 h. Then 2 M NaOH was

added and the aqueous phase was extracted three times with CH₂Cl₂.

1-Cyclopropyl-1-[7-(2,3-dichloropyridin-4-yl)-9-methoxy-2,3-di-

hydro-1,4-benzoxazepin-4(5H)-yl]methanone (54). Standard proto-

col 2 with 0.098 g (0.30 mmol) of 53 and 0.069 g (0.36 mmol) of 2,3-

dichloropyridine-4-boronic acid. FCC with EtOAc and hexanes (3:1,

L

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a

Scheme 4. Synthetic Route to 2,3,4,5-Tetrahydro-1,4-benzothiazepines

a

Reagents and conditions: (a) (i) NaNO₂, HCl, H₂O, 0 °C, (ii) KOAc, potassium ethyl xanthate, H₂O, 90 °C, (iii) NaOH, NaHSO₃, 85 °C, 62%;

(b) MeOH, H₂SO₄, reﬂux, 72%; (c) (i) 2-chloroethylamine, NaOMe, DMF, 0 °C to RT, (ii) tBuOK, THF, 0−45 °C, 63%; (d) BH₃-THF, −30 °C

to reﬂux; (e) propionyl chloride, DIPEA, CH₂Cl₂, 0 °C to RT, 49%; (f) 3,4-dimethoxyphenylboronic acid, Pd(dppf)Cl₂·CH₂Cl₂, DIPEA, H₂O/1,4-

dioxane, 95 °C, 59%; (g) cyclopropanecarbonyl chloride, DIPEA, CH₂Cl₂, 0 °C to RT, 45%; (h) 3,4-dimethoxyphenylboronic acid or 3-chloro-4-

methoxyphenylboronic acid, Pd(dppf)Cl₂·CH₂Cl₂, DIPEA, H₂O/1,4-dioxane, 95 °C, 85% and 70%.

Rf 0.2) gave 0.091 g (0.23 mmol, 77%) of 54 as an orange solid; mp

186−187 °C. ¹H NMR (RT, mixture of rotamers, 400 MHz, CD₂Cl₂)

δ = 8.34−8.23 (m, 1H), 7.29−7.20 (m, 1H), 7.04−6.89 (m, 2H), 4.77

(s, 1.2H), 4.67 (s, 0.8H), 4.28−4.21 (m, 0.8H), 4.14−4.08 (m, 2H),

4.03−3.97 (m, 1.2H), 3.88−3.82 (m, 3H), 1.91−1.81 (m, 0.6H),

1.78−1.65 (m, 0.4H), 0.89−0.82 (m, 2H), 0.78−0.69 (m, 2H). ¹³C

NMR (RT, mixture of rotamers, 126 MHz, CD₂Cl₂) δ = 172.6, 172.1,

152.0, 151.2, 150.4, 150.4, 149.2, 149.1, 146.8, 146.7, 132.7, 132.4,

132.0, 131.6, 128.6, 128.5, 124.9, 124.7, 122.6, 121.1, 113.0, 112.5,

72.6, 56.4, 51.1, 50.9, 48.8, 48.2, 11.4, 11.3, 7.4, 7.2. MS (EI+): m/z

calcd for (C₁₉H₁₈³⁵Cl₂N₂O₃) 392.0694, found 392.0699.

1-{5-[4-(Cyclopropanecarbonyl)-9-methoxy-2,3,4,5-tetrahydro-

1,4-benzoxazepin-7-yl]thiophen-2-yl}ethan-1-one (55). Standard

protocol 2 with 0.098 g (0.30 mmol) oof 53 and 0.061 g (0.36

mmol) of 5-acetyl-2-thienylboronic acid. FCC with EtOAc and

hexanes (2:1, Rf 0.2) gave 0.079 g (0.21 mmol, 71%) of 55 as an

orange solid; mp 85−87 °C. ¹H NMR (110 °C, 400 MHz, C₂D₂Cl₄) δ

= 7.58 (d, J = 3.9 Hz, 1H), 7.20 (d, J = 3.9 Hz, 1H), 7.15−7.03 (m,

2H), 4.70 (s, 2H), 4.21−4.09 (m, 2H), 4.04−3.92 (m, 2H), 3.88 (s,

3H), 2.49 (s, 3H), 1.81−1.64 (m, 1H), 0.98−0.84 (m, 2H), 0.79−0.64

(m, 2H). ¹³C NMR (110 °C, 101 MHz, C₂D₂Cl₄) δ = 189.7, 172.4,

152.4, 151.9, 149.8, 143.3, 133.0, 132.7, 129.1, 123.9, 119.8, 111.8,

72.5, 57.1, 49.8, 49.6, 26.4, 11.8, 7.3. MS (EI+): m/z calcd for

(C₂₀H₂₁NO₄S) 371.1191, found 371.1192.

160 °C under N₂. Then 0.52 mL (2.5 mmol) of Bredereck’s reagent

was added and heating continued for 1 h. Then 0.66 g (3.7 mmol) of

guanidinium carbonate and 0.34 g (2.5 mmol) of K₂CO₃were added

and the mixture was heated for further 4 h. After cooling, saturated

NaHCO₃solution was added and the mixture was extracted with

CH₂Cl₂three times. The combined organic layers were dried over

MgSO₄and concentrated in vacuo. FCC with CH₂Cl₂with 5% MeOH

(Rf 0.1) gave 0.13 g (0.39 mmol, 57%) of 56 as a yellow solid; mp

136−137 °C. ¹H NMR (RT, mixture of rotamers, 500 MHz, CD₂Cl₂)

δ = 8.34−8.26 (m, 1H), 7.63−7.47 (m, 2H), 7.06−6.98 (m, 1H),

5.25−5.13 (m, 2H), 4.79 (s, 1.1H), 4.68 (s, 0.9H), 4.24−4.18 (m,

0.9H), 4.13−4.06 (m, 2H), 4.01−3.95 (m, 1.1H), 3.92−3.88 (m, 3H),

1.92−1.83 (m, 0.6H), 1.74−1.68 (m, 0.4H), 0.89−0.81 (m, 2H),

0.77−0.69 (m, 2H). ¹³C NMR (RT, mixture of rotamers, 126 MHz,

CD₂Cl₂) δ = 173.0, 172.5, 164.62, 164.57, 163.8, 159.2, 159.1, 152.6,

152.1, 151.1, 150.9, 132.9, 132.7, 121.3, 119.8, 111.1, 110.6, 107.6,

107.5, 73.1, 72.8, 56.64, 56.58, 51.5, 51.3, 49.0, 48.8, 11.8, 11.6, 7.7,

7.6. MS (EI+): m/z calcd for (C₁₈H₂₀N₄O₃) 340.1535, found

340.1538.

1-[7-(2-Aminothiazol-4-yl)-9-methoxy-2,3-dihydro-1,4-benzoxa-

zepin-4(5H)-yl]-1-(cyclopropyl)methanone (57). To a solution of 1.0

g (3.1 mmol) of 53 and 0.11 g (0.16 mmol) of Pd(PPh₃)₂Cl₂in 12 mL

of anhydrous 1,4-dioxane under N₂were added 1.4 mL (4.0 mmol) of

tributyl(1-ethoxyvinyl)tin. The mixture was heated to 140 °C under

microwave irradiation with 300 W for 40 min. After cooling, 50 mL of

water was added and the mixture extracted with CH₂Cl₂three times.

The combined organic layers were dried over MgSO₄and

1-[7-(2-Aminopyrimidin-4-yl)-9-methoxy-2,3-dihydro-1,4-ben-

zoxazepin-4(5H)-yl]-1-(cyclopropyl)methanone (56). A solution of

0.20 g (0.69 mmol) of 60 in 5 mL of anhydrous DMF was heated to

M

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a

Scheme 5. Introduction of the Carbonyl Function at C-9

a

Reagents and conditions: (a) 2-aminoethanol, NaBH₄, MeOH, THF, RT, 93%; (b) di-tert-butyl dicarbonate, EtOAc, NaHCO₃solution, RT, 56%;

(c) PPh₃, DIAD, THF, 0 °C to RT, 85%; (d) (i) HCl, 1,4-dioxane, RT, (ii) propionyl chloride, DIPEA, CH₂Cl₂, 0 °C to RT, 74%; (e) 3,5-/3,4-

dimethoxyphenylboronic acid, Pd(dppf)Cl₂·CH₂Cl₂, DIPEA, H₂O, 1,4-dioxane, 95 °C, 72%/80%; (f) NaOH, MeOH, THF, 70 °C, 93%/87%; (g)

(i) 1-(2-aminoethyl)piperidine, EDC, DMAP, DIPEA, CH₂Cl₂, 0 °C to RT, 31%, (ii, 97 only) HCl, 1,4-dioxane, RT, 43%; (h) N,N-

dimethylethylenediamine, EDC, DMAP, DIPEA, CH₂Cl₂, 0 °C to RT, 69%; (i) N,N-diethylethylenediamine, EDC, DMAP, DIPEA, CH₂Cl₂, 0 °C to

RT, 46%.

a

Scheme 6. Preparation of 100−103

a

Reagents and conditions: (a) (i) (S)-3-amino-1-Boc-piperidine, EDC, DMAP, DIPEA, CH₂Cl₂, 0 °C to RT, (ii) HCl, 1,4-dioxane, RT, 31%; (b) (i)

(R)-3-amino-1-Boc-piperidine, EDC, DMAP, DIPEA, CH₂Cl₂, 0 °C to RT; (ii) HCl, 1,4-dioxane, RT, 55%; (c) formaldehyde solution, NaCNBH₃,

AcOH, MeCN, RT, 35% and 36%.

N

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concentrated in vacuo. FCC with EtOAc and hexanes (2:1, Rf 0.3)

gave 1.0 g of the crude enol ether. This intermediate was dissolved in a

mixture of 10 mL of THF and 10 mL of water and treated at 0 °C with

0.56 g (2.9 mmol) of N-bromosuccinimide. After 1 h at RT, the

mixture was extracted with EtOAc three times. The combined organic

layers were dried over MgSO₄and concentrated in vacuo to obtain

0.47 g of the crude α-bromoketone. Then 0.2 g (43%, 0.54 mmol) of

this residue and 0.20 g (2.6 mmol) of thiourea were dissolved in 5 mL

of anhydrous DMF and stirred overnight. After the addition of water,

the mixture was extracted with CH₂Cl₂three times. The combined

organic layers were dried over MgSO₄and concentrated in vacuo.

FCC with CH₂Cl₂with 3% MeOH (Rf 0.2) gave 0.090 g (0.26 mmol,

20%) of 57 as a yellow solid; mp 103−104 °C. ¹H NMR (100 °C, 400

MHz, DMSO-d₆) δ = 7.41−7.30 (m, 2H), 6.84 (s, 1H), 6.65 (s, 2H),

4.71 (s, 2H), 4.13−4.04 (m, 2H), 4.04−3.91 (m, 2H), 3.82 (s, 3H),

2.06−1.88 (m, 1H), 0.75−0.63 (m, 4H). ¹³C NMR (100 °C, 101

MHz, DMSO-d₆) δ = 171.2, 167.5, 150.7, 149.2, 147.2, 131.3, 129.8,

118.5, 110.3, 100.5, 71.2, 55.9, 48.6, 48.4, 10.5, 6.2. MS (ESI+): m/z

calcd for [(C₁₇H₂₀N₃O₃S)⁺] 346.1225, found 346.1219.

3.87 (s, 3H), 2.11−1.95 (m, 1H), 0.75−0.66 (m, 4H). ¹³C NMR (100

°C, 101 MHz, DMSO-d₆) δ = 188.5, 171.4, 160.4, 151.2, 147.4, 133.6,

133.5, 132.1, 131.9, 125.2, 124.4, 119.0, 113.1, 111.0, 71.2, 56.1, 55.9,

48.5, 48.4, 10.4, 6.3. MS (ESI+): m/z calcd for [(C₂₂H₂₄NO₅)⁺]

382.1654, found 382.1647.

4-[4-(Cyclopropanecarbonyl)-9-methoxy-2,3,4,5-tetrahydro-1,4-

benzoxazepin-7-yl]benzonitrile (62). Standard protocol 2 with 0.098

g (0.30 mmol) of 53 and 0.053 g (0.36 mmol) of 4-

cyanophenylboronic acid. FCC with EtOAc and hexanes (2:1, Rf

0.2) gave 0.091 g (0.26 mmol, 87%) of 62 as an orange solid; mp

157−158 °C. ¹H NMR (RT, mixture of rotamers, 400 MHz, CD₂Cl₂)

δ = 7.80−7.62 (m, 4H), 7.20−7.02 (m, 2H), 4.78 (s, 1.1H), 4.68 (s,

0.9H), 4.26−4.17 (m, 0.9H), 4.17−4.04 (m, 2H), 4.04−3.96 (m,

1.1H), 3.92−3.86 (m, 3H), 1.95−1.83 (m, 0.6H), 1.75−1.65 (m,

0.4H), 0.89−0.80 (m, 2H), 0.80−0.67 (m, 2H). ¹³C NMR (RT,

mixture of rotamers, 126 MHz, CD₂Cl₂) δ = 172.9, 172.4, 152.8,

152.3, 149.5, 149.3, 145.4, 145.3, 135.2, 134.8, 133.4, 133.3, 133.0,

132.9, 128.0, 127.9, 121.3, 119.8, 119.3, 119.2, 111.6, 111.3, 111.1,

110.9, 73.2, 73.0, 56.7, 51.5, 51.3, 49.1, 48.8, 11.8, 11.6, 7.7, 7.5. MS

(EI+): m/z calcd for (C₂₁H₂₀N₂O₃) 348.1474, found 348.1473.

1-Cyclopropyl-1-[9-methoxy-7-(4-methyl-3-nitrophenyl)-2,3-di-

hydro-1,4-benzoxazepin-4(5H)-yl]methanone (63). Standard proto-

col 2 with 0.80 g (2.5 mmol) of 53 and 0.51 g (2.8 mmol) of 4-methyl-

3-nitrophenylboronic acid. FCC with CH₂Cl₂with 1% MeOH Rf 0.3)

gave 0.56 g (1.5 mmol, 60%) of 63 as a yellow solid; mp 141−142 °C.

¹H NMR (110 °C, 400 MHz, C₂D₂Cl₄) δ = 8.04 (d, J = 2.0 Hz, 1H),

7.63 (dd, J = 8.0, 2.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.06−7.00 (m,

2H), 4.73 (s, 2H), 4.22−4.13 (m, 2H), 4.04−3.97 (m, 2H), 3.89 (s,

3H), 2.58 (s, 3H), 1.80−1.68 (m, 1H), 0.95−0.87 (m, 2H), 0.78−0.68

(m, 2H). ¹³C NMR (110 °C, 101 MHz, C₂D₂Cl₄) δ = 172.2, 152.2,

149.9, 149.1, 139.7, 133.9, 132.8, 132.5, 131.6, 130.7, 122.3, 120.0,

112.2, 72.2, 56.9, 49.7, 49.4, 19.1, 11.6, 7.0. MS (ESI+): m/z calcd for

[(C₂₁H₂₃N₂O₅)⁺] 383.1607, found 383.1600.

1-Cyclopropyl-1-{7-[4-(dimethylamino)phenyl]-9-methoxy-2,3-

dihydro-1,4-benzoxazepin-4(5H)-yl}methanone (64). Standard pro-

tocol 2 with 0.30 g (0.92 mmol) of 53 and 0.23 g (1.4 mmol) of 4-

(dimethylamino)phenylboronic acid. FCC with EtOAc and hexanes

(1:2, Rf 0.1) gave 0.17 g (0.46 mmol, 50%) of 64 as a white solid; mp

79−80 °C. ¹H NMR (RT, mixture of rotamers, 400 MHz, C₂D₂Cl₄) δ

= 7.52−7.39 (m, 2H), 7.09−6.93 (m, 2H), 6.78 (d, J = 8.2 Hz, 2H),

4.73 (s, 1.3H), 4.67 (s, 0.7H), 4.28−3.93 (m, 4H), 3.88 (s, 3H), 3.03−

2.93 (m, 6H), 1.91−1.80 (m, 0.7H), 1.69−1.62 (m, 0.3H), 0.96−0.87

(m, 2H), 0.81−0.69 (m, 2H). ¹³C NMR (RT, mixture of rotamers, 101

MHz, C₂D₂Cl₄) δ = 172.6, 172.0, 151.6, 151.1, 149.6, 146.6, 146.5,

136.8, 136.4, 131.9, 131.7, 128.1, 127.5, 127.4, 119.4, 118.1, 112.5,

110.2, 109.5, 72.4, 56.1, 51.0, 50.9, 48.6, 48.4, 40.4, 11.6, 11.4, 7.6, 7.4.

MS (ESI+): m/z calcd for [(C₂₂H₂₇N₂O₃)⁺] 367.2021, found

367.2017.

1-Cyclopropyl-1-[7-(3,5-dimethylisoxazol-4-yl)-9-methoxy-2,3-di-

hydro-1,4-benzoxazepin-4(5H)-yl]methanone (58). Standard proto-

col 2 with 0.098 g (0.30 mmol) of 53 and 0.080 g (0.36 mmol) of 3,5-

dimethylisoxazole-4-boronic acid pinacol ester. FCC with EtOAc and

hexanes (3:1, Rf 0.2) gave 0.089 g (0.26 mmol, 87%) of 58 as a white

1

solid; mp 175−176 °C. H NMR (110 °C, 400 MHz, C₂D₂Cl₄) δ =

6.74−6.66 (m, 2H), 4.69 (s, 2H), 4.20−4.10 (m, 2H), 4.05−3.95 (m,

2H), 3.84 (s, 3H), 2.35 (s, 3H), 2.21 (s, 3H), 1.80−1.64 (m, 1H),

0.96−0.87 (m, 2H), 0.76−0.66 (m, 2H). ¹³C NMR (110 °C, 101

MHz, C₂D₂Cl₄) δ = 172.4, 165.0, 158.4, 152.2, 148.4, 132.7, 126.0,

122.4, 116.3, 114.7, 72.4, 57.1, 50.1, 49.6, 11.7, 11.4, 10.6, 7.3. MS (EI

+): m/z calcd for (C₁₉H₂₂N₂O₄) 342.1580, found 342.1572.

1-[7-(Benzo[b]thiophen-2-yl)-9-methoxy-2,3-dihydro-1,4-benzox-

azepin-4(5H)-yl]-1-(cyclopropyl)methanone (59). Standard protocol

2 with 0.12 g (0.36 mmol) of 53 and 0.080 g (0.43 mmol) of

benzo[b]thien-2-ylboronic acid. FCC with EtOAc and hexanes (2:1,

Rf 0.2) gave 0.13 g (0.34 mmol, 94%) of 59 as a white solid; mp 83−

1

84 °C. H NMR (400 MHz, DMSO-d₆, 100 °C) δ = 7.95−7.87 (m,

1H), 7.87−7.79 (m, 1H), 7.79−7.70 (m, 1H), 7.43−7.25 (m, 4H),

4.79 (s, 2H), 4.20−4.08 (m, 2H), 4.06−3.92 (m, 2H), 3.89 (s, 3H),

2.12−1.95 (m, 1H), 0.78−0.66 (m, 4H). ¹³C NMR (101 MHz,

DMSO-d₆, 100 °C) δ = 171.4, 151.2, 148.3, 142.5, 140.0, 138.2, 131.9,

128.2, 124.1, 123.8, 122.9, 121.6, 119.2, 119.1, 110.6, 71.2, 56.1, 48.4,

48.4, 10.5, 6.3. MS (EI+): m/z calcd for (C₂₂H₂₁NO₃S) 379.1242,

found 379.1240.

1-[4-(Cyclopropanecarbonyl)-9-methoxy-2,3,4,5-tetrahydro-1,4-

benzoxazepin-7-yl]ethan-1-one (60). To a solution of 0.49 g (1.5

mmol) of 53 and 0.053 g (0.075 mmol) of Pd(PPh₃)₂Cl₂in 6.0 mL of

anhydrous 1,4-dioxane under N₂were added 0.66 mL of (2.0 mmol)

tributyl(1-ethoxyvinyl)tin. The mixture was heated to 140 °C under

microwave irradiation with 300 W for 40 min. After cooling, 30 mL of

10% aqueous HCl was added and the mixture extracted with CH₂Cl₂

three times. The combined organic layers were dried over MgSO₄and

concentrated in vacuo. FCC with EtOAc and hexanes (4:1, Rf 0.5)

gave 0.29 g (1.0 mmol, 67%) of 60 as a white solid; mp 76−77 °C. ¹H

NMR (500 MHz, C₂D₂Cl₄, 80 °C): δ = 7.47−7.38 (m, 2H), 4.73 (s,

2H), 4.30−4.16 (m, 2H), 4.08−3.95 (m, 2H), 3.87 (s, 3H), 2.53 (s,

3H), 1.81−1.63 (m, 1H), 0.93−0.87 (m, 2H), 0.79−0.70 (m, 2H). ¹³C

NMR (RT, mixture of rotamers, 126 MHz, C₂D₂Cl₄): δ = 196.9,

196.7, 172.8, 172.4, 152.4, 152.1, 151.5, 151.1, 132.2, 132.1, 130.8,

130.2, 123.5, 121.3, 111.4, 110.2, 72.1, 71.7, 56.3, 56.2, 50.4, 50.4, 48.1,

47.6, 26.5, 26.4, 11.5, 11.4, 7.7, 7.6. MS (ESI+): m/z calcd for

[(C₁₆H₂₀NO₄)⁺] 290.1392, found 290.1386.

5-[4-(Cyclopropanecarbonyl)-9-methoxy-2,3,4,5-tetrahydro-1,4-

benzoxazepin-7-yl]-2-methylbenzenaminium chloride (65). To a

suspension of 6 g of Raney nickel in 60 mL of water was added 9.2 g of

NaOH. Upon complete activation after 15 min, this suspension was

washed three times with water and then three times with EtOH.

Separately, 0.45 g (1.2 mmol) of 63 were dissolved in a mixture of 15

mL of MeOH and 25 mL of EtOH and then 2.2 mL of (44 mmol)

hydrazine monohydrate were added. The activated Raney nickel

suspension was then added to this solution, and the mixture was

reﬂuxed for 40 min. The suspension was ﬁltrated and the ﬁltrate

concentrated in vacuo to give 0.18 g (0.46 mmol, 38%) of 65 as a

1

purple solid; mp 221 °C (decomposition). H NMR (100 °C, 400

MHz, DMSO-d₆) δ = 7.19−7.05 (m, 4H), 7.05−6.94 (m, 1H), 5.62 (s,

3H), 4.75 (s, 2H), 4.19−4.02 (m, 2H), 4.02−3.92 (m, 2H), 3.85 (s,

3H), 2.19 (s, 3H), 2.10−1.92 (m, 1H), 0.78−0.66 (m, 4H). ¹³C NMR

(RT, mixture of rotamers, 126 MHz, DMSO-d₆) δ = 172.7, 172.0,

152.0, 151.8, 148.2, 139.1, 138.9, 134.7, 134.1, 133.2, 132.8, 132.5,

132.2, 132.1, 130.9, 130.7, 126.1, 125.9, 121.6, 121.4, 120.1, 119.4,

110.9, 110.4, 72.7, 72.1, 56.3, 50.9, 50.1, 48.7, 48.3, 17.3, 11.2, 11.1,

7.9, 7.6. MS (EI+): m/z calcd for (C₂₁H₂₄N₂O₃) 352.1787, found

352.1787.

5-[4-(Cyclopropanecarbonyl)-9-methoxy-2,3,4,5-tetrahydro-1,4-

benzoxazepin-7-yl]-2-methoxybenzaldehyde (61). Standard proto-

col 2 with 1.5 g (4.6 mmol) of 53 and with 0.96 g (5.4 mmol) of 3-

formyl-4-methoxyphenylboronic acid. FCC with EtOAc and hexanes

(3:1, Rf 0.2) gave 1.4 g of 61 as a yellow solid (3.7 mmol, 80%); mp

1

152−153 °C. H NMR (100 °C, 400 MHz, DMSO-d₆) δ = 10.42 (s,

1H), 7.97−7.88 (m, 2H), 7.32−7.26 (m, 1H), 7.20 (s, 1H), 7.16 (d, J

= 2.2 Hz, 1H), 4.78 (s, 2H), 4.14−4.06 (m, 2H), 4.01−3.94 (m, 5H),

O

DOI: 10.1021/acs.jmedchem.6b00774

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

N-{5-[4-(Cyclopropanecarbonyl)-9-methoxy-2,3,4,5-tetrahydro-

1,4-benzoxazepin-7-yl]-2-methylphenyl}ethanesulfonamide (66).

To a solution of 0.058 g (0.15 mmol) of 65 and 5 mg (0.04 mmol)

of DMAP in 2.0 mL of pyridine were added 0.015 mL of (0.16 mmol)

ethanesulfonyl chloride at 0 °C. The mixture was warmed to RT and

stirred overnight. Then 1 M HCl was added and the mixture was

extracted with EtOAc three times. The combined organic layers were

dried over MgSO₄and concentrated in vacuo. FCC with EtOAc and

hexanes (2:1, Rf 0.3) gave 0.040 g (0.090 mmol, 60%) of 66 as a white

(methylenedioxy)phenylboronic acid. FCC with EtOAc and hexanes

(3:2, Rf 0.4) gave 0.21 g (0.58 mmol, 94%) of 70 as a pale-brown

1

solid; mp 127−128 °C. H NMR (110 °C, 400 MHz, C₂D₂Cl₄) δ =

7.01−6.94 (m, 4H), 6.82 (dd, J = 7.7, 0.8 Hz, 1H), 5.94 (s, 2H), 4.71

(s, 2H), 4.15−4.10 (m, 2H), 4.01−3.96 (m, 2H), 3.86 (s, 3H), 1.81−

1.70 (m, 1H), 0.95−0.87 (m, 2H), 0.76−0.69 (m, 2H). ¹³C NMR

(RT, mixture of rotamers, 101 MHz, C₂D₂Cl₄) δ = 172.6, 172.0, 151.6,

151.1, 147.9, 147.3, 147.2, 146.9, 146.8, 136.4, 136.0, 134.7, 134.5,

132.0, 131.7, 120.4, 120.1, 118.7, 110.8, 110.1, 108.5, 108.5, 107.4,

107.4, 101.2, 101.1, 72.4, 56.2, 50.9, 48.6, 48.3, 11.6, 11.4, 7.6, 7.5. MS

(EI+): m/z calcd for (C₂₁H₂₁NO₅) 367.1420, found 367.1433.

1-Cyclopropyl-1-(9-methoxy-7-(4-methoxy-3,5-dimethylphenyl)-

2,3-dihydro-1,4-benzoxazepin-4(5H)-yl)methanone (71). Standard

protocol 2 with 0.20 g (0.61 mmol) of 53 and 0.12 g (0.68 mmol) of

3,5-dimethyl-4-methoxyphenylboronic acid. FCC with EtOAc and

hexanes (3:2, Rf 0.4) gave 0.071 g (0.19 mmol, 31%) of 71 as a pale-

brown solid; mp 146−147 °C. ¹H NMR (110 °C, 400 MHz, C₂D₂Cl₄)

δ = 7.16−7.12 (m, 2H), 7.02−6.97 (m, 2H), 4.71 (s, 2H), 4.16−4.10

(m, 2H), 4.01−3.96 (m, 2H), 3.87 (s, 3H), 3.73 (s, 3H), 2.31 (s, 6H),

1.81−1.70 (m, 1H), 0.94−0.88 (m, 2H), 0.75−0.68 (m, 2H). ¹³C

NMR (110 °C, 101 MHz, C₂D₂Cl₄) δ = 172.6, 156.9, 152.0, 148.1,

136.8, 135.9, 132.2, 131.0, 127.4, 120.2, 112.6, 72.5, 59.7, 57.1, 50.2,

49.6, 16.2, 11.9, 7.2. MS (EI+): m/z calcd for (C₂₃H₂₇NO₄) 381.1940,

found 381.1940.

1

solid; mp 155−156 °C. H NMR (110 °C, 400 MHz, C₂D₂Cl₄) δ =

7.59 (d, J = 1.9 Hz, 1H), 7.28 (dd, J = 7.9, 1.9 Hz, 1H), 7.23 (d, J = 7.9

Hz, 1H), 7.04−6.99 (m, 2H), 6.00 (br s, 1H), 4.72 (s, 2H), 4.19−4.09

(m, 2H), 4.03−3.95 (m, 2H), 3.88 (s, 3H), 3.14 (q, J = 7.4 Hz, 2H),

2.32 (s, 3H), 1.83−1.69 (m, 1H), 1.37 (t, J = 7.4 Hz, 3H), 0.94−0.86

(m, 2H), 0.78−0.68 (m, 2H). ¹³C NMR (RT, mixture of rotamers, 126

MHz, C₂D₂Cl₄) δ = 172.7, 172.1, 151.7, 151.2, 147.8, 147.6, 139.7,

135.7, 135.3, 135.1, 135.0, 132.1, 131.7, 131.5, 128.5, 124.3, 124.2,

120.4, 120.2, 119.0, 111.0, 110.4, 72.4, 72.2, 56.2, 51.6, 50.8, 48.5, 48.2,

46.7, 46.6, 17.6, 11.6, 11.4, 8.2, 7.7, 7.5. MS (EI+): m/z calcd for

(C₂₃H₂₈N₂O₅S) 444.1719, found 444.1727.

1-{5-[4-(Cyclopropanecarbonyl)-9-methoxy-2,3,4,5-tetrahydro-

1,4-benzoxazepin-7-yl]-2-methylphenyl}-3-ethylurea (67). To a

solution of 0.058 g (0.15 mmol) of 65 in 1.0 mL of CH₂Cl₂were

added 0.30 mL of (1.7 mmol) DIPEA and 0.080 mL of (1.3 mmol)

ethyl isocyanate, and the mixture was stirred for 72 h. Then 0.1 M HCl

was added and the mixture was extracted with EtOAc three times. The

combined organic layers were dried over MgSO₄and concentrated in

vacuo. FCC with EtOAc and hexanes (2:1, Rf 0.3) gave 0.030 g (0.071

1-(7-(3-Chloro-4-methoxyphenyl)-9-methoxy-2,3-dihydro-1,4-

benzoxazepin-4(5H)-yl)-1-(cyclopropyl)methanone (72). Standard

protocol 2 with 0.20 g (0.61 mmol) of 53 and 0.13 g (0.67 mmol)

of 3-chloro-4-methoxyphenylboronic acid. FCC with EtOAc and

hexanes (3:2, Rf 0.3) gave 0.16 g (0.42 mmol, 69%) of 72 as a pale-

brown solid; mp 130−131 °C. ¹H NMR (110 °C, 400 MHz, C₂D₂Cl₄)

δ = 7.52 (d, J = 2.3 Hz, 1H), 7.36 (dd, J = 8.5, 2.3 Hz, 1H), 7.00−6.93

(m, 3H), 4.71 (s, 2H), 4.16−4.11 (m, 2H), 4.01−3.96 (m, 2H), 3.89

(s, 3H), 3.87 (s, 3H), 1.79−1.69 (m, 1H), 0.94−0.88 (m, 2H), 0.76−

0.68 (m, 2H). ¹³C NMR (110 °C, 101 MHz, C₂D₂Cl₄) δ = 172.3,

154.9, 152.2, 148.5, 135.3, 134.5, 132.5, 128.8, 126.2, 123.6, 120.0,

113.4, 112.4, 72.5, 57.1, 56.7, 50.1, 49.6, 11.8, 7.3. MS (ESI+): m/z

calcd for [(C₂₁H₂₃³⁵ClNO₄)⁺] 388.1316, found 388.1317.

1

mmol, 47%) of 67 as a white solid; mp 190−191 °C. H NMR (110

°C, 400 MHz, C₂D₂Cl₄) δ = 7.63 (d, J = 1.9 Hz, 1H), 7.31−7.16 (m,

2H), 7.09−6.98 (m, 2H), 5.99 (br s, 1H), 4.71 (s, 2H), 4.59 (br s,

1H), 4.17−4.08 (m, 2H), 4.03−3.95 (m, 2H), 3.87 (s, 3H), 3.24 (q, J

= 7.2 Hz, 2H), 2.25 (s, 3H), 1.81−1.69 (m, 1H), 1.12 (t, J = 7.2 Hz,

3H), 0.94−0.86 (m, 2H), 0.79−0.68 (m, 2H). ¹³C NMR (110 °C, 101

MHz, C₂D₂Cl₄) δ = 172.4, 156.0, 152.1, 148.5, 139.6, 137.1, 136.2,

132.3, 131.2, 130.8, 123.8, 123.4, 120.2, 112.6, 72.4, 57.1, 50.2, 49.7,

35.5, 17.3, 15.4, 11.8, 7.3. MS (EI+): m/z calcd for (C₂₄H₂₉N₃O₄)

423.2158, found 423.2157.

1-Cyclopropyl-1-{7-[3-(hydroxymethyl)-4-methoxyphenyl]-9-me-

thoxy-2,3-dihydro-1,4-benzoxazepin-4(5H)-yl}methanone (73). To

a suspension of 0.078 g (2.0 mmol) of sodium borohydride in a

mixture of 7.5 mL of CH₂Cl₂and 2.5 mL of MeOH were added 0.43 g

(1.1 mmol) of 61, and the mixture was stirred for 2 h. Then 20 mL of

2 M HCl was added, and after 15 min of stirring, the mixture was

extracted with EtOAc three times. The combined organic layers were

dried over MgSO₄and concentrated in vacuo to give 0.42 g (1.9

1-Cyclopropyl-1-[7-(3,5-dimethoxyphenyl)-9-methoxy-2,3-dihy-

dro-1,4-benzoxazepin-4(5H)-yl]methanone (68). Standard protocol

2 with 0.12 g (0.37 mmol) of 53 and 0.078 g (0.43 mmol) of 3,5-

dimethoxyphenylboronic acid. FCC with EtOAc and hexanes (2:1, Rf

0.4) gave 0.090 g (0.23 mmol, 62%) of 68 as a white solid; mp 50−51

1

°C. H NMR (110 °C, 400 MHz, C₂D₂Cl₄) δ = 7.10−6.96 (m, 2H),

6.66 (d, J = 2.2 Hz, 2H), 6.44 (t, J = 2.2 Hz, 1H), 4.72 (s, 2H), 4.20−

4.09 (m, 2H), 4.04−3.95 (m, 2H), 3.87 (s, 3H), 3.81 (s, 6H), 1.85−

1.70 (m, 1H), 0.99−0.85 (m, 2H), 0.79−0.66 (m, 2H). ¹³C NMR

(RT, mixture of rotamers, 101 MHz, C₂D₂Cl₄) δ = 172.6, 172.1, 160.8,

160.8, 151.6, 151.1, 147.8, 147.7, 142.6, 142.3, 136.5, 136.0, 131.9,

131.5, 120.4, 119.1, 111.0, 110.4, 105.5, 105.1, 99.1, 98.6, 72.3, 72.2,

56.2, 55.4, 50.9, 48.5, 48.2, 11.6, 11.4, 7.6, 7.5. MS (EI+): m/z calcd for

(C₂₂H₂₅NO₅) 383.1733, found 383.1759.

1

mmol, 97%) of 73 as a white solid; mp 74−75 °C. H NMR (RT,

mixture of rotamers, 500 MHz, CDCl₃): δ = 7.52−7.39 (m, 2H),

7.14−6.87 (m, 3H), 4.82−4.66 (m, 4H), 4.29−4.21 (m, 0.7H), 4.19−

4.00 (m, 3.3H), 3.95−3.86 (m, 6H), 2.35 (s, 1H), 1.92−1.85 (m,

0.7H), 1.73−1.67 (m, 0.3H), 1.01−0.90 (m, 2H), 0.82−0.69 (m, 2H).

¹³C NMR (RT, mixture of rotamers, 101 MHz, CDCl₃): δ = 172.8,

172.1, 157.0, 156.9, 152.0, 151.3, 147.5, 147.1, 136.7, 136.4, 133.2,

132.4, 132.0, 129.5, 129.3, 127.43, 127.39, 127.3, 120.5, 118.9, 110.8,

110.5, 110.5, 110.1, 72.81, 72.75, 62.1, 62.0, 56.3, 56.2, 55.5, 51.2, 51.2,

48.9, 48.6, 11.6, 11.5, 7.7, 7.5. MS (ESI+): m/z calcd for

[(C₂₂H₂₆NO₅)⁺] 384.1811, found 384.1806.

1-Cyclopropyl-1-[9-methoxy-7-(4-methoxy-3-methylphenyl)-2,3-

dihydro-1,4-benzoxazepin-4(5H)-yl]methanone (69). Standard pro-

tocol 2 with 0.50 g (1.5 mmol) of 53 and 0.28 g (1.7 mmol) of 4-

methoxy-3-methylphenylboronic acid. FCC with EtOAc and hexanes

(3:2, Rf 0.3) gave 0.38 g (1.0 mmol, 68%) of 69 as a pale-brown solid;

1-Cyclopropyl-1-[7-(3,4-diﬂuorophenyl)-9-methoxy-2,3-dihydro-

1,4-benzoxazepin-4(5H)-yl]methanone (74). Standard protocol 2

with 0.12 g (0.37 mmol) of 53 and 0.068 g (0.43 mmol) of 3,4-

diﬂuorophenylboronic acid. FCC with EtOAc and hexanes (2:1, Rf

0.4) gave 0.090 g (0.25 mmol, 68%) of 74 as a white solid; mp 136−

1

mp 109−110 °C. H NMR (110 °C, 400 MHz, C₂D₂Cl₄) δ = 7.32−

7.27 (m, 2H), 7.02−6.96 (m, 2H), 6.87−6.82 (m, 1H), 4.71 (s, 2H),

4.15−4.10 (m, 2H), 4.00−3.95 (m, 2H), 3.87 (s, 3H), 3.83 (s, 3H),

2.26 (s, 3H), 1.81−1.69 (m, 1H), 0.94−0.87 (m, 2H), 0.75−0.68 (m,

2H). ¹³C NMR (RT, mixture of rotamers, 101 MHz, C₂D₂Cl₄) δ =

172.6, 172.0, 157.22, 157.17, 151.6, 151.1, 147.0, 146.9, 136.6, 136.2,

132.4, 132.1, 131.9, 131.7, 129.2, 129.1, 126.9, 126.8, 125.2, 125.1,

120.0, 118.6, 110.7, 110.1, 110.0, 72.4, 72.3, 56.2, 55.4, 50.9, 48.6, 48.4,

16.4, 16.3, 11.6, 11.4, 7.6, 7.4. MS (EI+): m/z calcd for (C₂₂H₂₅NO₄)

367.1784, found 367.1784.

1

137 °C. H NMR (110 °C, 400 MHz, C₂D₂Cl₄) δ = 7.34−7.26 (m,

1H), 7.26−7.09 (m, 2H), 7.04−6.91 (m, 2H), 4.72 (s, 2H), 4.22−4.10

(m, 2H), 4.04−3.94 (m, 2H), 3.88 (s, 3H), 1.83−1.66 (m, 1H), 0.95−

0.86 (m, 2H), 0.79−0.66 (m, 2H). ¹³C NMR (110 °C, 101 MHz,

1

2

C₂D₂Cl₄) δ = 172.4, 152.3, 150.6 (dd, J_CF= 248.6 Hz, J_CF= 12.8

Hz), 150.0 (dd, J_CF= 248.9 Hz, ²J_CF= 12.6 Hz), 149.0, 137.9, 134.8,

1

132.6, 122.9 (dd, ³J_CF= 5.5, ⁴J_CF= 3.4 Hz), 120.2, 117.5 (d, ²J_CF= 17.4

1-[7-(Benzo[d][1,3]dioxol-5-yl)-9-methoxy-2,3-dihydro-1,4-ben-

zoxazepin-4(5H)-yl]-1-(cyclopropyl)methanone (70). Standard pro-

tocol 2 with 0.20 g (0.61 mmol) of 53 and 0.11 g (0.67 mmol) of 3,4-

2

Hz), 115.9 (d, J_CF= 17.8 Hz), 112.5, 72.5, 57.1, 50.0, 49.6, 11.8, 7.3.

MS (EI+): m/z calcd for (C₂₀H₁₉F₂NO₃) 359.1333, found 359.1335.

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Article

1

1-Cyclopropyl-1-[7-(3,4-dichlorophenyl)-9-methoxy-2,3-dihydro-

1,4-benzoxazepin-4(5H)-yl]methanone (75). Standard protocol 2

with 0.098 g (0.30 mmol) of 53 and 0.068 g (0.36 mmol) of 3,4-

dichlorophenylboronic acid. FCC with EtOAc and hexanes (2:1, Rf

0.4) gave 0.095 g (0.24 mmol, 81%) of 75 as a pale-brown solid; mp

115−116 °C. ¹H NMR (RT, mixture of rotamers, 500 MHz, CD₂Cl₂)

δ = 7.75−7.60 (m, 1H), 7.59−7.46 (m, 1H), 7.46−7.36 (m, 1H),

7.17−6.96 (m, 2H), 4.77 (s, 1.2H), 4.66 (s, 0.8H), 4.23−4.16 (m,

0.8H), 4.12−4.05 (m, 2H), 4.01−3.95 (m, 1.2H), 3.93−3.84 (m, 3H),

1.94−1.82 (m, 0.6H), 1.74−1.68 (m, 0.4H), 0.89−0.82 (m, 2H),

0.79−0.67 (m, 2H). ¹³C NMR (RT, mixture of rotamers, 101 MHz,

CD₂Cl₂) δ = 172.9, 172.4, 152.7, 152.2, 149.1, 149.0, 141.2, 141.1,

134.6, 134.3, 133.4, 133.3, 133.0, 131.6, 131.4, 131.1, 131.0, 129.1,

129.0, 126.8, 120.9, 119.5, 111.3, 110.7, 73.2, 72.9, 56.7, 51.6, 51.3,

49.1, 48.8, 11.8, 11.6, 7.7, 7.5. MS (EI+): m/z calcd for

(C₂₀H₁₉NO₃³⁵Cl₂) 391.0742, found 391.0749.

as a white solid; mp 153−154 °C. H NMR (400 MHz, CDCl₃) δ =

7.46 (d, J = 2.3 Hz, 1H), 7.30 (dd, J = 8.5, 2.3 Hz, 1H), 6.92−6.87 (m,

3H), 4.72 (s, 2H), 4.41 (s, 2H), 4.10−4.05 (m, 2H), 3.87−3.81 (m,

6H), 3.81−3.76 (m, 2H). ¹³C NMR (RT, 101 MHz, CDCl₃) δ =

157.0, 153.4, 151.0, 146.5, 134.4, 132.9, 131.1, 127.6, 125.1, 121.7,

118.1, 111.2, 109.6, 71.5, 55.3, 49.4, 49.0. MS (EI+): m/z calcd for

(C₁₈H₁₉³⁵ClN₂O₄) 362.1033, found 362.1029.

1-[7-(3-Chloro-4-methoxyphenyl)-9-methoxy-2,3-dihydro-1,4-

benzoxazepin-4(5H)-yl]-2-hydroxyethan-1-one (80). A solution of

0.10 g (0.31 mmol) of 77 in 0.95 mL (10 mmol) of ethyl glycolate was

heated to 60 °C. After 24 and 48 h, EtOH was removed in vacuo. After

84 h, puriﬁcation by FCC with EtOAc and hexanes (5:1, Rf 0.2) gave

0.060 g (0.16 mmol, 51%) of 80 as a white solid; mp 160−161 °C. ¹H

NMR (RT, mixture of rotamers, 500 MHz, CDCl₃) δ = 7.57 (d, J = 2.3

Hz, 0.4H), 7.54 (d, J = 2.3 Hz, 0.6H), 7.42 (dd, J = 8.5, 2.3 Hz, 0.4H),

7.38 (dd, J = 8.5, 2.3 Hz, 0.6H), 7.12 (d, J = 2.2 Hz, 0.4H), 7.02−6.96

(m, 2H), 6.87 (d, J = 2.2 Hz, 0.6H), 4.76 (s, 0.8H), 4.42 (s, 1.2H),

4.35−4.31 (m, 1.2H), 4.21−4.06 (m, 4H), 3.98−3.89 (m, 6H), 3.72−

3.65 (m, 0.8H), 3.52 (t, J = 4.4 Hz, 0.4H), 3.48 (t, J = 4.4 Hz, 0.6H).

¹³C NMR (RT, mixture of rotamers, 126 MHz, CDCl₃) δ = 171.2,

5-[4-(Cyclopropanecarbonyl)-9-methoxy-2,3,4,5-tetrahydro-1,4-

benzoxazepin-7-yl]-2-methoxybenzaldehyde O-methyl Oxime (76).

To a suspension of 0.15 g (0.39 mmol) of 61 in 6 mL of EtOH were

added 0.13 g (1.5 mmol) of O-methylhydroxylamine hydrochloride

and 0.21 g (1.5 mmol) of K₂CO₃. After 12 h, the mixture was

concentrated in vacuo, treated with EtOAc and saturated NaCl

solution, and extracted with EtOAc three times. The combined organic

layers were dried over MgSO₄and concentrated in vacuo. FCC with

CH₂Cl₂with 2% MeOH (Rf 0.3) gave 0.16 g (0.39 mmol, 99%) of 76

170.6, 154.6, 154.5, 152.2, 151.6, 147.6, 147.4, 136.0, 135.8, 133.9,

133.7, 131.4, 131.1, 128.72, 128.71, 126.24, 126.21, 122.9, 122.8,

120.1, 119.0, 112.3, 112.2, 111.1, 110.4, 72.5, 72.1, 60.0, 56.31, 56.28,

56.2, 49.4, 49.3, 49.2, 48.7. MS (EI+): m/z calcd for (C₁₉H₂₀³⁵ClNO)

377.1030, found 377.1021.

1

as a white solid; mp 78−79 °C. H NMR (RT, mixture of rotamers,

7-(3-Chloro-4-methoxyphenyl)-9-methoxy-N-methyl-2,3-dihy-

dro-1,4-benzoxazepine-4(5H)-carbothioamide (81). To a solution of

0.15 g (0.47 mmol) of 77 in 3 mL of anhydrous THF was added 0.028

g (0.71 mmol) of a 60% suspension of NaH. After 15 min, a solution

of 0.068 g (0.94 mmol) of methyl isothiocyanate in 1.0 mL of

anhydrous THF was added and the mixture was stirred for 3 h at RT.

Saturated NaHCO₃solution was added and the mixture extracted with

CH₂Cl₂three times. The combined organic layers were dried over

MgSO₄and concentrated in vacuo. FCC with EtOAc and hexanes

(3:2, Rf 0.3) gave 0.13 g (0.32 mmol, 69%) of 81 as a white solid; mp

200−201 °C. ¹H NMR (RT, mixture of rotamers, 500 MHz, CDCl₃) δ

= 7.54 (d, J = 2.3 Hz, 1H), 7.38 (dd, J = 8.5, 2.3 Hz, 1H), 7.07−6.81

(m, 3H), 5.80−5.63 (m, 1H), 4.85−4.66 (m, 2H), 4.51−4.42 (m, 2H),

4.28−4.15 (m, 2H), 4.01−3.79 (m, 6H), 3.15−3.05 (m, 3H). ¹³C

NMR (RT, mixture of rotamers, 126 MHz, CDCl₃) δ = 182.9, 154.5,

152.2, 147.2, 135.6, 133.8, 130.3, 130.1, 128.7, 126.2, 124.0, 122.8,

120.4, 119.0, 112.6, 112.3, 111.0, 72.0, 71.9, 56.3, 56.1, 54.7, 54.4, 52.0,

51.8, 33.2, 33.1. MS (EI+): m/z calcd for (C₁₉H₂₁³⁵ClN₂O₃S)

392.0961, found 392.0953.

500 MHz, CD₂Cl₂): δ = 8.45 (s, 1H), 7.95 (d, J = 2.4 Hz, 1H), 7.60−

7.50 (m, 1H), 7.13−6.95 (m, 3H), 4.77 (s, 1.3H), 4.67 (s, 0.7H),

4.23−4.13 (m, 0.7H), 4.10−4.04 (m, 2H), 4.00−3.95 (m, 4.3H),

3.90−3.86 (m, 6H), 1.96−1.88 (m, 0.7H), 1.73−1.67 (m, 0.3H),

0.91−0.80 (m, 2H), 0.80−0.67 (m, 2H). ¹³C NMR (RT, mixture of

rotamers, 126 MHz, CD₂Cl₂): δ = 172.9, 172.3, 157.5, 152.5, 152.0,

148.2, 148.0, 144.74, 144.65, 136.6, 136.1, 133.7, 133.5, 133.2, 133.1,

129.9, 124.9, 124.7, 121.4, 120.6, 119.3, 111.9, 111.3, 110.7, 73.2, 73.0,

62.2, 56.7, 56.2, 51.6, 51.4, 49.2, 49.0, 11.8, 11.6, 7.7, 7.5. MS (ESI+):

m/z calcd for [(C₂₃H₂₇N₂O₅)⁺] 411.1920, found 411.1912.

7-(3-Chloro-4-methoxyphenyl)-9-methoxy-2,3,4,5-tetrahydro-

1,4-benzoxazepine (77). Standard protocol 2 with 1.5 g (5.8 mmol)

of 107 and 1.3 g (7.0 mmol) of 3-chloro-4-methoxyphenylboronic

acid. FCC with EtOAc with 5% triethylamine (Rf 0.1) gave 0.90 g (2.8

1

mmol, 48%) of 77 as a white solid; mp 117−118 °C. H NMR (RT,

500 MHz, CDCl₃) δ = 7.56 (d, J = 2.3 Hz, 1H), 7.40 (dd, J = 8.5, 2.3

Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 6.95 (d, J = 2.1 Hz, 1H), 6.89 (d, J

= 2.1 Hz, 1H), 4.14−4.10 (m, 2H), 4.02 (s, 2H), 3.94 (s, 3H), 3.93 (s,

3H), 3.29−3.24 (m, 2H). ¹³C NMR (RT, 101 MHz, CDCl₃) δ =

154.3, 151.8, 148.5, 136.7, 135.0, 134.4, 128.7, 126.1, 122.7, 119.5,

112.2, 109.8, 75.5, 56.33, 56.27, 53.2, 52.3. MS (EI+): m/z calcd for

(C₁₇H₁₈³⁵ClNO₃) 319.0975, found 319.0972.

1-[7-(3-Chloro-4-methoxyphenyl)-9-methoxy-2,3-dihydro-1,4-

benzoxazepin-4(5H)-yl]propane-1-thione (82). A solution of 0.052 g

(0.13 mmol) of 86 and 0.073 g (0.18 mmol) of Lawesson’s reagent in

1.0 mL of anhydrous THF was stirred at RT for 72 h. Water was added

and the mixture extracted with CH₂Cl₂three times. The combined

organic layers were dried over MgSO₄and concentrated in vacuo.

FCC with EtOAc and hexanes (3:1, Rf 0.7) gave 0.051 g (0.13 mmol,

7-(3-Chloro-4-methoxyphenyl)-9-methoxy-N,N-dimethyl-2,3-di-

hydro-1,4-benzoxazepine-4(5H)-carboxamide (78). Standard proto-

col 1 with 0.10 g (0.31 mmol) of 77 and 0.24 mL (2.6 mmol) of N,N-

dimethylcarbamoyl chloride. FCC with EtOAc and hexanes (5:1, Rf

0.1) gave 0.090 g (0.23 mmol, 74%) of 78 as a white solid; mp 85−86

1

96%) of 82 as a white solid; mp 82−83 °C. H NMR (110 °C, 400

MHz, C₂D₂Cl₄) δ = 7.61−6.82 (m, 5H), 5.30 (s, 0.9H), 4.85 (s, 1.1H),

4.68 (s, 1.1H), 4.32−4.03 (m, 2.9H), 3.94−3.83 (m, 6H), 2.98−2.64

(m, 2H), 1.35−1.21 (m, 3H). ¹³C NMR (RT, mixture of rotamers, 101

MHz, C₂D₂Cl₄) δ = 206.4, 205.1, 154.3, 154.2, 151.9, 151.3, 147.24,

147.19, 135.4, 134.8, 133.6, 133.5, 129.4, 129.0, 128.5, 126.2, 122.5,

122.4, 121.6, 119.2, 112.3, 112.2, 111.1, 110.4, 71.5, 70.8, 56.4, 56.4,

56.3, 56.2, 54.9, 54.4, 54.1, 37.0, 35.9, 13.9, 13.5. MS (EI+): m/z calcd

for (C₂₀H₂₂³⁵ClNO₃S) 391.1009, found 391.0988.

1

°C. H NMR (500 MHz, CD₂Cl₂) δ = 7.58 (d, J = 2.3 Hz, 1H), 7.44

(dd, J = 8.5, 2.3 Hz, 1H), 7.05−6.94 (m, 3H), 4.38 (s, 2H), 4.17−4.14

(m, 2H), 3.92 (s, 3H), 3.88 (s, 3H), 3.67−3.62 (m, 2H), 2.80 (s, 6H).

¹³C NMR (RT, 126 MHz, CD₂Cl₂) δ = 164.9, 154.7, 152.0, 148.7,

135.0, 134.5, 133.2, 128.9, 126.6, 122.8, 120.2, 112.7, 110.4, 73.0,

56.60, 56.57, 52.8, 52.7, 39.1. MS (EI+): m/z calcd for

(C₂₀H₂₃³⁵ClN₂O₄) 390.1346, found 390.1348.

7-(3-Chloro-4-methoxyphenyl)-9-methoxy-2,3-dihydro-1,4-ben-

zoxazepine-4(5H)-carboxamide (79). To a solution of 0.10 g (0.31

mmol) of 77 in 2 mL of CH₂Cl₂was added 0.42 mL of (3.1 mmol)

(trimethylsilyl)isocyanate and the mixture was stirred for 2.5 h. The

solvent was removed in vacuo. The residue was dissolved in CH₂Cl₂

and 7 mL of a solution of 4 M HCl in 1,4-dioxane was added, and the

mixture stirred for 1 h. After adjustment to pH 9 with 1 M NaOH, the

mixture was extracted with CH₂Cl₂three times. The combined organic

layers were dried over MgSO₄and concentrated in vacuo. FCC with

EtOAc and 3% MeOH (Rf 0.2) gave 0.098 g (0.27 mmol, 87%) of 79

1-[7-(3-Chloro-4-methoxyphenyl)-9-methoxy-2,3-dihydro-1,4-

benzoxazepin-4(5H)-yl]-2,2,2-triﬂuoroethan-1-one (83). To a sol-

ution of 0.040 g (0.13 mmol) of 77 and 5 mg (0.04 mmol) of DMAP

in 1.0 mL of CH₂Cl₂was added 0.070 mL of (0.50 mmol)

triﬂuoroacetic anhydride at 0 °C. The mixture was stirred and warmed

to RT. Then 0.70 mL of (41 mmol) DIPEA was added and the

mixture stirred for a further 12 h. Saturated NaHCO₃solution was

added, and the mixture was extracted with CH₂Cl₂three times. The

combined organic layers were dried over MgSO₄and concentrated in

vacuo. FCC with EtOAc and hexanes (1:2, Rf 0.2) gave 0.050 g (0.12

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mmol, 92%) of 83 as a white solid; mp 143−144 °C. H NMR (110

°C, 400 MHz, C₂D₂Cl₄) δ = 7.52 (d, J = 2.3 Hz, 1H), 7.36 (dd, J = 8.6,

2.3 Hz, 1H), 7.09−6.90 (m, 3H), 4.69 (s, 2H), 4.18−4.12 (m, 2H),

4.05−3.93 (m, 2H), 3.90 (s, 3H), 3.88 (s, 3H). ¹³C NMR (RT,

mixture of rotamers, 101 MHz, C₂D₂Cl₄) δ = 156.2, 156.2, 155.9,

155.8, 155.52, 155.45, 155.2, 154.3, 151.6, 147.4, 135.6, 135.5, 133.5,

133.4, 130.6, 129.9, 128.5, 126.2, 122.5, 120.1, 119.2, 117.6, 117.5,

114.8, 114.6, 112.31, 112.25, 111.1, 110.8, 72.6, 71.7, 56.3, 56.2, 51.3,

50.6, 50.0, 49.9. MS (EI+): m/z calcd for (C₁₉H₁₇³⁵ClF₃NO₄)

415.0798, found 415.0792.

1H), 7.09−6.97 (m, 3H), 4.56−4.48 (m, 2H), 4.08−4.02 (m, 2H),

3.92 (s, 3H), 3.89−3.82 (m, 5H), 3.68−3.59 (m, 3H). ¹³C NMR (RT,

mixture of rotamers, 101 MHz, C₂D₂Cl₄) δ = 156.4, 156.1, 154.8,

152.4, 152.3, 148.4, 135.4, 135.3, 134.4, 134.3, 133.9, 133.7, 128.9,

126.6, 122.9, 120.2, 119.8, 112.7, 110.8, 110.6, 73.2, 56.6, 53.0, 52.9,

50.7, 50.6, 50.5, 50.3. MS (EI+): m/z calcd for (C₁₉H₂₀³⁵ClNO₅)

377.1030, found 377.1026.

1-[7-(3,4-Dimethoxyphenyl)-9-methoxy-2,3-dihydro-1,4-benzo-

thiazepin-4(5H)-yl]propan-1-one (88). Standard protocol 2 with 0.20

g (0.61 mmol) of 111 and 0.22 g (1.2 mmol) of 3,4-

dimethoxyphenylboronic acid. FCC with EtOAc and hexanes (1:4,

Rf 0.3) gave 0.14 g (0.36 mmol, 59%) of 88 as a white solid; mp 82−

1-[7-(3-Chloro-4-methoxyphenyl)-9-methoxy-2,3-dihydro-1,4-

benzoxazepin-4(5H)-yl]-3,3,3-triﬂuoropropan-1-one (84). To a

solution of 0.066 g (0.21 mmol) of 77 and 5 mg (0.04 mmol) of

DMAP in 2.0 mL of CH₂Cl₂was added 0.035 mL of (0.40 mmol)

3,3,3-triﬂuoropropionic acid at 0 °C. After 5 min, 0.058 g (0.30 mmol)

of EDC·HCl was added, and the mixture warmed to RT and stirred for

further 12 h. Saturated NaHCO₃solution was added and the mixture

extracted with CH₂Cl₂three times. The combined organic layers were

dried over MgSO₄and concentrated. FCC with EtOAc and hexanes

(1:1, Rf 0.4) gave 0.082 g (0.19 mmol, 94%) of 84 as a white solid; mp

105−106 °C. ¹H NMR (RT, mixture of rotamers, 400 MHz, CDCl₃) δ

= 7.56 (d, J = 2.3 Hz, 0.4H), 7.54 (d, J = 2.3 Hz, 0.6H), 7.46−7.34 (m,

1H), 7.11 (d, J = 2.1 Hz, 0.4H), 7.07−6.92 (m, 2H), 6.88 (d, J = 2.1

Hz, 0.6H), 4.72 (s, 0.8H), 4.57 (s, 1.2H), 4.24−4.13 (m, 2H), 4.11−

4.01 (m, 1.2H), 4.00−3.84 (m, 6.8H), 3.31 (q, J = 9.9 Hz, 1.2H), 3.23

(q, J = 9.9 Hz, 0.8H). ¹³C NMR (RT, mixture of rotamers, 101 MHz,

1

83 °C. H NMR (RT, mixture of rotamers, 500 MHz, CD₂Cl₂) δ =

7.37 (d, J = 1.9 Hz, 0.5H), 7.20−7.06 (m, 2.5H), 7.02−6.92 (m, 2H),

4.82−4.64 (m, 2H), 4.08−3.89 (m, 8H), 3.89−3.86 (m, 3H), 2.93−

2.76 (m, 2H), 2.44 (q, J = 7.4 Hz, 0.8H), 2.27 (q, J = 7.4 Hz, 1.2H),

1.2 (t, J = 7.4 Hz, 3H). ¹³C NMR (RT, mixture of rotamers, 126 MHz,

CD₂Cl₂) δ = 173.6, 172.6, 159.4, 159.2, 149.9, 149.8, 149.7, 149.6,

144.6, 143.4, 141.4, 141.2, 133.6, 133.5, 123.8, 123.2, 122.7, 121.2,

119.4 119.79, 112.2, 112.1, 111.1, 111.0, 109.4, 108.8, 56.74, 56.67,

56.38, 56.30, 56.26, 52.33, 52.25, 49.7, 35.3, 34.0, 27.11, 27.06, 9.42,

9.40. MS (ESI+): m/z calcd for [(C₂₁H₂₆NO₄S)⁺] 388.1583, found

388.1575.

1-Cyclopropyl-1-[7-(3,4-dimethoxyphenyl)-9-methoxy-2,3-dihy-

dro-1,4-benzothiazepin-4(5H)-yl]methanone (89). Standard proto-

col 2 with 0.18 g (0.53 mmol) of 112 and 0.25 g (1.4 mmol) of 3,4-

dimethoxyphenylboronic acid. FCC with EtOAc and hexanes (1:1, Rf

0.1) gave 0.18 g (0.45 mmol, 85%) of 89 as a white solid; mp 92−93

°C. ¹H NMR (RT, mixture of rotamers, 400 MHz, CD₂Cl₂) δ = 7.36−

6.89 (m, 5H), 4.91 (s, 1H), 4.75 (s, 1H), 4.21−3.97 (m, 2H), 3.97−

3.82 (m, 9H), 2.96−2.80 (m, 2H), 2.00−1.88 (m, 0.5H), 1.70−1.64

(m, 0.5H), 0.90−0.81 (m, 2H), 0.79−0.65 (m, 2H). ¹³C NMR (RT,

mixture of rotamers, 101 MHz, CD₂Cl₂) δ = 173.4, 172.2, 159.3,

159.2, 149.80, 149.77, 149.7, 149.6, 144.7, 143.7, 141.4, 141.2, 133.62,

133.56, 123.8, 123.1, 122.6, 120.9, 119.81, 119.77, 112.08, 112.05,

111.01, 110.97, 109.3, 108.8, 56.71, 56.66, 56.4, 56.3, 56.2, 52.7, 52.6,

49.9, 35.4, 33.9, 12.1, 11.6, 8.0, 7.4. MS (ESI+): m/z calcd for

[(C₂₂H₂₆NO₄S)⁺] 400.1583, found 400.1577.

1-[7-(3-Chloro-4-methoxyphenyl)-9-methoxy-2,3-dihydro-1,4-

benzothiazepin-4(5H)-yl]-1-(cyclopropyl)methanone (90). Standard

protocol 2 with 0.18 g (0.53 mmol) of 112 and 0.26 g (1.4 mmol) of

3-chloro-4-methoxyphenylboronic acid. FCC with EtOAc and hexanes

(1:1, Rf 0.2) gave 0.15 g (0.37 mmol, 70%) of 90 as a white solid; mp

95−96 °C. ¹H NMR (110 °C, 400 MHz, C₂D₂Cl₄) δ = 7.55 (d, J = 2.4

Hz, 1H), 7.40 (dd, J = 8.5, 2.4 Hz, 1H), 7.22−7.09 (m, 1H), 7.05−

6.87 (m, 2H), 4.81 (s, 2H), 4.12−3.99 (m, 2H), 3.98−3.84 (m, 6H),

2.97−2.81 (m, 2H), 1.81−1.64 (m, 1H), 0.95−0.84 (m, 2H), 0.77−

0.63 (m, 2H). ¹³C NMR (110 °C, 101 MHz, C₂D₂Cl₄) δ = 172.4,

159.2, 155.2, 143.4, 139.4, 134.2, 128.9, 126.3, 124.7, 123.7, 121.7,

113.4, 109.8, 56.9, 56.7, 52.9, 50.6, 34.3, 12.0, 7.3. MS (EI+): m/z

calcd for (C₂₁H₂₂³⁵ClNO₃S) 403.1009, found 403.0998.

4-(tert-Butyl) 9-Methyl 7-Bromo-2,3-dihydro-1,4-benzoxazepine-

4,9(5H)-dicarboxylate (91). To a solution of 2.0 g (7.6 mmol) of

triphenylphosphine in 50 mL of anhydrous THF was added 1.5 mL

(7.6 mmol) of DIAD at 0 °C under N₂. After 20 min, a solution of 2.0

g (5.0 mmol) of 110 in 80 mL of anhydrous THF was added and the

mixture was stirred overnight. After concentration in vacuo, FCC with

EtOAc and hexanes (1:4, Rf 0.3) gave 1.7 g (4.3 mmol, 85%) of 114 as

a white solid; mp 89−90 °C. ¹H NMR (70 °C, 400 MHz, DMSO-d₆)

δ = 7.65 (d, J = 2.6 Hz, 1H), 7.60 (d, J = 2.6 Hz, 1H), 4.44 (s, 2H),

4.09−4.03 (m, 2H), 3.82 (s, 3H), 3.78−3.71 (m, 2H), 1.35 (s, 9H).

¹³C NMR (70 °C, 101 MHz, DMSO-d₆) δ = 164.5, 156.5, 153.8,

CDCl₃) δ = 162.7 (q, ³J_CF= 2.9 Hz), 161.9 (q, J_CF= 2.9 Hz), 154.7,

3

154.4, 152.3, 151.4, 147.6, 147.4, 136.1, 135.7, 133.9, 133.7, 131.2,

131.0, 128.8, 128.7, 126.2, 125.3, 122.9, 122.7, 122.6, 120.5, 118.3,

112.3, 112.2, 111.3, 110.4, 72.3, 72.2, 56.32, 56.31, 56.27, 56.23, 51.8,

51.6, 48.8, 48.3, 38.4 (q, ²J_CF= 29.4 Hz), 38.1 (q, ²J_CF= 29.4 Hz). MS

(EI+): m/z calcd for (C₂₀H₁₉³⁵ClF₃NO₄) 429.0954, found 429.0950.

1-[7-(3-Chloro-4-methoxyphenyl)-9-methoxy-2,3-dihydro-1,4-

benzoxazepin-4(5H)-yl]-2-ﬂuoropropan-1-one (85). To a solution of

0.050 g (0.16 mmol) of 77 and 5 mg (0.04 mmol) of DMAP in 1.0 mL

of CH₂Cl₂was added 0.019 mL (0.23 mmol) of 2-ﬂuoropropionic acid

at 0 °C. After 5 min, 0.061 g (0.32 mmol) of EDC·HCl was added and

the mixture warmed to RT and stirred for a further 12 h. Saturated

NaHCO₃solution was added and the mixture extracted with CH₂Cl₂

three times. The combined organic layers were dried over MgSO₄and

concentrated in vacuo. FCC with EtOAc and hexanes (1:1, Rf 0.3)

1

gave 0.50 g (0.13 mmol, 81%) of 85 as a colorless oil. H NMR (110

°C, 400 MHz, C₂D₂Cl₄) δ = 7.53 (d, J = 2.4 Hz, 1H), 7.37 (dd, J = 8.5,

2

3

2.4 Hz, 1H), 7.05−6.91 (m, 3H), 5.23 (dq, J_HF= 48.5 Hz, J_HH6.7

Hz, 1H), 4.75−4.59 (m, 2H), 4.25−4.06 (m, 2H), 4.03−3.76 (m, 8H),

1.52 (dd, J = 24.6, 6.6 Hz, 3H). ¹³C NMR (110 °C, 101 MHz,

C₂D₂Cl₄) δ = 168.1 (d, J_CF= 18.8 Hz), 155.0, 152.1, 148.5, 135.5,

134.4, 131.8, 128.8, 126.2, 123.6, 120.3, 113.4, 112.5, 87.2 (d, J_CF

2

1

=

2

178.2 Hz), 72.5, 57.1, 56.7, 49.9, 17.8 (d, J_CF= 22.9 Hz). MS (EI+):

m/z calcd for (C₂₀H₂₁³⁵ClFNO₄) 393.1143, found 393.1139.

1-[7-(3-Chloro-4-methoxyphenyl)-9-methoxy-2,3-dihydro-1,4-

benzoxazepin-4(5H)-yl]propan-1-one (86). Standard protocol 1 with

0.16 g (0.50 mmol) of 77 and 0.16 mL (0.65 mmol) of propionyl

chloride. FCC with EtOAc and hexanes (3:1, Rf 0.4) gave 0.075 g

1

(0.20 mmol, 40%) of 86 as a white solid; mp 66−67 °C. H NMR

(110 °C, 400 MHz, C₂D₂Cl₄) δ = 7.52 (d, J = 2.3 Hz, 1H), 7.37 (dd, J

= 8.5, 2.3 Hz, 1H), 7.06−6.86 (m, 3H), 4.59 (s, 2H), 4.15−4.06 (m,

2H), 3.94−3.84 (m, 8H), 2.47−2.24 (m, 2H), 1.10 (t, J = 7.4 Hz, 3H).

¹³C NMR (RT, mixture of rotamers, 101 MHz, C₂D₂Cl₄) δ = 172.9,

172.3, 154.3, 154.1, 151.9, 151.3, 147.6, 147.5, 135.1, 134.7, 133.7,

133.6, 132.1, 131.8, 128.5, 128.4, 126.2, 126.1, 122.5, 122.4, 120.0,

118.8, 112.3, 112.2, 110.7, 110.0, 72.4, 72.3, 56.29, 56.24, 56.22, 50.8,

48.3, 47.9, 26.7, 26.4, 9.19, 9.15. MS (EI+): m/z calcd for

(C₂₀H₂₂³⁵ClNO₄) 375.1237, found 375.1235.

Methyl 7-(3-Chloro-4-methoxyphenyl)-9-methoxy-2,3-dihydro-

1,4-benzoxazepine-4(5H)-carboxylate (87). Standard protocol 1

with 0.10 g (0.31 mmol) of 77 and 0.20 mL (2.6 mmol) of methyl

chloroformate. FCC with EtOAc and hexanes (1:1, Rf 0.3) gave 0.11 g

135.5, 134.9, 130.6, 126.6, 113.8, 79.2, 71.9, 51.9, 48.8, 48.4, 27.6. MS

(EI+): m/z calcd for (C₁₆H₂₀⁷⁹BrNO₅) 385.0525, found 385.0503.

Methyl 7-(3,5-Dimethoxyphenyl)-4-propionyl-2,3,4,5-tetrahydro-

1,4-benzoxazepine-9-carboxylate (92). Standard protocol 2 with 1.0

g (2.9 mmol) of 115 and 1.1 g (6.0 mmol) of 3,5-dimethoxyphe-

nylboronic acid. FCC with EtOAc and hexanes (2:1, Rf 0.3) gave 0.84

1

(0.29 mmol, 94%) of 87 as a white solid; mp 67−68 °C. H NMR

(110 °C, 400 MHz, C₂D₂Cl₄) δ = 7.62−7.58 (m, 1H), 7.50−7.41 (m,

g (2.1 mmol, 72%) of 92 as a colorless oil. H NMR (100 °C, 400

MHz, DMSO-d₆) δ = 7.84−7.66 (m, 2H), 6.76 (d, J = 2.2 Hz, 2H),

R

DOI: 10.1021/acs.jmedchem.6b00774

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

6.53 (t, J = 2.2 Hz, 1H), 4.71 (s, 2H), 4.20−4.13 (m, 2H), 3.95−3.86

(m, 2H), 3.86−3.79 (m, 9H), 2.38 (q, J = 7.3 Hz, 2H), 1.00 (t, J = 7.3

Hz, 3H). ¹³C NMR (RT, mixture of rotamers, 101 MHz, DMSO-d₆) δ

= 172.4, 172.0, 166.32, 166.30, 160.9, 156.9, 156.7, 140.7, 140.6, 134.8,

134.7, 133.3, 132.7, 131.7, 130.8, 126.9, 126.7, 125.5, 125.2, 104.9,

104.6, 99.6, 99.4, 72.5, 71.7, 55.3, 52.21, 52.19, 50.0, 49.3, 47.3, 25.8,

25.7, 9.3, 9.2. MS (EI+): m/z calcd for (C₂₂H₂₅NO₆) 399.1682, found

399.1662.

164.8, 164.4, 161.2, 161.0, 157.1, 156.9, 141.7, 141.6, 137.1, 136.9,

132.2, 131.7, 130.6, 130.0, 129.2, 126.0, 125.4, 105.3, 105.1, 100.0,

99.5, 73.3, 73.2, 57.3, 57.2, 55.5, 54.3, 51.0, 50.4, 48.1, 47.7, 36.62,

36.56, 26.8, 26.5, 26.1, 24.4, 9.21, 9.18. MS (EI+): m/z calcd for

(C₂₈H₃₇N₃O₅) 495.2733, found 495.2734.

1-{2-[7-(3,4-Dimethoxyphenyl)-4-propionyl-2,3,4,5-tetrahydro-

1,4-benzoxazepine-9-carboxamido]ethyl}piperidin-1-ium Chloride

(97). Standard protocol 3 with 0.090 g (0.23 mmol) of 95 and

0.033 mL (0.27 mmol) of 1-(2-aminoethyl)piperidine. FCC with

CH₂Cl₂with 10% MeOH (Rf 0.46) gave a colorless oil. 92 was then

precipitated from a solution in 1,4-dioxane as hydrochloride by

addition of 4 N solution of HCl in 1,4-dioxane. The precipitate was

washed with diethyl ether to obtain 0.043 g (0.10 mmol, 43%) of 97 as

a white solid; mp 98−99 °C. ¹H NMR (110 °C, 400 MHz, C₂D₂Cl₄) δ

= 12.45 (br s, 1H), 8.56 (br s, 1H), 8.30−8.00 (m, 1H), 7.69−7.39 (m,

1H), 7.19−7.01 (m, 2H), 6.91 (d, J = 8.7 Hz, 1H), 4.67 (s, 2H), 4.58−

4.31 (m, 2H), 4.16−3.80 (m, 10H), 3.66−3.32 (m, 2H), 3.29−3.11

(m, 2H), 2.98−2.48 (m, 2H), 2.45−2.04 (m, 4H), 1.90−1.44 (m, 4H),

1.10 (t, J = 7.0 Hz, 3H). ¹³C NMR (RT, mixture of rotamers, 101

MHz, C₂D₂Cl₄) δ = 172.9, 172.6, 165.8, 165.7, 156.5, 149.04, 148.95,

148.8, 148.6, 136.4, 136.1, 132.2, 131.9, 131.8, 131.4, 131.3, 130.8,

129.0, 128.4, 124.9, 123.9, 119.3, 111.54, 111.50, 110.3, 110.1, 73.2,

73.0, 57.5, 57.3, 56.1, 55.9, 54.9, 54.9, 50.6, 50.0, 47.6, 47.5, 35.6, 35.5,

26.7, 26.4, 22.5, 21.7, 9.2, 9.1. MS (ESI+): m/z calcd for

[(C₂₈H₃₈N₃O₅)⁺] 496.2811, found 496.2804.

Methyl 7-(3,4-Dimethoxyphenyl)-4-propionyl-2,3,4,5-tetrahydro-

1,4-benzoxazepine-9-carboxylate (93). Standard protocol 2 with

0.30 g (0.88 mmol) of 115 and 0.32 g (1.8 mmol) of 3,4-

dimethoxyphenylboronic acid. FCC with EtOAc and hexanes (3:1,

Rf 0.3) gave 0.28 g (0.70 mmol, 80%) of 93 as a white solid; mp 107−

1

108 °C. H NMR (RT, mixture of rotamers, 400 MHz, CDCl₃) δ =

7.83 (d, J = 2.5 Hz, 0.5H), 7.81 (d, J = 2.5 Hz, 0.5H), 7.72 (d, J = 2.5

Hz, 0.5H), 7.49 (d, J = 2.5 Hz, 0.5H), 7.19−7.00 (m, 2H), 7.00−6.86

(m, 1H), 4.71 (s, 1H), 4.61 (s, 1H), 4.23−4.14 (m, 2H), 4.09−4.03

(m, 1H), 3.98−3.87 (m, 10H), 2.46 (q, J = 7.4 Hz, 1H), 2.32 (q, J =

7.4 Hz, 1H), 1.18−1.08 (m, 3H). ¹³C NMR (RT, mixture of rotamers,

101 MHz, CDCl₃) δ = 172.9, 172.4, 166.72, 166.70, 157.5, 157.4,

149.3, 149.2, 149.0, 148.8, 136.64, 136.57, 133.6, 133.2, 132.28,

132.25, 132.2, 130.5, 128.5, 127.9, 125.8, 124.7, 119.5, 119.4, 111.6,

111.4, 110.3, 110.2, 73.12, 73.06, 56.1, 56.0, 52.4, 52.3, 51.1, 50.9,

48.33, 48.31, 26.8, 26.5, 9.2. MS (EI+): m/z calcd for (C₂₂H₂₅NO₆)

399.1682, found 399.1682.

N-[2-(Diethylamino)ethyl]-7-(3,5-dimethoxyphenyl)-4-propionyl-

2,3,4,5-tetrahydro-1,4-benzoxazepine-9-carboxamide (98). Stand-

ard protocol 3 with 0.050 g (0.13 mmol) of 94 and 0.028 mL (0.20

mmol) of N,N-diethylethylenediamine. FCC with CH₂Cl₂with 0.6%

MeOH and 3% triethylamine (Rf 0.3) gave 0.029 g (0.060 mmol,

46%) of 98 as a colorless oil. ¹H NMR (RT, mixture of rotamers, 400

MHz, CDCl₃) δ = 8.50 (s, 0.6H), 8.40 (s, 0.4H), 8.37 (d, J = 2.5 Hz,

0.6H), 8.29 (d, J = 2.5 Hz, 0.4H), 7.70 (d, J = 2.5 Hz, 0.4H), 7.49 (d, J

= 2.5 Hz, 0.6H), 6.76−6.70 (m, 2H), 6.50−6.43 (m, 1H), 4.73 (s,

0.6H), 4.62 (s, 1.4H), 4.29−4.19 (m, 2H), 4.11−4.05 (m, 1.4H),

3.95−3.89 (m, 0.6H), 3.88−3.81 (m, 6H), 3.61−3.52 (m, 2H), 2.76−

2.57 (m, 6H), 2.47 (q, J = 7.4 Hz, 1.4H), 2.33 (q, J = 7.4 Hz, 0.6H),

1.17−1.03 (m, 9H). ¹³C NMR (RT, mixture of rotamers, 101 MHz,

CDCl₃) δ = 173.0, 172.3, 164.9, 164.5, 161.2, 161.1, 157.1, 156.9,

141.7, 141.6, 137.0, 136.9, 132.2, 132.1, 131.7, 130.6, 130.0, 129.2,

126.1, 125.4, 105.3, 105.1, 100.0, 99.6, 73.3, 73.1, 55.5, 51.5, 51.4, 51.0,

50.4, 48.1, 47.7, 46.8, 46.6, 37.5, 37.4, 26.8, 26.5, 11.7, 9.2. MS (EI+):

m/z calcd for (C₂₇H₃₇N₃O₅) 483.2733, found 483.2720.

7-(3,5-Dimethoxyphenyl)-N-[2-(dimethylamino)ethyl]-4-propion-

yl-2,3,4,5-tetrahydro-1,4-benzoxazepine-9-carboxamide (99).

Standard protocol 3 with 0.11 g (0.29 mmol) of 94 and 0.039 mL

(0.35 mmol) of N,N-dimethylethylenediamine. FCC with CH₂Cl₂

with 1% MeOH and 1% triethylamine (Rf 0.3) gave 0.09 g (0.20

mmol, 69%) of 99 as a colorless oil. ¹H NMR (RT, mixture of

rotamers, 400 MHz, CDCl₃) δ = 8.53−8.46 (m, 0.7H), 8.40−8.34 (m,

1H), 8.28 (d, J = 2.5 Hz, 0.3H), 7.71 (d, J = 2.5 Hz, 0.3H), 7.49 (d, J =

2.5 Hz, 0.7H), 6.77−6.69 (m, 2H), 6.50−6.43 (m, 1H), 4.72 (s, 0.6H),

4.62 (s, 1.4H), 4.23−4.17 (m, 2H), 4.12−4.06 (m, 1.4H), 3.96−3.91

(m, 0.6H), 3.87−3.83 (m, 6H), 3.61−3.52 (m, 2H), 2.55−2.50 (m,

2H), 2.46 (q, J = 7.4 Hz, 1.4H), 2.34−2.27 (m, 6.6H), 1.16−1.10 (m,

3H). ¹³C NMR (RT, mixture of rotamers, 101 MHz, CDCl₃) δ =

173.0, 172.2, 164.8, 164.5, 161.2, 161.0, 157.0, 156.8, 141.7, 141.6,

137.2, 137.0, 132.4, 132.2, 131.9, 130.6, 129.9, 129.2, 126.2, 125.5,

105.3, 105.2, 100.0, 99.6, 73.3, 73.2, 57.8, 57.7, 55.5, 51.1, 50.5, 48.2,

47.8, 45.23, 45.19, 37.3, 26.8, 26.5, 9.2. MS (EI+): m/z calcd for

(C₂₅H₃₃N₃O₅) 455.2420, found 455.2426.

7-(3,5-Dimethoxyphenyl)-4-propionyl-2,3,4,5-tetrahydro-1,4-

benzoxazepine-9-carboxylic Acid (94). A solution of 0.30 g (0.75

mmol) of 92 in a mixture of 5 mL of MeOH, 5 mL of THF, and 10

mL of 1 M NaOH was heated to 70 °C for 45 min. Then the mixture

was acidiﬁed with 2 M HCl and extracted with EtOAc three times. The

combined organic layers were dried over MgSO₄and concentrated in

vacuo. FCC with EtOAc with 2% AcOH (Rf 0.5) gave 0.27 g (0.70

1

mmol, 93%) of 94 as a colorless oil. H NMR (90 °C, 400 MHz,

DMSO-d₆) δ = 7.80−7.68 (m, 2H), 6.76 (d, J = 2.2 Hz, 2H), 6.52 (t, J

= 2.2 Hz, 1H), 4.70 (s, 2H), 4.24−4.12 (m, 2H), 3.96−3.87 (m, 2H),

3.83 (s, 6H), 2.44−2.32 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). ¹³C NMR

(RT, mixture of rotamers, 126 MHz, DMSO-d₆) δ = 172.9, 172.4,

168.0, 161.4, 157.2, 157.0, 141.4, 141.2, 135.2, 135.1, 133.7, 133.1,

131.6, 130.7, 127.5, 127.2, 127.1, 127.0, 105.3, 105.0, 100.1, 99.9, 72.9,

72.2, 55.8, 50.5, 49.8, 47.9, 26.2, 9.7, 9.6. MS (EI+): m/z calcd for

(C₂₁H₂₃NO₆) 385.1525, found 385.1519.

7-(3,4-Dimethoxyphenyl)-4-propionyl-2,3,4,5-tetrahydro-1,4-

benzoxazepine-9-carboxylic Acid (95). A solution of 0.30 g (0.75

mmol) of 93 in a mixture of 5 mL of MeOH, 5 mL of THF, and 10

mL of 1 M NaOH was heated to 70 °C for 45 min. Then the mixture

was acidiﬁed with 2 M HCl and extracted with EtOAc three times. The

combined organic layers were dried over MgSO₄and concentrated in

vacuo. FCC with EtOAc with 2% AcOH (Rf 0.5) gave 0.25 g (0.65

mmol, 87%) of 95 as a white solid; mp 97−98 °C. ¹H NMR (110 °C,

400 MHz, C₂D₂Cl₄) δ = 8.16 (d, J = 2.5 Hz, 1H), 7.72−7.55 (m, 1H),

7.18−7.04 (m, 2H), 6.93 (d, J = 8.3 Hz, 1H), 4.68 (s, 2H), 4.39−4.25

(m, 2H), 4.02−3.93 (m, 2H), 3.90 (s, 3H), 3.87 (s, 3H), 2.36 (q, J =

7.4 Hz, 2H), 1.12 (t, J = 7.4 Hz, 3H). ¹³C NMR (RT, mixture of

rotamers, 101 MHz, C₂D₂Cl₄) δ = 173.0, 172.7, 165.2, 165.1, 156.8,

149.4, 149.2, 137.9, 137.7, 134.1, 132.7, 132.0, 131.6, 131.4, 131.3,

130.5, 129.8, 121.8, 121.0, 119.7, 111.9, 111.8, 110.5, 110.3, 74.6, 56.3,

56.1, 50.7, 50.0, 47.9, 47.4, 26.8, 26.6, 9.32, 9.28. MS (EI+): m/z calcd

for (C₂₁H₂₃NO₆) 385.1525, found 385.1518.

7-(3,5-Dimethoxyphenyl)-N-[2-(piperidin-1-yl)ethyl]-4-propionyl-

2,3,4,5-tetrahydro-1,4-benzoxazepine-9-carboxamide (96). Stand-

ard protocol 3 with 0.11 g (0.29 mmol) of 94 and 0.050 mL (0.35

mmol) of 1-(2-aminoethyl)piperidine. FCC with CH₂Cl₂with 0.6%

MeOH and 3% triethylamine (Rf 0.3) gave 0.055 g (0.11 mmol, 31%)

(S)-7-(3,5-Dimethoxyphenyl)-N-(piperidin-3-yl)-4-propionyl-

2,3,4,5-tetrahydro-1,4-benzoxazepine-9-carboxamide (100). Stand-

ard protocol 3 with 0.75 g (2.0 mmol) of 94 and 0.50 g (2.5 mmol) of

(S)-(+)-3-amino-1-Boc-piperidine. FCC with EtOAc and hexanes

(5:1, Rf 0.2) gave a white solid, which was dissolved in 3 mL of 1,4-

dioxane. Then 3 mL of a 4 N solution of HCl in 1,4-dioxane was

added and the mixture stirred overnight. Then 1 M NaOH was added

and the mixture was extracted with CH₂Cl₂three times. The combined

organic layers were dried over MgSO₄. After concentration in vacuo,

1

of 96 as a white solid; mp 88−89 °C. H NMR (RT, mixture of

rotamers, 400 MHz, CDCl₃) δ = 8.57−8.26 (m, 2H), 7.71 (d, J = 2.5

Hz, 0.3H), 7.49 (d, J = 2.5 Hz, 0.7H), 6.77−6.67 (m, 2H), 6.53−6.39

(m, 1H), 4.73 (s, 0.7H), 4.63 (s, 1.3H), 4.30−4.22 (m, 2H), 4.15−4.06

(m, 1.3H), 3.96−3.91 (m, 0.7H), 3.87−3.82 (m, 6H), 3.63−3.55 (m,

2H), 2.61−2.29 (m, 8H), 1.63−1.46 (m, 6H), 1.16−1.10 (m, 3H). ¹³C

NMR (RT, mixture of rotamers, 126 MHz, CDCl₃) δ = 173.0, 172.3,

S

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puriﬁcation by FCC with CH₂Cl₂with 5% MeOH and 2%

MgSO₄. FCC with CH₂Cl₂with 5% MeOH (Rf 0.2) gave 0.10 g (0.21

triethylamine (Rf 0.2) gave 0.29 g (0.62 mmol, 31%) of 100 as a

mmol, 36%) of 103 as a white solid; mp 75−76 °C; [α]²⁰+ 1.6 (c

D

1

white solid; mp 91−92 °C; [α]²⁰−4.5 (c 0.16, MeOH). H NMR

0.40, MeOH). ¹H NMR (RT, mixture of rotamers, 400 MHz, CDCl₃)

δ = 8.56 (br s, 0.7H), 8.43 (br s, 0.3H), 8.35 (d, J = 2.5 Hz, 0.7H), 8.27

(d, J = 2.5 Hz, 0.3H), 7.71 (d, J = 2.5 Hz, 0.3H), 7.49 (d, J = 2.5 Hz,

0.7H), 6.74 (d, J = 2.3 Hz, 0.6H), 6.71 (d, J = 2.3 Hz, 1.4H), 6.48 (t, J

= 2.2 Hz, 0.7H), 6.45 (t, J = 2.3 Hz, 0.3H), 4.72 (s, 0.6H), 4.62 (s,

1.4H), 4.34−4.28 (m, 1H), 4.25−4.18 (m, 2H), 4.13−4.08 (m, 1.4H),

3.98−3.90 (m, 0.6H), 3.86−3.82 (m, 6H), 2.62−2.30 (m, 5H), 2.28 (s,

3H), 2.23−2.13 (m, 1H), 1.78−1.70 (m, 2H), 1.67−1.57 (m, 2H),

1.16−1.09 (m, 3H). ¹³C NMR (RT, mixture of rotamers, 101 MHz,

CDCl₃) δ = 173.1, 172.2, 163.8, 163.4, 161.2, 161.1, 157.0, 156.8,

141.6, 141.6, 137.2, 137.1, 132.5, 132.1, 132.0, 130.5, 129.9, 129.2,

126.4, 125.8, 105.3, 105.2, 100.0, 99.6, 73.3, 60.4, 56.0, 55.5, 51.1, 50.5,

48.3, 47.9, 46.7, 45.5, 45.4, 28.4, 26.8, 26.5, 22.1, 9.2. MS (ESI+): m/z

calcd for [(C₂₇H₃₆N₃O₅)⁺] 482.2649, found 482.2650.

D

(RT, mixture of rotamers, 500 MHz, C₂D₂Cl₄) δ = 8.40 (br s, 1H),

8.34−8.21 (m, 1H), 7.69 (d, J = 2.6 Hz, 0.4H), 7.51 (d, J = 2.6 Hz,

0.6H), 6.76−6.70 (m, 2H), 6.50−6.43 (m, 1H), 4.77−4.56 (m, 2H),

4.31−3.87 (m, 5H), 3.87−3.82 (m, 6H), 3.21−3.05 (m, 1H), 2.92−

2.69 (m, 3H), 2.46−2.39 (m, 1.2H), 2.32−2.26 (m, 0.8H), 1.91−1.55

(m, 4H), 1.13−1.05 (m, 3H). ¹³C NMR (RT, mixture of rotamers, 126

MHz, C₂D₂Cl₄) δ = 172.8, 172.2, 163.7, 163.5, 160.9, 156.8, 156.7,

141.2, 136.5, 132.2, 131.9, 131.8, 130.5, 129.5, 128.8, 125.9, 125.3,

105.2, 104.9, 99.8, 99.4, 73.1, 55.5, 50.9, 50.7, 50.1, 47.9, 47.5, 46.1,

46.0, 45.5, 29.8, 26.6, 26.4, 23.2, 23.1, 9.1. MS (EI+): m/z calcd for

(C₂₆H₃₃N₃O₅) 467.2420, found 467.2419.

(R)-7-(3,5-Dimethoxyphenyl)-N-(piperidin-3-yl)-4-propionyl-

2,3,4,5-tetrahydro-1,4-benzoxazepine-9-carboxamide (101). Stand-

ard protocol 3 with 0.75 g (2.0 mmol) of 94 and 0.50 g (2.5 mmol) of

(R)-(−)-3-amino-1-Boc-piperidine. FCC with EtOAc and hexanes

(5:1, Rf 0.2) gave a white solid, which was dissolved in 3 mL of 1,4-

dioxane. Then 3 mL of a 4 N solution of HCl in 1,4-dioxane was

added and the mixture stirred overnight. Then 1 M NaOH was added,

and the mixture was extracted with CH₂Cl₂three times. The combined

organic layers were dried over MgSO₄. It was concentrated in vacuo,

and puriﬁcation by FCC with CH₂Cl₂with 5% MeOH and 2%

4-Bromo-2-{[(2-hydroxyethyl)amino]methyl}-6-methoxyphenol

(104). To a solution of 9.9 g (43 mmol) of 5-bromo-3-

methoxysalicylaldehyde in 200 mL of THF and 20 mL of MeOH,

3.3 g (54 mmol) of 2-aminoethanol were added, and the mixture was

stirred for 25 min. Over 1.5 h, three equal portions of 1.5 g (40 mmol)

of NaBH₄were added and the mixture stirred overnight. The solvent

was evaporated under reduced pressure and the residue dissolved in

EtOAc. Upon addition of 200 mL of water, the product partially

precipitated as white solid and was collected by ﬁltration. The EtOAc/

water mixture was extracted with EtOAc. The combined organic layers

were dried over MgSO₄and concentrated in vacuo. FCC of precipitate

and concentrate of organic phases with EtOAc and MeOH (4:1, Rf

0.3) gave 11 g (40 mmol, 93%) of 104 as a white solid; mp 153−154

°C. ¹H NMR (400 MHz, DMSO-d₆) δ = 6.96 (d, J = 2.2 Hz, 1H), 6.88

(d, J = 2.2 Hz, 1H), 3.80 (s, 2H), 3.75 (s, 3H), 3.46 (t, J = 5.7 Hz,

2H), 2.54 (t, J = 5.7 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ =

148.3, 145.9, 126.3, 122.7, 113.5, 108.8, 59.7, 55.8, 50.2, 49.5. MS (EI

+): m/z calcd for (C₁₀H₁₄⁷⁹BrNO₃) 275.0157, found 275.0157.

tert-Butyl (5-Bromo-2-hydroxy-3-methoxybenzyl)(2-

hydroxyethyl)carbamate (105). To a suspension of 11 g (40

mmol) of 104 in 100 mL of EtOAc and 58 mL of saturated

NaHCO₃solution, 12 g (53 mmol) of di-tert-butyl dicarbonate were

added, and the mixture was stirred overnight. The suspension turned

into a clear two-phase system, and the organic layer was separated. The

aqueous phase was extracted three times with EtOAc, and the

combined organic layers were dried over MgSO₄and concentrated in

vacuo. FCC with EtOAc and hexanes (3:2, Rf 0.5) gave 11 g (28

mmol, 69%) of 105 as a white solid; mp 95−96 °C. ¹H NMR (70 °C,

400 MHz, DMSO-d₆) δ = 8.75 (s, 1H), 7.01 (d, J = 2.3 Hz, 1H), 6.81

(d, J = 2.3 Hz, 1H), 4.53−4.32 (m, 3H), 3.81 (s, 3H), 3.53−3.43 (m,

2H), 3.32−3.19 (m, 2H), 1.40 (s, 9H). ¹³C NMR (70 °C, 101 MHz,

DMSO-d₆) δ = 154.9, 148.3, 143.3, 127.1, 122.3, 113.5, 109.4, 78.6,

58.9, 56.1, 49.0, 45.2, 27.7. MS (EI+): m/z calcd for (C₁₅H₂₂⁷⁹BrNO₅)

375.0681, found 375.0685.

tert-Butyl 7-Bromo-9-methoxy-2,3-dihydro-1,4-benzoxazepine-

4(5H)-carboxylate (106). To a solution of 9.7 g (26 mmol) of 105

and 11 g (42 mmol) of triphenylphosphine in 260 mL of CH₂Cl₂, 8.7

mL of (40 mmol) DIAD were added, and the mixture was stirred

overnight. Water was added, and the mixture was extracted with

EtOAc three times. The combined organic layers were dried over

MgSO₄and concentrated in vacuo. FCC with EtOAc and hexanes

(1:4, Rf 0.3) gave 8.6 g (24 mmol, 91%) of 106 as a white solid; mp

96−97 °C. ¹H NMR (70 °C, 400 MHz, DMSO-d₆) δ = 7.09 (d, J = 2.3

Hz, 1H), 6.98 (d, J = 2.3 Hz, 1H), 4.38 (s, 2H), 4.05−3.93 (m, 2H),

3.78 (s, 3H), 3.74−3.67 (m, 2H), 1.35 (s, 9H). ¹³C NMR (70 °C, 101

MHz, DMSO-d₆) δ = 154.6, 152.8, 148.2, 134.9, 124.4, 116.0, 114.7,

79.8, 72.3, 56.9, 49.9, 49.3, 28.5. MS (EI+): m/z calcd for

(C₁₅H₂₀⁷⁹BrNO₄) 357.0576, found 357.0575.

triethylamine (Rf 0.2) gave 0.50 g (1.1 mmol, 55%) of 101 as a white

1

solid; mp 91−92 °C; [α]²⁰+ 4.4 (c 0.16, MeOH). H NMR (RT,

D

mixture of rotamers, 500 MHz, C₂D₂Cl₄) δ = 8.44 (br s, 0.6H), 8.35

(br s, 0.4H), 8.32 (d, J = 2.5 Hz, 0.6H), 8.27 (d, J = 2.5 Hz, 0.4H),

7.69 (d, J = 2.5 Hz, 0.4H), 7.51 (d, J = 2.5 Hz, 0.6H), 6.75 (d, J = 2.3

Hz, 0.8H), 6.73 (d, J = 2.3 Hz, 1.2H), 6.48 (t, J = 2.3 Hz, 0.6H), 6.46

(t, J = 2.3 Hz, 0.4H), 4.79−4.57 (m, 2H), 4.30−3.89 (m, 5H), 3.87−

3.82 (m, 6H), 3.10−3.03 (m, 1H), 2.83−2.67 (m, 3H), 2.47−2.38 (m,

1.2H), 2.35−2.24 (m, 0.8H), 1.91−1.55 (m, 4H), 1.12−1.07 (m, 3H).

¹³C NMR (RT, mixture of rotamers, 126 MHz, C₂D₂Cl₄) δ = 172.8,

172.2, 163.7, 163.4, 160.88, 160.85, 156.8, 156.7, 141.2, 136.5, 132.3,

131.9, 131.8, 130.5, 129.5, 128.8, 125.9, 125.3, 105.2, 104.9, 99.8, 99.4,

73.1, 55.5, 50.7, 50.1, 47.9, 47.5, 46.1, 45.7, 45.5, 29.8, 26.6, 26.4, 23.2,

9.2, 9.1. MS (EI+): m/z calcd for (C₂₆H₃₃N₃O₅) 467.2420, found

467.2420.

(S)-7-(3,5-Dimethoxyphenyl)-N-(1-methylpiperidin-3-yl)-4-pro-

pionyl-2,3,4,5-tetrahydro-1,4-benzoxazepine-9-carboxamide (102).

To a solution of 0.16 g (0.34 mmol) of 100 in 1 mL of acetonitrile,

0.14 mL (1.7 mmol) of a 35% solution of formaldehyde in water, and

34 mg (0.54 mmol) of NaCNBH₃were added. The mixture was

stirred for 1 h, then 2 M NaOH was added and the mixture was

extracted with CH₂Cl₂three times. The combined organic layers were

dried over MgSO₄. FCC with CH₂Cl₂with 5% MeOH (Rf 0.2) gave

0.060 g (0.12 mmol, 35%) of 102 as a white solid; mp 75−76 °C;

[α]²⁰_D−1.7 (c 0.41, MeOH). ¹H NMR (RT, mixture of rotamers, 500

MHz, CDCl₃) δ = 8.56 (br s, 0.7H), 8.43 (br s, 0.3H), 8.36 (d, J = 2.5

Hz, 0.7H), 8.28 (d, J = 2.5 Hz, 0.3H), 7.71 (d, J = 2.5 Hz, 0.3H), 7.49

(d, J = 2.5 Hz, 0.7H), 6.74 (d, J = 2.3 Hz, 0.6H), 6.72 (d, J = 2.3 Hz,

1.4H), 6.48 (t, J = 2.2 Hz, 0.7H), 6.45 (t, J = 2.2 Hz, 0.3H), 4.72 (s,

0.6H), 4.62 (s, 1.4H), 4.34−4.28 (m, 1H), 4.24−4.19 (m, 2H), 4.14−

4.08 (m, 1.4H), 3.97−3.91 (m, 0.6H), 3.87−3.83 (m, 6H), 2.65−2.30

(m, 5H), 2.29−2.26 (m, 3H), 2.23−2.13 (m, 1H), 1.78−1.71 (m, 2H),

1.67−1.58 (m, 2H), 1.15−1.11 (m, 3H). ¹³C NMR (RT, mixture of

rotamers, 101 MHz, CDCl₃) δ = 173.0, 172.2, 163.8, 163.4, 161.2,

161.0, 157.0, 156.8, 141.7, 141.6, 137.2, 137.1, 132.5, 132.1, 132.0,

130.5, 129.9, 129.2, 126.4, 125.8, 105.3, 105.2, 100.0, 99.6, 73.3, 60.5,

60.4, 56.0, 55.5, 51.1, 50.5, 48.3, 47.9, 46.7, 45.44, 45.36, 28.5, 28.4,

26.8, 26.5, 22.1, 22.0, 9.20, 9.18. MS (EI+): m/z calcd for

(C₂₇H₃₅N₃O₅) 481.2577, found 481.2591.

(R)-7-(3,5-Dimethoxyphenyl)-N-(1-methylpiperidin-3-yl)-4-pro-

pionyl-2,3,4,5-tetrahydro-1,4-benzoxazepine-9-carboxamide (103).

To a solution of 0.27 g (0.58 mmol) of 101 in 1.7 mL of acetonitrile,

0.23 mL (2.9 mmol) of a 35% solution of formaldehyde in water, and

58 mg (0.92 mmol) of NaCNBH₃was added. The mixture was stirred

for 1 h, then 2 M NaOH was added and the mixture was extracted

with CH₂Cl₂three times. The combined organic layers were dried over

7-Bromo-9-methoxy-2,3,4,5-tetrahydro-1,4-benzoxazepine

(107). To a suspension of 10 g (28 mmol) of 106 in 120 mL of MeOH

was added a mixture of 80 mL of 36% HCl and 120 mL of 1,4-dioxane.

The mixture was reﬂuxed for 2 h and then concentrated in vacuo.

Saturated Na₂CO₃solution was carefully added, and the mixture was

extracted with CH₂Cl₂three times. The combined organic layers were

T

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Article

dried over MgSO₄and concentrated in vacuo to give 6.7 g (26 mmol,

94%) of 107 as a white solid; mp 119−120 °C. H NMR (400 MHz,

DMSO-d₆) δ = 7.05 (d, J = 2.4 Hz, 1H), 6.98 (d, J = 2.4 Hz, 1H),

3.93−3.83 (m, 2H), 3.81−3.70 (m, 5H), 3.06−2.94 (m, 2H). ¹³C

NMR (126 MHz, DMSO-d₆) δ = 153.1, 149.0, 140.0, 124.1, 115.1,

115.1, 75.8, 56.9, 52.9, 52.8. MS (EI+): m/z calcd for

(C₁₀H₁₂⁷⁹BrNO₂) 257.0051, found 257.0052.

The combined organic layers were dried over MgSO₄and

concentrated in vacuo. A short FCC with CH₂Cl₂with 5% MeOH

and 2% triethylamine gave the secondary amine as crude intermediate.

The standard protocol for the N-acylation was applied to equal

portions of this intermediate with propionyl chloride to obtain 111 or

with cyclopropanecarbonyl chloride to obtain 112.

1-(7-Bromo-9-methoxy-2,3-dihydro-1,4-benzothiazepin-4(5H)-

yl)propan-1-one (111). FCC with EtOAc and hexanes (1:3, Rf 0.2)

gave 0.36 g (1.1 mmol, 49%) of 111 as a white solid; mp 118−119 °C.

¹H NMR (100 °C, 400 MHz, DMSO-d₆) δ = 7.20 (d, J = 2.1 Hz, 1H),

7.08 (d, J = 2.1 Hz, 1H), 4.62 (s, 2H), 3.93−3.86 (m, 2H), 3.82 (s,

3H), 2.98−2.91 (m, 2H), 2.30 (q, J = 7.4 Hz, 2H), 0.97 (t, J = 7.4 Hz,

3H). ¹³C NMR (RT, mixture of rotamers, 126 MHz, DMSO-d₆) δ =

172.8, 171.9, 158.5, 158.2, 144.3, 142.6, 125.8, 124.6, 124.0, 123.7,

119.9, 119.6, 113.4, 113.0, 56.4, 50.9, 50.6, 50.3, 47.9, 33.6, 31.7, 25.8,

25.6, 9.2, 9.1. MS (ESI+): m/z calcd for [(C₁₃H₁₇⁷⁹BrNO₂S)⁺]

330.0163, found 330.0158.

1

5-Bromo-2-mercapto-3-methoxybenzoic Acid (108). A suspen-

sion of 9.6 g (30 mmol) of 4-bromo-2-carboxy-6-methoxybenzenami-

nium bromide,²⁵2.4 g (60 mmol) NaOH, and 2.1 g (30 mmol) of

NaNO₂in 60 mL of water was added over 0.5 h to a mixture of 20 mL

of conc HCl with ice, and the temperature was kept at 0 °C by the

addition of ice. After 0.5 h at 0 °C, potassium acetate was used to

adjust to neutral pH. The resulting yellow solution was added to a

stirred solution of 23 g (140 mmol) of potassium ethyl xanthate in 40

mL of water at 90 °C. After 0.5 h at 90 °C, the solution was cooled to

0 °C. Concentrated HCl was added until acidic pH. The resulting

precipitate was collected by ﬁltration and dissolved in 100 mL of 10%

NaOH. The solution was heated to 85 °C for 2 h. Then 3.1 g (30

mmol) of NaHSO₃were added and 85 °C was maintained for 0.25 h.

The solution was ﬁltered, cooled to 0 °C, and acidiﬁed with conc HCl.

The precipitate was separated by ﬁltration and dissolved in a mixture

of EtOAc and THF (1:1). This organic layer was washed with

saturated NaCl solution twice, dried over MgSO₄, and concentrated in

vacuo. Puriﬁcation by FCC with CH₂Cl₂with 4% EtOH and 5%

AcOH (Rf 0.2) gave 4.9 g (19 mmol, 62%) of 108 as a white solid; mp

208−209 °C. ¹H NMR (400 MHz, CD₂Cl₂) δ = 7.90 (d, J = 2.0 Hz),

7.20 (d, J = 2.0 Hz), 5.28 (br s, 1H), 3.96 (s, 3 H). ¹³C NMR (101

MHz, CD₂Cl₂) δ = 169.2, 155.3, 129.0, 127.1, 127.0, 118.0, 117.3, 57.4

MS (EI+): m/z calcd for (C₈H₅O₂⁷⁹BrS) 243.9193, found 243.9195.

Methyl 5-Bromo-2-mercapto-3-methoxybenzoate (109). A sol-

ution of 3.0 g (12 mmol) of 108 in 25 mL of anhydrous MeOH and

1.0 mL of 96% sulfuric acid was reﬂuxed under N₂for 12 h and then

concentrated in vacuo. Ice was added, and the mixture was extracted

with EtOAc three times. After drying over MgSO₄and concentration

in vacuo, FCC with hexanes and EtOAc (9:1, Rf 0.4) gave 2.3 g (8.6

1-(7-Bromo-9-methoxy-2,3-dihydro-1,4-benzothiazepin-4(5H)-

yl)-1-(cyclopropyl)methanone (112). FCC with EtOAc and hexanes

(1:3, Rf 0.2) gave 0.34 g (1.0 mmol, 45%) of 112 as a white solid; mp

1

87−88 °C. H NMR (100 °C, 400 MHz, DMSO-d₆) δ = 7.34−7.19

(m, 1H), 7.08 (d, J = 2.0 Hz, 1H), 4.73 (s, 2H), 4.11−3.93 (m, 2H),

3.83 (s, 3H), 2.96−2.90 (m, 2H), 1.95−1.85 (m, 1H), 0.73−0.66 (m,

4H). ¹³C NMR (RT, mixture of rotamers, 101 MHz, DMSO-d₆) δ =

173.1, 172.0, 158.9, 158.8, 144.7, 144.0, 126.2, 125.1, 124.4, 124.0,

120.35, 120.33, 113.9, 113.5, 56.94, 56.91, 51.6, 51.4, 51.1, 49.0, 34.3,

32.4, 11.7, 11.1, 8.0, 7.5. MS (ESI+): m/z calcd for

[(C₁₄H₁₇⁷⁹BrNO₂S)⁺] 342.0163, found 342.0157.

Methyl 5-Bromo-2-hydroxy-3-{[(2-hydroxyethyl)amino]methyl}-

benzoate (113). To a solution of 1.7 g (6.5 mmol) of methyl 5-

bromo-3-formyl-2-hydroxybenzoate²⁹in a mixture of 4 mL of MeOH

and 36 mL of anhydrous THF, 0.48 mL of (8.1 mmol) 2-

aminoethanol was added. The solution was stirred for 0.5 h, and

then 0.22 g (5.8 mmol) of NaBH₄was added in portions over 15 min.

After 1 h of stirring, the solution was concentrated and water and

EtOAc were added. The product partially precipitated as white solid,

the remaining product was extracted from the water phase at alkaline

pH with EtOAc. After drying over MgSO₄and evaporation of the

solvent, 1.8 g (6.0 mmol, 93%) of 113 were obtained; mp 150−151

1

mmol, 72%) of 109 as a yellow solid; mp 60−61 °C. H NMR (500

MHz, DMSO-d₆) δ = 7.69−7.65 (m, 1H), 7.45−7.42 (m, 1H), 5.42−

5.39 (m, 1H), 3.95 (s, 3H), 3.85 (s, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ = 165.2, 154.8, 127.7, 126.3, 125.1, 117.3, 116.7, 57.3,

52.6. MS (EI+): m/z calcd for (C₉H₉O₃⁷⁹BrS) 275.9456, found

275.9452.

1

°C. H NMR (500 MHz, DMSO-d₆): δ = 7.69 (d, J = 2.7 Hz, 1H),

7.57 (d, J = 2.7 Hz, 1H), 3.86−3.80 (m, 5H), 3.49 (t, J = 5.7 Hz, 2H),

2.61 (t, J = 5.7 Hz, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ = 167.3,

159.1, 135.7, 130.6, 129.8, 116.1, 108.3, 59.7, 52.4, 50.4, 48.3. MS (EI

+): m/z calcd for (C₁₁H₁₄⁷⁹BrNO₄) 303.0106, found 303.0095.

Methyl 5-Bromo-3-{[(tert-butoxycarbonyl)(2-hydroxyethyl)-

amino]methyl}-2-hydroxybenzoate (114). To a dispersion of 2.8 g

(9.2 mmol) of 113 in a mixture of 100 mL of EtOAc and 60 mL of

saturated NaHCO₃solution, 2.8 g (13 mmol) of di-tert-butyl

dicarbonate was added and the mixture was stirred for 12 h. The

organic layer was separated and the aqueous phase extracted with

EtOAc three times. The combined organic layers were dried over

MgSO₄and concentrated in vacuo. FCC with EtOAc and hexanes

7-Bromo-9-methoxy-3,4-dihydro-1,4-benzothiazepin-5(2H)-one

(110). To a solution of 2.3 g (8.3 mmol) of 109 and 1.0 g (9.0 mmol)

of 2-chloroethylamine hydrochloride in 17 mL of anhydrous DMF at 0

°C under N₂, 1.0 g (19 mmol) of NaOMe was added and the mixture

stirred for 12 h. Water was added, the pH adjusted with 2 M NaOH to

12, and the mixture extracted with CH₂Cl₂three times. The combined

organic layers were dried over MgSO₄and concentrated in vacuo. The

residue was dissolved in 60 mL of anhydrous THF and cooled to 0 °C.

Then 7.2 g (64 mmol) of t-BuOK was added and the mixture stirred at

45 °C for 1 h. Saturated ammonium chloride solution was added and

the mixture extracted with EtOAc three times. The combined organic

layers were dried over MgSO₄and concentrated in vacuo. FCC with

CH₂Cl₂with 4% MeOH (Rf 0.3) gave 1.5 g (5.2 mmol, 63%) of 110

as a yellow solid; mp 185−186 °C. ¹H NMR (400 MHz, CD₂Cl₂) δ =

7.33 (d, J = 2.0 Hz, 1H), 7.16 (d, J = 2.0 Hz, 1H), 7.02 (br s, 1H), 3.87

(s, 3H), 3.32−3.26 (m, 2H), 3.10−3.05 (m, 2H). ¹³C NMR (101

MHz, CD₂Cl₂) δ = 170.9, 160.3, 144.1, 124.4, 123.7, 117.1, 116.9,

57.0, 40.2, 37.4. MS (ESI+): m/z calcd for [(C₁₀H₁₁⁷⁹BrNO₂S)⁺]

287.9694, found 287.9688.

1-(7-Bromo-9-methoxy-2,3-dihydro-1,4-benzothiazepin-4(5H)-

yl)propan-1-one (111) and 1-(7-Bromo-9-methoxy-2,3-dihydro-1,4-

benzothiazepin-4(5H)-yl)-1-(cyclopropyl)methanone (112). A sol-

ution of 1.3 g (4.5 mmol) of 110 in 16 mL of anhydrous THF and

cooled to −30 °C under N₂. Then 45 mL of 1 M BH₃-THF solution

(45 mmol) was added and the mixture reﬂuxed for 40 h. A mixture of

15 mL of MeOH, 15 mL of conc HCl, and 30 mL of water was added

at RT, and the mixture was reﬂuxed for 1 h. pH was adjusted to 12

with K₂CO₃, and the mixure was extracted with EtOAc three times.

1

(1:2, Rf 0.5) gave 2.1 g (5.2 mmol, 56%) of 114 as a colorless oil. H

NMR (70 °C, 400 MHz, DMSO-d₆) δ = 7.81 (d, J = 2.6 Hz, 1H), 7.45

(d, J = 2.6 Hz, 1H), 4.44 (s, 2H), 3.94 (s, 3H), 3.51 (t, J = 6.1 Hz,

2H), 3.31 (t, J = 6.1 Hz, 2H), 3.10 (br s, 1H), 1.38 (s, 9H). ¹³C NMR

(70 °C, 101 MHz, DMSO-d₆) δ = 168.2, 156.8, 154.7, 135.7, 129.9,

129.7, 114.0, 109.6, 78.7, 59.0, 52.5, 49.5, 45.3, 27.7. MS (EI+): m/z

calcd for (C₁₆H₂₂⁷⁹BrNO₆) 403.0630, found 403.0637.

Methyl 7-Bromo-4-propionyl-2,3,4,5-tetrahydro-1,4-benzoxaze-

pine-9-carboxylate (115). To a solution of 0.89 g (2.3 mmol) of

91 in 5 mL of 1,4-dioxane, 10 mL of a 4 M solution of HCl in 1,4-

dioxane was added. After 5 h, the supernatant was removed and the

white precipitate washed with diethyl ether. The solid was dissolved in

10 mL of CH₂Cl₂and cooled to 0 °C. Then 1.6 mL of (9.2 mmol) of

DIPEA and 0.40 mL (4.6 mmol) of propionyl chloride were added.

The mixture was warmed to RT and stirred for 12 h. NaHCO₃

solution was added and the mixture extracted three times with

CH₂Cl₂. The combined organic layers were dried over MgSO₄and

concentrated in vacuo. FCC with EtOAc and hexanes (1:1, Rf 0.3)

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

gave 0.58 g (1.7 mmol, 74%) of 91 as a white solid; mp 112−113 °C.

¹H NMR (100 °C, 400 MHz, DMSO-d₆) δ = 7.80−7.57 (m, 2H), 4.62

(s, 2H), 4.17−4.11 (m, 2H), 3.90−3.86 (m, 2H), 3.83 (s, 3H), 2.34 (q,

J = 7.4 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H). ¹³C NMR (RT, mixture of

rotamers, 126 MHz, CD₂Cl₂) δ = 172.4, 172.1, 164.9, 156.8, 156.6,

135.7, 135.6, 134.9, 134.7, 131.2, 130.9, 126.8, 126.6, 114.3, 114.2,

72.7, 71.8, 52.42, 52.40, 49.9, 48.5, 47.1, 46.8, 25.70, 25.68, 9.2, 9.1.

MS (EI+): m/z calcd for (C₁₄H₁₆⁷⁹BrNO₄) 341.0263, found 341.0260.

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ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.6b00774.

(4) Chan, W.-I.; Hannah, R. L.; Dawson, M. A.; Pridans, C.; Foster,

D.; Joshi, A.; Gottgens, B.; Van Deursen, J. M.; Huntly, B. J. P. The

̈

transcriptional coactivator Cbp regulates self-renewal and differ-

entiation in adult hematopoietic stem cells. Mol. Cell. Biol. 2011, 31,

5046−5060.

NMR spectra, PX table, DSF data, and ITC determi-

nations (PDF)

(5) (a) Iyer, N. G.; Ozdag, H.; Caldas, C. p300//CBP and cancer.

Oncogene 2004, 23, 4225−4231. (b) Katsumoto, T.; Yoshida, N.;

Kitabayashi, I. Roles of the histone acetyltransferase monocytic

leukemia zinc finger protein in normal and malignant hematopoiesis.

Cancer Sci. 2008, 99, 1523−1527. (c) Zhang, Y.; Zeleznik-Le, N.;

Emmanuel, N.; Jayathilaka, N.; Chen, J.; Strissel, P.; Strick, R.; Li, L.;

Neilly, M. B.; Taki, T.; Hayashi, Y.; Kaneko, Y.; Schlegelberger, B.;

Rowley, J. D. Characterization of genomic breakpoints in MLL and

CBP in leukemia patients with t(11;16). Genes, Chromosomes Cancer

2004, 41, 257−265.

(6) Bowers, E. M.; Yan, G.; Mukherjee, C.; Orry, A.; Wang, L.;

Holbert, M. A.; Crump, N. T.; Hazzalin, C. A.; Liszczak, G.; Yuan, H.;

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Marmorstein, R.; Mahadevan, L. C.; Alani, R. M.; Cole, P. A. Virtual

ligand screening of the p300/CBP histone acetyltransferase:

identification of a selective small molecule inhibitor. Chem. Biol.

2010, 17, 471−482.

Molecular formula strings (CSV)

Accession Codes

102 with CBP: 5J0D. The authors will release the atomic

coordinates and experimental data upon article publication.

AUTHOR INFORMATION

Corresponding Authors

*For F.B.: phone, +49 89 218077301; fax, +49-89-218077803;

E-mail, franz.bracher@cup.uni-muenchen.de.

*For S.K.: phone, +44 1865 612933; E-mail, Stefan.Knapp@

sgc.ox.ac.uk.

■

Author Contributions

Tobias Popp, compound preparation and characterization,

writing of paper; Cynthia Tallant, protein production and

structure determination; Catherine Rogers, FRAP; Oleg

Fedorov, compound screening, supervision; Paul Brennan,

(7) Shrimp, J. H.; Sorum, A. W.; Garlick, J. M.; Guasch, L.; Nicklaus,

M. C.; Meier, J. L. Characterizing the covalent targets of a small

molecule inhibitor of the lysine acetyltransferase P300. ACS Med.

Chem. Lett. 2016, 7, 151−155.

supervision of research; Susanne Muller, supervision of

̈

research; FRAP, editing of paper and provision of ﬁgures;

Stefan Knapp, supervision of research, writing/editing of paper;

Franz Bracher, supervision of research, writing/editing of paper.

(8) (a) Best, J. L.; Amezcua, C. A.; Mayr, B.; Flechner, L.; Murawsky,

C. M.; Emerson, B.; Zor, T.; Gardner, K. H.; Montminy, M.

Identification of small-molecule antagonists that inhibit an activator:-

coactivator interaction. Proc. Natl. Acad. Sci. U. S. A. 2004, 101,

17622−17627. (b) Buhrlage, S. J.; Bates, C. A.; Rowe, S. P.; Minter, A.

R.; Brennan, B. B.; Majmudar, C. Y.; Wemmer, D. E.; Al-Hashimi, H.;

Mapp, A. K. Amphipathic small molecules mimic the binding mode

and function of endogenous transcription factors. ACS Chem. Biol.

2009, 4, 335−344. (c) Li, B. X.; Xiao, X. Discovery of a small-molecule

inhibitor of the KIX−KID interaction. ChemBioChem 2009, 10, 2721−

2724. (d) Majmudar, C. Y.; Højfeldt, J. W.; Arevang, C. J.; Pomerantz,

W. C.; Gagnon, J. K.; Schultz, P. J.; Cesa, L. C.; Doss, C. H.; Rowe, S.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We are grateful for support by the Structural Genomics

Consortium (SGC). The Project was funded through running

budgets of Bracher’s chair and of the SGC. Moreover, we thank

Julia Polyak for technical assistance.

́

P.; Vasquez, V.; Tamayo-Castillo, G.; Cierpicki, T.; Brooks, C. L.;

ABBREVIATIONS USED

■

Sherman, D. H.; Mapp, A. K. Sekikaic acid and lobaric acid target a

dynamic interface of the coactivator CBP/p300. Angew. Chem., Int. Ed.

2012, 51, 11258−11262.

BET, bromodomain and extraterminal domain; CBP/CREBBP,

cAMP responsive element binding protein binding protein;

DIAD, diisopropyl azodicarboxylate; DIPEA, N,N-diisopropyl

ethylamine; DSF, diﬀerential scanning ﬂuorimetry; EDC, 1-

ethyl-3-(3-dimethylaminopropyl)carbodiimide; FRAP, ﬂuores-

cence recovery after photobleaching; HAT, histone acetyl

transferase; ITC, isothermal titration calorimetry; Kac, N-

acetylated lysine; MOZ, monocytic leukemia zinc-ﬁnger; P300,

adenovirus E1A-associated 300 kDa protein; T_m, melting point

(9) (a) Picaud, S.; Wells, C.; Felletar, I.; Brotherton, D.; Martin, S.;

Savitsky, P.; Diez-Dacal, B.; Philpott, M.; Bountra, C.; Lingard, H.;

Fedorov, O.; Muller, S.; Brennan, P. E.; Knapp, S.; Filippakopoulos, P.

RVX-208, an inhibitor of BET transcriptional regulators with

selectivity for the second bromodomain. Proc. Natl. Acad. Sci. U. S.

A. 2013, 110, 19754−19759. (b) Hewings, D. S.; Wang, M.; Philpott,

M.; Fedorov, O.; Uttarkar, S.; Filippakopoulos, P.; Picaud, S.;

Vuppusetty, C.; Marsden, B.; Knapp, S.; Conway, S. J.; Heightman,

T. D. 3,5-dimethylisoxazoles act as acetyl-lysine-mimetic bromodo-

main ligands. J. Med. Chem. 2011, 54, 6761−6770.

(10) Vidler, L. R.; Brown, N.; Knapp, S.; Hoelder, S. Druggability

analysis and structural classification of bromodomain acetyl-lysine

binding sites. J. Med. Chem. 2012, 55, 7346−7359.

(11) (a) Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W. B.;

Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.;

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X

DOI: 10.1021/acs.jmedchem.6b00774

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Development of Selective CBP/P300 Benzoxazepine Bromodomain Inhibitors

DOI: 10.1021/acs.jmedchem.6b00774

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Full text of DOI:10.1021/acs.jmedchem.6b00774

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