Bicyclic Diazepinones as Dual Ligands of the α2δ-1 Subunit of Voltage-Gated Calcium Channels and the Norepinephrine Transporter

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Bicyclic Diazepinones as Dual Ligands of the α2δ‑1 Subunit of

Voltage-Gated Calcium Channels and the Norepinephrine

Transporter

́

Jos é Luis D í az, F é lix Cuevas, Gonzalo Pazos, Paula Alvarez-Bercedo, Ana I. Oliva,

́

M. Angeles Sarmentero, Daniel Font, Agust í n Jim é nez-Aquino, Mar í a Mor ó n, Adriana Port,

Rosal í a Pascual, Albert Dordal, Enrique Portillo-Salido, Raquel F. Reinoso, Jos é Miguel Vela,

and Carmen Almansa *

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ABSTRACT: The synthesis and pharmacological activity of a new

series of bicyclic diazepinones with dual activity toward the α2δ-1

subunit of voltage-gated calcium channels (Ca_vα2δ-1) and the

norepinephrine transporter (NET) are reported. Exploration of the

positions amenable for substitution on a nonaminoacidic Ca_vα2δ-1

scaﬀold allowed the identiﬁcation of favorable positions for the

attachment of NET pharmacophores. Among the patterns

explored, attachment of the 2-ethylamino-9-methyl-6-phenyl-

6,7,8,9-tetrahydro-5H-pyrimido[4,5-e][1,4]diazepin-5-one framework to the meta-position of the phenyl ring of the 3-

methylamino-1-phenylpropoxy and 3-methylamino-1-thiophenylpropoxy moieties provided dual compounds with excellent NET

functionality. Alternative bicyclic frameworks were also explored, and some lead molecules were identiﬁed, which showed a balanced

dual proﬁle and exhibited good ADMET properties.

(Ca_vβ), α₂δ (Ca_vα₂δ), and γ (Ca_vγ). The α₁subunits are the

key porous forming units of the channel complex, regulating

Ca²⁺conduction and inﬂux. The α₂δ, β, and γ subunits are

auxiliary but key for increasing the expression of the α₁

subunits in the plasma membrane and modulating their

function. VGCC are subdivided into ﬁve diﬀerent subtypes:

low voltage-activated T-type (Ca_v3.1, Ca_v3.2, and Ca_v3.3) and

high voltage-activated L-type (Ca_v1.1 through Ca_v1.4), N-type

(Ca_v2.2), P/Q-type (Ca_v2.1), and R-type (Ca_v2.3). Both the

Ca_v1 and Ca_v2 subfamilies contain an auxiliary α2δ subunit,⁸

which is also subdivided in four diﬀerent subtypes: α2δ-1,

initially cloned from skeletal muscle and showing a fairly

ubiquitous distribution; α2δ-2 and α2δ-3 subunits, cloned

from brain; and α2δ-4, recently cloned and largely non-

neuronal.

INTRODUCTION

■

The treatment of pain constitutes an important challenge since

current therapies are suboptimal and hampered by side eﬀects,

which contribute to noncompliance or limited eﬃcacy.^1,2

Considering that most pain states involve multiple mediators

and signaling pathways, both central and peripheral, multi-

modal analgesic approaches are often used in the clinical

practice³and recommended in management guidelines.

Additive or synergistic eﬀects between interventions can

enhance analgesia, and eﬃcacy may be achieved at lower

doses, potentially reducing the risk of adverse eﬀects.⁴

Combinations of available drugs are often used in the clinic,⁵

but developing single compounds with multimodal activity

may oﬀer some advantages, such as better eﬃcacy, superior

treatment compliance (one instead of various pills at

potentially diﬀerent dosage intervals/frequencies), lowering

the risk of drug−drug interactions, simpler pharmacokinetics,

and less variability among patients, both in drug exposure and

response to treatment.⁶With this view in mind, we decided to

explore the preparation of dual ligands acting at the α2δ-1

subunit of voltage-gated calcium channels (Ca_vα2δ-1) and at

the norepinephrine (NE) transporter (NET, SLC6A2), two

mechanisms with clear rationale in the treatment of pain.

Voltage-gated calcium channels (VGCC)⁷are required for

many key functions in the body and are assembled through

interactions of diﬀerent subunits, namely, α₁(Ca_vα₁), β

The Ca_vα2δ-1 subunit (and the Ca_vα₂δ-2 but not Ca_vα₂δ-3

and Ca_vα₂δ-4 subunits) is the therapeutic target⁹of pregabalin

(1) and gabapentin (2), two widely used drugs (Figure 1) for

treating neuropathic pain¹⁰and ﬁbromyalgia¹¹as well as other

Received: October 28, 2020

Published: February 16, 2021

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inhibitors (SSRIs, represented by ﬂuoxetine, 7), selective NE

reuptake inhibitors (NRIs, represented by reboxetine, 8, and

atomoxetine, 9), and the dual inhibitors of SERT and NET

(SNRIs, represented by duloxetine, 10). This compound class

is primarily used for the treatment of depression and other

psychiatric disorders but has also relevant eﬀects in the

treatment of pain, mainly attributed to the NET.²⁷In fact, the

NRI reboxetine (8) has been shown to reverse allodynia in

animal models²⁸and the SNRI duloxetine (10) produces a

decrease in pain sensitivity, whereas the SSRI, ﬂuoxetine (7), is

ineﬀective. Duloxetine has been approved for the treatment of

diabetic neuropathy, ﬁbromyalgia, and chronic musculoskeletal

pain by the Food and Drug Administration (FDA), reinforcing

the involvement of monoamine transporters in pain etiology.²⁹

Figure 1. Structure of described blockers of the Ca_vα2δ-1.

nonpain indications, such as epilepsy¹²or restless leg

syndrome.¹³The binding of these drugs to the Ca_vα2δ-1

subunit results in a reduction in the calcium-dependent release

of multiple neurotransmitters, and its upregulation in the spinal

dorsal horn and dorsal root ganglia after nerve injury correlates

with neuropathic pain development.

An important eﬀort has been devoted along the years to

ﬁnding new Ca_vα2δ-1 ligands for the treatment of pain.¹⁴Most

of the described compounds have aminoacidic structures

closely related to 1 and 2, and mirogabalin (3)^15,16has been

the only newly approved compound in more than 15 years. A

few nonaminoacidic structures have also been reported, but

none of them have progressed into clinical development.

Among others,^17,18Merck reported several Ca_vα2δ-1 ligands

represented by compound 4,¹⁹⁻²¹a structure that was later

modiﬁed by GlaxoSmithKline²²to aﬀord compounds such as

5, which showed good analgesic activity in the complete

Freund’s adjuvant pharmacodynamic model of pain. Finally,

Kyowa Hakko Kirin Co., ﬁled two patent families,^23,24

describing pyrimidodiazepinone tricyclic derivatives repre-

sented by compound 6.

On the other hand, NET is a monoamine transporter mostly

expressed in the peripheral and central nervous systems, which

recycles primarily NE (but also serotonin and dopamine) from

synaptic spaces into presynaptic neurons. It is closely linked to

the serotonin transporter (SERT), which exerts similar

functions with serotonin. NET inhibition provides an increased

availability of NE for binding to postsynaptic receptors that

regulate adrenergic neurotransmission. Numerous studies have

demonstrated that activation of spinal α2-adrenergic receptors

exerts a strong antinociceptive eﬀect²⁵and the tricyclic

antidepressants (TCAs), represented by amitriptyline, have

been used in the treatment of chronic pain for decades.²⁶The

TCAs are nonselective for NET and are the basis for the

development of more speciﬁc inhibitors that modulate both

the noradrenergic and the serotonergic systems with diﬀerent

degrees of selectivity, giving rise to selective serotonin reuptake

Figure 2. Structures of representative SSRI (ﬂuoxetine), NRI

(reboxetine and atomoxetine), and SNRI (duloxetine).

In the search for dual ligands acting at the Ca_vα2δ-1 and

NET, we decided to focus on the nonaminoacidic Ca_vα2δ-1

ligands since the SAR data published on the aminoacidic

structures indicated that achieving relevant activity was

restricted to a narrow structural framework. The pyrimidodia-

zepinone nucleus of compound 6 was selected for fusing or

merging with the more homogeneous NET pharmacophore,

common to compounds 7−10. Although several approaches

have been used for the design of dual compounds,^6,30such as

mutual prodrugs³¹or bivalent ligands,³²the best scenario for

achieving the desired drug-like properties is provided by

“adding” the pharmacophores required for the individual

activities or even better “merging” them by taking advantage of

commonalities in their structures. This was not an obvious task

since both target pharmacophores did not seem to have much

in common, but as depicted in Figure 3, we hoped that this

strategy would allow the identiﬁcation of dual compounds that

showed advantages over the individual therapies and their

mere combination. We report here the structure−activity

relationship (SAR) studies that led to the identiﬁcation of a

new series of bicyclic diazepinones^33,34with dual aﬃnity

toward the Ca_vα2δ-1 and NET.

RESULTS AND DISCUSSION

■

Chemistry. The synthesis of the compounds described

herein (Tables 1, 2, and 3) was accomplished, as depicted in

Schemes 1−5. The most simple oxadiazolyl derivatives (Table

1) were prepared from methylthiopyrimidyl starting materials,

as shown in Scheme 1. Pyrimidine intermediates 13 were

obtained by SNAr amination of chloropyrimidine 11 with

methyl or ethyl ethylenediamine (12) in the presence of a

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Figure 3. Strategy for developing dual Ca_vα2δ-1 and NET ligands.

a

Scheme 1. Preparation of compounds 15, 16, 17 and 23.

a

Reagents and conditions: (a) for R = Me: KO^tBu, THF, rt, and 24 h; for R = Et: Et₃N, ACN, 80 °C, and 48 h; (b) CuI, K₃PO₄, N¹,N²-

dimethylethane-1,2-diamine, dioxane, 100 °C, and 16 h; (c) m-CPBA, DCM, rt, and 1 h; (d) NHR₁R₂, THF, rt, and 16 h; (e) Et₃N, ACN, 0 °C to

rt, and 20 h; (f) NaBH(OAc)₃, DCE, rt, and 1.5 h; (g) Boc₂O, DCM, 0 °C to rt, and 20 h; (h) m-CPBA, DCM, rt, and 3.5 h; (i) EtNH₂, H₂O, rt,

and 72 h; (j) HCl, dioxane, rt, and 16 h; and (k) Pd₂(dba)₃, Xantphos, Cs₂CO₃, dioxane, 110 °C, and 24 h.

base, a reaction that proceeded with concomitant intra-

molecular cyclization. Copper-catalyzed coupling of 13 with

iodoaryl 14 in the presence of N¹,N²-dimethylethane-1,2-

diamine and K₃PO₄as bases provided the methylthio

derivatives 15, which were oxidized with m-CPBA and

subsequently substituted with amines NHR₁R₂. The reduced

derivative 23 was prepared in a seven-step sequence starting

from aldehyde 18. This involved reaction of 18 with N¹-

methylethane-1,2-diamine (12-1), intramolecular reductive

amination using NaBH(OAc)₃, protection of the amino

group with Boc, oxidation of the methylthio group to the

sulfone, substitution with ethylamine, Boc deprotection, and

ﬁnal Pd-catalyzed N-arylation of 21 with the bromoaryl

compound 22 using the catalytic system Pd₂(dba)₃, Xantphos,

and Cs₂CO₃.

Compounds 31−34 (Table 2) with a 3-methylamino-1-

phenylpropoxy (MAP) moiety were prepared according to the

sequence shown in Scheme 2. Key intermediates 27-1 (n = 0,

X = I) were prepared by the Mitsunobu reaction of 24 with

iodophenols 25 followed by the reaction with methylamine

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a

Scheme 2. Preparation of Compounds 31−34

a

Reagents and conditions: (a) PPh₃, DIAD, THF, rt, and 20 h; (b) methylamine, H₂O, EtOH, 130 °C, and 1 h; (c) Boc₂O, DCM, rt, and 2 h; (d)

NaH, DMF, rt, and 3 h; (e) CuI, K₃PO₄, N¹,N²-dimethylethane-1,2-diamine, dioxane, 110 °C, and 16 h; (f) m-CPBA, DCM, rt, and 1 h; (g)

ethylamine, THF, rt, and 16 h; (h) HCl, dioxane, rt, and 45 min; (i) paraformaldehyde, NaBH(OAc)₃, DIPEA, AcOH, DCE, rt, and 70 h; and (j)

methylamine, NaCN, EtOH, 100 °C, and 16 h.

and Boc protection of the amino group. Intermediates 27-2 (n

= 1, X = Br) were obtained by alkylation of 28³⁵with

bromobenzyl reagents 29 using NaH in DMF. Starting from

compounds 27, several ﬁnal derivatives were obtained.

Synthesis of compounds 31 involved copper-catalyzed N-

arylation of 13-1 with 27, oxidation of the thioether group to

the sulfone, substitution with ethylamine, and ﬁnal N-Boc

deprotection, using the conditions described above. Com-

pound 32, with a dimethylamino substituent, was obtained in

good yield by reductive amination of compound 31a with

paraformaldehyde. A similar methodology to that used for

preparing compounds 31 was used for the synthesis of

compound 33 using the tricyclic scaﬀold 35, obtained as

described in the literature.²³In the case of 34, the pyrimidine

ethylamino substituent was already present in intermediate 37,

synthesized by an amidation reaction of 36 with methylamine.

For the synthesis of pure enantiomers of compounds 31a and

33, commercial enantiopure intermediates 24 were used. No

epimerization occurred in the Mitsunobu or alkylation

reaction, and an enantiomeric excess of >96% was obtained.

Throughout the manuscript, the suﬃx R or S after the number

indicates that the compound is the corresponding pure

enantiomer, while no suﬃx refers to a racemic derivative.

The 3-methylamino-1-(thiophen-2-yl)propoxy (MAT) de-

rivatives described in Table 3 were prepared by a diﬀerent

methodology, outlined in Schemes 3, 4, 5. The Mitsunobu-

based route of Scheme 2 was not successful when working with

enantiopure derivative 38 since epimerization was observed in

the aryl ether product. A better alternative was the O-arylation

of 38 with 3-ﬂuorobromobenzene 39 using NaH as the base,

which aﬀorded intermediate 40 in good yield and no decrease

in the enantiomeric excess in relation to the starting material

used (typically, values of >96% ee were obtained in the ﬁnal

compounds). The copper-catalyzed N-arylation reaction

required protection of the methylamino group, and initially,

N-Boc-protected derivatives 41-1 were prepared. However, in

some cases, ether cleavage in the ﬁnal deprotection step under

acidic conditions was observed. Although this could be solved

in some instances by using ZnBr₂in DCM instead of HCl, the

trimethylsilylethoxycarbonyl (Teoc)-protecting group was

alternatively used. N-Teoc-protected intermediates 41-2 were

easily prepared by treating 40 with 4-nitrophenyl (2-

(trimethylsilyl)ethyl)carbonate. The N-arylation with 41-2 of

diﬀerent bicyclic scaﬀolds was performed as described above,

and the ﬁnal deprotection step, using CsF in DMF at 90 °C or

under MW irradiation conditions, provided the ﬁnal

compounds. In the case of enantiopure derivatives, good

enantiomeric excess was always obtained.

In this way, the pyrimido[4,5-e][1,4]diazepin-5-one deriva-

tives (Scheme 3) were obtained. Reactions of 41-1 and 41-2

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a

Scheme 3. Preparation of Pyrimido[4,5-e][1,4]diazepin-5-one Derivatives

a

Reagents and conditions: (a) NaH, DMA, 90 °C, and 3 h; (b) R = Boc: Boc₂O, DCM, rt, and 3 h; R = Teoc: 4-nitrophenyl-(2-

(trimethylsilyl)ethyl)carbonate, DIPEA, DCM, rt, and 20 h; (c) CuI, K₃PO₄, N¹,N²-dimethylethane-1,2-diamine, dioxane, 100 °C, and 20 h; (d) R

= Boc: ZnBr₂, DCM, rt, 24 h; (e) R = Teoc: CsF, DMF, 90 °C, and 90 min; (f) m-CPBA, DCM, rt, and 1 h; (g) NHR₁R₂, THF, rt, and 20 h or

MW at 100 °C and 30 min; (h) Pd/C, Et₃SiH, THF, 0 °C to rt, and 4 h; and (i) MeMgBr, THF, 0 °C, and 1 h.

with the methylthio-bicycle 13-1 provided compounds 42.

Compound 43 was prepared from 42-1 by deprotection of the

Boc group using ZnBr₂in DCM. Compound 42-1 was

oxidized to the sulfone derivative 44-1, which was reacted with

ethylamine and deprotected with ZnBr₂to provide compound

45a, while the methyl derivative 47 resulted from methyl

magnesium bromide attack to sulfone 44-1 and subsequent

Boc deprotection. For the preparation of compounds 45b, 45c,

46, and 48, the Teoc-protecting group was preferred. The

reaction of sulfone 44-2 with the corresponding amines, at rt

or under MW irradiation at 100 °C, and deprotection with CsF

in DMF provided derivatives 45b and 45c. The unsubstituted

46 was prepared by reduction of the thioether 42-2 with

triethylsilane in the presence of Pd/C. The triﬂuoromethyl

derivative 48 was prepared by the reaction of 41-2 with bicycle

49, which in turn was obtained from ethyl 4-chloro-2-

(triﬂuoromethyl)pyrimidine-5-carboxylate, using the condi-

tions described above for the preparation of 13-1.

The pyridodiazepin-5(2H)-one derivatives were synthesized

(Scheme 4) from key intermediates 54 using in all cases the

Teoc-protected derivative 41-2. Compounds 54 were obtained

from the two regioisomeric acid chlorides 50 by subsequent

treatment with N-Boc-protected diamine 51 in the presence of

Et₃N, deprotection of the Boc group, and intramolecular

cyclization in the presence of CsF and Et₃N in DMF.

Treatment of 54 with ethylamine under microwave irradiation

gave the aminopyridyl scaﬀolds 55, which were derivatized as

described above to provide ﬁnal compounds 56 and 57.

Halogenolysis of 54-2 to provide 58 was performed with

triethylsilane and Pd/C in THF, and its ﬁnal derivatization

provided compound 59. The Suzuki reaction of 54-2 with

methylboronic acid provided scaﬀold 60, which was further

derivatized to compound 61. The methoxy group was

introduced with NaOMe in MeOH to give scaﬀold 62,

which was transformed to ﬁnal compound 63.

Final compounds 65, 67, 69, and 71, with alternative central

scaﬀolds, were obtained (Scheme 5) by the reaction of 41-2

with the corresponding bicyclic intermediates 64, 66, 68, and

70, respectively, using the conditions described above. The

pyrido derivatives 64 and 66 were prepared, in a similar way to

that described above for the preparation of 13-1, from the

corresponding ﬂuoropyridinyl esters. Scaﬀold 70 was com-

mercially available, and 68 was obtained by reductive

amination of commercially available 72 with paraformalde-

hyde.

SAR Studies. The biological activity of the compounds

synthesized was evaluated in human α2δ-1- and α2δ-2-

enriched membranes using [³H]-gabapentin as the radioligand.

Both targets were evaluated since in principle we aimed at

ﬁnding selective agents for the α2δ-1 subunit. This was based

on previous studies on the distribution and diﬀerential eﬀects

of the two subtypes,³⁶suggesting that the analgesic eﬀects of

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a

Scheme 4. Preparation of Pyridodiazepin-5(2H)-one Derivatives

a

Reagents and conditions: (a) Et₃N, THF, rt, and 2.5 h; (b) HCl, dioxane, rt, and 2 h; (c) CsF, Et₃N, DMF, 75 °C, and 16 h; (d) EtNH₂, H₂O,

135 °C, MW, and 2 h; (e) CuI, K₃PO₄, N¹,N²-dimethylethane-1,2-diamine, dioxane, 100−130 °C, and 20 h; (f) CsF, DMF, 90 °C, and 90 min or

MW at 90 °C and 90 min; (g) Et₃SiH, Pd/C, THF, rt, and 16 h; (h) MeB(OH)₂, Pd(PPh₃)₄, K₂CO₃, dioxane, 130 °C, and 48 h; and (i) NaOMe,

MeOH, 110 °C, MW, and 2 h.

compounds like pregabalin are due to the interaction with α2δ-

1, while their major side eﬀects such as sedation, dizziness, and

ataxia may be caused by the interaction with α2δ-2. NET

activity was evaluated by measuring the binding aﬃnity to

human ΝΕΤ-enriched membranes using [³H]-nisoxetine as the

radioligand and the functional activity in cells overexpressing

NET and using 4-(4-(dimethylamino)styryl)-N-methylpyridi-

nium iodide (ASP⁺) as the ﬂuorescent substrate. The results

for the new compounds prepared are shown in Tables 1, 2, and

3, where the activities of reference compounds 1, 6, and 8 are

also presented for comparison purposes.

For the design of the dual Ca_vα2δ-1 and NET ligands, we

envisaged a merging approach from the scaﬀold represented by

compound 6 and the NET pharmacophore common to

compounds 7−10. The SAR data around 6 were scarce since

they were only described in a patent publication,²³so we ﬁrst

focused on exploring the key features for Ca_vα2δ-1 binding and

the most suitable positions, allowing substitution to attach the

NET pharmacophore. The tricyclic scaﬀold of 6 was simpliﬁed

by eliminating the fused pyrrolidine ring and keeping only a

methyl group attached to the benzodiazepinone nitrogen (I,

Figure 4). Maintaining an ethylamino group at the 2-position

of the pyrimidine, we ﬁrst explored the phenyl ring

substitution. Several simple substituents were introduced in

ortho-, meta-, and para-positions, and in general, they were

poorly tolerated (results not shown), but a preferential trend

for the meta-substitution was observed. The oxadiazolyl

derivative in the meta-position was identiﬁed as the best

substituent, and therefore, framework (II) was selected for

further SAR studies (Table 1).

This study showed that the ethylamino group in position 2

was important for the activity since 16a was the most potent

compound, while shortening to methylamino (16c) provided a

drop in aﬃnity and the methylthio (15-1) and methylethyla-

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a

Scheme 5. Preparation of Compounds 65, 67, 69, and 71

a

Reagents and conditions: (a) CuI, K₃PO₄, N¹,N²-dimethylethane-1,2-diamine, dioxane, 100 °C, and 20 h; (b) CsF, DMF, 90 °C, and 90 min; and

(c) paraformaldehyde, NaBH(OAc)₃, DIPEA, AcOH, DCE, rt, 48 h.

Table 1. Initial Modiﬁcations of Compound 6

a

b

c

d

comp

Ca_vα2δ-1 (K_i, nM)

Ca_vα2δ-2 (K_i, nM)

NET (K_i, nM)

NET (IC₅₀, nM)

f

e

1

6

8

15-1

16a

16b

16c

16d

17

19 12

99

2148

2

>10,000

e

11

>10,000

2

>10,000

e

>10,000

2974

39 15

1.8

1

e

>10,000

e

81 15

e

g

e

>10,000

NT

e

244 97

>10,000

e

g

e

>10,000

NT

70

g

e

178 72

1026 999

f

e

23

8

a

Binding aﬃnity (K_i, nM) in human α2δ-1-enriched membranes from hamster tumor CHO-K1 cells (human Cav2.2/β3/α2δ1 calcium channel cell

b

line, ChanTest) using [³H]-gabapentin as the radioligand. Each value is the mean SD of two determinations. Binding aﬃnity (K_i, nM) in human

α2δ-2-enriched membranes from human embryonic kidney HEK-293 cells using [³H]-gabapentin as the radioligand. Each value is the mean SD

c

of at least two determinations. Binding aﬃnity (K_i, nM) in human norepinephrine transporter (NET)-enriched membranes from human

d

embryonic kidney HEK-293 cells using [³H]-nisoxetine as the radioligand. Each value is the mean SD of at least two determinations. Functional

activity (IC₅₀, nM) in HEK-293 cells overexpressing human NET and using ASP⁺as the ﬂuorescent substrate. Each value is the mean SD of two

e

f

g

or three determinations. Less than 50% inhibition at 10 μM. % Inhibition at 10 μM. Not tested.

mino (16b) derivatives were inactive. Elongating to methox-

yethylamino (16d) and other substituents, including larger

alkyl (butyl and pentyl), benzyl or phenyl groups provided a

complete loss in aﬃnity (Ca_vα2δ1 K_i> 10,000 nM). In the

same way, enlarging position 9 was detrimental since the ethyl

derivative 17 showed reduced aﬃnity in relation to 16a.

Finally, the benzodiazepinone oxygen atom was proven to be

necessary since the reduced derivative 23 was substantially less

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Table 2. Initial Exploration of the MAP Moiety Attachment to the Ca_vα2δ-1 Framework

a

b

c

comp

n, position

Ca_vα2δ-1 (K_i, nM)

NET (K_i, nM)

NET (IC₅₀, nM)

203 37

54

31aR

31aS

31b

31c

31d

31e

31f

0, meta

0, para

0, ortho

1, meta

1, para

1, ortho

1417 617

2238 990

610 242

29 17

2

d

>10,000

995 581

716 86

d

3493 17

706 356

6246 2797

>10,000

556 85

184 73

41

>10,000

>10,000^e

7

1

d

>10,000

32

2450 2166

1862 721

d

33R

33S

34

187

0

>10,000

336 131

842 70

1533 300

d

>10,000

a

Binding aﬃnity (K_i, nM) in human α2δ-1-enriched membranes from hamster tumor CHO-K1 cells (human Cav2.2/β3/α2δ1 calcium channel cell

b

line, ChanTest) using [³H]-gabapentin as the radioligand. Each value is the mean SD of at least two determinations. Binding aﬃnity (K_i, nM) in

human norepinephrine transporter (NET)-enriched membranes from human embryonic kidney HEK-293 cells using [³H]-nisoxetine as the

c

radioligand. Each value is the mean SD of at least two determinations. Functional activity (IC₅₀, nM) in HEK-293 cells overexpressing human

d

NET and using ASP⁺as the ﬂuorescent substrate. Each value is the mean SD of two or three determinations. Less than 50% inhibition at 10

μM.

potent than 16a. The properties of compound 16a were

considered to be a good starting point since it exhibited a good

ADME proﬁle, with adequate in vitro metabolic stability in

human and rodent liver microsomes (intrinsic clearance below

5 μL/min/mg protein),³⁷no potential for drug−drug

interactions based on the low inhibition (<50% at 1 μM) of

recombinant human cytochrome P450 isoforms (rhCYP 1A2,

2C9, 2C19, 2D6, and 3A4),³⁸and IC₅₀above 10 μM in the

human ether-a-go-go-related gene (hERG)³⁹patch clamp

assay, a well-known predictor of cardiac toxicity. Compound

16a also exhibited selectivity for the Ca_vα2δ-2, for which it

showed a K_i= 2974 nM. All the remaining new compounds

reported here showed K_ivalues above 10 μM for the Ca_vα2δ-2,

so these results are not reported in Tables 2 and 3.

The previous results indicated that substitution in positions

2 and 9 would not be tolerated, so we decided to explore the

attachment of a NET pharmacophore onto the phenyl ring of

the 2-ethylamino-9-methyl-6-phenyl-6,7,8,9-tetrahydro-5H-

pyrimido[4,5-e][1,4]diazepin-5-one scaﬀold. As the most

representative NET framework, the 3-methylamino-1-phenyl-

propoxy (MAP) moiety was chosen, and the results of the

initial exploration are provided in Table 2. Other shorter (3-

methylamino-1-phenylethoxy) or longer (3-methylamino-1-

phenylbutoxy) chains were also explored, but they provided

weaker aﬃnities (results not shown). We were pleased to see

that the compound resulting from the MAP attachment in the

meta-position, 31a, provided dual activity, showing micromolar

aﬃnity for the Ca_vα2δ-1 and, interestingly, higher aﬃnity for

the NET. The S enantiomer showed nanomolar potency for

NET, both in the binding and functional tests, and was clearly

superior to the R isomer, a result consistent with the usual

behavior of NET inhibitors.^40,41On the contrary, pending the

MAP moiety to the ortho- or para-position provided

compounds 31b and 31c, with only residual aﬃnities.

Next, we explored the introduction of ﬂexibility through a

methylene linker between the MAP moiety and the phenyl

ring. The meta-derivative 31d exhibited dual aﬃnity but

reduced NET aﬃnity in relation to 31a. The para-derivative

31e was highly potent on the NET but poorly active on the

Ca_vα2δ-1, and the ortho-derivative 31f was again inactive. A

dimethylamino group in place of the methylamino group on

the MAP chain (compound 32) proved to be clearly

detrimental for NET inhibition. This result was consistent

with previous ﬁndings in the monoamine transporter ﬁeld,

where dimethylation reduced NET aﬃnity while maintaining

or increasing SERT activity.⁴²Attaching the MAP chain to the

Ca_vα2δ-1 scaﬀold of 6 provided derivative 33, with both

enantiomers showing two-digit nanomolar aﬃnity for the

Ca_vα2δ-1 but poor activity at NET. Finally, the open derivative

34 was inactive for both targets. These results indicated that

the proposed approach was indeed adequate to obtain dual

derivatives, but that simultaneous improvement of activity in

both targets would be challenging, as suggested by the

comparison of 31aS versus 33S, where an increase in NET

activity resulted in a decrease in Ca_vα2δ-1 activity and vice

versa.

The phenyl group of the meta-MAP derivative 31a was next

changed to a 2-thiophenyl group to provide compound 45a,

where the S enantiomer was again the eutomer for NET

activity, while both enantiomers kept similar Ca_vα2δ-1

aﬃnities. The 3-methylamino-1-(thiophen-2-yl)propoxy

(MAT) chain of 45aS seemed to provide superior Ca_vα2δ-1

aﬃnity versus its MAP counterpart, 31aS, and was the

framework selected to study the variation of the bicyclic core

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(Table 3). It should be noted that in many cases, the racemic

compounds were ﬁrst prepared and for the most active

derivatives, the S enantiomers (which were consistently the

eutomers for NET, following the trends of 31aR/S and 45aR/

S) were then synthesized. For the sake of the length of the

paper, we report only the S enantiomers when available.

Surprisingly, intermediate 43S showed a very high NET

activity both in the binding and functional assay as well as

submicromolar Ca_vα2δ-1 activity, a result unexpected in view

of the low Ca_vα2δ-1 aﬃnity of 15-1. This result indicated that

the 2-position, where the ethylamino group was identiﬁed as

key for Ca_vα2δ-1 activity in the ﬁrst study (Table 1), could be

further explored. The dimethylamino derivative 45b showed a

worsening in all activities, and the amino 45cS and

unsubstituted 46S were again fairly potent in both targets.

The methyl (47S) and triﬂuoromethyl (48) derivatives showed

an impairment at Ca_vα2δ-1.

Table 3. Activity Data of MAT Derivatives Depicted in

Schemes 3, 4, and 5

Deletion of one nitrogen atom of the pyrimido bicycle

provided the pyrido derivatives 56S and 57S, with dual

aﬃnities comparable to those of 45aS. Many analogues of both

pyridine isomers were prepared, and only a handful of the most

active compounds (59S, 61S, and 63S) are reported in Table

3. The other two pyrido isomers 65 and 67 showed a

decreased aﬃnity at NET, similar to the benzo derivative 69.

The monocyclic diazepinone 71 showed a substantial decrease

in aﬃnity, with NET activity retained in the S enantiomer and

Ca_vα2δ-1 activity mainly in the R enantiomer.

Several of the compounds reported in Tables 2 and 3 show

quite a balanced dual proﬁle, but in general, the compounds

are more potent in NET than in the Ca_vα2δ-1, with several

compounds, such as 31aS and 43S, with outstanding NET

results. These ﬁndings were surprising since we were expecting

the opposite as many of the NET inhibitors described in the

literature correspond to small ligands circumscribed to

structures related to those depicted in Figure 2. When

analyzing the NET structural information published by the

group of Gouaux, ﬁrst for LeuT, bacterial homolog of biogenic

amine transporters (BATs), engineered to achieve human

BAT-like pharmacology,⁴²and later on for the human SERT,⁴³

we guessed that a cavity located near the primary binding site,

corresponding to a low aﬃnity or allosteric site in the

homologous proteins, might allow the accommodation of

larger derivatives, such as 31aS and 43S.

To check this hypothesis and get more knowledge about the

key ligand−receptor interactions, which might help in a later

optimization program, we built a NET homology model using

as a template the six crystallized SERT structures (details given

in the Experimental Section) and docked into it compound

31aS. We observed that the MAP moiety was positioned in the

expected primary binding site, orienting the bicyclic

diazepinone framework in a contiguous site in the extracellular

vestibule. We embedded the resulting docked structure in an

explicit lipid bilayer, solvated it, and ran a 1 ms MD simulation.

The main interactions observed in the resulting trajectory are

schematically drawn in Figure 5. The MAP amine group

interacts with the carboxylate of the conserved Asp75 and with

Phe72, with which it establishes cation−π interactions. Ser318

and Ser419 on each side of the ligand basic amine interact with

it directly (Ser318) or through water bridges (Ser419),

deﬁning together with Asp75 and Phe72 what has been

previously deﬁned⁴³as subsite A. Subsite B is occupied by the

phenyl group of the MAP moiety engaging in π−π stacking

interactions with Phe323, mostly edge to face, and in

a

b

c

See footnotes of Table 2. See footnotes of Table 2. See footnotes

d

of Table 2. Less than 50% inhibition at 1 μM.

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Figure 4. Modiﬁcation sequence from 6 to 16a.

Figure 5. Docking of compound 31aS in the NET homology model generated from the SERT structures l5I6X, 5I6Z, 5I71, 5I73, 5I74, and 5I75.

(A) RMSD of the protein and the docked ligand in the 1 μs molecular dynamics simulations. (B) 2D representation of the interactions of 31aS in

the active site of NET. (C) Image of NET embedded in the lipid bilayer and solvated. (D) Compound 31aS in the NET binding site; the

aminoacids belonging to the diﬀerent subsites have been colored in orange for subsite A, purple for B, cyan for C, and yellow for the residues

belonging to the allosteric site.

hydrophobic interactions with Val148, Val325, and Tyr152.

These residues together with Phe323 start deﬁning subsite C

by π stacking with the phenyl that constitutes the intersection

of the original Ca_vα2δ-1 scaﬀold with the NET pharmaco-

phore. This phenyl ring additionally establishes hydrophobic

interactions with Ala477 and Val148 and forms an edge-to-face

aromatic interaction with Tyr151. Subsite C connects the

primary binding site with a site in the extracellular vestibule,

which is able to harbor the Ca_vα2δ-1 pharmacophore. It is

lined by transmembrane helices 1, 6, and 10 and located

halfway between the sites found in LeuT and SERT. The

pyrimidodiazepinone ring system is stabilized by hydrophobic

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CombiFlash RF system with disposable columns. NMR spectra

were recorded on a Bruker Fourier 300, Bruker Avance I 400, or

Bruker Avance I 500, operating at frequencies of 300, 400, and 500

MHz for ¹H and 75, 100, and 125 MHz for ¹³C, respectively.

Chemical shifts (δ) are in parts per million. Accurate mass

measurements were carried out using a 6540 UHD high-resolution

mass spectrometer QTOF system and obtained by electron spray

ionization (ESI) in positive mode. Data were acquired and processed

using MassHunter software. Commercially available reagents and

solvents (HPLC grade) were used without further puriﬁcation for all

the analytical tests. Analytical HPLC purity data for all ﬁnal

compounds were obtained on an Agilent HP 1200-MS 6120 system

using method 1: Agilent Eclipse XDB-C18 column (4.6 × 150 mm, 5

μm), gradient 5−95% B (A = H₂O (0.05% TFA), B = acetonitrile)

over 7 min, then isocratic 95% B for 5 min, injection volume: 1 μL,

and ﬂow: 1 mL/min. UV spectra were recorded at 254 nm using an

Agilent HP 1200 VWD detector. Data were integrated and reported

using Agilent ChemStation software. All compounds showed purity

higher than 95% as determined by this method. Chiral SFC analysis of

intermediates for ee determination was performed on a Waters

ACQUITY UPC²system using method 2: CHIRALPAK IB column

(4.6 × 100 mm, 3 μM), IPA/CO₂5:95, and 230 nm. Chiral analytical

HPLC of ﬁnal compounds for ee determination was performed in an

Agilent 1260 system. The coated or immobilized polysaccharide

columns were acquired from Chiral Technologies Europe. Three

methods were used: method 3: CHIRALCEL AS-H column (4.6 ×

250 mm, 5 μm), isocratic n-heptane/ethanol + 0.33% DEA 80:20,

injection volume: 5 μL, and ﬂow: 0.8 mL/min; method 4:

CHIRALCEL AS-H column (4.6 × 250 mm, 5 μm), isocratic n-

heptane/ethanol + 0.33% DEA 70:30, injection volume: 5 μL, and

ﬂow: 0.8 mL/min; and method 5: CHIRALPAK AD-H column (4.6

× 250 mm, 5 μm), isocratic n-heptane/iso-propanol + 0.33% DEA

70:30, injection volume: 5 μL, and ﬂow: 0.8 mL/min. All compounds

active in biological assays were electronically ﬁltered for structural

attributes common to pan-assay interference compounds (PAINS)

and were found to be negative.⁴⁵

Determination of Physicochemical Properties. pK_awas

calculated using ACDLabs 9.0.3 and cLogP using ChemDraw Ultra

10.0.3. Kinetic solubility was measured by HPLC after centrifugation

of a sample obtained by adding phosphate buﬀer at pH = 7.4 (1 mL)

to 10 μL of a 10 mM solution of the test compound in DMSO and

stirring for 4 h.

General Procedures for the preparation of compound. s9-

Methyl-2-(methylthio)-6,7,8,9-tetrahydro-5H-pyrimido[4,5-e][1,4]-

diazepin-5-one (13-1). A solution of potassium tert-butoxide (1.0 g,

8.42 mmol) in THF (0.2% water content, 25 mL) was stirred under

air at rt for 10 min. Then, a solution of ethyl 4-chloro-2-

(methylthio)pyrimidine-5-carboxylate (11, 1.0 g, 4.21 mmol) and

N¹-methylethane-1,2-diamine (12-1, 329 mg, 4.21 mmol) in THF

(0.2% water content, 18 mL) was added dropwise and the mixture

was stirred at rt for 20 h. Additional amounts of potassium tert-

butoxide were added after 4 h (4.21 mmol) and 16 h (6.31 mmol) to

complete the reaction. The reaction mixture was concentrated under

vacuum, DCM was added, and the solution was washed successively

with water, NH₄Cl saturated solution, and brine. The organic phase

was concentrated under vacuum to aﬀord 13-1 (388 mg, 41% yield).

¹H NMR (300 MHz, CD₃OD): δ 8.65 (s, 1H), 4.86 (s, 3H), 3.73 (m,

interactions with Ala77, Phe317, and Ala477 and by ionic

interactions of Arg81 with the nitrogen atoms in positions 1

and 3 and of the 2-ethylamino group. Additional hydrogen

bonding is established with Gln314 and one or both the 2-

ethylamino group and the nitrogen in position 3 of the

pyrimidine ring. This H bond interaction between the ligand

and the amide of Gln314 may explain the worsening in NET

aﬃnity of the dimethylamino derivative 45b, and altogether,

the mentioned polar interactions in this subsite explain the

convenience of polar substitution in the bicycle and the

complete loss of NET aﬃnity of the benzodiazepinone 69.

Further insight into the intriguing binding of this compound

series in the NET will be reported in due course.

Besides exhibiting promising dual aﬃnities, several com-

pounds among those reported in Table 3 showed a good

ADMET proﬁle. For example, compounds 45cS and 59S

showed a balanced dual proﬁle accompanied by a good

physicochemical proﬁle (MW: 438.5 and 422.5, respectively;

cLogP: 2.4 and 3.2, respectively; and kinetic solubility above

10 μM in both cases), an adequate metabolic stability in

human, mouse, and rat liver microsomes (intrinsic clearance

below 5 μL/min/mg protein), no potential for drug−drug

interactions based on the low inhibition (<50% at 1 μM) of

recombinant human cytochrome P450 isoforms (rhCYP 1A2,

2C9, 2C19, 2D6, and 3A4), and IC₅₀above 10 μM in the

human ether-a-go-go-related gene (hERG) patch clamp assay.

These results prompted the initiation of a lead optimization

program toward the ﬁnding of a preclinical candidate dual

compound for the treatment of pain, which identiﬁcation will

be reported soon.

CONCLUSIONS

■

In summary, the synthesis and pharmacological activity of a

new series of bicyclic diazepinones with dual activity toward

the Ca_vα2δ-1 and the NET have been studied. A non-

aminoacidic Ca_vα2δ-1 scaﬀold described in the literature was

simpliﬁed, and its diﬀerent positions were explored in order to

identify Ca_vα2δ-1 key binding features and the best positions

for the attachment of NET pharmacophores. Among the

patterns explored, substitution with the 3-methylamino-1-

phenylpropoxy (MAP) and 3-methylamino-1-thiophenylpro-

poxy (MAT) moieties in the meta-position of the phenyl ring

of the 2-ethylamino-9-methyl-6-phenyl-6,7,8,9-tetrahydro-5H-

pyrimido[4,5-e][1,4]diazepin-5-one scaﬀold was the most

interesting. The dual compounds showed excellent NET

functionality despite being substantially larger than prototypic

NET inhibitors described in the literature. The existence of a

cavity located near the NET primary binding site, which might

allow the accommodation of larger derivatives, can explain this

fact.

Changing the central scaﬀold by other bicycles provided

balanced dual compounds that also exhibited good ADMET

properties and were the basis for a lead optimization program,

which allowed the identiﬁcation of a suitable preclinical

candidate for the treatment of pain that will be published soon.

2H), 3.51 (m, 2H), 2.55 (s, 3H).

9-Methyl-6-(3-(5-methyl-1,3,4-oxadiazol-2-yl)phenyl)-2-(methyl-

thio)-6,7,8,9-tetrahydro-5H-pyrimido[4,5-e][1,4]diazepin-5-one

(15-1). To a solution of compound 13-1 (71 mg, 0.31 mmol) and 2-

(3-iodophenyl)-5-methyl-1,3,4-oxadiazole (14)⁴⁶(145 mg, 0.50

mmol) in dioxane (3 mL), CuI (30 mg, 0.15 mmol), K₃PO₄(134

mg, 0.63 mmol), and N¹,N²-dimethylethane-1,2-diamine (17 μL, 0.15

mmol) were added, and the mixture was heated at 100 °C under an

Ar atmosphere for 16 h. The reaction mixture was cooled to rt and

ﬁltered through a pad of Celite, and the solvent was removed under

vacuum. Puriﬁcation by ﬂash chromatography, silica gel, gradient

hexane to 100% acetone, aﬀorded 15-1 (15 mg, 13% yield). ¹H NMR

(500 MHz, CDCl₃): δ 8.82 (bs, 1H), 7.97 (t, J = 1.5 Hz, 1H), 7.92

EXPERIMENTAL SECTION

■

Unless otherwise noted, all materials were obtained from commercial

suppliers and used without further puriﬁcation. Reference compounds

were obtained as follows: pregabalin (1)⁴⁴and compound 6²³were

prepared following the routes described in the literature, and

reboxetine (8) was acquired from Waterstonetech (WS50505).

Flash chromatography was performed on a Teledyne Isco

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(dt, J = 1.5, 7.7 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1H), 7.53−7.51 (m, 1H),

4.09−4.07 (m, 2H), 3.87−3.85 (m, 2H), 3.35 (s, 3H), 2.64 (s, 3H),

2.59 (s, 3H). HPLC, method 1: RT, 5.03 min; purity, 98.0%. HRMS

calcd for C₁₈H₁₉N₆O₂S, 383.1285 [M + H]⁺; found, 383.1275 [M +

H]⁺.

was added, and the mixture was stirred at rt for 16 h. The reaction

mixture was concentrated under vacuum, 10% Na₂CO₃aqueous

solution was added and extracted with DCM, and the organic phase

was concentrated to dryness to aﬀord compound 21 (61 mg, 96%

yield). H NMR (300 MHz, CD₃OD): δ 7.57 (s, 1H), 3.70 (s, 2H),

3.47 (m, 2H), 3.34 (q, J = 7 Hz, 2H), 3.11 (m, 2H), 3.08 (s, 3H),

1.18 (t, J = 7 Hz, 3H).

1

2-(Ethylamino)-9-methyl-6-(3-(5-methyl-1,3,4-oxadiazol-2-yl)-

phenyl)-6,7,8,9-tetrahydro-5H-pyrimido[4,5-e][1,4]diazepin-5-one

(16a). To a solution of 15-1 (66 mg, 0.17 mmol) in DCM (1.5 mL),

m-CPBA (55 mg, 0.22 mmol) was added, and the mixture was stirred

at rt for 1 h. NaHCO₃saturated solution was added, and the mixture

was extracted with DCM. The organic layer was washed with brine

and dried with Na₂SO₄, and the solvent was removed under vacuum.

The crude product was dissolved in THF (1 mL), ethylamine (0.43

mL of a 2 M solution in THF, 0.86 mmol) was added, and the

mixture was stirred at rt for 16 h. The crude residue was concentrated

under vacuum and puriﬁed by ﬂash chromatography, silica gel,

gradient hexane to 100% acetone, to aﬀord compound 16a (40 mg,

61% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.72 (bs, 1H), 7.95 (t, J

= 1.5 Hz, 1H), 7.90 (dt, J = 1.7, 7.0 Hz, 1H), 7.47−7.54 (m, 2H),

5.78 (bs, 1H), 4.04−4.02 (m, 2H), 3.78−3.75 (m, 2H), 3.47 (m, 2H),

3.23 (s, 3H), 2.60 (s, 3H), 1.24 (t, J = 7.2 Hz, 3H). HPLC, method 1:

RT, 4.88 min; purity, 98.4%. HRMS calcd for C₁₉H₂₂N₇O₂, 380.1829

[M + H]⁺; found, 380.1831[M + H]⁺.

N-Ethyl-9-methyl-6-(3-(5-methyl-1,3,4-oxadiazol-2-yl)phenyl)-

6,7,8,9-tetrahydro-5H-pyrimido[4,5-e][1,4]diazepin-2-amine (23).

A mixture of Pd₂(dba)₃(5 mg, 0.0053 mmol), Xantphos (5 mg,

0.012 mmol), compound 21 (30 mg, 0.14 mmol), 2-(3-

bromophenyl)-5-methyl-1,3,4-oxadiazole (22, 25 mg, 0.10 mmol),

and Cs₂CO₃(48 mg, 0.14 mmol) in dry dioxane (0.5 mL) under

argon was heated at 110 °C in a sealed tube for 24 h. The reaction

mixture was cooled to rt and ﬁltered through a pad of Celite, and the

ﬁltrate was concentrated under vacuum. Puriﬁcation by ﬂash

chromatography, silica gel, gradient hexane to 100% acetone, aﬀorded

1

compound 23 (6 mg, 15% yield). H NMR (300 MHz, CDCl₃): δ

7.78 (bs, 1H), 7.39 (m, 1H), 7.34−7.29 (m, 2H), 6.88−6.85 (m, 1H),

5.14 (bs, 1H), 4.48 (s, 2H), 3.80−3.76 (m, 4H), 3.42−3.35 (m, 2H),

3.12 (s, 3H), 2.62 (s, 3H), 1.20 (t, J = 7.2 Hz, 3H). HPLC, method 1:

RT, 5.51 min; purity, 95.1%. HRMS calcd for C₁₉H₂₄N₇O, 366.2037

[M + H]⁺; found, 366.2031 [M + H]⁺.

4-((2-Aminoethyl)(methyl)amino)-2-(methylthio)pyrimidine-5-

carbaldehyde (19). To a solution of 4-chloro-2-(methylthio)-

pyrimidine-5-carbaldehyde (18, 712 mg, 3.77 mmol) in acetonitrile

(40 mL) at 0 °C, Et₃N (2.47 mL, 17.74 mmol) and N¹-methylethane-

1,2-diamine (12-1, 0.42 mL, 4.91 mmol) were added, and the mixture

was stirred at rt for 20 h. NaHCO₃saturated solution was added and

extracted with EtOAc. The organic phase was concentrated under

vacuum, and the residue was puriﬁed by ﬂash chromatography, silica

gel, gradient DCM to 30% MeOH, to aﬀord compound 19 (627 mg,

(R)-1-(3-Chloro-1-phenylpropoxy)-3-iodobenzene (26R-meta).

To a solution of (S)-3-chloro-1-phenylpropan-1-ol (24S, 400 mg,

2.34 mmol) in THF (11 mL), 3-iodophenol (774 mg, 3.52 mmol)

and PPh₃(922 mg, 3.52 mmol) were added. The solution was cooled

at 0 °C, DIAD (0.692 mL, 3.52 mmol) was added dropwise with rapid

stirring, and the reaction mixture was stirred at rt for 20 h. The

solvent was removed under vacuum. Puriﬁcation by ﬂash chromatog-

raphy, silica gel, gradient hexane to 100% ethyl acetate, aﬀorded

1

compound 26R-meta (779 mg, 89% yield). H NMR (300 MHz,

1

73% yield). H NMR (300 MHz, CDCl₃): δ 2.56 (s, 3H), 3.34 (s,

CDCl₃): δ 7.41−7.36 (m, 4H), 7.32 (m, 1H), 7.29 (m, 1H), 7.25 (m,

1H), 6.91 (t, J = 8.5 Hz, 1H), 6.82 (m, 1H), 5.37 (m, 1H), 3.84−3.77

(m, 1H), 3.65−3.58 (m, 1H), 2.53−2.43 (m, 1H), 2.28−2.18 (m,

1H).

3H), 3.51 (m, 2H), 4.04 (m, 2H), 8.06 (m, 1H), 8.16 (s, 1H).

tert-Butyl 9-Methyl-2-(methylthio)-5,7,8,9-tetrahydro-6H-

pyrimido[4,5-e][1,4]diazepine-6-carboxylate (20). To a solution of

compound 19 (210 mg, 0.92 mmol) in DCE (38 mL), NaBH(OAc)₃

(393 mg, 1.85 mmol) was added, and the reaction mixture was stirred

at rt for 1.5 h. NaHCO₃saturated solution was added and extracted

with DCM. The organic phase was concentrated under vacuum, and

the residue was puriﬁed by ﬂash chromatography, silica gel, gradient

DCM to 20% MeOH, to aﬀord 9-methyl-2-(methylthio)-6,7,8,9-

tetrahydro-5H-pyrimido[4,5-e][1,4]diazepine (152 mg, 78% yield).

¹H NMR (300 MHz, CDCl₃): δ 8.11 (s, 1H), 4.03 (m, 2H), 3.52 (m,

tert-Butyl (R)-(3-(3-Iodophenoxy)-3-phenylpropyl)(methyl)-

carbamate (27-1R-meta). To a solution of 26R-meta (779 mg,

2.09 mmol) in EtOH (2.3 mL), methylamine (40% solution in water,

5.4 mL, 62.7 mmol) was added, and the mixture was heated at 130 °C

in a sealed tube for 1 h. The reaction mixture was cooled at rt, water

was added, the mixture was extracted with DCM, and the organic

phases were concentrated under vacuum. The crude product was

dissolved in DCM (17 mL), di-tert-butyldicarbonate (502 mg, 2.30

mmol) was added at 0 °C, and the reaction mixture was stirred at rt

for 2 h. Water was added, and the mixture was extracted with DCM

and washed with NaHCO₃saturated solution and brine. The solvent

was removed under vacuum, and the residue was puriﬁed by ﬂash

chromatography, silica gel, gradient cyclohexane to 100% EtOAc, to

2H), 3.34 (s, 3H), 2.55 (s, 3H).

To a solution of the previous compound (145 mg, 0.69 mmol) in

DCM (6.5 mL) at 0 °C, di-tert-butyldicarbonate (150 mg, 0.69

mmol) was added, and the mixture was stirred at rt for 20 h. NaHCO₃

saturated solution was added, extracted with DCM, and concentrated

under vacuum to aﬀord compound 20 that was used in the next step

1

aﬀord compound 27-1R-meta (838 mg, 86% yield). H NMR (300

1

without further puriﬁcation (214 mg, quant yield). H NMR (300

MHz, CDCl₃): δ 7.41−7.26 (m, 5H), 7.25 (m, 1H), 7.21 (m, 1H),

6.88 (t, J = 8.5 Hz, 1H), 6.77 (m, 1H), 5.10 (m, 1H), 3.56−3.26 (m,

2H), 2.87 (s, 3H), 2.28−2.01 (m, 2H), 1.52−1.33 (bs, 9H). Chiral

SFC analysis, method 2: RT, 2.63 min; 96% ee.

MHz, CD₃OD): δ rotamers 7.75 and 7.69 (s, 1H), 4.51 and 4.45 (s,

2H), 3.79 (m, 4H), 3.20 (s, 3H), 2.49 (s, 3H), 1.45 and 1.38 (s, 9H).

N-Ethyl-9-methyl-6,7,8,9-tetrahydro-5H-pyrimido[4,5-e][1,4]-

diazepin-2-amine (21). To a solution of compound 20 (227 mg, 0.73

mmol) in DCM (4 mL), m-CPBA (252 mg, 1.46 mmol) was added,

and the mixture was stirred at rt for 3.5 h. NaHCO₃saturated solution

was added, and the mixture was extracted with DCM. The organic

layer was dried with MgSO₄, and the solvent was removed under

vacuum. The crude residue was treated with ethylamine (70%

solution in water, 2.1 mL, 26.10 mmol), and the mixture was stirred at

rt for 72 h. The reaction mixture was concentrated under vacuum and

puriﬁed by ﬂash chromatography, silica gel, gradient DCM to 20%

MeOH, to aﬀord tert-butyl 2-(ethylamino)-9-methyl-5,7,8,9-tetrahy-

dro-6H-pyrimido[4,5-e][1,4]diazepine-6-carboxylate (94 mg, 47%

tert-Butyl (3-((3-Bromobenzyl)oxy)-3-phenylpropyl)(methyl)-

carbamate (27-2-meta). To a solution of tert-butyl (3-hydroxy-3-

phenylpropyl)(methyl)carbamate (28, 115 mg, 0.43 mmol) in DMF

(6 mL) cooled at 0 °C, NaH (35 mg, 60% in mineral oil, 0.86 mmol)

was added, and the solution was stirred at rt for 30 min. Then, a

solution of 1-bromo-3-(bromomethyl)benzene (162 mg, 0.65 mmol)

in DMF (2 mL) was added at 0 °C and the mixture was stirred at rt

for 3 h. Water was added, and the mixture was extracted with ethyl

acetate. The organic phase was dried over Na₂SO₄, ﬁltered, and

concentrated under vacuum. Puriﬁcation by ﬂash chromatography,

silica gel, gradient cyclohexane to 100% ethyl acetate, aﬀorded

1

yield). H NMR (300 MHz, CDCl₃): δ 1.20 (t, J = 7.2 Hz, 3H),

compound 27-2-meta (159 mg, 80% yield). H NMR (500 MHz,

1.43 (s, 9H), 3.10 (s, 3H), 3.40 (m, 2H), 3.57 (m, 2H), 3.73 (m, 2H),

4.29 and 4.41 (s, 2H), 5.06 (bs, 1H), 7.59 and 7.69 (s, 1H).

To a solution of the previous compound (94 mg, 0.30 mmol) in

dioxane (0.3 mL), HCl (4 M solution in dioxane, 1.0 mL, 4.28 mmol)

CDCl₃): δ 7.48 (s, 1H), 7.44−7.37 (m, 3H), 7.37−7.30 (m, 3H),

7.26−7.19 (m, 2H), 4.40 (d, J = 12.1 Hz, 1H), 4.34 (m, 1H), 4.25 (d,

J = 12.1 Hz, 1H), 3.41−3.22 (m, 2H), 2.85 (s, 3H), 2.07 (m, 1H),

1.94 (m, 1H), 1.45 (s, 9H).

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tert-Butyl (R)-Methyl(3-(3-(9-methyl-2-(methylthio)-5-oxo-

5,7,8,9-tetrahydro-6H-pyrimido[4,5-e][1,4]diazepin-6-yl)phenoxy)-

3-phenylpropyl)carbamate (30aR). To a solution of 13-1 (150 mg,

0.67 mmol) and 27-1R-meta (313 mg, 0.67 mmol) in dioxane (10

mL), CuI (38 mg, 0.20 mmol), K₃PO₄(284 mg, 1.33 mmol), and

N¹,N²-dimethylethane-1,2-diamine (17 mg, 0.20 mmol) were added,

and the mixture was heated at 110 °C under an Ar atmosphere for 16

h. The reaction mixture was cooled to rt, and the solvent was removed

under vacuum to give a crude product that was puriﬁed by ﬂash

chromatography, silica gel, gradient cyclohexane to 100% ethyl

in dioxane (3 mL), CuI (16 mg, 0.08 mmol), K₃PO₄(118 mg, 0.55

mmol), and N¹,N²-dimethylethane-1,2-diamine (9 μL, 0.08 mmol)

were added, and the mixture was heated at 110 °C under an Ar

atmosphere for 16 h. The reaction mixture was cooled to rt, and the

solvent was removed under vacuum to give a crude product that was

puriﬁed by ﬂash chromatography, silica gel, gradient DCM to 25%

MeOH, to aﬀord tert-butyl (3-(3-(2-(ethylamino)-N-methylpyrimi-

dine-5-carboxamido)phenoxy)-3-phenylpropyl)(methyl)carbamate

1

(51 mg, 35% yield). H NMR (300 MHz, CDCl₃): δ 8.65 (s, 2H),

7.34−7.19 (m, 5H), 6.81−6.74 (m, 2 H), 6.72 (m, 1H), 6.13 (m,

1H), 5.10 (dd, J = 4.2, 8.1 Hz, 1H), 3.97−3.83 (m, 2H), 3.47−3.35

(m, 2H), 2.97 (d, J = 4.8 Hz, 3H), 2.84 (s, 3H), 2.23−2.02 (m, 2H),

1.40 (s, 9H), 1.12 (t, J = 7.2 Hz, 3H).

1

acetate, to aﬀord compound 30aR (267 mg, 70% yield). H NMR

(400 MHz, CDCl₃): δ 8.78 (s, 1H), 7.35 (m, 4H), 7.28 (m, 1H), 7.20

(m, 1H), 6.82 (m, 2H), 6.72 (m, 1H), 5.13 (m, 1H), 3.90 (m, 2H),

3.71 (m, 2H), 3.41 (m, 2H), 3.28 (s, 3H), 2.86 (s, 3H), 2.57 (s, 3H),

2.14 (m,2 H), 1.42 (s, 9H).

To a solution of the previous compound (40 mg, 0.07 mmol) in

dioxane (0.2 mL), HCl (4 M in dioxane, 0.27 mL, 1.07 mmol) was

added, and the mixture was stirred at rt for 30 min. The reaction

mixture was concentrated to dryness under vacuum. To obtain the

free base, DCM was added and washed with Na₂CO₃(10% aqueous

solution), and the aqueous phase was extracted with DCM. The

combined organic layers were concentrated under vacuum to aﬀord

(R)-2-(Ethylamino)-9-methyl-6-(3-(3-(methylamino)-1-

phenylpropoxy)phenyl)-6,7,8,9-tetrahydro-5H-pyrimido[4,5-e][1,4]-

diazepin-5-one (31aR). To a solution of 30aR (187 mg, 0.33 mmol)

in DCM (6 mL), m-CPBA (123 mg, 0.49 mmol) was added, and the

mixture was stirred at rt for 1 h. NaHCO₃saturated solution was

added, and the mixture was extracted with DCM. The organic layer

was washed with brine and dried with Na₂SO₄, and the solvent was

removed under vacuum. The crude product was dissolved in THF (6

mL), ethylamine (1.66 mL of a 2 M solution in THF, 3.32 mmol) was

added, and the mixture was stirred at rt for 16 h. The crude residue

was concentrated under vacuum and puriﬁed by ﬂash chromatog-

raphy, silica gel, gradient cyclohexane to 100% ethyl acetate, to aﬀord

tert-butyl (R)-(3-(3-(2-(ethylamino)-9-methyl-5-oxo-5,7,8,9-tetrahy-

dro-6H-pyrimido[4,5-e][1,4]diazepin-6-yl)phenoxy)-3-

phenylpropyl)(methyl)carbamate (175 mg, 94% yield). ¹H NMR

(500 MHz, CDCl₃): δ 8.71 (s, 1H), 7.35 (m, 4H), 7.26 (m, 1H), 7.17

(m, 1H), 6.83 (m, 2H), 6.68 (m, 1H), 5.11 (m, 1H), 3.87 (m, 2H),

3.63 (m, 2H), 3.48 (m, 2H), 3.40 (m, 2H), 3.20 (s, 3H), 2.85 (s, 3H),

2.16 (m, 1H), 2.09 (m, 1H), 1.42 (s, 9H), 1.25 (t, J = 7 Hz, 3H).

To a solution of the previous compound (128 mg, 0.22 mmol) in

dioxane (0.4 mL), HCl (4 M in dioxane, 0.800 mL, 3.20 mmol) was

added, and the mixture was stirred at rt for 45 min. The reaction

mixture was concentrated to dryness under vacuum. To obtain the

free base, DCM was added and washed with Na₂CO₃(10% aqueous

solution), and the aqueous phase was extracted with DCM. The

combined organic layers were concentrated under vacuum to aﬀord

1

34 (30 mg, quant yield). H NMR (400 MHz, CD₃OD): δ 8.65 (s,

2H), 7.38−7.36 (m, 2H), 7.33−7.30 (m, 2H), 7.25−7.22 (m, 2H),

6.86−6.84 (m, 1H), 6.75−6.74 (m, 2H), 5.31 (dd, J = 4.7, 8.0 Hz,

1H), 3.99−3.88 (m, 2H), 2.88 (s, 3H), 2.75−2.66 (m, 2H), 2.36 (s,

3H), 2.22−2.15 (m, 1H), 2.06−1.99 (m, 1H), 1.08 (t, J = 7.2 Hz,

3H). HPLC, method 1: RT, 5.48 min; purity, 97.5%. HRMS calcd for

C₂₄H₃₀N₅O₂, 420.2394 [M + H]⁺; found, 420.2380 [M + H]⁺.

2-(Ethylamino)-N-methylpyrimidine-5-carboxamide (37). To a

solution of ethyl 2-(methylthio)pyrimidine-5-carboxylate (36, 110

mg, 0.55 mmol) in DCM (4 mL), m-CPBA (166 mg, 0.72 mmol) was

added, and the mixture was stirred at rt for 1 h. A NaHCO₃aqueous

saturated solution was added, and the mixture was extracted with

DCM. The organic layer was washed with brine, dried over Na₂SO₄,

and ﬁltered, and the solvent was removed under vacuum. The residue

was dissolved in THF (4 mL), ethylamine (2 M solution in THF, 1.38

mL, 2.77 mmol) was added, and the mixture was stirred at rt for 3 h.

The solvent was removed under vacuum. The residue was treated

with methylamine (3 mL of a 33% solution in EtOH, 24.19 mmol)

and NaCN (3 mg, 0.061 mmol), and the mixture was heated at 100

°C for 16 h. The solvent was removed under vacuum. Puriﬁcation by

ﬂash chromatography, silica gel, gradient DCM to 20% MeOH,

1

31aR (101 mg, 89% yield). H NMR (400 MHz, CD₃OD): δ 8.58

1

aﬀorded 37 (90 mg, 90% yield). H NMR (300 MHz, CD₃OD): δ

(bs, 1H), 7.48−7.46 (m, 2H), 7.42−7.38 (m, 2H), 7.34−7.26 (m,

2H), 6.92−6.91 (m, 1H), 6.89−6.85 (m, 2H), 5.42 (dd, J = 4.8, 8.0

Hz, 1H), 3.94−3.92 (m, 2H), 3.77−3.76 (m, 2H), 3.52−3.47 (m,

2H), 3.29 (s, 3H), 2.85−2.73 (m, 2H), 2.44 (s, 3H), 2.31−2.20 (m,

1H), 2.14−2.06 (m, 1H), 1.29 (t, J = 7.2 Hz, 3H). HPLC, method 1:

RT, 4.95 min; purity, 97.2%. HRMS calcd for C₂₆H₃₂N₆O₂, 461.266

[M + H]⁺; found, 461.267 [M + H]⁺.

8.68 (s, 2H), 3.44 (q, J = 7.2 Hz, 2H), 2.89 (s, 3H), 1.22 (t, J = 7.2

Hz, 3H).

(S)-3-(3-Bromophenoxy)-N-methyl-3-(thiophen-2-yl)propan-1-

amine (40S). To a solution of (S)-3-(methylamino)-1-(thiophen-2-

yl)propan-1-ol (38S, 1.2 g, 7.01 mmol) in dimethylacetamide (20

mL), NaH (420 mg, 60% in mineral oil, 10.51 mmol) was added, and

the mixture was stirred at rt for 30 min. 1-Bromo-3-ﬂuorobenzene

(39, 2.45 g, 14.01 mmol) was added, and the mixture was heated at 90

°C for 3 h. Water was added, and the mixture was extracted with ethyl

acetate. The organic phase was dried over Na₂SO₄and concentrated

under vacuum to aﬀord compound 40S that was used in the next step

6-(3-(3-(Dimethylamino)-1-phenylpropoxy)phenyl)-2-(ethylami-

no)-9-methyl-6,7,8,9-tetrahydro-5H-pyrimido[4,5-e][1,4]diazepin-

5-one (32). To a solution of 31a (57 mg, 0.124 mmol) in DCE (3

mL), paraformaldehyde (16 mg, 0.495 mmol), DIPEA (0.108 mL,

0.62 mmol), NaBH(OAc)₃(105 mg, 0.49 mmol), and AcOH (7 μL,

0.12 mmol) were added, and the reaction mixture was stirred at rt for

70 h. NaHCO₃aqueous saturated solution was added, extracted with

DCM, washed with brine, dried over Na₂SO₄, ﬁltered, and

concentrated under vacuum. Puriﬁcation by ﬂash chromatography,

silica gel, gradient DCM to 20% MeOH, aﬀorded 32 (63 mg, quant

1

without further puriﬁcation. H NMR (400 MHz, CDCl₃): δ 7.25

(dd, J = 1.2, 5.1 Hz, 1H), 7.13 (m, 1H), 7.10−7.06 (m, 2H), 7.03 (m,

1H), 6.96 (dd, J = 3.4, 5.0 Hz, 1H), 6.87 (dt, J = 2.2, 7.2 Hz, 1H),

5.57 (dd, J = 5.2, 7.5 Hz, 1H), 2.78 (m, 2H), 2.47 (s, 3H), 2.32 (m,

1H), 2.24 (bs, 1H), 2.13 (m, 1H).

1

tert-Butyl (S)-(3-(3-Bromophenoxy)-3-(thiophen-2-yl)propyl)-

(methyl)carbamate (41-1S). To a solution of 40S (1.42 g, 4.38

mmol) in DCM (25 mL) at 0 °C, di-tert-butyldicarbonate (1.25 g,

5.73 mmol) was added, and the mixture was stirred at rt for 3 h.

Water was added, and the mixture was extracted with DCM, washed

with NaHCO₃aqueous saturated solution and brine, and concen-

trated under vacuum. Puriﬁcation by ﬂash chromatography, silica gel,

gradient cyclohexane to 100% ethyl acetate, aﬀorded 41-1S (1.3 g,

70% yield global two steps). ¹H NMR (300 MHz, CDCl₃): δ 7.25 (d,

J = 5 Hz, 1H), 7.10 (m, 1H), 7.06 (m, 2H), 7.02 (m, 1H), 6,96 (m,

1H), 6.85 (td, J = 7, 2.5 Hz, 1H), 5.41 (m, 1H), 3.43 (m, 2H), 2.87

yield). H NMR (400 MHz, CD₃OD): δ 8.50 (bs, 1H), 7.40−7.38

(m, 2H), 7.34−7.30 (m, 2H), 7.26−7.18 (m, 2H), 6.84−6.83 (m,

1H), 6.81−6.76 (m, 2H), 5.31 (dd, J = 4.7, 7.9 Hz, 1H), 3.85−3.84

(m, 2H), 3.69−3.67 (m, 2H), 3.42 (q, J = 7.1, 16.0 Hz, 2H), 3.21 (s,

3H), 2.59−2.41 (m, 2H), 2.24 (s, 6H), 2.21−2.13 (m, 1H), 2.05−

1.96 (m, 1H), 1.21 (t, J = 7.1 Hz, 3H). HPLC, method 1: RT, 4.97

min; purity, 96.1%. HRMS calcd for C₂₇H₃₅N₆O₂, 475.2816 [M +

H]⁺; found, 475.2817 [M + H]⁺.

2-(Ethylamino)-N-methyl-N-(3-(3-(methylamino)-1-

phenylpropoxy)phenyl)pyrimidine-5-carboxamide (34). To a sol-

ution of 37 (50 mg, 0.27 mmol) and 27-1-meta (213 mg, 0.41 mmol)

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(s, 3H), 2.31 (m, 1H), 2.18 (m, 1H), 1.42 (s, 9H). Chiral SFC

analysis, method 2: RT, 1.94 min; 98% ee.

reaction mixture was stirred at rt for 48 h. Water was added and

stirred until complete solution with the help of ultrasound irradiation.

The layers were separated, and the aqueous phase was extracted with

DCM. The organic layer was dried over Na₂SO₄and concentrated

under vacuum. Puriﬁcation by ﬂash chromatography, silica gel,

gradient DCM to 40% MeOH, aﬀorded a salt that was treated with

NaHCO₃aqueous saturated solution and extracted with DCM. The

solvent was removed under vacuum to aﬀord 45aS (345 mg, 52%

2-(Trimethylsilyl)ethyl (S)-(3-(3-Bromophenoxy)-3-(thiophen-2-

yl)propyl)(methyl)carbamate (41-2S). To a solution of 40S (2 g,

6.13 mmol) in DCM (14 mL), DIPEA (1.07 mL, 6.13 mmol) and a

solution of 4-nitrophenyl (2-(trimethylsilyl)ethyl) carbonate (1.73 g,

6.13 mmol) in DCM (6 mL) were added, and the mixture was stirred

at rt for 20 h. The reaction mixture was washed with NaHCO₃

aqueous saturated solution and 2 M NaOH until the bright yellow

color almost disappeared. The organic layer was dried over Na₂SO₄,

ﬁltered, and concentrated under vacuum. Puriﬁcation by ﬂash

chromatography, silica gel, gradient cyclohexane to 100% ethyl

acetate, aﬀorded 41-2S (2.05 g, 62% yield global two steps). ¹H NMR

(400 MHz, CDCl₃): δ 7.21 (m, 1H), 7.11−7.05 (m, 3H), 7.03−6.98

(m, 1H), 6.92 (m, 1H), 6.83 (m, 1H), 5.42 (m, 1H), 4.19−3.93 (m,

2H), 3.57 (m, 1H), 3.37 (m, 1H), 2.89 (m, 3H), 2.29 (m, 1H), 2.16

(m, 1H), 0.99 (m, 1H), 0.81 (m, 1H), 0.03 (s, 9H).

1

yield). H NMR (400 MHz, CD₃OD): δ 8.52 (s, 1H), 7.32 (dd, J =

1.1, 5.2 Hz, 1H), 7.24 (t, J = 8.2 Hz, 1H), 7.10 (dd, J = 0.7, 3.5 Hz,

1H), 6.95 (dd, J = 3.5, 5.1 Hz, 1H), 6.92−6.88 (m, 2H), 6.83 (ddd, J

= 0.7, 1.7, 8.2 Hz, 1H), 5.67 (dd, J = 5.7, 7.5 Hz, 1H), 3.89−3.87 (m,

2H), 3.73−3.71 (m, 2H), 3.45−3.40 (m, 2H), 3.22 (s, 3H), 2.72−

2.66 (m, 2H), 2.36 (s, 3H), 2.36−2.25 (m, 1H), 2.14−2.06 (m, 1H),

1.22 (t, J = 7.2 Hz, 3H). HPLC, method 1: RT, 4.82 min; purity,

99.3%. HRMS calcd for C₂₄H₃₀N₆O₂S, 467.2224 [M + H]⁺; found,

467.2233 [M + H]⁺.

tert-Butyl (S)-Methyl(3-(3-(9-methyl-2-(methylthio)-5-oxo-

5,7,8,9-tetrahydro-6H-pyrimido[4,5-e][1,4]diazepin-6-yl)phenoxy)-

3-(thiophen-2-yl)propyl)carbamate (42-1S). To a solution of 13-1

(150 mg, 0.67 mmol) and 41-1S (371 mg, 0.87 mmol) in dioxane (10

mL), CuI (38 mg, 0.20 mmol), K₃PO₄(284 mg, 1.34 mmol), and

N¹,N²-dimethylethane-1,2-diamine (22 μL, 0.20 mmol) were added,

and the mixture was heated at 100 °C in a sealed tube for 20 h. The

reaction mixture was cooled to rt and concentrated under vacuum.

Puriﬁcation by ﬂash chromatography, silica gel, gradient cyclohexane

to 100% ethyl acetate, aﬀorded 42-1S (320 mg, 84% yield). ¹H NMR

(300 MHz, CDCl₃): δ 8.78 (s,1H), 7.25 (m, 2H), 7.03 (m, 1H), 6.95

(m, 1H), 6.87 (m, 2H), 6.82 (m, 1H), 5.43 (m, 1H), 3.93 (m, 2H),

3.75 (m, 2H), 3.39 (m, 2H), 3.29 (s, 3H), 2.86 (s, 3H), 2.56 (s, 3H),

2.25 (m, 2H), 1.42 (s, 9H).

2-(Dimethylamino)-9-methyl-6-(3-(3-(methylamino)-1-(thio-

phen-2-yl)propoxy)phenyl)-6,7,8,9-tetrahydro-5H-pyrimido[4,5-e]-

[1,4]diazepin-5-one (45b). To a solution of 42-2 (105 mg, 0.17

mmol) in DCM (5 mL), m-CPBA (59 mg, 0.26 mmol) was added,

and the mixture was stirred at rt for 1 h. NaHCO₃aqueous saturated

solution was added, and the mixture was extracted with DCM. The

organic layer was washed with brine and dried with Na₂SO₄, and the

solvent was removed under vacuum. The crude product was dissolved

in THF (1 mL), and dimethylamine (40% solution in water, 0.43 mL)

was added. The mixture was stirred at rt for 20 h. The solvent was

removed under vacuum. Puriﬁcation by ﬂash chromatography, silica

gel, gradient DCM to 25% MeOH, aﬀorded 2-(trimethylsilyl)ethyl (3-

(3-(2-(dimethylamino)-9-methyl-5-oxo-5,7,8,9-tetrahydro-6H-

pyrimido[4,5-e][1,4]diazepin-6-yl)phenoxy)-3-(thiophen-2-yl)-

propyl)(methyl)carbamate (90 mg, 86% yield). ¹H NMR (500 MHz,

CDCl₃): δ 8.75 (s, 1H), 7.21−7.17 (m, 2H), 7.03−6.68 (m, 1H),

6.93−6.91 (m, 1H), 6.86−6.84 (m, 2H), 6.78−6.74 (m, 1H), 5.44−

5.39 (m, 1H), 4.17−4.03 (m, 2H), 3.88−3.86 (m, 2H), 3.65−3.63

(m, 2H), 3.50−3.34 (m, 2H), 3.19 (s, 6H), 3.18 (s, 3H), 2.87 (s, 3H),

2.34−2.24 (m, 1H), 2.21−2.11 (m, 1H), 1.00−0.87 (m, 2H), 0.01 (s,

9H).

2-(Trimethylsilyl)ethyl (S)-Methyl(3-(3-(9-methyl-2-(methylthio)-

5-oxo-5,7,8,9-tetrahydro-6H-pyrimido[4,5-e][1,4]diazepin-6-yl)-

phenoxy)-3-(thiophen-2-yl)propyl)carbamate (42-2S). To a solu-

tion of 13-1 (350 mg, 1.56 mmol) and 41-2S (1.1 g, 2.34 mmol) in

dioxane (15 mL), CuI (89 mg, 0.47 mmol), K₃PO₄(662 mg, 3.12

mmol), and N¹,N²-dimethylethane-1,2-diamine (50 μL, 0.47 mmol)

were added, and the mixture was heated at 100 °C in a sealed tube for

20 h. The reaction mixture was cooled to rt and concentrated under

vacuum. Puriﬁcation by ﬂash chromatography, silica gel, gradient

cyclohexane to 100% ethyl acetate, aﬀorded 42-2S (815 mg, 85%

To a solution of the previous compound (90 mg, 0.14 mmol) in

DMF (4 mL), CsF (112 mg, 0.73 mmol) was added, and the mixture

was heated at 90 °C for 90 min. The solvent was removed under

vacuum. Puriﬁcation by ﬂash chromatography, silica gel, gradient

1

yield). H NMR (500 MHz, CDCl₃): δ 8.78 (s, 1H), 7.26 (m, 2H),

1

7.04 (m, 1H), 6.96 (m, 1H), 6.88 (m, 2H), 6.84 (m, 1H), 5.45 (m,

1H), 4.15 (m, 2H), 3.95 (m, 2H), 3.76 (m, 2H), 3.45 (m, 2H), 3.30

(s, 3H), 2.91 (s, 3H), 2.57 (s, 3H), 2−33 (m, 1H), 2−19 (m, 1H),

1.00 (m, 1H), 0.93 (m, 1H), 0.05 (s, 9H).

DCM to 40% MeOH, aﬀorded 45b (55 mg, 85% yield). H NMR

(400 MHz, CD₃OD): δ 8.58 (s, 1H), 7.32 (dd, J = 1.2 5.1 Hz, 1H),

7.24 (t, 1H), 7.10 (dd, J = 1.1, 3.3 Hz, 1H), 6.95 (dd, J = 3.6, 5.1 Hz,

1H), 6.92−6.88 (m, 2H), 6.83 (ddd, J = 0.7, 1.9, 8.0 Hz, 1H), 5.68

(dd, J = 5.4, 7.5, 1H), 3.88−3.86 (m, 2H), 3.72−3.70 (m, 2H), 3.21

(s, 3H), 3.18 (s, 6H), 2.85−2.72 (m, 2H), 2.41 (s, 3H), 2.35−2.27

(m, 1H), 2.17−2.10 (m, 1H). HPLC, method 1: RT, 4.73 min; purity,

100%. HRMS calcd for C₂₄H₃₁N₆O₂S, 467.2224 [M + H]⁺; found,

467.2230 [M + H]⁺.

(S)-2-Amino-9-methyl-6-(3-(3-(methylamino)-1-(thiophen-2-yl)-

propoxy)phenyl)-6,7,8,9-tetrahydro-5H-pyrimido[4,5-e][1,4]-

diazepin-5-one (45cS). A similar procedure to that used for the

preparation of 45b was used for the preparation of 45cS, using, in step

1, ammonia 30% solution in water under MW irradiation at 100 °C

for 30 min. ¹H NMR (400 MHz, CD₃OD): δ 8.52 (s, 1H), 7.30 (d, J

= 4.9 Hz, 1H), 7.23 (t, 1H), 7.09 (d, J = 3.6 Hz, 1H), 6.94−6.87 (m,

3H), 6.81 (d, J = 7.9 Hz, 1H), 5.66 (dd, J = 5.8, 7.7, 1H), 3.85−3.83

(m, 2H), 3.68−3.66 (m, 2H), 3.17 (s, 3H), 2.75−2.62 (m, 2H), 2.35

(s, 3H), 2.33−2.24 (m, 1H), 2.14−2.06 (m, 1H). ¹³C NMR (100

MHz, CD₃OD): δ 168.08, 163.19, 162.62, 158.73, 158.41, 144.71,

144.59, 129.64, 126.31, 125.38, 124.98, 118.88, 114.54, 114.50,

103.44, 74.57, 54.53, 49.64, 47.55, 37.84, 37.62, 34.78. HRMS calcd

for C₂₂H₂₆N₆O₂S, 439.1900 [M + H]⁺; found, 439.1902 [M + H]⁺.

(S)-9-Methyl-6-(3-(3-(methylamino)-1-(thiophen-2-yl)propoxy)-

phenyl)-6,7,8,9-tetrahydro-5H-pyrimido[4,5-e][1,4]diazepin-5-one

(46S). A mixture of 42-2S (1 g, 1.63 mmol) and 5% Pd/C (139 mg,

0.065 mmol) in THF (15 mL) was cooled at 0 °C, and triethylsilane

(1.3 mL, 8.15 mmol) was added. The reaction mixture was stirred at 0

(S)-2-(Ethylamino)-9-methyl-6-(3-(3-(methylamino)-1-(thio-

phen-2-yl)propoxy)phenyl)-6,7,8,9-tetrahydro-5H-pyrimido[4,5-e]-

[1,4]diazepin-5-one (45aS). To a solution of 42-1S (970 mg, 1.70

mmol) in DCM (20 mL), m-CPBA (588 mg, 2.55 mmol) was added,

and the mixture was stirred at rt for 1 h. NaHCO₃aqueous saturated

solution was added, and the mixture was extracted with DCM. The

organic layer was washed with brine and dried with Na₂SO₄, and the

solvent was removed under vacuum. The crude product was dissolved

in THF (15 mL), and ethylamine (2 M solution in THF, 4.26 mL,

8.51 mmol) was added. The mixture was stirred at rt for 20 h. The

solvent was removed under vacuum. Puriﬁcation by ﬂash chromatog-

raphy, silica gel, gradient cyclohexane to 100% ethyl acetate, aﬀorded

tert-butyl (S)-(3-(3-(2-(ethylamino)-9-methyl-5-oxo-5,7,8,9-tetrahy-

dro-6H-pyrimido[4,5-e][1,4]diazepin-6-yl)phenoxy)-3-(thiophen-2-

1

yl)propyl)(methyl)carbamate (817 mg, 85% yield). H NMR (400

MHz, CDCl₃): δ 8.70 (s, 1H), 7.23−7.19 (m, 2H), 7.01 (d, J = 3.0

Hz, 1H), 6.93 (dd, J = 3.6, 5.0 Hz, 1H), 6.87−6.85 (m, 2H), 6.78

(ddd, J = 0.8, 2.2, 8.2 Hz, 1H), 5.41 (dd, J = 5.2, 7.8, 1H), 5.16 (bs,

1H), 3.90−3.88 (m, 2H), 3.67−3.65 (m, 2H), 3.50−3.43 (m, 2H),

3.41−3.33 (m, 2H), 3.20 (s, 3H), 2.84 (s, 3H), 2.32−2.26 (m, 1H),

2.20−2.11 (m, 1H), 1.41 (s, 9H), 1.23 (t, J = 7.3 Hz, 3H).

A solution of the previous compound (800 mg, 1.41 mmol) in

DCM (15 mL) was added to ZnBr₂(1.9 g, 8.47 mmol, dried under

vacuum at 250 °C for 2 h) under an atmosphere of Ar, and the

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°C in a sealed tube for 5 min and then at rt for 4 h. The reaction

mixture was ﬁltered through a pad of Celite, and the ﬁltrate was

concentrated under vacuum. Puriﬁcation by ﬂash chromatography,

silica gel, gradient hexane to 100% acetone, aﬀorded 2-

(trimethylsilyl)ethyl (S)-methyl(3-(3-(9-methyl-5-oxo-5,7,8,9-tetrahy-

dro-6H-pyrimido[4,5-e][1,4]diazepin-6-yl)phenoxy)-3-(thiophen-2-

yl)propyl)carbamate (750 mg, 81% yield). ¹H NMR (500 MHz,

CDCl₃): δ 8.91 (s, 1H), 8.69 (s, 1H), 7.27−7.23 (m, 2H), 7.04 (m,

1H), 6.95 (m, 1H), 6.88 (m, 2H), 6.84 (m, 1H), 5.45 (m, 1H), 4.20−

4.05 (m, 2H), 3.94 (m, 2H), 3.77 (m, 2H), 3.51−3.39 (m, 2H), 3.28

(s, 3H), 2.90 (s, 3H), 2.32 (m, 1H), 2.19 (m, 1H), 0.99 (m, 1H), 0.92

(m, 1H), 0.04 (s, 9H).

in THF (27 mL) and Et₃N (5.64 mL, 40.50 mmol) were added. The

mixture was stirred at 0 °C for 10 min and then at rt for 2.5 h. Water

was added, the mixture was extracted with DCM, the DCM extract

was isolated and dried over Na₂SO₄, and the solvent was removed

under vacuum. Puriﬁcation by ﬂash chromatography, silica gel,

gradient cyclohexane to 100% ethyl acetate, aﬀorded 52-2 (2.9 g, 76%

yield). H NMR (300 MHz, CD₃OD): δ 8.01 (d, J = 7.83 Hz, 1H),

7.34 (d, J = 7.83 Hz, 1H), 3.60−3.64 (m, 2H), 3.50−3.54 (m, 2H),

2.93 (s, 3H), 1.43 (s, 9H).

1

2,6-Dichloro-N-(2-(methylamino)ethyl)nicotinamide Hydro-

chloride (53-2). To a solution of 52-2 (2.9 g, 8.33 mmol) in dioxane

(15 mL), HCl (4 M solution in dioxane, 31.2 mL, 125 mmol) was

added, and the mixture was stirred at rt for 2 h. The reaction mixture

was concentrated to dryness under vacuum to aﬀord 53-2 (2.3 g,

To a solution of the previous compound (720 mg, 1.27 mmol) in

DMF (12 mL), CsF (963 mg, 6.34 mmol) was added, and the mixture

was heated at 90 °C for 90 min. Water was added and stirred until

complete solution with the help of ultrasound irradiation. DCM was

added, and the mixture was ﬁltered through a pad of Celite and

concentrated under vacuum. Puriﬁcation by ﬂash chromatography,

silica gel, gradient DCM to 35% MeOH/NH₃aq (1:0.01), aﬀorded

1

quant yield). H NMR (300 MHz, CDCl₃): δ 8.08 (d, J = 8.01 Hz,

1H), 7.56 (d, J = 8.01 Hz, 1H), 3.72 (t, J = 6.02 Hz, 2H), 3.28 (t, J =

6.02 Hz, 2H), 2.79 (s, 3H).

8-Chloro-1-methyl-1,2,3,4-tetrahydro-5H-pyrido[2,3-e][1,4]-

diazepin-5-one (54-2). To a mixture of 53-2 (1.2 g, 3.74 mmol) and

CsF (2.84 g, 18.69 mmol) in DMF (125 mL) under an Ar

atmosphere, Et₃N (1.25 mL, 8.97 mmol) was added, and the mixture

was heated at 75 °C for 16 h. The reaction mixture was cooled to rt,

and the solvent was removed under vacuum. Puriﬁcation by ﬂash

chromatography, silica gel, gradient DCM to 40% MeOH, aﬀorded

54-2 (729 mg, 92% yield). ¹H NMR (300 MHz, CDCl₃): δ 8.18 (d, J

= 8.0 Hz, 1H), 7.26 (bs, 1H), 6.69 (d, J = 8.0 Hz, 1H), 3.65 (m, 2 H),

3.54 (m, 2H), 3.19 (s, 3H).

1

46S (510 mg, 87% yield). H NMR (400 MHz, CD₃OD): δ 8.72 (s,

1H), 8.56 (s, 1H), 7.32 (dd, J = 0.9, 4.9 Hz, 1H), 7.27 (t, 1H), 7.10

(d, J = 3.3 Hz, 1H), 6.96−6.91 (m, 3H), 6.87 (d, J = 8.0 Hz, 1H),

5.68 (dd, J = 5.7, 7.7, 1H), 3.95−3.93 (m, 2H), 3.84−3.82 (m, 2H),

3.28 (s, 3H), 2.76−2.63 (m, 2H), 2.36 (s, 3H), 2.34−2.24 (m, 1H),

2.15−2.06 (m, 1H). HPLC, method 1: RT, 4.38 min; purity, 99.2%.

HRMS calcd for C₂₂H₂₅N₅O₂S, 424.1802 [M + H]⁺; found, 424.1808

[M + H]⁺.

8-(Ethylamino)-1-methyl-1,2,3,4-tetrahydro-5H-pyrido[2,3-e]-

[1,4]diazepin-5-one (55-2). A mixture of 54-2 (95 mg, 0.45 mmol)

and ethylamine (70% solution in water, 2.75 mL, 34.1 mmol) was

irradiated with microwaves at 130 °C for 2 h. The solvent was

removed under vacuum, and the residue was puriﬁed by ﬂash

chromatography, silica gel, gradient DCM to 40% MeOH, to aﬀord

55-2 (84 mg, 85% yield). ¹H NMR (300 MHz, CDCl₃): δ 8.15 (d, J =

8.5 Hz, 1H), 6.19 (bs, 1H), 5.85 (d, J = 8.5 Hz, 1H), 4.58 (bs, 1H),

3.58 (m, 2 H), 3.49 (m, 2H), 3.38 (m, 2H), 3.17 (s, 3H), 1.26 (t, J =

7.2 Hz, 3H).

(S)-2,9-Dimethyl-6-(3-(3-(methylamino)-1-(thiophen-2-yl)-

propoxy)phenyl)-6,7,8,9-tetrahydro-5H-pyrimido[4,5-e][1,4]-

diazepin-5-one (47S). To a solution of 42-1S (250 mg, 0.44 mmol)

in DCM (7 mL), m-CPBA (151 mg, 0.66 mmol) was added, and the

mixture was stirred at rt for 1 h. NaHCO₃aqueous saturated solution

was added, and the mixture was extracted with DCM. The organic

layer was washed with brine and dried with Na₂SO₄, and the solvent

was removed under vacuum. The crude product was dissolved in THF

(7 mL) and cooled at 0 °C, and MeMgBr (3 M solution in Et₂O,

0.176 mL, 0.52 mmol) was added. The mixture was stirred at 0 °C for

1 h. Some drops of water were added, and the solvent was removed

under vacuum. Puriﬁcation by ﬂash chromatography, silica gel,

gradient cyclohexane to 80% acetone, aﬀorded tert-butyl (S)-(3-(3-

(2,9-dimethyl-5-oxo-5,7,8,9-tetrahydro-6H-pyrimido[4,5-e][1,4]-

diazepin-6-yl)phenoxy)-3-(thiophen-2-yl)propyl)(methyl)carbamate

A similar procedure was used for the preparation of 55-1 starting

from 50-1.

(S)-8-(Ethylamino)-1-methyl-4-(3-(3-(methylamino)-1-(thio-

phen-2-yl)propoxy)phenyl)-1,2,3,4-tetrahydro-5H-pyrido[2,3-e]-

[1,4]diazepin-5-one (57S). A mixture of CuI (18 mg, 0.09 mmol) and

N¹,N²-dimethylethane-1,2-diamine (10 μL, 0.09 mmol) in dioxane

(0.25 mL) was stirred at rt for 20 min. To this mixture, a solution of

55-2 (41 mg, 0.19 mmol) and 41-2S (96 mg, 0.19 mmol) in a mixture

of dioxane (1.25 mL) and DMSO (0.2 mL) and K₃PO₄(119 mg, 0.56

mmol) were added. The reaction mixture was heated at 130 °C in a

sealed tube for 20 h. The reaction mixture was cooled at rt and

concentrated under vacuum. Puriﬁcation by ﬂash chromatography,

silica gel, gradient hexane to 100% ethyl acetate, aﬀorded 2-

(trimethylsilyl)ethyl (S)-(3-(3-(8-(ethylamino)-1-methyl-5-oxo-

1,2,3,5-tetrahydro-4H-pyrido[2,3-e][1,4]diazepin-4-yl)phenoxy)-3-

(thiophen-2-yl)propyl)(methyl)carbamate (51 mg, 45% yield). ¹H

NMR (300 MHz, CDCl₃): δ 7.95 (d, J = 8.5 Hz, 1H), 7.27−7.19 (m,

2H), 7.03 (br, 1H), 6.95−6.90 (m, 3H), 6.78 (d, J = 8.0 Hz, 1H),

5.87 (d, J = 8.4 Hz, 1H), 5.46−5.42 (m, 1H), 4.63−4.59 (m, 1H),

4.17−4.10 (m, 2H), 3.91−3.88 (m, 2H), 3.60−3.57 (m, 2H), 3.45−

3.31 (m, 4H), 3.09 (s, 3H), 2.90 (s, 3H), 2.35−2.28 (m, 1H), 2.22−

2.12 (m, 1H), 1.26 (t, J = 7.2 Hz, 3H), 0.97 (bs, 2H), 0.04 (s, 9H).

To a solution of the previous compound (51 mg, 0.08 mmol) in

DMF (0.6 mL), CsF (64 mg, 0.42 mmol) was added, and the mixture

was irradiated with MW at 90 °C for 90 min. The reaction mixture

was cooled at rt, ﬁltered through a pad of Celite, and concentrated

under vacuum to aﬀord 57S (38 mg, 98% yield). ¹H NMR (400 MHz,

CDCl₃): δ 7.94 (d, J = 8.5 Hz, 1H), 7.22−7.18 (m, 2H), 7.01 (d, J =

3.4 Hz, 1H), 6.94−6.87 (m, 3H), 6.79 (dd, J = 2.3, 8.4 Hz, 1H), 5.86

(d, J = 8.5 Hz, 1H), 5.55 (dd, J = 5.6, 7.6 Hz, 1H), 4.59 (br, 1H),

3.89−3.87 (m, 2H), 3.58−3.56 (m, 2H), 3.39−3.32 (m, 2H), 3.07 (s,

3H), 2.77−2.67 (m, 2H), 2.41 (s, 3H), 2.32−2.21 (m, 1H), 2.12−

2.04 (m, 1H), 1.24 (t, 3H). HPLC, method 1: RT, 4.84 min; purity,

1

(160 mg, 68% yield). H NMR (400 MHz, CDCl₃): δ 8.87 (s,1H),

7.23 (m, 2H), 7.03 (m, 1H), 6.94 (m, 1H), 6.87 (m, 2H), 6.82 (m,

1H), 5.43 (m, 1H), 3.91 (m, 2H), 3.73 (m, 2H), 3.38 (m, 2H), 3.27

(s, 3H), 2.85 (s, 3H), 2.57 (s, 3H), 2.30 (m, 1H), 2.18 (m, 1H), 1.42

(s, 9H).

A solution of the previous compound (160 mg, 0.30 mmol) in

DCM (5 mL) was added to ZnBr₂(402 mg, 1.78 mmol, dried under

vacuum at 250 °C for 2 h) under an atmosphere of Ar, and the

reaction mixture was stirred at rt for 24 h. Water was added, and the

mixture was stirred until the solids completely dissolved with the help

of ultrasound irradiation. The layers were separated, and the aqueous

phase was extracted with DCM. The organic layer was dried over

Na₂SO₄and concentrated under vacuum. Puriﬁcation by ﬂash

chromatography, silica gel, gradient DCM to 35% MeOH, aﬀorded

a salt that was treated with NaHCO₃aqueous saturated solution and

extracted with DCM. The solvent was removed under vacuum to

aﬀord 47S (44 mg, 31% yield). ¹H NMR (400 MHz, CD₃OD): δ 8.74

(s, 1H), 7.40 (dd, J = 1.2, 5.0 Hz, 1H), 7.34 (t, 1H), 7.18 (dd, J = 1.1,

3.5 Hz, 1H), 7.04−6.98 (m, 3H), 6.94 (ddd, J = 0.8, 1.9, 7.9 Hz, 1H),

5.76 (dd, J = 5.7, 7.5 Hz, 1H), 4.01−3.99 (m, 2H), 3.90−3.88 (m,

2H), 3.36 (s, 3H), 2.84−2.71 (m, 2H), 2.58 (s, 3H), 2.44 (s, 3H),

2.42−2.31 (m, 1H), 2.21−2.14 (m, 1H). HPLC, method 1: RT, 4.50

min; purity, 96.1%. HRMS calcd for C₂₃H₂₇N₅O₂S, 438.1957 [M +

H]⁺; found, 438.1959 [M + H]⁺.

tert-Butyl (2-(2,6-Dichloronicotinamido)ethyl)(methyl)-

carbamate (52-2). To a solution of 2,6-dichloronicotinoyl chloride

(50-2, 2.45 g, 11.67 mmol) in THF (18 mL) at 0 °C, a solution of

tert-butyl (2-aminoethyl)(methyl)carbamate (51, 1.90 g, 10.90 mmol)

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96.0%. HRMS calcd for C₂₅H₃₁N₅O₂S, 466.2271 [M + H]⁺; found,

466.2276 [M + H]⁺.

A similar procedure was used for the preparation of 56S, 59S, 61S,

63S, 65, 67, 69, 71R, and 71S from the corresponding starting

materials. In some examples, puriﬁcation by ﬂash chromatography was

needed (silica gel, gradient DCM to 40% MeOH).

(S)-1-Methyl-4-(3-(3-(methylamino)-1-(thiophen-2-yl)propoxy)-

phenyl)-3,4-dihydro-1H-pyrido[2,3-e][1,4]diazepin-5(2H)-one

(59S). ¹H NMR (400 MHz, CDCl₃): δ 8.33 (dd, J = 2.1, 4.8 Hz, 1H),

8.04 (dd, J = 2.1, 7.6 Hz, 1H), 7.27−7.22 (m, 2H), 7.02 (d, J = 3.1

Hz, 1 H), 6.96−6.91 (m, 3H), 6.85 (dd, J = 2.3, 8.2 Hz, 1H), 6.80

(dd, J = 4.8, 7.8 Hz, 1H), 5.57 (dd, J = 5.4, 7.7 Hz, 1H), 3.90−3.87

(m, 2H), 3.61−3.58 (m, 2H), 3.08 (s, 3H), 2.77−2.67 (m, 2H), 2.42

(s, 3H), 2.35−2.20 (m, 1H), 2.14−2.03 (m, 1H). ¹³C NMR (100

MHz, CDCl₃): δ 168.78, 158.63, 155.56, 151.22, 144.97, 143.72,

140.87, 129.85, 126.59, 124.89, 124.83, 119.64, 118.90, 114.68,

114.51, 114.34, 74.99, 56.81, 49.17, 48.17, 38.76, 38.75, 36.53. HRMS

calcd for C₂₃H₂₆N₄O₂S, 423.1853 [M + H]⁺; found, 423.1849 [M +

H]⁺.

extracted with DCM. The organic layer was dried over Na₂SO₄and

ﬁltered, and the solvent was removed under vacuum. The residue was

treated with diethyl ether, and the solid obtained was ﬁltered and

washed with diethyl ether to aﬀord 68 (410 mg, 92% yield). ¹H NMR

(500 MHz, CDCl₃): δ 8.37 (bs, 1H), 7.67 (m, 1H), 7.38 (m, 1H),

6.98 (m, 1H), 6.89 (m, 1H), 3.36 (m, 2H), 3.30 (m, 2H), 2.84 (s,

3H).

NET Modeling. The human sequence of the sodium-dependent

noradrenaline transporter (spP23975) was aligned to the SERT

sequences of the crystallized structures 5I6X, 5I6Z, 5I71, 5I73, 5I74,

and 5I75,⁴³and 100 homology models were generated using the

MODELLER⁴⁷program within Discovery Studio.⁴⁸The model with

the lower PDF total energy was selected, and its loops were optimized

with MODELLER using the DOPE method. The best homology

model obtained was typed with CHARMM, residue pK’s were

calculated, the protein was ionized at pH 7, and compound 31aS was

docked with LibDock,⁴⁹deﬁning the binding site as an 8 Å sphere

centered on the paroxetine structure of the aligned 5I6X crystal

structure. The obtained complex was inserted into an SPC−POPC

orthorhombic box, using the Maestro’s system builder,⁵⁰with a 10 Å

buﬀer between the system and the edge of the box, and sodium and

chloride ions were added to neutralize and reach a ﬁnal concentration

of 0.15 M. Both systems were typed with the OPLS force ﬁeld and

equilibrated by a standard eight-step protocol for transmembrane

proteins, and ﬁnally, a 1 μs production simulation in the NPgT

ensemble was run and analyzed with Desmond.⁵¹

Human α2δ-1 Subunit of Cav2.2 Calcium Channel Assay.

Human α2δ-1-enriched membranes (3 μg) from hamster tumor

CHO-K1 cells (human Cav2.2/β3/α2δ calcium channel cell line,

catalog #CT6159, ChanTest) were incubated with 15 nM radio-

labeled [³H]-gabapentin in assay buﬀer containing HEPES/KOH 10

mM (pH 7.4). Nonspeciﬁc binding (NSB) was measured by adding

10 μM pregabalin. The ﬁnal volume was 100 μL. The binding of the

tested compounds was measured at ﬁve diﬀerent concentrations per

duplicate, to determine aﬃnity values (K_i). After 60 min of incubation

at 27 °C, the binding reaction was terminated by ﬁltering through

MultiScreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine

in a vacuum manifold station followed by three washes with ice-cold

ﬁltration buﬀer containing 50 mM Tris−HCl (pH 7.4). Filter plates

were dried at 60 °C for 2 h, and 30 μL of scintillation cocktail was

added to each well before radioactivity reading. Readings were

performed in a TriLux 1450 MicroBeta radioactive counter

(PerkinElmer).

Human α2δ-2 Subunit of Cav2.2 Calcium Channel Assay.

Human α2δ-2-enriched membranes (6 μg) from human embryonic

kidney HEK-293 cells were incubated with 15 nM radiolabeled [³H]-

gabapentin in assay buﬀer containing HEPES/KOH 10 mM (pH 7.4).

Nonspeciﬁc binding (NSB) was measured by adding 10 μM

pregabalin. The ﬁnal volume was 100 μL. The binding of the tested

compounds was measured at ﬁve diﬀerent concentrations, duplicate,

to determine aﬃnity values (K_i). After 60 min of incubation at 27 °C,

the binding reaction was terminated by ﬁltering through MultiScreen

GF/C (Millipore) presoaked in 0.5% polyethyleneimine in a vacuum

manifold station followed by three washes with ice-cold ﬁltration

buﬀer containing 50 mM Tris−HCl (pH 7.4). Filter plates were dried

at 60 °C for 2 h, and 30 μL of scintillation cocktail was added to each

well before radioactivity reading. Readings were performed in a

TriLux 1450 MicroBeta radioactive counter (PerkinElmer).

NET Binding Assay. NET-enriched membranes (5 μg) were

incubated with 5 nM radiolabeled [³H]-nisoxetine in assay buﬀer

containing 50 mM Tris−HCl, 120 mM NaCl, and 5 mM KCl (pH

7.4). NSB (nonspeciﬁc binding) was measured by adding 10 μM

desipramine. The binding of the test compound was measured at

either one concentration (% inhibition at 1 or 10 μM) or ﬁve diﬀerent

concentrations to determine aﬃnity values (K_i). After 60 min of

incubation at 4 °C, the binding reaction was terminated by ﬁltering

through MultiScreen GF/C (Millipore) presoaked in 0.5% poly-

ethyleneimine in a vacuum manifold station followed by three washes

with ice-cold ﬁltration buﬀer containing 50 mM Tris−HCl and 0.9%

NaCl (pH 7.4). Filter plates were dried at 60 °C for 1 h, and 30 μL of

1-Methyl-1,2,3,4-tetrahydro-5H-pyrido[2,3-e][1,4]diazepin-5-one

(58). To a solution of 54-2 (550 mg, 2.60 mmol) in THF (12 mL),

Pd/C (5% wt, 221 mg, 0.10 mmol) was added, and the mixture was

cooled at 0 °C in a sealed tube. Triethylsilane (1.51 g, 13.0 mmol)

was added, and the reaction mixture was stirred at 0 °C for 5 min and

then at rt for 16 h. The reaction mixture was ﬁltered through a pad of

Celite, and the ﬁltrate was concentrated under vacuum. Puriﬁcation

by ﬂash chromatography, silica gel, gradient DCM to 30% MeOH,

1

aﬀorded 58 (378 mg, 82% yield). H NMR (300 MHz, CDCl₃): δ

8.32 (dd, J = 2.0, 4.6 Hz, 1H), 8.21 (dd, J = 2.0, 7.6 Hz, 1H), 7.01 (bs,

1H), 6.75 (dd, J = 4.6, 7.6 Hz, 1H), 3.62 (m, 2 H), 3.54 (m, 2H), 3.16

(s, 3H).

1,8-Dimethyl-1,2,3,4-tetrahydro-5H-pyrido[2,3-e][1,4]diazepin-

5-one (60). To a mixture of 54-2 (50 mg, 0.23 mmol), methylboronic

acid (16 mg, 0.27 mmol), Pd(Ph₃)₄(27 mg, 0.024 mmol), and

K₂CO₃(98 mg, 0.71 mmol) under Ar, degassed dioxane (1.2 mL) was

added, and the mixture was heated at 130 °C in a sealed tube for 48 h.

The reaction mixture was cooled to rt and ﬁltered through a pad of

Celite. The ﬁltrate was concentrated under vacuum, and the residue

was puriﬁed by ﬂash chromatography, silica gel, gradient hexane to

100% acetone, to aﬀord 60 (45 mg, 77% yield). ¹H NMR (400 MHz,

CDCl₃): δ 8.15 (d, J = 7.8 Hz, 1H), 6.61 (d, J = 7.8 Hz, 1H), 6.56 (bs,

1H), 3.61 (m, 2 H), 3.53 (m, 2H), 3.19 (s, 3H), 2.45 (s, 3H).

8-Methoxy-1-methyl-1,2,3,4-tetrahydro-5H-pyrido[2,3-e][1,4]-

diazepin-5-one (62). To a solution of 54-2 (500 mg, 2.36 mmol) in

MeOH (17 mL), NaOMe (766 mg, 14.17 mmol) was added, and the

mixture was irradiated with MW at 110 °C for 2 h. The solvent was

removed under vacuum, and the residue was puriﬁed by ﬂash

chromatography, silica gel, gradient DCM to 30% MeOH, to aﬀord

62 (440 mg, 90% yield). ¹H NMR (400 MHz, CD₃OD): δ 8.09 (d, J

= 8.5 Hz, 1H), 6.10 (d, J = 8.5 Hz, 1H), 3.91 (s, 3H), 3.63 (m, 2 H),

3.47 (m, 2H), 3.22 (s, 3H).

1-Methyl-1,2,3,4-tetrahydro-5H-pyrido[3,2-e][1,4]diazepin-5-one

(64). To a solution of ethyl 3-ﬂuoropicolinate (190 mg, 1.12 mmol) in

dimethylacetamide (2.2 mL), K₂CO₃(310 mg, 2.24 mmol) and N¹-

methylethane-1,2-diamine (83 mg, 1.12 mmol) were added, and the

mixture was heated at 150 °C in a sealed tube for 64 h. The reaction

mixture was cooled at rt and concentrated under vacuum. Puriﬁcation

by ﬂash chromatography, silica gel, gradient DCM to 20% MeOH,

aﬀorded compound 64 (56 g, 28% yield). ¹H NMR (500 MHz,

CDCl₃): δ 8.33 (m, 1H), 7.98 (bs, 1H), 7.32 (m, 1H), 7.27 (m, 1H),

3.45 (m, 2H), 3.37 (m, 2H), 2.89 (s, 3H).

A similar procedure was used for the preparation of 66 from methyl

3-ﬂuoroisonicotinate.

1-Methyl-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one

(68). To a suspension of 1,2,3,4-tetrahydro-5H-benzo[e][1,4]-

diazepin-5-one (72, 410 mg, 2.53 mmol) in DCE (20 mL), DIPEA

(653 mg, 5.06 mmol), paraformaldehyde (296 mg, 9.35 mmol),

NaBH(OAc)₃(1.98 g, 9.35 mmol), and acetic acid (152 mg, 2.53

mmol) were added, and the reaction mixture was stirred at rt for 48 h.

NaHCO₃aqueous saturated solution was added, and the mixture was

2182

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J. Med. Chem. 2021, 64, 2167−2185

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

scintillation cocktail was added to each well before radioactivity

reading. Readings were performed in a TriLux 1450 MicroBeta

radioactive counter (PerkinElmer).

Albert Dordal − ESTEVE Pharmaceuticals, 08038 Barcelona,

Spain

Enrique Portillo-Salido − ESTEVE Pharmaceuticals, 08038

Barcelona, Spain

Raquel F. Reinoso − ESTEVE Pharmaceuticals, 08038

Barcelona, Spain

José Miguel Vela − ESTEVE Pharmaceuticals, 08038

Barcelona, Spain

NET Functional Assay. The day before the experiment, 5000

cells/well were seeded in 384 poly-^D-lysine-treated well plates

(Corning) in a complete medium and maintained at 37 °C at 5%

CO₂. On the experiment day, the cell culture medium was removed

and trypan blue (20 μL) was added (600 μM ﬁnal concentration).

This was immediately followed by the application of the test

compounds (10 μL/well) at diﬀerent ﬁnal concentrations (from 10⁻⁴

to 10⁻¹¹M). Then, the plates were left at 37 °C for 20 min. After this

time, ASP⁺(4-(4-(dimethylamino) styryl)-N-methylpyridinium io-

dide, 20 μL) was added (10 mM ﬁnal well concentration). The

reaction was incubated for 90 min at 37 °C. Finally, the ﬂuorescence

was read with the ﬂuorescent reader EnVision (PerkinElmer).

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01867

Author Contributions

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01867.

■

sı

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

We thank Magda Bordas, Raquel Enrech, Joan Andreu Morató,

Mónica Carro, Edmundo Ortega, Manuel Merlos, Pilar Pérez,

■

Analytical data for all the ﬁnal compounds and

intermediates and HPLC traces for ﬁnal compounds

(PDF)

Molecular formula strings of compounds (CSV)

NET homology data for compound 31aS (PDB)

́

Javier Burgueno, Marta Pujol, Enrique Hernandez, Javier Farré,

̃

́

Inés Alvarez, Xavier Monroy, Maite Seraﬁni, Eva Ayet, and

Sandra Yeste for their expert contribution to analytical, in vitro

and in vivo studies and Carlos Pérez and Eduardo Villarroel for

their contribution to compound management. This work was a

part of activities in R&D Projects IDI20130945 and

IDI20150917 supported by the Spanish Ministerio de

Economía y Competitividad (MINECO), through the Centro

para el Desarrollo Tecnológico Industrial (CDTI), co-ﬁnanced

by the European Union through the European Regional

Development Fund (ERDF; Fondo Europeo de Desarrollo

Regional, FEDER).

AUTHOR INFORMATION

Corresponding Author

■

Carmen Almansa − ESTEVE Pharmaceuticals, 08038

Barcelona, Spain; orcid.org/0000-0001-5665-4685;

Phone: 0034650697372; Email: calmansa@

welab.barcelona

Authors

José Luis Díaz − ESTEVE Pharmaceuticals, 08038 Barcelona,

Spain; orcid.org/0000-0001-8812-4689

Félix Cuevas − Institute of Chemical Research of Catalonia

(ICIQ), Barcelona Institute of Science and Technology,

43007 Tarragona, Spain

ABBREVIATIONS USED

■

ADME, absorption, distribution, metabolism, and excretion;

ASP⁺, 4-(4-(dimethylamino)styryl)-N-methylpyridinium io-

dide; Ca_vα2δ-1, α2δ-1 subunit of voltage-gated calcium

channels; CNS, central nervous system; FDA, Food and

Drug Administration; hERG, human ether-a-go-go-related

gene; MAP, 3-methylamino-1-phenylpropoxy; MAT, 3-meth-

ylamino-1-thiophenylpropoxy; NE, norepinephrine; NET,

norepinephrine transporter; NRI, norepinephrine reuptake

inhibitor; PI, positive ionizable; rhCYP, recombinant human

cytochrome P450; SAR, structure−activity relationship; SERT,

serotonin transporter; SNRI, serotonin and norepinephrine

reuptake inhibitor; SSRI, selective serotonin reuptake inhib-

itor; Teoc, trimethylsilylethoxycarbonyl; VGCC, voltage-gated

calcium channels

Gonzalo Pazos − Institute of Chemical Research of Catalonia

(ICIQ), Barcelona Institute of Science and Technology,

43007 Tarragona, Spain

́

Paula Alvarez-Bercedo − Institute of Chemical Research of

Catalonia (ICIQ), Barcelona Institute of Science and

Technology, 43007 Tarragona, Spain

Ana I. Oliva − Institute of Chemical Research of Catalonia

(ICIQ), Barcelona Institute of Science and Technology,

43007 Tarragona, Spain

́

M. Angeles Sarmentero − Institute of Chemical Research of

Catalonia (ICIQ), Barcelona Institute of Science and

Technology, 43007 Tarragona, Spain

Daniel Font − Institute of Chemical Research of Catalonia

(ICIQ), Barcelona Institute of Science and Technology,

43007 Tarragona, Spain

Agustín Jiménez-Aquino − Institute of Chemical Research of

Catalonia (ICIQ), Barcelona Institute of Science and

Technology, 43007 Tarragona, Spain

María Morón − Institute of Chemical Research of Catalonia

(ICIQ), Barcelona Institute of Science and Technology,

43007 Tarragona, Spain

Adriana Port − ESTEVE Pharmaceuticals, 08038 Barcelona,

Spain

Rosalía Pascual − ESTEVE Pharmaceuticals, 08038

Barcelona, Spain

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Article Doi

Bicyclic Diazepinones as Dual Ligands of the α2δ-1 Subunit of Voltage-Gated Calcium Channels and the Norepinephrine Transporter

DOI: 10.1021/acs.jmedchem.0c01867

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