NL2035306B1 - 1,3-isoindolinedione derivatives and heteroaromatic analogues for stimulating endothelial cell-pericyte interaction - Google Patents

Abstract

The present invention relates to pharmaceutical compound, and formulations comprising the compound, wherein the compound has a structure according to formula I or ||: A4 A3' „@ N-… A \A1 ° (I) A4 A5 / O N—RX \ A 6 ° (II) The compound and formulations are suitable for use in as a medicament in the prevention or treatment of a disease or a disorder characterized by pericyte dysfunction, detachment and/or loss, in a therapeutic treatment for blood vessel stabilization, in a treatment of promoting, restoring or preserving pericyte attachment to blood vessels, and/or in a treatment of a vascular-associated disease in a patient.

Description

P352801NL -1- 1,3-ISOINDOLINEDIONE DERIVATIVES AND HETEROAROMATIC ANALOGUES FOR STIMULATING

ENDOTHELIAL CELL-PERICYTE INTERACTION

Field of the Invention

The present invention relates to the field of medicine. More particularly, it relates to the use of 1,3- isoindolinedione derivatives and heteroaromatic analogues thereof to stimulate endothelial cell-pericyte interaction and pericyte coverage of blood vessels.

Background of the invention

Blood vessels are intricate networks of hollow tubes that continuously adapt structurally and functionally to ensure optimal delivery of nutrients and oxygen to every cell of the body. They are composed of endothelial cells forming the inner lining of the vessel walls and of mural cells referred to as Vascular Smooth Muscle cells (VSMCs) and pericytes (PCs). VSMCs surround arteries and veins whereas PCs are located within the basal membrane of capillaries, venules and terminal arterioles. PCs have important roles in blood vessel formation and stabilization, regulate endothelial cell survival and control blood barrier to solutes and immune cells as well as blood flow. Many serious diseases affecting millions of people worldwide are caused by or associated with PC death or migration away from blood vessels. These pathological responses can lead to chronic inflammation, excessive vessel growth or/and blood vessel rarefaction that ultimately can lead to hypoperfusion and tissue damage.

PC dysfunction has been reported to cause/contribute to the development of a broad range of conditions, such as diabetes (nephropathy and retinopathy), chronic kidney disease (CKD), cardiomyopathy, central nervous system (CNS) disorders such as stroke, epilepsy, spinal cord injury, dementia notably Alzheimer's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, radiation necrosis and small inherited vessel diseases such as Cerebral autosomal dominant arteriopathy with (Cadasil) and Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (Carasil), Cerebral Amyloid Angiopathy (CAA), Retinal Vasculopathy with Cerebral

Leukoencephalopathy and systemic manifestations (RVCL-S), Hereditary Hemorrhagic Telangiectasia (HHT) and Cerebral Cavernous Malformations (CCM). Most of these conditions currently still lack the availability of effective treatment that is suitable for chronic treatment. The present inventors and others, have previously provided proof of concept data that by promoting PC attachment to blood vessels, they could prevent blood vessel weakness caused by HHT! or by brain irradiation23. Thalidomide was particularly effective in this in both mice and patients suggesting that it may be useful in other diseases with similar underlying pathology.

Thalidomide and its analogs lenalidomide and pomalidomide are immunomodulatory imide drugs (IMiDs), which are highly effective treatment modalities for Multiple Myeloma and del(5q) Myelodysplasic

Syndrome. The class of compounds exerts pleiotropic effects including immunomodulatory, potent anti- inflammatory, anti-angiogenic and direct anti-myeloma activities. Mechanistically, these drugs act as a molecular bridge linking selected neo-substrates to the ubiquitin proteasome system for degradation. All

IMiDs are characterized by a glutarimide ring that binds to Cereblon (CRBN), a substrate of the E3 ubiquitin

P352801NL -2- ligase CRL4 complex to target proteins for degradation. The glutarimide moiety is responsible for most of their pharmacological activities but also for their adverse teratogenic and hematologic effects. Notably, accumulative evidence indicates that the immuno-modulatory, anti-angiogenic and direct anti-proliferative activities of all IMiDs depend only on the glutarimide motive and not on the phthalimide motive.

IMiDs cannot be used to treat chronic diseases. Patients treated with IMiDs can develop serious side effects including possible birth defects, fatigue, weakness, anemia, neuropathy, neutropenia, thrombocytopenia, blood clots causing stroke or heart attack, increase risk of death in people with chronic lymphocytic leukemia (CLL), risk of malighancies, severe liver problems, severe skin reactions, thyroid problems and risk in early death in people who have Mantle Cell Lymphoma (MCL).

Hence, it is an object of the present invention to provide compounds and/or treatments to promote endothelial cells-pericyte interaction and pericyte coverage of blood vessels that, unlike currently known

IMiDs, can be used in chronic treatments.

Summary of the Invention

Accordingly, the present invention is directed to a pharmaceutical compound, wherein the compound has a structure according to formula | or II: 2

JAS

AF ar IV >) {3

AX J

; © (I) wherein

At, A2, A3 and A? each individually represent CR! or N;

A5 and AS each individually represent S, CR’ or N; wherein R' represents hydrogen, halogen, OH, C+-Cs alkyl, NO2, or NR2R3, wherein R2 and R? are each independently selected from H, C1-Cs alkyl, C1-Cs-alkyl-carbonyl, and C+-Cs- alkoxy-carbonyl; wherein Rx represents Y or CHR4R5, wherein Y represents a 4-, 5-, or 6-membered heterocycle comprising 1, 2, or 3 heteroatoms, wherein Y is optionally substituted with one or more substituents selected from the group consisting of halide, saturated or unsaturated C+-Cs hydrocarbon, Ci-Cs-alkoxy-carbonyl, or C+-Cs-alkyl-carbonyl, preferably acetyl or propionyl;

P352801NL -3- wherein R# represents H, Ci-Cs-alkyl, Ci-Ce-cycloalkyl, C1-Ce-cycloalkylalkyl, heteroaryl, C1-Cs- hetercaralkyl, C1-Cs-heterocycloalkyl, or C1-Cs-heterocycloalkylalkyl, wherein R5 represents H, OH, COOH, C+-Cs alkyl, preferably COOH, i-propyl or t-butyl; and wherein

R+ and R5 are not both H.

A reference to compounds of formula (I) and (Il) and sub-references as descried herein also includes pharmaceutically acceptable ionic forms, salts, solvates, isomers, including geometric and stereochemical isomers, tautomers, N- oxides, esters, prodrugs, isotopes and protected forms thereof, preferably pharmaceutically acceptable salts or prodrugs.

In a second aspect, the invention relates to a pharmaceutical compound, wherein the compound has a structure according to formula | or II: 3

AF oS ; is i NN

SE of a (I a so # TN, EE © (I) wherein

A1, A2, A3 and A? each individually represent CR! or N;

A5 and A® each individually represent S, CR! or N; wherein R' represents hydrogen, halogen, OH, C+1-Cs alkyl, NO2, or NR2R3, wherein R2 and R? are each independently selected from H, C1-Cs alkyl, C1-Cs-alkyl-carbonyl, and C4-Cs- alkoxy-carbonyl; wherein Rx represents H, Y or CHR*RS, wherein Y represents a 4-, 5-, or 6-membered heterocycle comprising 1, 2, or 3 heteroatoms, wherein Y is optionally substituted with one or more substituents selected from the group consisting of halide, saturated or unsaturated C:-Cs hydrocarbon, C1-Cs-alkoxy-carbonyl, or Cs-Ce-alkyl-carbonyl, preferably acetyl or propionyl; wherein R+ represents H, Ci-Cs-alkyl, C+-Cs-cycloalkyl, C+-Cs-cycloalkylalkyl, heteroaryl, C1-Cs- heteroaralkyl, C1+-Cs-heterocycloalkyl, or C+-Cs-heterocycloalkylalkyl, wherein R5 represents H, OH, COOH, C+-Cs alkyl, preferably COOH, i-propyl or t-butyl; optionally wherein R+ and R5 are not both H, for use as a medicament in a therapeutic treatment for stimulating endothelial cell-pericyte interaction and promoting PC coverage of blood vessels.

P352801NL -4-

In a third aspect, the invention relates to a pharmaceutical compound, wherein the compound has a structure according to formula | or II: 4

At

II

Fae 3 3 0

G

RE A

Saf (I) 5 wherein

A1, A2, A3 and A+ each individually represent CR! or N;

A5 and AS each individually represent S, CR! or N; wherein Rt represents hydrogen, halogen, OH, C+-Cs alkyl, NO2, or NR2R3, wherein R2 and R3 are each independently selected from H, C1-Cs alkyl, C1+-Cs-alkyl-carbonyl, and C1-Cs- alkoxy-carbonyl; wherein Rx represents H, Y or CHR*RS, wherein Y represents a 4-, 5-, or 6-membered heterocycle comprising 1, 2, or 3 heteroatoms, wherein Y is optionally substituted with one or more substituents selected from the group consisting of halide, saturated or unsaturated C1-Cs hydrocarbon, Ci-Ce-alkoxy-carbonyl, or C+-Cs-alkyl-carbonyl, preferably acetyl or propionyl; wherein R* represents H, Ci-Cs-alkyl, C-Cs-cycloalkyl, C:+-Cs-cycloalkylalkyl, heteroaryl, C1-Cs- hetercaralkyl, C1-Cs-heterocycloalkyl, or C1-Cs-heterocycloalkylalkyl, wherein R5 represents H, OH, COOH, C+-Cs alkyl, preferably COOH, i-propyl or t-butyl; optionally wherein R+ and R® are not both H, for use as a medicament in the prophylactic or curative treatment of a disease or a disorder characterized by pericyte dysfunction, detachment and/or loss.

Brief description of the Figures

Figure 1 shows a comparative assessment of prevention of pericyte detachment and vascular leakage induced by radiation therapy by selected phthalimides.

Figure 2 shows a comparative assessment of preventing late vascular rarefaction and neuron loss induced by radiation therapy in the somatosensorial cortex (SS) and in the hippocampal region (Hp) by selected phthalimides.

Figure 3 demonstrate blood vessel stabilization properties by selected phthalimides as illustrated using a mouse model of Hereditary Hemorrhagic Telangiectasia (HHT) type 1.

P352801NL -5-

Figure 4 shows selected analogues of phthalimide that restore endothelial cell-pericyte interaction and blood vessel stability in accordance with an embodiment of the present invention.

Detailed Description of the Invention

The present invention provides new 1,3-isoindolinedione derivatives and heteroaromatic analogs thereof, as well as 1,3-isoindolinedione derivatives and heteroaromatic analogs thereof for use in stimulating endothelial cell-PC interaction and promoting PC coverage of blood vessels, and for use in the prevention or treatment of a disease or a disorder characterized by pericyte dysfunction, pericyte detachment and/or pericyte loss.

It was surprisingly found that these compounds are capable of preventing pericyte (PC) detachment, and promoting endothelial cells-pericyte interaction and pericyte coverage of blood vessels. As such, the compounds of the invention can advantageously be used in prophylactic or curative treatments of diseases or disorders characterized by pericyte dysfunction, detachment and/or loss, including curative therapeutic treatments comprising promotion of endothelial cell-PC interaction and blood vessel stabilization, as well as prophylactic and/or curative therapeutic treatments comprising promotion, restoration or preservation of

PC attachment to blood vessels.

The present inventors found that thalidomide but not its analogue lenalidomide is able to prevent pericyte (PC) detachment (Figure 1A-B) and Blood Brain Barrier (BBB) breakdown (Figure 1C) induced by radiation therapy in mice, as a model of early vessel injury associated with late neuron loss and cognitive decline.

The present inventors have also found that prophylactic treatment with thalidomide but not with its analogue lenalidomide (Figure 2A), by preventing PC detachment and BBB leakage, was able to prevent late capillary rarefaction and to prevent neuron loss in the cortical or hippocampal region induced by radiation therapy in the cortical (Figure 2B-F) or hippocampal region (Figure 2B, 2G-J). It was further observed that thalidomide and pomalidomide, contrary to their analogue lenalidomide, stimulate endothelial cell-PC interaction and blood vessel coverage in a mouse model of HHT using an ex vivo electrophysiology approach to quantify endothelial cell-PC interactions (Figure 3). Consequently, it was inferred that the carbonyl group (C=O) of the phthaloyl ring plays an important role in the ability of thalidomide to promote pericyte recruitment to the endothelium. It was also found that apremilast, an oral small-molecule inhibitor of phosphodiesterase 4 (PDE4) that shares the phthalimide ring with thalidomide but lacks the glutarimide ring and thus fails to bind to cereblon, could also rescue endothelial cell-PC interaction in a mouse model of HHT1 at high concentrations, confirming that the glutarimide ring is not required to promote PC coverage of the vasculature (Figure 3). Hence, it would appear that the isoindolinedione motive, as found in some

IMiDs such as thalidomide, but not in lenalidomide, appears to be the active fragment in compounds promoting PC attachment to blood vessels. It was furthermore observed that the glutarimide motive always present in the IMiDs is not essential to blood vessel stabilization and that major side effects associated with the known IMiDs may advantageously be suppressed by leaving out or replacing this structural motive.

Additionally, it was found that the claimed effects are obtained not only for phthalimide derivatives containing a Ce-aromatic moiety but also by other aromatic or heteroaromatic moieties.

P352801NL -6-

Accordingly, each At, A2, A3 and A* in the 5- or 6-membered aromatic moiety of the compounds as defined herein may individually represent CR or N, and A5 and AS each individually represent S, CR! or N; wherein R' represents hydrogen, halogen, OH, C1-Cs alkyl, NO2, or NR2R3, wherein R2 and R3 are each independently selected from H, C+-Cs alkyl, C+-Cs-alkyl-carbonyl, and C+-Cs-alkoxy-carbonyl.

In an embodiment, At or A2 represents C-NH2 or C-NHC(O)CHs; preferably A! represents C-NHz, and A2, A3 and A+ each individually represent CH, C-CH: or C-Cl, preferably CH. In an embodiment, A2 represents C-NHz, and At, A3 and A+ each individually represent CH, C-CH: or C-Cl, preferably CH.

In one embodiment, one of A5 and AS is S, the remainder of A+, AS and AS being CR! or CH.

The compounds according to the present invention can advantageously be used in treatments, e.g. prophylactic and/or curative therapeutic treatments, of a disease or a disorder characterized by pericyte dysfunction, detachment and/or loss; these involve therapeutic treatments, e.g. curative treatments, comprising promotion of endothelial cell-PC interaction and blood vessel stabilization, and prophylactic and/or curative therapeutic treatments comprising promoting, restoring or preserving pericyte attachment to blood vessels. As used herein, blood vessel stabilization refers inter alia to the protection of BBB integrity, control of blood flow, limitation of inflammation and hypoperfusion, and reduction of tissue damage, or combinations thereof.

Without wishing to be bound to any particular theory, it appears that the major side effects of thalidomide and its analogues such as lenalidomide and pomalidomide are caused at least in part by their aptness and capability to bind to the cereblon protein, which is the sole or primary target protein resulting in the breakdown of multiple neo-substrates mediated by the CRLACRBN ligase complex. Accordingly, the present inventors realized that at least some major side effects of phthalimide derivatives can be suppressed by selecting the Rxgroup such that the compound does essentially not bind to the cereblon protein. This insight allows rapid in vitro screening of toxicity of compounds that exhibit the beneficial effects of the invention.

The compounds as defined herein are N-substituted on the maleimide moiety with an Rx group which is chosen such that the compounds of the present invention do not lead to the binding to cereblon. This effectively excludes glutarimide as present in thalidomide, and hence thalidomide as such. In an embodiment, the lack of binding to cereblon (CRBN) of a compound according to the invention corresponds to the compound exhibiting a half-maximal inhibitory concentration (ICso) higher than 100 uM, preferably higher than 200 pM, more preferably higher than 500 uM or 1000 pM as determined in a competitive binding assay, as compared to the binding of thalidomide to cereblon.

Accordingly, in an embodiment the compound of formula | does not include thalidomide, pomalidomide, lenalidomide or any other compound comprising a glutarimide moiety such as present in these compounds.

The moiety RX is as defined herein and is further selected such that it does not interfere with the pharmaceutical activity.

In an embodiment, R* is Y, wherein Y represents a 4-, 5-, or 6-membered heterocycle comprising 1, 2, or 3 heteroatoms. Preferably, the heteroatoms are independently selected from N, O and S. Preferably,

Y represents a 4-, 5-, or 6-membered heterocycle comprising 1 heteroatom.

P352801NL -7-

Particular good results are obtained with compounds wherein R* represents a substituted or unsubstituted 4-, 5- or 8-membered heterocycle such as a morpholine, azetidine, pyrrolidine or piperidine group.

Accordingly, in an embodiment RX represents a 4-, 5-, or 6-membered heterocycle comprising 1, 2, or 3 heteroatoms, preferably 1 heteroatom, preferably 1 nitrogen atom.

Azetidines as group Rx display particularly good results. Accordingly, in a preferred embodiment of the invention, R* represents

ÔN-R wherein R® represents Ci-Cs-alkyl, C2-Cs-alkenyl, C2-Cs-alkynyl, C1-Cs-alkoxyalkyl, C1-Cs-alkoxy- carbonyl, aryl, or C1-Ce-alkyl-carbonyl, preferably acetyl or propionyl.

Advantageously, these compounds are synthetically relatively easily accessible by a condensation reaction of the corresponding amino acid H2N-C(R3)2-CO:H and the corresponding maleimide derivative.

In an alternative embodiment, R* represents CHR*R?, wherein R+ represents hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, tert-butyl, neopentyl, isopentyl, cyclopropyl-methyl, or thienyl-methyl.

In an embodiment, R5 represents COOH, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, tert-butyl, preferably COOH, methyl, i-propyl or t-butyl.

In exemplary embodiments, the compound has a structure according to formula (1-2) {2 oo / 0 (8) wherein each Z is independently selected from hydrogen, halogen, OH, C+-Cs alkyl, NO2, and NR2R?, wherein R2 and R3 are each independently selected from H, C+-Cs alkyl, C1-Cs-alkyl-carbonyl, and C1-Cs- alkoxy-carbonyl, preferably wherein each Z is independently selected from H, halogen, NHz, and

NHC (O)CHs; wherein mis 1, 2, 3 or 4, preferably 1 or 2; and wherein R® represents saturated or unsaturated C+-Cs hydrocarbon, C1-Cs-alkoxy-carbonyl, or C1-Cs-alkyl- carbonyl, preferably acetyl or propionyl.

In some exemplary embodiments, A’ or A2 is C-NH2 and Rx is a an N-substituted azetidine group. In a preferred embodiment, the compound has a structure according to formula I-b or formula I-c:

P352801NL -8- 2 §

A

{z 1 | © fl

EY ae {

NH, “ 3 (l-b) 0

Ff ose % = Ay * 3 Ni {z DEN | : ) N Rs 4 ij (lc) wherein each Z is independently selected from hydrogen, halogen, OH, C1-Cs alkyl, and NO:2, preferably wherein each Z is independently selected from H, CHs and CI; wherein m is 1, 2, or 3; and wherein RS represents saturated or unsaturated C+-Cs hydrocarbon, C+-Cs-alkoxy-carbonyl, or C1-Cs-alkyl- carbonyl, preferably acetyl or propionyl.

In another aspect of the invention there is provided a formulation comprising a pharmaceutical compound as defined herein, the formulation further comprising a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.

Table 1 lists exemplary compounds according to the invention.

Table 1

O 2 Oo in 2 JEN ene A £3

SIT Ne N Fe hy NL FI Ne x A Se

B 1 5, YY 0 No, © 0 @ o Q O et - 0 Ne, SN © JEN 3 <3 es nd . AN or i $i 3 en &F xy NL GN pe

Noo a No Xx Ni Ds hd Ny SN a i N

Se A ò HHT Ty o iH, © ò ®

P352801NL -9-

A

£3 > ad ge .

TO id nt g Ngati N oR \

NH © ety Nei 3 3 : ¥ \ } 3 3

J NH ö G £ © & € 0 Gt

FEIN een =. o we SN oo TN on, EN - ee ANE AY Sy I ) No AY Sy ta No x ; % i ù ds Py

Ni, UO fi, 0 Bn, U (10) (11) (12) 0 Pp 2

SN A 4 © , SEN ol i EE a a NH, Q NH, B (13) (14) (15) 3 Oo i { i ~ i 4 . Ri 5

LAA OT owed MN

TO en pon Te > (16) (17) (18) 0 o} 0 ad . 2 0 0

FT pe Ng

NH, © > NH, © NH, © (19) (20) (21)

P352801NL -10- 0 3 >

N—N LL NC NK yy N= NA /

Se ey a Are 0 wi, O Soa, © )

NH, NH, NB (22) (23) @4) 0 0 7

CF wed] a NC}

A on, eN Aeg ol N

T$ v A |g

Ni © ò ie NH, © 0 26) (27) (NET OTN

N Meg Ned x Sag “3 JON 28) (29) co 0 | 0 7 ed Spe & Fama 1 7 | O

DE NK 5 NONA

Ne pon OH

Ni, © 0 NH, © © NH, © oo) 33) (33) 0 3 = gek ST

N— NH HEI LiL NN © 2 ie X Yond

NH, © Np © (34) (35)

Hence, in an embodiment a compound (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (18), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), (33), (34) or (35) as displayed in Table 1 is provided, and/or a compound (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), 5 (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32),

P352801NL -11- (33), (34) or {35) for use as a medicament, particularly as a medicament in the prophylactic or curative treatment of a disease or a disorder characterized by pericyte dysfunction, detachment and/or loss, preferably in a treatment of promoting, restoring or preserving pericyte attachment to blood vessels. In an exemplary embodiment, a compound (1), (2), (5), (6), (7), (9), (10), (11), (15), (16), (17), (18), (19), (20), 21), (22), (23), or (24), as displayed in Table 1 is provided, as well as a formulation comprising such compound, for use as medicament, particularly for use as a medicament as defined herein.

Table 2 lists further exemplary compounds according to formula I-a, I-b and I-c.

Table 2 53 BY “ J RY i I; ADNR ner Ff

Comp. NIR IR : 1.005 C-H | C(O)O-t-butyl H : ae [en ow ow oom

Tae [en {ea Goce

P352801NL -12- 1.020 C-NHC(O)CHa ICH [CH CH | C(O)CH=CH: 1.021 C-NHC(O)CHs C-H CH CH C(O)O-t-butyl H

TE [EEE Con 1.023 C-NHC(O)CHs CH | C(O)-n-propyl 1.024 C-NHC(O)CHa CH | C(O)-neopentyl 1.025 C-NH: CHs CH CH C(O)CH: H 7 HCE oa SOR

P352801NL -13-

Hence, in an embodiment compounds 1.001 to 1.088 as disclosed in Table 2 are provided. In another embodiment compounds 1.001 to 1.088 as disclosed in Table 2, as well as a formulation comprising one or more of these compounds and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier, for use as a medicament are provided, particularly for use as a medicament in the treatment of a disease or a disorder characterized by pericyte dysfunction, detachment and/or loss, preferably in a treatment of promoting, restoring or preserving pericyte attachment to blood vessels.

P352801NL -14-

Table 3 lists exemplary compounds according to formula [-a.

Table 3 3

MW

Co in

Sa Lr

TL N oe CL ER

TOT

2.004 CNH: | CH CH | CH C(O)CH=CH: H 2.006 C-H | C(O)phenyl H 2.008 CNH: | CH CH | CH C(O)-neopentyl H 2.010 C-H | C(O)CH2CHs H 2.012 CH [CNH CH | CH C(O)CH=CH: H 2.014 C-H | C(O)phenyl H 2.022 C-H | C(O)phenyl H ;

P352801NL -15-

CH | GOEREE

CH | EO eagen

TOF

Open)

COT espe

ORT

COT

Open)

Ee {cicero 2.048 C-Cl | C(O)-neopentyl H

COHEN as a {en earn fH 2.052 C-H | C(O)CH=CH2 H

Cowen as [om {en fear os 2.056 C-H | C(O)-neopentyl H

P352801NL -16-

ST | SO

ST | Gowen

SOE

CO

Come i

CORR

CO aes en {a ci [or nea [ee 2.086 C-H | C(O)phenyl CHa

Hence, in an embodiment compounds 2.001 to 2.088 as disclosed in Table 3 are provided. In another embodiment compounds 2.001 to 2.088 as disclosed in Table 3, as well as a formulation comprising one or more of these compounds and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier, for use as a medicament are provided, particularly for use as a medicament in the treatment of a disease or a disorder characterized by pericyte dysfunction, detachment and/or loss, preferably in a treatment of promoting, restoring or preserving pericyte attachment to blood vessels.

Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. a nucleic acid sequence, vector, modified cell or isolated peptide as provided herein), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art.

A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example,

P352801NL -17- suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like. Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art. Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art. Adjuvants are pharmacological and/or immunological agents that modify the effect of other agents in a formulation.

Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary Skill in the art.

The compounds of the invention may be used in therapeutic treatments, including prophylactic and curative therapeutic treatments, of diseases or disorders characterized by pericyte dysfunction or pericyte loss, including curative therapeutic treatments comprising promotion of endothelial cell-pericyte interaction and blood vessel stabilization, as well as prophylactic and/or curative therapeutic treatments comprising restoration, promotion or preservation of pericyte attachment to blood vessels.

In an embodiment, the disease or disorder characterized by pericyte dysfunction, detachment and/or loss is selected from diabetes mellitus complications, chronic kidney disease (CKD), small vessel diseases,

Central Nervous System (CNS) diseases.

In an embodiment, the disease or disorder characterized by pericyte dysfunction, pericyte detachment and/or pericyte loss is selected from diabetic nephropathy (DN), diabetic retinopathy (DR), diabetic cardiomyopathy (DCM), and diabetic neuropathy.

In an embodiment, the disease or disorder characterized by pericyte dysfunction, detachment and/or loss is selected from hypertensive kidney diseases, IgA nephropathy, congenital nephropathy syndrome, lupus nephritis, polycystic kidney disease and allograft nephropathy.

In an embodiment, the disease or disorder characterized by pericyte dysfunction, detachment and/or loss is selected from cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (Cadasil), Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (Carasil), Cerebral Amyloid Angiopathy (CAA), Retinal Vasculopathy with Cerebral

Leukoencephalopathy and systemic manifestations (RVCL-S), Hereditary Hemorrhagic Telangiectasia (HHT), and Cerebral Cavernous Malformations (CCM).

In an embodiment, the disease or disorder characterized by pericyte dysfunction, detachment and/or loss is selected from stroke, epilepsy, spinal cord injury, vascular dementia, Alzheimer's disease,

Huntington's disease, Parkinson's disease, traumatic brain injury, multiple sclerosis, amyotrophic lateral sclerosis, and radiation necrosis.

In a further aspect the invention provides a compound or a formulation as defined herein for use as a medicament for stimulating endothelial cell-pericyte interaction and promoting pericyte coverage of blood vessels in a patient, and in a method for stimulating endothelial cell-PC interaction and promoting and PC

P352801NL -18- coverage of blood vessels in a patient, the method comprising administering compound or a formulation as defined herein.

In another aspect the invention relates to the compounds and formulations as defined herein for use as a medicament in a treatment of promoting endothelial cell-pericyte interaction, particularly of blood vessel stabilization, and/or a treatment of promoting or preserving pericyte attachment to blood vessels, as well as methods for the curative therapeutic treatment of promoting endothelial cell-pericyte interaction and blood vessel stabilization, and/or a treatment of promoting or preserving pericyte attachment to blood vessels.

In one embodiment, the treatment is a prophylactic therapeutic treatment.

In one embodiment, the treatment is curative therapeutic treatment.

The blood vessel stabilization properties and associated medical applicability of the compounds and formulations in accordance with the present invention can be established by an in vivo model using preclinical mouse models of HHT. By coupling electrophysiology and light microscopy, the ability of pericytes to modulate blood vessel constriction in response to an electrical stimulus can be quantified? (See also Figures 1 and 3). These experiments can be performed in whole retinas of control mice and of heterozygous or iKO endoglin mice (HHT1 mouse model) and in iKO endoglin mice treated with selected compounds (cf. Figures 3-4).

By comparing the activity of thalidomide and its analogue lenalidomide in the in vivo model of radio- necrosis and late brain damage, the essential role of both carbonyls groups in the phthalimide could be established (cf. Figures 1 and 2). Figure 1 shows how PC dysfuncticn induced by radiotherapy includes pericyte detachment that leads to BBB leakage, and in figure 2, to late blood capillary rarefaction and neuron loss.

Restoration of endothelial cell-PC interaction can be used to treat chronic diseases with vascular alterations. The compounds ofthe present invention are advantageously capable of sustaining blood vessel integrity limiting capillary rarefaction; promoting/maintaining blood barrier, notably blood-brain barrier (BBB) and blood-retina barrier (BRB), limiting vascular leakage and inflammation; reducing vessel overgrowth and the development of vascular malformations notably telangiectasia, low or high flow vascular malformations; restoring blood flow limiting tissue hypoperfusion and late damage. Thus, the compound according to the present invention, or a pharmaceutically acceptable salt or prodrug thereof, can be used in a medical treatment of vascular-associated diseases.

Accordingly, in one aspect of the invention there is provided the compounds and formulation as defined herein for use as a medicament in a prophylactic or curative treatment of a vascular-associated disease.

In another aspect of the invention there is provided method for the treatment of a vascular-associated disease in a patient, the method comprising administering to the patient a pharmaceutical compound, or a formulation comprising a pharmaceutical compound, wherein the compound has a structure according to formula | or II:

P352801NL -19- {3

AY

ES

> 0 2

IL, Pf eR 0) wherein

A", A2, A3 and A+ each individually represent CR! or N;

A5 and AS each individually represent S, CR! or N; wherein Rt represents hydrogen, halogen, OH, C+-Cs alkyl, NO2, or NR2R3, wherein R2 and R3 are each independently selected from H, C+-Cs alkyl, C+-Ce-alkyl-carbonyl, and C+-Cs- alkoxy-carbonyl; wherein Rx represents H, Y or CHR*RS, wherein Y represents a 4-, 5-, or 6-membered heterocycle comprising 1, 2, or 3 heteroatoms, wherein Y is optionally substituted with one or more substituents selected from the group consisting of halide, saturated or unsaturated C1-Cs hydrocarbon, Ci-Ce-alkoxy-carbonyl, or C+-Cs-alkyl-carbonyl, preferably acetyl or propionyl; wherein R* represents H, Ci-Cs-alkyl, C-Cs-cycloalkyl, C:+-Cs-cycloalkylalkyl, heteroaryl, C1-Cs- heteroaralkyl, C1-Cs-heterocycloalkyl, or C1-Cs-heterocycloalkylalkyl, wherein R5 represents H, OH, COOH, C+-Cs alkyl, preferably COOH, i-propyl or t-butyl; optionally wherein R+ and R5 are not both H.

In a further aspect of the present invention there is provided a method for promoting, restoring or preserving pericyte attachment to blood vessels in a subject, preferably in a method of treatment of a vascular-associated disease, the method comprising administering to the subject the compound or formulation as defined above.

In a further aspect of the present invention there is provided a method for stabilizing blood vessels in a subject, preferably in a method of treatment of a vascular associated disease, the method comprising administering to the subject the compound or formulation as defined above.

Specific examples vascular-associated diseases include diabetes mellitus complications (nephropathy and retinopathy), chronic kidney disease (CKD), cardiomyopathy, central nervous system (CNS) disorders such as stroke, epilepsy, spinal cord injury, dementia notably Alzheimer's disease,

Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, radiation necrosis and small vessel diseases such as Cerebral autosomal dominant arteriopathy with (Cadasil and Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (Carasil), Cerebral Amyloid

P352801NL -20-

Angiopathy (CAA), Retinal Vasculopathy with Cerebral Leukoencephalopathy and systemic manifestations (RVCL-S), Hereditary Hemorrhagic Telangiectasia (HHT), Cerebral Cavernous Malformations (CCM). Most of these conditions currently still lack the availability of effective treatment that is suitable for chronic treatment.

Detailed description of the Figures

Figure 1 shows that thalidomide but not its derivative lenalidomide prevents PC detachment and vascular leakage induced by radiation therapy. {A} Diagram of the injection protocol of vehicle, thalidomide or lenalidomide in combination with brain or eye radiation therapy. (B) Retinal PCs stimulated electrically induce localized vascular constriction. Top panels show representative bright field images of one blood capillary before, during and after PC electrical stimulation showing vessel constriction. The graph represents that the mean intensity inducing PC contraction and vascular constriction in control and irradiated mice treated with vehicle alone, thalidomide or lenalidomide. (C} Whole brains photographed after cadaverine Alexa Fluor-555 circulation and quantification of tracer accumulation in control and irradiated mice treated with vehicle alone, thalidomide or lenalidomide after 2 hours of tracer circulation.

Circulation time was 2 hours. All error bars show mean + s.e.m. ‘P<0.05; “P<0.01; *"P<0.001; ““P<0.0001 result from 1-way ANOVA and Dunnett's post hoc tests comparing the mean of each group to the control group. ns: non-significant.

Figure 2 shows that thalidomide but not its derivative lenalidomide prevents late vascular rarefaction and neuron loss induced by radiation therapy in the somatosensorial cortex (SS) and in the hippocampal region (Hp). (A) Diagram of the injection protocol of vehicle, thalidomide or lenalidomide in combination with brain radiation therapy. (B} Confocal image of a brain section stained for NeuN positive neurons showing the somatosensorial (SS) cortex and hippocampal region (Hp). (C-D) Confocal images of the somatosensorial cortex showing endothelial cells stained by GLUT1 (C) and neurons stained by NeuN (D) in control and irradiated mice treated with vehicle alone, thalidomide and lenalidomide. Animals were sacrificed 9 months after receiving a single dose of radiation therapy as indicated in A. (E)} Quantification of the vessel density in the somatosensorial cortex. (F} Number of neurons in the cortical region. (G-H)

Confocal images of the hippocampal region showing endothelial cells stained by GLUT1 (G) and neurons stained by NeuN (H) in control and irradiated mice treated with vehicle alone, thalidomide and lenalidomide. (1) Quantification of the vessel density in the hippocampal region. (F} Number of neurons in the hippocampal region.

Figure 3 reveals that thalidomide and selected FDA-approved analogues exert distinct blood vessel stabilization properties as illustrating using a mouse model of Hereditary Hemorrhagic Telangiectasia (HHT) type 1. (A) Scheme of tamoxifen (Tx) injection for the induction of Eng gene deletion (HHT1) and treatment plan with thalidomide and derivatives. (B) Chemical structures of thalidomide (THA), lenalidomide (LENA), pomalidomide (POMA) and apremilast (APRE). {C} Mean intensity inducing PC contraction in control and in Eng-iKO® mice treated with vehicle alone or that received thalidomide at 4, 40 or 200 mmol kg, lenalidomide at 40, 200 or 400 mmol.kg"t, pomalidomide at 40 mmel.kg’ or apremilast at 40 or 400

P352801NL -21- mmol.kg”’. Figure 4 shows (A) selected analogues of phthalimide that restore endothelial cell-pericyte interaction and blood vessel stability in accordance with the present invention, as demonstrated by (B) the mean intensity inducing PC contraction in control and in Eng-iKO® mice as shown in figure 3C.

The methods and treatment described herein may be a chronic treatment comprising chronic administration of the pharmaceutical compound or formulation. It may also comprise a temporary or acute treatment. In addition, it may be appreciated that the treatment may be curative and/or prophylactic.

Another aspect of the present invention is directed to the use in organoids and organ-on-a-chip (OoC) models. Organoid and OoC are in vitro miniaturized model systems of organs that have gained enormous interest for modeling disease, for personalized medicine, drug testing and cell therapy. They can be established for an increasing variety of organs including brain, kidney, liver, gut, thyroid, prostate, airways either from tissue resident adult stem cells, from biopsies, from embryonic stem cells or induced pluripotent stem cells. Despite considerable success, limitations of current organoid and OoC systems to achieve real life applications remain the limited level of maturity and function and this is also true for the vascular network. Blood vessels of organoids and OoC appear highly immature and leaky with a marked reduction of PC coverage

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The term "and/or" includes any and all combinations of one or more of the associated listed items. It will be understood that the terms "comprises" and/or "comprising" specify the presence of stated features but do not preclude the presence or addition of one or more other features.

For the purpose of clarity and concise description features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.

If for a compound included herein no absolute stereochemistry has specifically been indicated, the compound may be a racemic mixture, a mixture of diastereoisomers or an enantiomerically pure compound.

Typically, if no absolute stereochemistry has specifically been indicated, the compound is a racemic mixture. Moreover, if no relative stereochemistry has specifically been indicated, the compound is a mixture of diastereoisomers.

The present invention is illustrated by the following non-limiting examples and experiments.

P352801NL -22-

Examples and experiments

Synthesis of compounds lllustrative compounds were synthesized by a condensation of the appropriate anhydride (e.g. with the appropriate phthalic anhydride) with the appropriate amine and the following general procedures.

General procedure A for the condensation reaction using 3-nitrophthalic anhydride and substituted amines.

Equimolar amounts of 3-nitrophthalic anhydride and amine were dissolved in acetic acid and irradiated at 130 °C for 3 hours in a microwave synthesiser. The reaction mixture was subsequently concentrated in vacuo, taken up in dichloromethane and washed with sat. ag. NaHCO: and brine, respectively. The organics were dried over MgSO: and the solvents were evaporated to dryness in vacuo.

The crude product was finally purified by column chromatography.

General procedure B for the condensation reaction using 3-nitrophthalic anhydride and substituted amines.

Equimolar amounts of 3-nitrophthalic anhydride and amine were suspended in THF to which was then added triethylamine (2 equivalents). The suspension was stirred at room temperature until TLC indicated full consumption. The reaction mixture was subsequently concentrated in vacuo, taken up in a 0.6:1 mixture of glacial acetic acid and toluene and irradiated in a MW synthesizer at 130 °C for 0.5 hours.

The resulted solution was then concentrated in vacuo and subsequently purified by column chromatography.

General procedure C for the nitro reduction using Tin (lI) chloride dihydrate.

The corresponding 3-nitrophthalimide was taken up in ethyl acetate, to which was then added an excess amount of Tin (II) chloride dihydrate. The resulting solution was stirred at either 40 °C or at refluxing temperature until completion (TLC). The reaction mixture was then quenched by the addition of saturated aqueous NaHCO:3, layers separated and the aqueous fraction was extracted from ethyl acetate (3x). The combined organics were dried over MgSO:4 and the solvents were evaporated to dryness in vacuo. The crude product was finally purified by column chromatography.

General procedure D for the condensation reaction using ethyl 4-nitro-1,3-dioxoisoindoline-2- carboxylate and substituted amines.

To a stirring solution of ethyl 4-nitro-1,3-dioxoisoindoline-2-carboxylate (1 equivalent) and triethylamine (2 equivalents) in THF was added the appropriate amine in one portion (1 equivalent). The resulting suspension was stirred at room temperature overnight, after which a clear solution was obtained.

The crude reaction mixture was concentrated onto silica and purified by column chromatography.

General procedure E for the N-acylation of 3-(4-nitro-1,3-dioxoiseindelin-2-ylhazetidin-1-ium chloride using various acyl chlorides.

To a stirring suspension of 3-(4-nitro-1,3-dioxoisoindolin-2-yhazetidin-1-ium chloride in dichloromethane were added triethylamine (2.2 equiv.) and the corresponding acyl chloride (1.2 equiv.).

The reaction was stirred at room temperature until a clear solution was obtained, and TLC indicated there

P352801NL -23- was no remaining starting material. The reaction mixture was then concentrated in vacuo and the crude product was purified by column chromatography. 2-Isopropyl-4-nitroiscindoline-1,3-dione: @

Nel E

Ro ~~ % : NO, ©

The compound was synthesized according to general procedure A using the following conditions: 4- nitroiscbenzofuran-1,3-dione (0.10 g, 0.52 mmol), isopropylamine (0.045 mL, 1 equiv.) were taken up in glacial acetic acid (2.5 mL) and irradiated at 130 °C for 3 hours in a microwave synthesiser. Yield: 58% (0.07 g, 0.30 mmol) as a colourless solid. 'H NMR (500 MHz, CDCI) ô 8.12 — 8.05 (m, 2H), 7.91 (dd, J= 8.1, 7.4 Hz, 1H), 4.57 (hept, J= 7.0 Hz, 1H), 1.50 (d, J = 7.0 Hz, 6H). 4-amino-2-isopropylisoindoline-1,3-dione (14): {

Ni, ©

The compound was synthesized according to general procedure C using the following conditions: 2- isopropyl-4-nitroisoindaline-1,3-dione (0.07 g, 0.3 mmol), tin(ll) chloride dihydrate (0.68 g, 10 equiv.) were stirred in ethyl acetate (3 mL) at 40 °C overnight. Yield: 84% (0.05 g, 0.25 mmol) as a bright yellow solid. 'H (400 MHz, CDCls) 6 7.37 (dd, J = 8.3, 7.1 Hz, 1H), 7.10 (dd, J= 7.1, 0.7 Hz, 1H), 6.83 (dd, J = 8.3, 0.7

Hz, 1H), 5.06 (brs, 2H), 4.46 (hept, J = 6.9 Hz, 1H), 1.45 (d, J = 7.0 Hz, 6H). 13C NMR (101 MHz, CDCls) 5170.5, 168.7, 145.0, 135.0, 132.9, 121.1, 112.7, 111.7, 42.6, 20.3. LC-MS (ESI) m/z calcd for

C1H32N202 [M + H]* 204.1, found 205.1. HPLC (method A): 9.181 min, purity 99%. 2-isobutyl-4-nitroisoindoline-1,3-dione: 0 no, 0 7

The compound was synthesized according to general procedure A using the following conditions: 4- nitroisobenzofuran-1,3-dione (0.15 g, 0.78 mmol), isobutylamine (0.068 g, 1.2 equiv.) were taken up in glacial acetic acid (2 mL) and irradiated at 130 °C for 3 hours in a microwave synthesiser. Yield: 89% (0.17 g, 0.69 mmol) as a colourless solid. 'H NMR (400 MHz, CDCl) 6 8.15-8.13 (m, 1H), 8.15 — 8.09

P352801NL -24 - (m, 1H), 7.94 (dd, J =8.2, 7.4 Hz, 1H), 3.55 (d, J = 7.4 Hz, 2H), 2.24 - 2.05 (m, 1H), 0.96 (d, J = 6.8 Hz, 6H). 4-amino-2-isobutylisoindoline-1,3-dione (15): a

Ng, ©

The compound was synthesized according to general procedure C using the following conditions: 2- isobutyl-4-nitroiscindoline-1,3-dione (0.171 g, 0.69 mmol) and tin(ll) chloride dihydrate (1.6 g, 10 equiv.) were stirred in ethyl acetate (3 mL) at 40 °C for 72 hours. Yield: 87% (0.13 g, 0.60 mmol) as a bright yellow solid. ’H NMR (400 MHz, CDCls) 6 7.39 (dd, J= 8.3, 7.1 Hz, 1H), 7.13 (dd, J = 7.1, 0.7 Hz, 1H), 6.85 (dd, J = 8.3, 0.7 Hz, 1H), 5.02 (s, 2H), 3.43 (d, J = 7.4 Hz, 2H), 2.08 (dh, J= 13.7, 6.9 Hz, 1H), 0.92 (d, J=6.7 Hz, 6H). 3C NMR (101 MHz, CDCl) 6 170.62, 169.01, 145.07, 135.16, 132.86, 121.15, 112.87, 111.57, 45.04, 27.99, 20.25. LC-MS (ESI) m/z calcd for C12H14N202 [M + H]* 218.1, found 219.0.

HPLC (method A): 9.737 min, purity 97%. 4-nitro-2-phenylisoindoline-1,3-dione:

Q

YO Ne 3

Say Nn

NO, {3

The compound was synthesized according to general procedure A using the following conditions: 4- nitroisobenzofuran-1,3-dione (0.15 g, 0.78 mmol), aniline (0.045 mL, 1 equiv.) were taken up in glacial acetic acid (2.5 mL) and irradiated at 130 °C for 3 hours in a microwave synthesiser. Yield: 75% (0.16 g, 0.58 mmol) as a colourless crystalline solid. "TH NMR (400 MHz, CDCl) § 8.22 (dd, J = 7.5, 1.0 Hz, 1H), 8.16 (dd, J=8.2,0.9 Hz, 1H), 7.99 (dd, J= 8.1, 7.5 Hz, 1H), 7.57 — 7.48 (m, 2H), 7.47 — 7.40 (m, 3H). 4-amino-2-phenylisoindoline-1,3-dione (16): 9 nf \

Nh, ©

The compound was synthesized according tc general procedure C using the following conditions: 4-nitro- 2-phenylisoindoline-1,3-dione (0.15 g, 0.56 mmol} and tin(ll} chloride dihydrate (0.51 g, 4 equiv.) were stirred in ethyl acetate (15 mL) at 40 °C for 16 hours. Yield: 79% (0.11 g, 0.44 mmol) as a bright yellow solid. TH NMR (400 MHz, CDCl) ò 7.42-7.26 (m, 6H), 7.14 (dd, J=7.1, 0.7 Hz, 1H), 6.80 (dd, J = 8.3, 0.7

Hz, 1H), 5.13 (br s, 2H). 13C NMR (101 MHz, CDCls) ò 169.26, 167.61, 145.84, 135.66, 132.51, 131.93,

P352801NL -25- 129.19, 127.97, 126.68, 121.39, 113.23, 111.00. LC-MS (ESI) m/z calcd for C14H1oN202 [M + H]* 238.1, found 239.0. HPLC (method A): 9.41 min, purity 98%. tert-butyl (S)-3-(4-nitro-1,3-dioxoisoindolin-2-yl}pyrrolidine-1-carboxylate: 3 fo gu VAN 0 : NO, Ù B)

The compound was synthesized according to general procedure B using the following conditions: tert

Butyl (S)-3-aminopyrrolidine-1-carboxylate (0.50 g, 2.68 mmol), 4-nitroisobenzofuran-1,3-dione (0.57 g, 1.1 equiv.), triethylamine (0.7 mL, 2 equiv.). Yield: 93% (0.90 g, 2.50 mmol) as a light brown oil. "H NMR (400 MHz, CDCls) 6 8.22 — 8.08 (m, 2H), 7.96 (t, J = 7.7 Hz, 1H), 4.89 (p, J = 8.2 Hz, 1H), 3.78 — 3.84 (m, 3H), 3.49 - 3.37 (m, 1H), 2.61 (p, J = 9.1 Hz, 1H), 2.16 (d, J = 22.8 Hz, 1H), 1.47 (d, J = 7.3 Hz, 9H). (S)-3-{4-nitro-1,3-dioxoisoindolin-2-yl)pyrrolidin-1-ium chloride:

NN Fa

NG TN

Ng, © tert-Butyl (S)-3-(4-nitro-1,3-dioxoiscindolin-2-yl)pyrrolidine-1-carboxylate (0.90 g, 2.49 mmol) was taken up in methanolic HCI (4N, 25 mL) and stirred at room temperature for 1 hour. After stirring for 1 hour the reaction mixture was filtered and the solids were washed with EtOAc and subsequently dried in vacuo, affording the title compound as a white solid. Yield: 81% (0.60 g, 2.02 mmol). "H NMR (400 MHz, DMSO-

Ds) 8 9.73 (brs, 1H), 9.31 (brs, 1H), 8.30 (dd, J= 8.1, 0.8 Hz, 1H), 8.18 (dd, J= 7.5, 0.8 Hz, 1H}, 8.08 (dd, J = 8.0, 7.6 Hz, 1H), 4.97 — 4.85 (m, 1H), 3.56 — 3.41 (m, 3H), 3.35 — 3.25 (m, 1H), 2.38 — 2.20 (m, 2H). (S)-2-(1-acetylpyrrolidin-3-yl)-4-nitroisoindoline-1,3-dione: 0

To a stirring suspension of (S)-3-(4-nitro-1,3-dioxoiscindolin-2-yl}pyrrolidin-1-ium chloride (0.15 g, 0.50 mmol) in DCM (10 mL) were added triethylamine (0.05 mL, 2 equiv.) and acetyl chloride (0.14 mL, 1.4 equiv.), and the resulted solution was stirred for 2 hours. The reaction mixture was then concentrated in vacuo, affording the crude title compound as a crystalline white solid. Yield: 0.20 g, 0.50 mmol, quant. 'H

NMR (400 MHz, CDCls) 6 8.18 — 8.09 (m, 2H), 7.96 (q, J = 7.6 Hz, 1H), 4.95 (dp, J = 15.6, 8.1 Hz, 1H),

P352801NL -26- 4.02 -3.72 (m, 3H), 3.68 — 3.42 (m, 1H), 2.81 — 2.52 (m, 1H), 2.43 — 2.28 (m, 1H), 2.09 (d, J = 14.0 Hz, 3H). (S)-2-(1-acetylpyrrolidin-3-yl)-4-aminoisoindoline-1,3-dione (25): < Ni, U &

The compound was synthesized according to general procedure C using the following conditions: (S)-2- (1-acetylpyrrolidin-3-yl)-4-nitroisoindcline-1,3-dione (0.15 g, 0.50 mmol) and tin(ll) chloride dihydrate (0.45 g. 4 equiv.) were stirred in ethyl acetate (60 mL) at a reflux for 8 hours. The crude product was purified by column chromatography, eluting with 3% MeOH in DCM as eluent, affording the title compound as a yellow film. Yield: 63% (0.13 g, 0.47 mmol) as a bright yellow solid. 'H NMR (400 MHz, DMSO-De) § 7.42 (ddd, J= 8.5, 7.0, 2.4 Hz, 1H), 7.00 —- 6.91 (m, 2H), 6.48 (d, J = 5.1 Hz, 2H), 4.73 (dp, J = 27.4, 8.0 Hz, 1H), 3.80 — 3.54 (m, 3H), 3.50 (dt, J = 9.9, 7.7 Hz, 0.5H), 3.30 (dt, J = 11.8, 8.1 Hz, 0.5H), 2.47 —- 2.27 (m, 1H), 2.13 (ddtd, J = 32.1, 12.1, 7.6, 4.4 Hz, 1H), 1.94 (d, J = 14.1 Hz, 3H). "*C NMR (101 MHz, DMSO) 5 169.29, 168.15, 167.96, 167.89, 167.85, 146.55, 146.52, 135.20, 135.16, 132.19, 132.15, 121.43, 121.39, 110.67, 110.65, 108.82, 108.75, 48.56, 48.09, 47.37, 46.79, 45.70, 43.96, 29.05, 27.44, 22.32, 21.88. 'H

NMR (500 MHz, DMSO-D, 100 °C) 6 7.43 (dd, J= 8.4, 7.0 Hz, 1H), 7.01 (d, J= 8.4 Hz, 1H),6.97 (d, J = 7.0 Hz, 1H), 6.24 (s, 2H), 4.85 — 4.62 (m, 1H), 4.02 — 3.59 (m, 3H), 3.53 (s, 0.5H), 3.36 (s, 0.5H), 2.48 — 2.32 (m, 1H), 2.30 — 2.05 (m, 1H), 1.96 {s, 3H). LC-MS (ESI) m/z calcd for C14H1sN3O3 [M + H]t 273.1, found 274.0, HPLC (method A}: 6.92 min, purity 99%. tert-butyl (R}-3-(4-nitro-1,3-dioxoisoindolin-2-yl}pyrrolidine-1-carboxylate: o

ESA oo CS

Tu Ven

The compound was synthesized according tc general procedure B using the following conditions: fert-

Butyl (R)-3-aminopyrrolidine-1-carboxylate (0.50 g, 2.68 mmol}, 4-nitroisobenzofuran-1,3-dione (0.57 g, 1.1 equiv.), triethylamine (0.7 mL, 2 equiv.). Yield: 83% (0.81 g, 2.23 mmol) as a light brown oil. "TH NMR (400 MHz, CDCl3) 6 8.13 (dd, J= 7.7, 1.9 Hz, 2H), 7.96 (t, J = 7.8 Hz, 1H), 4.89 (p, J= 8.4 Hz, 1H), 3.83 —3.63 (m, 3H), 3.49 — 3.38 (m, 1H), 2.62 (p, J=9.1 Hz, 1H), 2.19 (brs, 1H), 1.48 (d, J = 7.7 Hz, 9H).

P352801NL -27- (R)-3-(4-nitro-1,3-dioxoisoindolin-2-yl)pyrrolidin-1-ium chloride: 2 at ARE no, © tert-Butyl (R)-3-(4-nitro-1,3-dioxoisoindolin-2-yhpyrrolidine-1-carboxylate (0.80 g, 2.21 mmol) was taken up in methanolic HCI (4N, 25 mL) and stirred at room temperature for 1 hour. After stirring for 1 hour the reaction mixture was filtered and the solids were washed with EtOAc and subsequently dried in vacuo, affording the title compound as a white solid. Yield: 84% (0.55 g, 1.85 mmol). '"H NMR (400 MHz, DMSO-

De) 0 9.64 (s, 1H), 8.24 (s, 1H), 8.30 (dd, J= 8.1, 0.8 Hz, 1H), 8.18 (dd, J= 7.5, 0.9 Hz, 1H), 8.08 (dd, J = 8.0, 7.6 Hz, 1H), 4.92 (tt, J = 8.5, 6.0 Hz, 1H), 3.60 — 3.40 (m, 3H), 3.34 — 3.25 (m, 1H), 2.40 — 2.19 (m, 2Hj). (R)-2-(1-acetylpyrrolidin-3-yl)-4-nitroisoindoline-1,3-dione: £3

NO, 3 a

To a stirring suspension of (R)-3-(4-nitro-1,3-dioxoisoindolin-2-ylypyrrolidin-1-ium chloride (0.15 g, 0.50 mmol) in DCM (10 mL) were added triethylamine (0.05 mL, 2 equiv.) and acetyl chloride (0.14 mL, 1.4 equiv.), and the resulted solution was stirred for 2 hours. The reaction mixture was then concentrated in vacuo, affording the crude title compound as a crystalline white solid. Yield: 0.20 g, 0.50 mmol, quant. 'H

NMR (400 MHz, CDCl) 6 8.13 (ddd, J=7.8, 6.3, 1.7 Hz, 2H), 8.01 —- 7.91 (m, 1H), 4.95 (dp, J= 15.6, 8.2

Hz, 1H), 4.02 — 3.69 (m, 3H), 3.66 — 3.44 (m, 1H), 2.74 — 2.54 (m, 1H), 2.40 — 2.28 (m, 1H), 2.09 (d, J = 14.1 Hz, 3H). (R}-2-(1-acetylpyrrolidin-3-yl}-4-aminoisoindoline-1,3-dione (27): 0

SEN i Ey ni, © &

The compound was synthesized according tc general procedure C using the following conditions: (R)-2- (1-acetylpyrrolidin-3-yl}-4-nitroiscindoline-1,3-dione (0.15 g, 0.50 mmal} and tin(ll) chloride dihydrate (0.45 g, 4 equiv.) were stirred in ethyl acetate (60 mL) at a reflux for 16 hours. The crude product was purified by column chromatography, eluting with 3% MeOH in DCM as eluent, affording the title compound as a yellow film. Yield: 99% (0.13 g, 0.49 mmol) as a bright yellow solid. 'H NMR (400 MHz, DMSO-Ds) 6 7.42 (ddd, J= 8.3, 7.0, 2.5 Hz, 1H), 7.00 — 6.91 (m, 2H), 6.48 (d, J = 5.1 Hz, 2H), 4.73 (dp, J = 27 4, 8.0 Hz, 1H), 3.82 — 3.54 (m, 3H), 3.50 (dt, J = 9.9, 7.8 Hz, 0.5H), 3.30 (dt, J = 11.8, 8.1 Hz, 0.5H), 2.48 — 2.27 (m,

P352801NL -28- 1H), 2.24 — 1.99 (m, 1H), 1.94 (d, J = 14.1 Hz, 3H). "*C NMR (101 MHz, DMSO-Ds) § 169.29, 168.14, 167.96, 167.85, 146.55, 146.52, 135.20, 135.16, 132.19, 132.14, 121.44, 121.39, 110.67, 110.65, 108.80, 108.75, 48.56, 48.09, 47.36, 46.79, 45.70, 43.96, 29.05, 27.44, 22.32, 21.88. "H NMR (500 MHz, DMSO-

De, 100 °C) 6 7.43 (dd, J=8.4, 7.0 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H), 6.97 (d, 4 = 7.1 Hz, 1H), 6.25 (br s, 2H), 4.85-4.61 (m, 1H), 3.998 — 3.59 (m, 3H), 3.59 — 3.44 (m, 0.5H), 3.44 — 3.25 (m, 0.5H), 2.47 — 2.31 (m, 1H), 2.29 — 2.09 (m, 1H), 1.96 (br s, 3H). LC-MS (ESI) m/z calcd for C14H1sN3Os [M + H]* 273.1, found 274.0. HPLC (method A): 7.18 min, purity 98%. tert-butyl 4-(4-nitro-1,3-dioxoisoindolin-2-yl)piperidine-1-carboxylate: £3

NO, t

The compound was synthesized according to general procedure B using the following conditions: fert- butyl 4-aminopiperidine-1-carboxylate (0.74 g, 3.71 mmol}, 4-nitroisobenzofuran-1,3-dione (0.72 9, 1.0 equiv), triethylamine (0.67 mL, 1.3 equiv.). Yield: 83% (1.16 g, 3.09 mmol) as a colourless oil. 'H NMR (400 MHz, DMSO-Ds) & 8.26 (dd, J = 8.0, 0.9 Hz, 1H), 8.14 (dd, J=7.5, 0.9 Hz, 1H), 8.08 — 8.00 (m, 1H), 4.20 (tt, J=12.2, 4.0 Hz, 1H), 4.13 — 3.93 (m, 2H), 3.00 — 2.68 (m, 2H), 2.08 (qd, J = 12.6, 4.4 Hz, 2H), 1.76 — 1.67 (m, 2H), 1.42 (s, 9H). 4-{4-nitro-1,3-dioxoisoindolin-2-yljpiperidin-1-ium chloride: 0

Neg ee

No, © fert-Butyl 4-(4-nitro-1,3-dioxoiscindolin-2-yl)piperidine-1-carboxylate (0.50 g, 1.33 mmol) was taken up in methanolic HCI (4N, 20 mL) and stirred at room temperature for 1 hour. After stirring for 1 hour the reaction mixture was filtered and the solids were washed with EtOAc and subsequently dried in vacuo, affording the title compound as a white solid. Yield: 56% (0.23 g, 0.75 mmol). 1H NMR (400 MHz, DMSO-

Ds) 8 9.17 (s, 1H), 8.59 (s, 1H), 8.27 (dd, J = 8.1, 0.8 Hz, 1H), 8.16 (dd, J= 7.5, 0.8 Hz, 1H}, 8.10 — 8.01 (m, 1H), 4.34 (tt, J = 12.1, 3.7 Hz, 1H), 3.36 (d, J = 12.5 Hz, 2H), 3.04 (gq, J= 12.4 Hz, 2H), 2.48 - 2.35 {m, 2H), 1.91 (d, J = 12.8 Hz, 2H).

P352801NL -29- 2-(1-acetylpiperidin-4-yl)-4-nitroisoindoline-1,3-dione: aw ld

NG, ©

To a stirring suspension of 4-(4-nitro-1,3-dioxoisoindolin-2-yhpiperidin-1-ium chloride (0.15 g, 0.48 mmol) in DCM (20 mL) were added triethylamine (0.16 mL, 2.4 equiv.) and acetyl chloride (0.08 mL, 2.3 equiv.), and the solution was stirred for 1 hour. The reaction mixture was then washed with brine (2x), the organics dried over MgSOa and evaporated to dryness in vacuo, affording the title compound as a white crystalline solid. Yield: 99% (0.15 g, 0.48 mmol). "H NMR (400 MHz, CDCls) ò 8.14 — 8.07 (m, 2H), 7.96 — 7.88 (m, 1H), 4.84 (ddd, J = 11.1, 4.4, 2.2 Hz, 1H), 4.38 (it, J = 12.2, 4.1 Hz, 1H), 4.02 — 3.92 (m, 1H), 3.16 (td, J=13.4,2.6 Hz, 1H), 2.60 (td, J = 13.2, 2.6 Hz, 1H), 2.52 — 2.28 (m, 2H), 2.15 (s, 3H), 1.85 - 1.71 (m, 2H). 2-(1-acetylpiperidin-4-yl}-4-aminoisoindoline-1,3-dione (29): 3 9! ON =X

The compound was synthesized according to general procedure C using the following conditions: 2-(1- acetylpiperidin-4-yl}-4-nitroisoindoline-1,3-dione (0.15 g, 0.48 mmol) and tin(ll) chloride dihydrate (0.43 g, 4 equiv.) were stirred in ethyl acetate (30 mL) at a reflux for 6 hours. The crude product was purified by column chromatography, eluting with a gradient of 0-2% MeOH in DCM as eluent, affording the title compound as a yellow solid. Yield: 99% (0.14 g, 0.47 mmol) as a bright yellow solid. *H NMR (400 MHz,

CDCl) § 7.41 (dd, $= 8.3, 7.2 Hz, 1H), 7.12 (dd, J=7.1, 0.5 Hz, 1H), 6.85 (dd, J = 8.3, 0.5 Hz, 1H), 5.26 (s,2H), 4.81 (ddd, J=11.1,4.4, 22 Hz, 1H), 4.27 (it, J=12.2,4.1 Hz, 1H), 3.95 (ddd, J=11.4, 4.4, 2.1

Hz, 1H), 3.14 (td, J = 13.4, 2.6 Hz, 1H), 2.59 (td, J = 13.2, 2.6 Hz, 1H), 2.39 (dad, J = 39.9, 12.6, 4.4 Hz, 2H), 2.14 (s, 3H), 1.75 (dd, J = 11.7, 4.2, 2.3 Hz, 2H). 13C NMR (101 MHz, CDCls) § 170.16, 168.97, 168.42, 145.41, 135.35, 132.54, 121.19, 112.81, 111.11, 48.44, 46.23, 41.40, 29.58, 28.98, 21.63.

LC-MS (ESI) m/z calcd for C1sHizN2Os [M + H]* 287.1, found 288.0. HPLC (method A): 7.68 min, purity 99%.

P352801NL -30- tert-butyl 3-methyl-3-(4-nitro-1,3-dioxoisoindolin-2-yl}azetidine-1-carboxylate:

NO, D >

The compound was synthesized according to general procedure B using the following conditions: fert- butyl 3-amino-3-methylazetidine-1-carboxylate (0.50 g, 2.68 mmol), 4-nitroiscobenzofuran-1,3-dione (0.52 g, 1.0 equiv.), triethylamine (0.75 mL, 2 equiv.). Yield: 79% (1.18 g, 3.09 mmol) as a white solid. "H NMR (400 MHz, DMSO-Ds) 6 8.26 (dd, J = 8.0, 0.9 Hz, 1H), 8.13 (dd, J = 7.4, 0.9 Hz, 1H), 8.04 (dd, J = 8.0, 7.5 Hz, 1H), 4.37 (d, J = 8.7 Hz, 2H), 3.82 (d, J = 8.7 Hz, 2H), 1.59 (s, 3H), 1.38 (s, 9H). 3-methyl-3-{4-nitro-1,3-dioxoisoindolin-2-yljazetidin-1-ium chloride:

Q

A NHC

NO,

To a stirring solution of fert-butyl 3-methyl-3-(4-nitro-1,3-dioxoisoindolin-2-ylyazetidine-1-carboxylate (0.75 g, 2.07 mmol} in EtOAc (10 mL) was carefully added 4N HCI in dioxane (10 mL) and the resulted solution was left to stir for 2 hours. The suspension was then filtered and the solids were washed with pet ether and subsequently dried, affording the title compound as a white solid. Yield: 89% (0.66 g, 2.10 mmol). tH

NMR (400 MHz, DMSO-De) ò 10.15 (s, 1H), 9.47 (s, 1H), 8.31 (dd, J= 8.0, 0.9 Hz, 1H), 8.16 (dd, J = 7.5, 0.9 Hz, 1H), 8.07 (dd, J = 8.1, 7.5 Hz, 1H), 4.53 (d, J = 10.4 Hz, 2H), 3.93 (d, J = 11.7 Hz, 1H), 1.73 (s, 3H). 2-(1-acetyl-3-methylazetidin-3-yl}-4-nitroisoindoline-1,3-dione: £3

NJ a N 0 No, ©

To a stirring suspension of 3-methyl-3-(4-nitro-1,3-dioxoiscindolin-2-ylyazetidin-1-ium chloride (0.30 g, 1.01 mmol) in dichloromethane (10 mL) were added subsequently triethylamine (0.34 mL, 2.4 equiv.) and acetyl chloride (0.17 mL, 2.3 equiv.). The resulted solution was stirred for 1 hour, after which TLC showed full conversion. The reaction mixture was diluted with DCM and washed with 0.5N HCI (ag) and brine.

The organics were dried over MgSO: and solvents removed in vacuo, affording the title compound as an off-white solid. Yield: 94% (0.29 g, 0.95 mmol). 'H NMR (400 MHz, DMSO-Ds) § 8.28 (dd, J = 8.0, 0.9 Hz, 1H), 8.15 (dd, J= 7.5, 0.9 Hz, 1H), 8.05 (dd, J= 8.0, 7.5 Hz, 1H), 4.62 (d, J= 8.9 Hz, 1H), 4.34 (d, /= 9.9

Hz, 1H), 4.16 (d, J = 9.1 Hz, 1H), 3.86 (d, J = 9.9 Hz, 1H), 1.78 (s, 3H), 1.60 (s, 3H).

P352801NL -31- 2-(1-acetyl-3-methylazetidin-3-yl}-4-aminoisoindoline-1,3-dione (33): 7 3

Ni, £

The compound was synthesized according to general procedure C using the following conditions: 2-(1- acetyl-3-methylazetidin-3-yD-4-nitroisoindoline-1,3-dione (0.26 g, 0.84 mmol) and tin{ll) chloride dihydrate (0.76 g, 4 equiv.) were stirred in ethyl acetate (25 mL) at a reflux for 16 hours. The crude product was purified by column chromatography, eluting with 100% EtOAc as eluent, affording the title compound as a bright yellow solid. Yield: 85% (0.22 g, 0.80 mmol). "H NMR (400 MHz, CDCl:) 6 7.41 (dd, J = 8.3, 7.1 Hz, 1H), 7.09 (dd, J= 7.1, 0.7 Hz, 1H), 6.89 (dd, J = 8.4, 0.8 Hz, 1H), 5.41 (brs, 2H), 4.71 (d, J = 9.3 Hz, 1H), 4.53 (d, J= 10.6 Hz, 1H), 4.21 (d, J= 9.3 Hz, 1H), 4.15 (d, J = 10.6 Hz, 1H), 1.91 (s, 3H), 1.70 (s, 3H).

BC NMR (101 MHz, CDCls) ò 170.98, 169.45, 167.82, 145.70, 135.46, 132.61, 121.44, 112.60, 110.84, 60.80, 58.46, 49.50, 24.73, 19.02. LC-MS (ESI) m/z calcd for C14H1sN3O3 [M + HJ 273.1, found 274.0.

HPLC (method A): 7.34 min, purity 99%. ethyl 4-nitro-1,3-dioxoisoindoline-2-carboxylate: 0

Ty 3 STN

No, © N

To a stirring suspension of 3-nitrophthalimide (2.0 g, 10.4 mmol) in 17 mL DCM were added triethylamine (6.5 mL, 4.5 equiv.) and DMAP (0.13 g, 0.1 equiv}. This mixture was then cooled to 0 °C using an ice bath before the dropwise addition of ethylchloroformate (5 mL, 5 equiv.). After the addition was completed the solution was maintained at 0 °C for 30 minutes, after which the reaction mixture was diluted with ethyl acetate (250 mL) and washed subsequently with HCI (aq., 3% v/v) and brine, after which the organics were dried over MgSQs and concentrated in vacuo. The crude was purified by recrystallization from a mixture of EtOAc/Hexane. Yield: 70% (1.91 g, 7.23 mmol) as a light brown solid. "TH NMR (400 MHz,

CDCls) ò 8.24 (dd, J= 7.6, 0.9 Hz, 1H), 8.19 (dd, J=8.0, 1.0 Hz, 1H), 8.03 (t, J= 7.8 Hz, 1H), 4.51 (gq, J = 7.1 Hz, 2H), 1.45 (t, J = 7.1 Hz, 3H). ethyl 1,3-dioxo-1,3-dihydro-2H-pyrrelo[3,4-c]pyridine-2-carboxylate: 2

Ge ©

NS oy a

To a stirring suspension of 1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dicne (2.5 g, 16.9 mmol) and triethylamine (3.1 mL, 1.3 equiv.) in DMF (8 mL) was added at 0 °C in dropwise fastion ethyl chloroformate (2.0 mL, 1.2 equiv.). After the addition was completed the solution was maintained at 8 °C for 30 minutes, after which

P352801NL -32- the reaction mixture maintained at room temperature for 1 hour. The reaction mixture was then poured onto ice, extracted with EtOAc (3x 200 mL) and the combined organics washed with brine. The organics were dried over MgSO. and concentrated in vacuo. The crude was purified by column chromatography eluting with 40/60 EtOAc in pentane, affording the title compound as a yellow crystalline solid. Yield: 30% (1.119, 5.04 mmol). "TH NMR (400 MHz, CDCls) & 9.30 (s, 1H), 9.17 (d, J=4.9 Hz, 1H), 7.88 (d, J= 4.8

Hz, 1H), 4.52 (q, J=7.1 Hz, 2H), 1.48 (t, J = 7.1 Hz, 3H). 2-(1-acetylazetidin-3-yl)-1 H-pyrrolo[3,4-c]pyridine-1,3{2H)-dione (4): > o °

The compound was synthesized according to general procedure D using the following reagents: ethyl 1,3- díoxo-1,3-dihydro-2H-pyrrolo[3,4-clpyridine-2-carboxylate (0.15 g, 0.68 mmol), triethylamine (0.19 mL, 2 equiv.), and 1-acetylazetidin-3-aminium chloride (0.1 g, 1 equiv.). The reaction mixture was stirred at room temperature for 2 hours and was then heated to 50 °C for 2 hours. Yield: 30% (0.05 g, 0.21 mmol) as an off-white solid. "H NMR (400 MHz, CDCl; 6 9.15 (d, J= 1.1 Hz, 1H), 8.08 (d, J=4.9 Hz, 1H), 7.76 (dd, J=4.8, 1.2 Hz, 1H), 5.09 (it, J = 8.7, 6.2 Hz, 1H), 4.70 (dd, J = 8.7, 6.1 Hz, 1H), 4.49 (dd, J= 10.1, 6.3 Hz, 1H), 4.42 (t, J = 8.7 Hz, 1H), 4.32 (t, J = 9.4 Hz, 1H}, 1.92 (s, 3H). 13C NMR (101 MHz, CDCls) & 170.82, 166.56, 166.26, 156.15, 145.15, 138.97, 125.40, 117.09, 54.53, 52.63, 38.79, 19.06. LC-MS (ESI) m/z calcd for C12H11N3O3 [M + H]* 245.1, found 246.1. HPLC (method B): 7.78 min, purity 96%. (S)-4-methyl-2-(4-nitro-1,3-dioxoisocindolin-2-yl)pentancic acid: 2 #

NO, 2 Q

The compound. was synthesized according to general procedure D using the following reagents: ethyl 4- nitro-1,3-dioxoiscindoline-2-carboxylate (0.4 g, 1.5 mmol), triethylamine (0.42 mL, 2 equiv.), and L-leucine (0.20 g, 1 equiv.). After stirring at room temperature overnight the reaction mixture was subsequently heated to 130 °C for 15 minutes. Yield: 52% (0.24 g, 0.78 mmol) as colourless oil. 'H NMR (400 MHz,

CDClz) 6 8.17 — 8.11 {m, 2H), 7.94 (t, J = 7.8 Hz, 1H), 5.00 (ddd, J = 11.5, 4.4, 1.4 Hz, 1H), 1.97 (dddd, J =14.5,10.3, 4.4, 1.2 Hz, 1H), 1.55 — 1.43 (m, 1H), 1.34 — 1.20 (m, 1H), 1.02 — 0.87 (m, 6H}. (S)-2-{4-amino-1,3-dioxoisoindolin-2-yl}-4-methylpentanoic acid (17): 20 NH, QQ

P352801NL -33-

The compound was synthesized according to general procedure C using the following conditions: (S)-4- methyl-2-(4-nitro-1,3-dioxoisoindolin-2-yl)pentancic acid (0.23 g, 0.75 mmol) and tin(ll} chloride dihydrate (0.68 g, 4 equiv.) were stirred in ethyl acetate (10 mL) at 40 °C for 16 hours. Product purified by flash column chromatography using a C18 cartridge, eluting with a gradient of 10-90% CH3CN in H20. Yield: 63% (0.13 g, 0.47 mmol) as a bright yellow solid. "H NMR (400 MHz, CDCls) 6 7.40 (t, J = 7.7 Hz, 1H), 715, J=7.1Hz, 1H), 6.87 (d, J=8.3 Hz, 1H), 4.92 (dd, J= 11.5, 4.3 Hz, 1H), 2.31 (ddd, J = 15.1, 11.6, 4.1 Hz, 1H), 1.90 (ddd, J = 14.4, 10.2, 4.4 Hz, 1H), 1.57 — 1.40 (m, 1H), 0.92 (t, J = 6.8 Hz, 6H). °C

NMR (101 MHz, CDCl) ò 175.88, 169.50, 168.03, 145.10, 135.55, 132.46, 121.68, 113.50, 111.34, 50.15, 37.20, 25.14, 23.26, 21.10. LC-MS (ESI) m/z calcd for C14H1sN204 [M + H]* 276.1, found 277.0.

HPLC (method A): 8.43 min, purity 97%. (S)-4,4-dimethyl-2-{4-nitro-1,3-dioxoisoindolin-2-yl)pentanoic acid:

Rpg, Se

SUH

NO, d

The compound was synthesized according to general procedure D using the following reagents: ethyl 4- nitro-1,3-dioxoisoindoline-2-carboxylate (0.91 g, 3.44 mmol), triethylamine (0.96 mL, 2 equiv}, and (S)-2- amino-4,4-dimethylpentanoic acid (0.50 g, 1 equiv.). After stirring at room temperature under a Nz atmosphere for 4 hours the reaction mixture was subsequently irradiated in a MW synthesizer at 130 °C for 10 minutes. Yield: 92% (1.02 g, 3.18 mmol) as light yellow oil. "TH NMR (400 MHz, CDCl) & 11.24 — 10.69 (m, 1H), 8.19 = 8.11 (m, 2H), 7.95 (t, J = 7.9 Hz, 1H), 5.05 — 4.97 (m, 1H), 2.32 — 2.17 {m, 2H), 0.93 (d,J=1.7 Hz, 9H). (S)-2-{4-amino-1,3-dioxoisoindolin-2-yl)-4,4-dimethylpentanoic acid (30):

The compound was synthesized according to general procedure C using the following conditions: (S)-4,4- dimethyl-2-(4-nitro-1,3-dioxcisoindolin-2-yl)pentanocic acid (0.25 g, 0.78 mmol) and tin(ll) chloride dihydrate (0.70 g, 4 equiv.) were stirred in ethyl acetate (20 mL) at reflux for 6 hours. Crude product was purified by column chromatography eluting with a solution of 1% MeOH and 0.5% AcOH in dichloromethane, affording the title compound as a yellow solid. Yield: 88% (0.20 g, 0.70 mmol). 'H NMR (400 MHz, CDCl:)} 6 7.39 (dd, J= 8.4, 7.1 Hz, 1H), 7.14 (d, J = 7.1 Hz, 1H), 6.84 (d, J = 8.2 Hz, 1H), 4.93 (dd, J=9.6, 2.9 Hz, 1H), 2.40 — 2.11 (m, 2H), 0.92 (s, 9H). 13C NMR (101 MHz, CDClz) § 176.43, 169.57, 168.07, 145.63, 135.51, 132.52, 121.43, 113.18, 111.10, 49.08, 41.30, 30.51, 29.26. LC-MS (ESI) m/z calcd for CisH1sNzO4 [M + H]* 290.1, found 291.0. HPLC (method A): 9.80 min, purity 88%.

P352801NL -34- (S)-3-cyclopropyl-2-(4-nitro-1,3-dioxoisoindolin-2-yl}propanoic acid:

Q

Sg SO

NG, DD

The compound was synthesized according to general procedure D using the following reagents: ethyl 4- nitro-1,3-dioxoiscindoline-2-carboxylate (1.02 g, 3.87 mmol), triethylamine (1.08 mL, 2 equiv.), and (S)-2- amino-3-cyclopropylpropanoic acid (0.50 g, 1 equiv.). After stirring at room temperature under a Nz atmosphere for 2 hours the reaction mixture was subsequently irradiated in a MW synthesizer at 130 °C for 5 minutes. Yield: 25% (0.30 g, 1.0 mmol) as a colourless oil. "TH NMR (400 MHz, CDCls) & 11.11 — 10.84 (m, 1H), 8.18 (dd, J = 3.2, 0.9 Hz, 1H), 8.17 — 8.13 (m, 1H), 8.02 — 7.93 (m, 1H), 5.05 (dd, J = 10.3, 5.4 Hz, 1H), 2.24 2.12 (m, 2H), 0.73 — 0.57 (m, 1H), 0.49 — 0.32 (m, 2H), 0.12 (td, J=9.1, 8.7, 4.9 Hz, 1H), 0.08 — 0.00 (m, 1H). (S)-2-{4-amino-1,3-dioxoisoindolin-2-yl}-3-cyclopropylpropanoic acid (31):

A Nn ee eH

NH, a 0

The compound was synthesized according to general procedure C using the following conditions: (S)-3- cyclopropyl-2-(4-nitro-1,3-dioxcisoindolin-2-yl}preopanoic acid (0.30 g, 1.0 mmol} and tin(ll) chloride dihydrate (0.89 g, 4 equiv.) were stirred in ethyl acetate (40 mL) at reflux for 6 hours. Crude product was purified by column chromatography eluting with a solution of 2% MeOH and 0.5% AcOH in dichloromethane, affording the title compound as a yellow solid. Yield: 89% (0.24 g, 0.88 mmol). 'H (400

MHz, CDCls) 6 7.40 (dd, J=8.3, 7.1 Hz, 1H), 7.15 (dd, J = 7.2, 0.7 Hz, 1H), 6.86 (dd, J = 8.3, 0.7 Hz, 1H), 4.96 (dd, J= 10.8, 4.8 Hz, 1H), 2.17 (ddd, J = 14.4, 10.8, 7.2 Hz, 1H), 2.04 (ddd, J= 14.4, 7.0, 4.8

Hz, 1H), 0.73 — 0.58 (m, 1H), 0.47 — 0.32 (m, 2H), 0.14 — 0.01 (m, 2H). 13C NMR (101 MHz, CDCl:) § 175.43, 169.56, 168.07, 145.63, 135.51, 132.45, 121.44, 113.18, 110.97, 52.03, 33.45, 8.25, 4.76, 3.64.

LC-MS (ESI) m/z calcd for C1aH14N204 [M + H]* 275.1, found 275.0. HPLC (method A): 8.81 min, purity 99%. (S)-2-{4-nitro-1,3-dioxoisoindolin-2-yl}-3-(thiophen-2-yl}propanoic acid: {3 ew ee Nes

Sg OH

NG, DD

The compound was synthesized according tc general procedure D using the following reagents: ethyl 4- nitro-1,3-dioxoisoindoline-2-carboxylate (0.77 g, 2.92 mmol), triethylamine (0.81 mL, 2 equiv.), and (S)-2-

P352801NL -35- amino-3-(thiophen-2-yl)propanoic acid (0.50 g, 1 equiv.). After stirring at room temperature under a Nz atmosphere for 2 hours the reaction mixture was subsequently irradiated in a MW synthesizer at 130 °C for 5 minutes. Yield: 78% {0.79 g, 2.28 mmol) as a light brown solid. *H NMR (400 MHz, CDCls) ò 10.51 (s, 1H), 8.14 (dd, J=8.1,1.0 Hz, 1H), 8.10 (dd, J = 7.5, 0.9 Hz, 1H), 7.92 (dd, J = 8.1, 7.5 Hz, 1H), 7.09 (dd, J=4.7, 1.7 Hz, 1H), 6.87 — 6.80 (m, 2H), 5.22 (dd, J = 11.5, 4.8 Hz, 1H), 3.97 — 3.71 {m, 2H). (S)-2-{(4-amino-1,3-dioxoisoindolin-2-yl}-3-(thiophen-2-yl)propanoic acid (32): 0 Im

L ge A Í

NH, OQ

The compound was synthesized according to general procedure C using the following conditions: (S)-2- (4-nitro-1,3-dioxoiscindolin-2-yl}-3-(thiophen-2-ylypropanoic acid (0.35 g, 1.0 mmol} and tin(ll} chloride dihydrate (0.92 g, 4 equiv.) were stirred in ethyl acetate (40 mL) at reflux for 6 hours. Crude product was purified by column chromatography eluting with a gradient of 2-10% MeOH in dichloromethane, followed by washing of the resulted solids with hot dichloromethane, affording the title compound as a yellow solid.

Yield: 46% (0.15 g, 0.47 mmol). *H NMR (400 MHz, DMSO-Ds) ò 13.36 (brs, 1H), 7.43 (dd, /=8.5,7.0

Hz, 1H), 7.26 (dd, J= 5.0, 1.4 Hz, 1H), 6.96 (dd, J = 14.9, 7.7 Hz, 2H), 6.90 — 6.82 (m, 2H), 6.51 (br s, 2H), 4.92 (dd, J= 9.7, 6.4 Hz, 1H), 3.70 — 3.55 (m, 2H). *C NMR (101 MHz, DMSO-Ds) 6 170.00, 168.65, 167.46, 146.73, 139.26, 135.51, 131.81, 126.94, 126.19, 124.93, 121.79, 110.98, 108.25, 52.57, 28.26.

LC-MS (ESI) m/z calcd for C1sH1zN204S [M + H]* 316.1, found 317.0. HPLC (method A): 8.11 min, purity 99%. tert-butyl 3-(4-nitro-1,3-dioxoisoindolin-2-yljazetidine-1-carboxylate: 0

Sg SN wd

No, UO \

The compound was synthesized according to general procedure D using the following reagents: ethyl 4- nitro-1,3-dioxoisoindoline-2-carboxylate (0.1 g, 0.38 mmol), triethylamine (0.11 mL, 2 equiv.), and tert- butyl 3-aminocazetidine-1-carboxylate hydrochloride (0.07 g. 1 equiv.}. Product purified by column purification using a gradient of 0-1% MeOH in dichloromethane. Yield: 82% (0.11 g, 0.31 mmol) as white solid. *H NMR (400 MHz, CDCl3) ò 8.18 — 8.15 (m, 1H), 8.15 - 8.13 (m, 1H), 7.96 (dd, J = 8.2, 7.4 Hz, 1H), 5.05 (tt, J = 8.5, 6.2 Hz, 1H), 4.51 (dd, J = 9.0, 6.3 Hz, 2H), 4.26 (t, J = 8.9 Hz, 2H), 1.48 (s, 9H).

P352801NL -36- tert-butyl 3-(4-amino-1,3-dioxoisoindolin-2-yl)azetidine-1-carboxylate (21):

D eee vay

Se yg es 0 —_— }

Ng, © >

The compound was synthesized according to general procedure C using the following conditions: tert butyl 3-(4-nitro-1,3-dioxoisoindolin-2-yl)azetidine-1-carboxylate (0.09 9, 0.27 mmol) and tin(ll) chloride dihydrate (0.37 g, 6 equiv.) were stirred in ethyl acetate (15 mL) at 40 °C for 3 hours. Product purified by flash column chromatography using a C18 cartridge, eluting with a gradient of 10-90% CHsCN in H20.

Yield: 40% (0.03 g, 0.11 mmol) as a bright yellow solid. 'H NMR (400 MHz, CDCl3) § 7.41 (dd, J = 8.2, 7.3 Hz, 1H), 7.12 (d, J=7.1 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 4.96 (tt, J = 8.6, 6.2 Hz, 1H), 4.50 (dd, J = 8.9,6.2 Hz, 2H), 4.20 (t, J = 8.7 Hz, 2H), 1.47 (5, 9H). 13C NMR (101 MHz, CDCls) ò 169.76, 168.10, 156.58, 145.65, 135.58, 132.40, 121.45, 112.97, 110.86, 79.92, 54.11, 38.30, 28.51. LC-MS (ESI) m/z calcd for CisHisN3O4 [M + H]* 317.1, found 218.0 [M — Boc + H]t. HPLC (method A): 9.92 min, purity 94%. 4-amino-2-{azetidin-3-yl}isoindoline-1,3-dione hydrochloride salt (34):

P

Ll N— NH -xXHCI

So ie

NH, © tert-butyl 3-(4-amino-1,3-dioxoisoindolin-2-yl)azetidine-1-carboxylate (0.10 g, 0.32 mmol) was stirred in a 4N HCI solution in MeOH (20 mL) for 2 hours. The resulting suspension was concentrated in vacuo, and co-evaporated with EtOAc (2x), yielding the title compound as an HCI salt. Yield: 88% (0.08 g, 0.28 mmol) as a bright yellow solid. 'H NMR (400 MHz, DMSO-Ds) ò 9.63 (brs, 1H}, 9.18 (brs, 1H), 7.45 (dd,

J=8.5,7.0Hz 1H), 7.02 (dd, J = 8.5, 0.7 Hz, 1H), 6.98 (dd, J = 7.0, 0.7 Hz, 1H), 6.36 (br s, 3H), 5.01 (p,

J =8.3 Hz, 1H), 4.59 — 4.46 (m, 2H), 4.25 — 4.08 (m, 2H). 13C NMR (101 MHz, DMSO-Ds) & 168.90, 167.72, 146.78, 135.42, 132.09, 121.68, 110.92, 108.65, 49.84, 39.99. LC-MS (ESI) m/z calcd for

C11H11N302 [M + H]* 217.1, found 218.0 [M + H]*. HPLC (method B): 1.19 min, purity 95%. tert-butyl (S)-3-{4-nitro-1,3-dioxoisoindolin-2-yl)piperidine-1-carboxylate: 3 ’ 0 x

The compound was synthesized according tc general procedure D using the following reagents: ethyl 4- nitro-1,3-dioxoisoindoline-2-carboxylate (0.66 g, 2.50 mmol), triethylamine (0.70 mL, 2 equiv.}, and fert-

P352801NL - 37 - butyl {S)-3-amincpiperidine-1-carboxylate (0.50 g, 1 equiv.). Yield: 89% (0.93 g, 2.48 mmol) as a colourless oil. '"H NMR (400 MHz, CDCls) ò 8.17 — 8.08 (m, 2H), 7.94 (dd, J= 8.2, 7.4 Hz, 1H), 4.88 — 4.68 (m, 1H), 4.22 (tt, J= 11.9, 4.3 Hz, 1H), 3.865 — 3.30 (m, 1H), 2.88 — 2.55 (m, 1H), 2.32 (tt, J = 12.5, 6.3 Hz, 1H), 1.93 - 1.78 (m, 2H), 1.68 — 1.56 (m, 1H), 1.46 (d, J = 1.4 Hz, SH). (S)-3-(4-nitro-1,3-dioxoisoindolin-2-yl)piperidin-1-ium chloride:

Se I NNO

No, ©

To a stirring solution of fert-Butyl (S)-3-(4-nitro-1,3-dicoxoisoindelin-2-yhpiperidine-1-carboxylate (0.90 g, 2.40 mmol) in methanol (10 mL) was carefully added 4N HCI in dioxane (10 mL) and subsequently stirred for 1 hour. The reaction mixture was then concentrated in vacuo, affording the title compound as a white solid. Yield: 75% (0.56 g, 1.81 mmol). *H NMR (400 MHz, DMSO-Ds) ò 9.57 (brs, 1H), 9.42 (brs, 1H), 8.29 (dd, J = 8.0, 0.9 Hz, 1H), 8.17 (dd, J = 7.5, 0.9 Hz, 1H), 8.09 — 8.04 (m, 1H), 4.47 (ddt, J= 16.2, 10.1, 3.7 Hz, 1H), 3.34 (br s, 2H), 3.25 (d, J=12.5 Hz, 1H), 2.83 (t, J = 12.9 Hz, 1H), 2.17 (gd, J= 13.1, 12.3, 4.3 Hz, 1H), 2.03 — 1.68 (m, 3H). (S)-2-{1-acetylpiperidin-3-yl})-4-nitroisoindoline-1,3-dione: 0

Q

To a stirring suspension of (S)-3-(4-nitro-1,3-dioxoiscindolin-2-yl)piperidin-1-ium chloride (0.16 g, 0.50 mmol) in DCM (12.5 mL) were added triethylamine (0.17 mL, 2.4 equiv.) and acetyl chloride (0.08 mL, 2.3 equiv.), and the solution was stirred for 1 hour. The reaction mixture was then concentrated onto silica and purified by column chromatography, eluting with 1% MeQH in dichloromethane as eluent affording the title compound as a colourless oil. Yield: 81% (0.15 g, 0.46 mmol). 'H NMR (400 MHz, CDCls) § 8.12 (dd, J= 8.5, 7.7 Hz, 2H), 8.01 — 7.87 (m, 1H), 4.73 - 4.61 (m, 1H), 4.28 — 4.15 (m, 1H), 3.90 — 3.72 (m, 1H), 3.33 (t, J=12.0 Hz, 0.5H), 3.11 (td, J=13.3, 2.6 Hz, 0.5H), 2.81 — 2.23 (m, 2H), 212d, J=11.2

Hz, 3H), 1.99 - 1.83 (m, 2H), 1.70 — 1.50 (m, 1H).

P352801NL -38- (S)-2-(1-acetylpiperidin-3-yl}-4-aminoisoindoline-1,3-dione (26):

Ng Nl 3

The compound was synthesized according to general procedure C using the following conditions: (S)-2- (1-acetylpiperidin-3-yl}-4-nitroisoindoline-1,3-dione (0.15 g, 0.46 mmol) and tin(ll) chloride dihydrate (0.41 g, 4 equiv.) were stirred in ethyl acetate (30 mL) at a reflux for 16 hours. The crude product was purified by column chromatography, eluting with EtOAc as eluent, affording the title compound as a yellow solid.

Yield: 93% (0.12 g, 0.43 mmol) as a bright yellow solid. 'H NMR (400 MHz, DMSO-Ds) ò 7.42 (ddd, J = 8.4,7.0,2.4 Hz, 1H), 7.00 - 6.91 (m, 2H), 6.47 (brs, 2H), 4.44 — 4.33 (m, 1H), 3.98 — 3.75 (m, 2H), 3.58 (dd, J=12.9,11.4 Hz, 0.5H), 3.08 (t, J= 11.9 Hz, 0.5H), 2.97 (td, J= 13.3, 2.6 Hz, 0.5H), 243 (id, J = 13.1,2.8 Hz, 0.5H), 2.33 - 2.16 (m, 1H), 2.00 (d, J = 22.8 Hz, 3H), 1.89 — 1.68 (m, 2H), 1.60 — 1.44 (m, 0.5H), 1.43 — 1.29 (m, 0.5H). 13C NMR (101 MHz, DMSO-Ds) & 169.29, 168.31, 168.22, 167.87, 146.51, 135.18, 132.16, 132.09, 121.40, 121.37, 110.68, 110.64, 108.69, 47.98, 47.56, 46.68, 45.86, 43.11, 40.91, 27.59, 27.49, 25.27, 24.44, 21.35, 21.32. "H NMR (500 MHz, DMSO-Ds, 100 °C) 6 7.43 (dd, J = 8.4,7.0Hz, 1H), 7.01 (d, J= 8.4 Hz, 1H), 6.97 (d, J = 7.0 Hz, 1H), 6.24 (s, 2H), 4.00 — 3.87 (m, 1H), 2.31 (qd, J=12.6, 4.2 Hz, 1H), 2.01 (s, 3H), 1.89 — 1.78 (m, 2H), 1.49 (br s, 1H). LC-MS (ESI) m/z calcd for

C15H17N3O3 [M + H]* 287.1, found 288.1. HPLC (method A): 7.99 min, purity 99%. tert-butyl (R}-3-(4-nitro-1,3-dioxoisoindolin-2-yl}piperidine-1-carboxylate: 3

Spey Set

Np, © 0

G J

The compound was synthesized according to general procedure D using the following reagents: ethyl 4- nitro-1,3-dioxoisoindoline-2-carboxylate (0.66 g, 2.50 mmol), triethylamine (0.70 mL, 2 equiv.), and fert- butyl (R)-3-aminopiperidine-1-carboxylate (0.50 g, 1 equiv.). Yield: 82% (0.77 g, 2.05 mmol) as a colourless oil. *H NMR (400 MHz, CDCls) 6 8.17 — 8.08 (m, 2H), 7.94 (dd, J= 8.2, 7.3 Hz, 1H), 4.78 (br s, 1H), 4.30 — 4.19 (m, 1H), 4.01 (brs, 1H), 3.49 (d, J= 14.9 Hz, 1H), 2.72 (brs, 1H), 2.33 (qd, /=12.8, 4.0

Hz, 1H), 1.85-1.78 (m, 2H), 1.61 (tt, J= 13.3, 3.9 Hz, 1H), 1.46 (d, J = 1.3 Hz, SH).

P352801NL -39- (R)-3-(4-nitro-1,3-dioxoisoindolin-2-yl)piperidin-1-ium chloride: &

Sot NH

NO, ©

To a stirring solution of fert-Butyl (R)-3-(4-nitro-1,3-dioxoisoindolin-2-yhpiperidine-1-carboxylate (0.77 g, 2.05 mmol) in EtOAc (10 mL) was carefully added 4N HCI in dioxane (10 mL) and subsequently stirred for 1 hour. The reaction mixture was then filtered and the solids washed with EtOAc, affording the title compound as a white solid. Yield: 75% (0.48 g, 1.54 mmol). "H NMR (400 MHz, DMSO-Ds) § 9.57 (br s, 1H), 9.40 (br s, 1H), 8.29 (dd, J = 8.0, 0.9 Hz, 1H), 8.17 (dd, J = 7.5, 0.9 Hz, 1H), 8.11 — 8.02 (m, 1H), 4.47 (tdd, J = 12.3, 8.2, 4.0 Hz, 1H), 3.42 — 3.33 (m, 3H), 3.29 - 3.21 (m, 1H), 2.83 (q, J = 11.9 Hz, 1H), 217(qd, J=13.1, 12.3, 4.3 Hz, 1H), 1.96 — 1.73 (m, 3H). (R)-2-(1-acetylpiperidin-3-yl}-4-nitroisoindoline-1,3-dione:

NN en 4 a

To a stirring suspension of (8}-3-(4-nitro-1,3-dioxoisoindolin-2-yhpiperidin-1-ium chloride (0.16 g, 0.50 mmol) in DCM (12.5 mL) were added triethylamine (0.17 mL, 2.4 equiv.) and acetyl chloride (0.08 mL, 2.3 equiv.), and the solution was stirred for 1 hour. The reaction mixture was then diluted with dichloromethane and subsequently washed with 0.5N HCI (aq.) and brine. The organics were dried over

MgSO. and solvents were removed in vacuo, affording the title compound as a colourless film. Yield: 99% (0.16 g, 0.50 mmol). 'H NMR (400 MHz, CDCl:) 6 8.12 (dd, J= 8.5, 7.7 Hz, 2H), 8.03 — 7.89 (m, 1H), 4.73 —4.81(m, 1H), 4.30 -4.13 (m, 1H), 3.89 — 3.74 (m, 1.5H), 3.33 (t, J= 12.0 Hz, 0.5H), 3.10 (id, J = 13.3, 2.5 Hz, 0.5H), 2.55 (td, J=13.2, 3.0 Hz, 0.5H), 2.50 - 2.31 (m, 1H), 2.12 (d, J= 10.9 Hz, 3H), 2.01 - 1.83 (m, 2H), 1.71 — 1.50 (m, 1H). {R)-2-(1-acetylpiperidin-3-yl}-4-aminoisoindoline-1,3-dione (28): ae € NN

G

The compound was synthesized according to general procedure C using the following conditions: (R)-2- (1-acetylpiperidin-3-yl)-4-nitroisoindoline-1,3-dione (0.16 g, 0.50 mmol) and tin{ll) chloride dihydrate (0.45 g, 4 equiv.) were stirred in ethyl acetate (30 mL) at a reflux for 16 hours. The crude product was purified

P352801NL -40- by column chromatography, eluting with EtOAc as eluent, affording the title compound as a yellow solid.

Yield: 95% (0.14 g, 0.47 mmol) as a bright yellow solid. 'H NMR (400 MHz, DMSO-Ds) 6 7.53 — 7.29 (m, 1H), 7.05 — 6.83 (m, 2H), 6.47 (brs, 2H), 4.44 — 4.33 (m, 1H), 3.99 — 3.77 (m, 2H}, 3.58 (dd, J= 12.9, 11.4 Hz, 0.5H), 3.08 (t, J = 11.8 Hz, 0.5H), 2.97 (td, J = 13.5, 2.6 Hz, 0.5H), 2.43 (id, J = 13.0, 2.8 Hz, 0.5H),2.26 (qd, J=12.7, 4.2 Hz, 1H), 2.00 (d, J = 22.8 Hz, 3H), 1.89 — 1.69 (m, 2H), 1.63 — 1.43 (m, 0.5H), 1.43 — 1.29 (m, 0.5H). *C NMR (101 MHz, DMSO-Ds) ò 169.29, 168.31, 168.22, 167.87, 146.51, 135.18, 132.16, 132.09, 121.40, 121.37, 110.68, 110.64, 108.75, 108.69, 47.98, 47.56, 46.68, 45.86, 43.11,40.91, 27.59, 27.49, 25.27, 24.44, 21.34, 21.31. 'H NMR (500 MHz, DMSO-Ds, 100 °C) ò 7.43 (dd,

J=84,7.0Hz, 1H), 7.01 (d, J= 8.4 Hz, 1H), 6.97 (d, J = 7.0 Hz, 1H), 6.24 (brs, 2H), 4.01 — 3.88 (m, 1H), 2.31 (gd, J = 12.3, 3.9 Hz, 1H), 2.01 (s, 3H), 1.80 — 1.78 (m, 2H), 1.50 (br s, 1H). LC-MS (ESI) m/z calcd for C1sH17N3O3 [M + H]* 287.1, found 288.1. HPLC (method A): 8.07 min, purity 99%. 3-{4-nitro-1,3-dioxoisoindolin-2-yl}azetidin-1-ium chloride:

Q

© TC NHC no, © tert-Butyl 3-(4-nitro-1,3-dioxoisoindolin-2-yhazetidine-1-carboxylate (0.29 g, 0.83 mmol) was stirred in. a freshly prepared solution of HCI in MeOH (2M, 25 mL) at room temperature for 2 hours. After completion (TLC) the solvents were removed in vacuo and the solids were washed with hot MeOH. Yield: 67% (0.16 g, 0.55 mmol) as light yellow crystals. 'H NMR (400 MHz, DMSO-Ds) 6 9.24 (br s, 2H), 8.33 (d, J=8.0

Hz, 1H), 8.22 (d, J= 7.4 Hz, 1H), 8.10 (t, J = 7.8 Hz, 1H), 5.16 — 5.04 (m, 1H), 4.51 (dd, J = 11.6, 7.3 Hz, 2H), 4.22 (dd, J= 11.5, 9.1 Hz, 2H). 2-(1-acetylazetidin-3-yl)-4-nitroisoindoline-1,3-dione: {x

No, ©

The compound was synthesized according to general procedure E using the following conditions: 3-(4- nitro-1,3-dioxoisoindolin-2-yl)azetidin-1-ium chloride (0.054 g, 0.198 mmol), triethylamine (0.05 mL, 2 equiv.), and acetic anhydride (0.02 mL, 1.3 equiv.) were stirred in dichloromethane (5 mL) at room temperature until completion. Yield: 92% (0.05 g, 0.18 mmol) as a colourless solid. *H NMR (400 MHz,

CDCl3) ò 8.20 — 8.13 (m, 2H), 7.99 (dd, J= 8.3, 7.3 Hz, 1H), 5.14 (it, J=8.7,6 2 Hz, 1H), 4.76 (dd, J = 8.6,6.1 Hz, 1H), 4.58 — 4.42 (m, 2H), 4.36 (t, J = 9.2 Hz, 1H), 1.95 (s, 3H).

P352801NL -41- 2-(1-acetylazetidin-3-yl}-4-aminoisoindoline-1,3-dione (5): a

Nn

Sg ~~ v 3

Ni, ©

The compound was synthesized according to general procedure C using the following conditions: 2-(1- acetylazetidin-3-yl)-4-nitroiscindoline-1,3-dione (0.05 g, 0.17 mmol) and tin{ll) chloride dihydrate (0.15 g, 4 equiv.) were stirred in ethyl acetate (20 mL) at 40 °C for 16 hours. Product purified by flash column chromatography using a C18 cartridge, eluting with a gradient of 0-50% CHsCN in H2O. Yield: 77% (0.03 g, 0.13 mmol) as a bright yellow solid. "H NMR (400 MHz, Acetone-Ds) ö 7.46 (dd, J= 8.4, 7.0 Hz, 1H), 7.07-6.99 (m, 2H), 5.04 (tt, J=8.7, 6.1 Hz, 1H), 4.71 (dd, J/= 8.5, 8.0 Hz, 1H), 4.52 — 4.41 (m, 2H), 4.19 (t, J=9.2 Hz, 1H), 1.84 (s, 3H). LC-MS (ESI) m/z calcd for C13H13N3O3 [M + H]* 259.1, found 260.0.

HPLC (method A): 6.54 min, purity 99%. 4-nitro-2-(1-propionylazetidin-3-yl)isoindoline-1,3-dione: £3

No, O

The compound was synthesized according to general procedure E using the following conditions: 3-(4- nitro-1,3-dioxoiscindolin-2-yl)azetidin-1-ium chloride (0.05 g, 0.18 mmol), triethylamine (0.05 mL, 2.2 equiv.), and proprionyl chloride (0.02 mL, 1.2 equiv.) were stirred in dichloromethane (2 mL) at room temperature until completion. Yield: 86% (0.05 g, 0.15 mmol) as a colourless solid. '"H NMR (400 MHz,

CDCl3) ò 8.20 — 8.12 (m, 2H), 7.99 (dd, J= 8.3, 7.3 Hz, 1H), 5.14 (it, J=8.6,6.1 Hz, 1H), 4.78 — 4.69 (m, 1H), 4.56 — 4.41 (m, 2H), 4.35 (t, J = 8.9 Hz, 1H), 2.28 — 2.08 (m, 2H), 1.16 (t, J = 7.5 Hz, 3H). 4-amino-2-(1-propionylazetidin-3-yl}isoindoline-1,3-dione (18): a

Seg i Se

Ni, ©

The compound was synthesized according to general procedure C using the following conditions: 4-nitro- 2-(1-propionylazetidin-3-yl}isoindoline-1,3-dione (0.05 g, 0.15 mmol) and tin(ll} chloride dihydrate (0.14 g, 4 equiv.) were stirred in ethyl acetate (5 mL) at 40 °C for 72 hours. Product purified by column chromatography, eluting with ethyl acetate. Yield: 88% (0.04 g, 0.15 mmol) as a bright yellow solid. 'H

NMR (400 MHz, CDCla) 6 7.42 (dd, J= 8.3, 7.1 Hz, 1H), 7.13 (dd, J=7.1, 0.7 Hz, 1H), 6.89 (dd, J = 8.4, 0.7 Hz, 1H), 5.04 (tt, J = 8.7, 6.2 Hz, 1H), 4.67-4.60 (m, 4H), 4.36 (t, J = 9.0 Hz, 2H), 2.19 (q, J = 7.6 Hz, 2H), 1.17 (t, J= 7.5 Hz, 3H). 13C NMR (101 MHz, CDCl:) & 174.27, 169.67, 188.04, 145.87, 135.65,

P352801NL -42- 132.22, 121.62, 112.91, 110.50, 38.30, 24.97, 9.01. LC-MS (ESI) m/z calcd for C14H1sN3O: [M + H]J* 273.1, found 274.0. HPLC (method A): 7.50 min, purity 89%. 2-(1-butyrylazetidin-3-yl)-4-nitroisoindoline-1,3-dione: 9 : No, 0 :

The compound was synthesized according to general procedure E using the following conditions: 3-(4- nitro-1,3-dioxoiscindolin-2-yl}azetidin-1-ium chloride (0.05 g, 0.18 mmol), triethylamine (0.05 mL, 2.2 equiv.), and butyryl chloride (0.02 mL, 1.2 equiv.) were stirred in dichloromethane (2 mL) at room temperature until completion. Yield: 84% (0.05 g, 0.15 mmol) as a colourless solid. 'H NMR (400 MHz,

CDCl:) 5 8.22 8.12 (m, 2H), 7.99 (dd, J= 8.3, 7.3 Hz, 1H), 5.14 (tt, J=8.7,6.2 Hz, 1H), 4.74 (dd, J = 8.6,6.2 Hz, 1H), 4.55 -4.41 (m, 2H), 4.35 (t, J= 9.4 Hz, 1H), 2.22 — 2.05 (m, 2H), 1.70 (p, J = 7.1 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H). 4-amin0-2-{1-butyrylazetidin-3-yl}isoindoline-1,3-dione (23): £3 ba Be oN UN “SL

Y 3 x 16 ni, © N

The compound was synthesized according to general procedure C using the following conditions: 2-(1- butyrylazetidin-3-yl)-4-nitroisoindoline-1,3-dione (0.05 g, 0.15 mmol) and tin(ll) chloride dihydrate (0.14 g, 4 equiv.) were stirred in ethyl acetate (5 mL) at 40 °C for 72 hours. Product purified by column chromatography, eluting with ethyl acetate. Yield: 97% (0.04 g, 0.15 mmol} as a bright yellow solid. 'H

NMR (400 MHz, CDCl) 6 7.41 (dd, y= 8.3, 7.1 Hz, 1H), 7.12 (dd, J= 7.1, 0.7 Hz, 1H), 6.89 (dd, J = 8.4, 0.7 Hz, 1H), 5.03 (tt, J= 8.7, 6.1 Hz, 1H), 4.89 (br ss, 2H), 4.62 (br s, 2H), 4.36 (t, J = 9.0 Hz, 2H), 2.14 (dd, J=84, 6.6 Hz, 2H), 1.69 (h, J = 7.4 Hz, 2H}, 0.97 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, methanol-

Da) & 175.67, 170.72, 169.83, 148.14, 136.37, 133.55, 122.55, 112.48, 110.73, 56.16, 53.93, 39.31, 34.27, 19.40, 14.08. LC-MS (ESI) m/z calcd for CisH17N3Os [M + H]* 287.1, found 288.0. HPLC (method

A): 8.22 min, purity 98%.

P352801NL -43- 2-(1-(3,3-dimethylbutanoyljazetidin-3-yl}-4-nitroisoindoline-1,3-dione:

D

Ol,

No, © >

The compound was synthesized according to general procedure E using the following conditions: 3-(4- nitro-1,3-dioxoiscindolin-2-yl)azetidin-1-ium chloride (0.10 g, 0.35 mmol), triethylamine (0.11 mL, 2.2 equiv.), and 3,3-dimethylbutyryl chloride (0.08 mL, 1.1 equiv.) were stirred in acetonitrile (2 mL) at room temperature until completion. Yield: 93% (0.11 g, 0.33 mmol) as a colourless solid. "H NMR (400 MHz,

CDCl:) © 8.20 — 8.12 (m, 2H), 7.99 (dd, J=8.2, 7.3 Hz, 1H), 5.11 (tt, J= 8.7, 86.2 Hz, 1H), 4.79 - 4.70 (m, 1H), 4.58 — 4.41 (m, 2H), 4.41 — 4.31 (m, 1H}, 2.05 (d, J = 5.0 Hz, 2H), 1.08 (s, SH). 4-amino-2-(1-(3,3-dimethylbutanoyljazetidin-3-yl)isoindoline-1,3-dione (24):

U ee AB

Nit, ò >

The compound was synthesized according to general procedure C using the following conditions: 2-(1- (3.3-dimethylbutanoyl)azetidin-3-yl)-4-nitroisoindoline-1,3-dione (0.05 g, 0.15 mmol) and tin(ll) chloride dihydrate (0.13 g, 4 equiv.) were stirred in ethyl acetate (10 mL) at 40 °C for 72 hours. Product purified by column chromatography, eluting with a gradient of 0-5% MeOH in dichloromethane. Yield: 98% (0.05 g, 0.14 mmol) as a bright yellow solid. 'H NMR (400 MHz, CDCls) 6 7.44 — 7.35 (m, 1H), 7.10 (dd, J = 7.1, 0.7 Hz, 1H), 6.88 (dd, J = 8.4, 0.7 Hz, 1H), 5.00 (tt, J = 8.8, 6.2 Hz, 1H), 4.87 — 4.54 (m, 4H), 4.46 — 4.24 (m, 2H), 2.04 (s, 2H), 1.06 (s, 9H). 3C NMR (101 MHz, CDCl3) 6 172.36, 169.60, 188.07, 145.84, 135.62, 132.24, 121.60, 112.89, 110.53, 44.49, 37.83, 31.62, 29.97. LC-MS (ESI) m/z calcd for C17H21N3O3 [M +

H]* 315.2, found 316.1. HPLC (method A): 9.25 min, purity 96%. 2-(1-(cyclopropanecarbonyljazetidin-3-yl}-4-nitroiscindoline-1,3-dione: 3 erk EN Ô

NO, © u

The compound was synthesized according tc general procedure E using the following conditions: 3-(4- nitro-1,3-dioxoisoindolin-2-yl)azetidin-1-ium chloride (0.07 g, 0.25 mmol), triethylamine (0.08 mL, 2.2 equiv.), and cyclopropanecarbonyl chloride (0.03 g, 1.2 equiv.) were stirred in acetonitrile (3 mL) at room temperature until completion. Yield: 78% (0.06 g, 0.19 mmol) as a colourless solid. '"H NMR (400 MHz,

CDCl3) 6 8.20 — 8.13 (m, 2H), 7.98 (dd, J=8.2, 7.4 Hz, 1H), 5.17 (it, J=8.7,6.2 Hz, 1H), 4.88 (dd, J =

P352801NL -44- 8.5,6.2 Hz, 1H), 4.60 (t, J = 8.6 Hz, 1H), 4.53 (dd, J= 10.0, 6.3 Hz, 1H), 4.37 (t, J = 9.4 Hz, 1H), 1.44 (it,

J= 7.98, 4.8 Hz, 1H), 1.06 — 0.96 (m, 2H), 0.86 — 0.74 (m, 2H). 4-amino-2-(1-(cyclopropanecarbonyl)azetidin-3-yl}isoindoline-1,3-dione (19): © < Ni, © =

The compound was synthesized according to general procedure C using the following conditions: 2-(1- (cyclopropanecarbonyl)}azetidin-3-yl)-4-nitroisoindoline-1,3-dicne (0.06 g, 0.198 mmol) and tin(ll) chloride dihydrate (0.18 g, 4 equiv.) were stirred in ethyl acetate (10 mL) at 40 °C for 48 hours. Product purified by column chromatography, eluting with 1.5% MeOH in dichloromethane. Yield: 73% (0.04 g, 0.14 mmol) as a bright yellow solid. "H NMR (400 MHz, CDCl3) § 7.42 (dd, J=8.3, 7.1 Hz, 1H), 7.13 (dd, J= 7.1, 0.7 Hz, 1H}, 6.88 (dd, J = 8.3, 0.7 Hz, 1H), 5.08 (tt, J= 8.7, 6.2 Hz, 1H), 4.76-4.70 (m, 4H), 4.44 (t, J = 9.0 Hz, 2H), 1.44 (tt, J=7.9, 4.6 Hz, 1H), 1.01 (dt, J = 4.6, 3.3 Hz, 2H), 0.79 (dt, J = 8.0, 3.4 Hz, 2H). 13C NMR (101 MHz, CDCl3) 6 174.11, 169.75, 168.01, 145.80, 135.69, 132.37, 121.58, 113.02, 110.63, 53.92, 38.52, 10.45, 7.64. LC-MS (ESI) m/z calcd for C1sH1sN3Oa [M + HJ" 285.1, found 286.0. HPLC (method

A): 7.70 min, purity 98%. 2-(1-acryloylazetidin-3-yl}-4-nitroisoindoline-1,3-dione: 9

No, ©

The compound was synthesized according to general procedure E using the following conditions: 3-(4- nitro-1,3-dioxoisoindolin-2-yl}azetidin-1-ium chloride (0.07 g, 0.25 mmol), triethylamine (0.08 mL, 2.2 equiv.), and acryloyl chloride (0.02 mL, 1.2 equiv.) were stirred in acetonitrile (3 mL) at room temperature until completion. Yield: 46% (0.03 g, 0.11 mmol) as a colourless solid. 'H NMR (400 MHz, CDCls) § 8.20 -8.12 (m, 2H), 7.99 (dd, J = 8.3, 7.3 Hz, 1H), 6.37 (dd, J= 17.0, 1.9 Hz, 1H), 6.22 (dd, J= 17.0, 10.3 Hz, 1H), 5.72 (dd, J = 10.3, 1.9 Hz, 1H), 5.18 (tt, J= 8.7, 6.2 Hz, 1H), 4.87 — 4.79 (m, 1H), 4.65 — 4.52 (m, 2H), 4.44 (t, J=9.1 Hz, 1H).

P352801NL -45- 2-(1-acryloylazetidin-3-yl}-4-aminoisoindoline-1,3-dione (20): 0 ni, 0

The compound was synthesized according to general procedure C using the following conditions: 2-(1- acryloylazetidin-3-yl)-4-nitroiscindoline-1,3-dione (0.03 g, 0.11 mmol) and tin(ll} chloride dihydrate (0.10 g, 4 equiv.) were stirred in ethyl acetate (3 mL) at 40 °C for 48 hours. Product purified by column chromatography, eluting with 1.5% MeOH in dichloromethane. Yield: 82% (0.03 g, 0.09 mmol) as a bright yellow solid. "TH NMR (400 MHz, CDCl:) 6 7.41 (dd, J=8.4, 7.1 Hz, 1H), 7.12 (d, J=7.0 Hz, 1H), 6.92 — 6.85 (m, 1H), 6.37 (dd, J = 17.0, 1.9 Hz, 1H), 6.22 (dd, J = 17.0, 10.3 Hz, 1H), 5.70 (dd, J = 10.3, 1.9 Hz, 1H), 5.08 (it, J = 8.7, 6.2 Hz, 1H), 4.71 (brs, 2H), 4.53-4.4.45 (m, 4H). '3C NMR (101 MHz, CDCl) & 169.57, 168.16, 165.75, 145.82, 135.72, 132.24, 127.95, 125.95, 121.63, 113.04, 38.53. LC-MS (ESI) m/z calcd for C14H13NaO3 [M + H]* 271.1, found 272.0. HPLC (method A}: 7.31 min, purity 99%. 2-(1-benzoylazetidin-3-yl)-4-nitroiscindoline-1,3-dione:

Q

Eek ©

Y 1 STR

NO © SD

The compound was synthesized according to general procedure E using the following conditions: 3-(4- nitro-1,3-dioxoisoindolin-2-yhazetidin-1-ium chloride (0.05 g, 0.18 mmol), triethylamine (0.05 mL, 2.2 equiv.), and benzoyl chloride (0.03 mL, 1.2 equiv.) were stirred in dichloromethane (2 mL) at room temperature until completion. Yield: 99% (0.06 g, 0.17 mmol) as a colourless solid. TH NMR (400 MHz,

CDCl3) ò 8.17 — 8.10 (m, 2H), 7.96 (dd, J = 8.3, 7.3 Hz, 1H), 7.72 — 7.63 (m, 2H), 7.52 — 7.36 (m, 3H), 5.18 (it, J=8.7,6.1 Hz, 1H), 4.91 — 4.83 (m, 1H), 4.74 — 4.65 (m, 1H), 4.62 — 4.53 (m, 2H). 4-amino-2-(1-benzoylazetidin-3-yl}isoindoline-1,3-dione (22): bo 5 mel , 3 3 ST ni, © Le

The compound was synthesized according to general procedure C using the following conditions: 2-(1- benzoylazetidin-3-yl)-4-nitroiscindoline-1,3-dione (6.06 g, 0.18 mmol) and tin(ll) chloride dihydrate (0.16 g, 4 equiv.) were stirred in ethyl acetate (5 mL) at 40 °C for 48 hours. Product purified by column chromatography, eluting with a gradient of 50-75% ethyl acetate in pentane. Yield: 84% (0.05 g, 0.15 mmol) as a bright yellow solid. "TH NMR (400 MHz, DMSO-Ds) ò 7.69 — 7.62 (m, 2H), 7.58 — 7.39 (m, 4H), 6.97 (dd, J=7.7,6.2 Hz, 2H), 6.51 (brs, 2H), 5.00 (tt, J= 8.6, 5.8 Hz, 1H), 4.72 — 4.57 (m, 2H), 4.50 (dd,

P352801NL -46-

J= 10.2, 5.9 Hz, 1H}, 4.34 (t, J = 9.5 Hz, 1H). LC-MS (ESD m/z calcd for C1sH15N3O3 [M + HJ* 321.1, found 322.0. HPLC (method A): 8.91 min, purity 99%. tert-butyl (1-acetylazetidin-3-yljcarbamate:

HNN <£

XD

SA

3-N-Boc-amino-azetidine hydrochloride (2.0 g, 9.58 mmol) was suspended in dichloromethane (40 mL) and the suspension was cooled to 0 °C using an icebath. To this was added triethylamine (4.0 mL, 3 equiv.) followed by a dropwise addition of acetyl chloride (1.02 mL, 1.5 equiv. in 10 mL dichloromethane).

Upon completion of the addition (TLC) the reaction mixture was allowed to warm to room temperature at which it was then maintained for 1.5 hours. HzO was added, layers separated and the organics were washed with brine and dried over MgSOa. The crude product was purified by column chromatography eluting first with 50/50 EtOAc in pentane and then 100% EtOAc. Yield: 83% (1.71 g, 8.0 mmol) as a colourless oil. "TH NMR (400 MHz, CDClas) 6 6.07 (brs, 1H), 4.43 — 4.18 (m, 2H), 4.16 — 4.03 (m, 1H), 3.97 — 3.82 (m, 1H), 3.78 — 3.85 (m, 1H), 1.71 (s, 3H), 1.30 (s, 9H). 1-acetylazetidin-3-aminium chloride:

CIN nl tert-Butyl (1-acetylazetidin-3-yl)carbamate (1.71 g, 8.0 mmol} was taken up in 40 mL DCM to which was carefully added HCI (4.0 M in dioxane, 27 mL). This solution was stirred at room temperature for 1 hour after which diethylether (40 mL) was added, resulting in the precipitation of the product. The flask was left overnight to complete the precipitation after which the solids were filtered and washed with ether. Yield: 99% (1.21 g, 8.0 mmol), as a off-white solid. "H NMR (400 MHz, DMSO-Ds) & 8.81 (s, 3H), 4.37 - 4.28 (m, 1H), 4.12 (dd, J = 9.6, 4.5 Hz, 1H}, 4.06 — 3.88 (m, 2H}, 3.84 (dd, J= 9.8, 4.4 Hz, 1H), 1.75 (s, 3H). 2-(1-acetylazetidin-3-yl}-5-nitroisoindoline-1,3-dione: 3

NX on &

JE ON

{3

Step one: To a stirring suspension of 5-nitroisobenzofuran-1,3-dione (0.13 g, 0.66 mmol), and 1- acetylazetidin-3-aminium chloride (0.15 g, 1.5 equiv.) in THF (5 mL) was added triethylamine (0.11 mL, 1.2 equiv.). The addition of triethylamine induced instant precipitation. The suspension was stirred at room temperature for 4 hours. The reaction mixture was filtered, solids washed with THF and subsequently dried in vacuo. Step two: The solids were suspended in acetic anhydride (5 mL) to which was then added sodium acetate trihydrate (0.08 g, 1 equiv.). The reaction mixture was heated to 80 °C

P352801NL - 47 - and maintained for 40 minutes, after which TLC indicated full conversion. The reaction mixture was poured onto ice, extracted from EtOAc (3x 50 mL). The combined organics were dried over MgSO. and solvents were subsequently removed in vacuo. Yield: 41% (0.07 g, 0.25 mmol} over two steps, as a light brown solid. 'H NMR (400 MHz, CDCls) § 8.69 — 8.59 (m, 2H), 8.07 (dd, J= 8.1, 0.7 Hz, 1H), 5.11 (it, J = 87,81Hz, 1H), 4.72 (t, J=7.6 Hz, 1H), 4.80 — 4.39 (m, 2H), 4.33 (t, J= 9.8 Hz, 1H), 1.92 (s, 3H). 2-(1-acetylazetidin-3-yl)-56-aminoiscindoline-1,3-dione (6):

G

U

The compound was synthesized according to general procedure C using the following conditions: 2-(1- acetylazetidin-3-yl}-5-nitroisoindoline-1,3-dione (0.07 g, 0.25 mmol) and tin(ll} chloride dihydrate (0.22 g, 4 equiv.) were stirred in ethyl acetate (12 mL) at 40 °C for 24 hours. Product purified by column chromatography, eluting with 2% MeOH in chloroform. Yield: 62% (0.04 g, 0.16 mmol} as a bright yellow solid. "H NMR (400 MHz, DMSQ-Ds) 6 7.50 (d, J = 8.2 Hz, 1H), 6.92 (d, J = 2.0 Hz, 1H), 6.80 (dd, J= 8.2, 2.1 Hz, 1H), 6.52 (br s, 2H), 4.90 (tt, J = 8.7, 6.0 Hz, 1H), 4.52 (dd, J = 8.6, 6.0 Hz, 1H), 4.37 (td, J= 8.7, 0.9Hz, 1H), 4.26 (dd, J =9.7, 6.1 Hz, 1H), 4.09 (t, J = 9.2 Hz, 1H), 1.79 (s, 3H). 13C NMR (101 MHz,

DMSO-Ds) ò 169.47, 168.03, 167.55, 155.13, 134.34, 125.04, 116.81, 116.37, 106.92, 54.71, 52.39, 37.66, 18.87. LC-MS (ESI) mz calcd for C13H13N3O3 [M + H]* 259.1, found 260.1, HPLC (method B): 9.13 min, purity 88%. 5-(1-acetylazetidin-3-yl)-4H-thieno[2,3-c]pyrrole-4,6(5H)-dione (9): 0

Ng | EL < iN A, {3

Step one: To a stirring suspension of thieno[2,3-c]furan-4,6-dione (0.09 g, 0.9 equiv.), and 1- acetylazetidin-3-aminium chloride (0.1 g, 0.66 mmol) in THF (5 mL) was added triethylamine (0.18 mL, 2 equiv.). The addition of triethylamine induced instant precipitation. The suspension was stirred overnight at room temperature after which mixture was concentrated in vacuo. Step two: The crude was suspended in acetic anhydride (5 mL) to which was then added sodium acetate trihydrate (0.10 g, 1 equiv.). The reaction mixture was heated to 80 °C and maintained for 1 hour, after which LC-MS indicated full conversion. The reaction mixture was concentrated in vacuo and residual acetic anhydride was removed by co-evaporation with toluene (3x). The crude product was purified by flash column chromatography, eluting with a gradient of 0-5% MeOH in chloroform. Yield: 42% (0.07 g, 0.28 mmol) over two steps, as a white solid. "TH NMR (400 MHz, CDCls) 6 7.86 (d, J=4.7 Hz, 1H), 7.35 (d, J= 4.7 Hz, 1H), 5.03 (tt, J = 8.7,6.3 Hz, 1H), 4.73 (dd, J = 8.6, 6.1 Hz, 1H), 4.51 (dd, J= 10.0, 6.4 Hz, 1H), 4.43 (t, J = 8.6, 1H), 4.33 (t, J=9.4 Hz, 1H), 1.96 (s, 3H). 13C NMR (101 MHz, CDCls) ô 170.60, 163.16, 162.03, 144.38, 140.61,

P352801NL -48 - 138.50, 121.39, 55.02, 53.04, 38.74, 19.04. LC-MS (ESI) nvz calcd for C11H1oN203S [M + H]* 250.0, found 251.0. HPLC (method B}: 9.31 min, purity 99%.

N-(2-(1-acetylazetidin-3-yl}-1,3-dioxoisoindolin-4-yljacetamide (7}:

JEN Cy, BS ol o : ò

To a stirring solution of 2-(1-acetylazetidin-3-y[}-4-nitroisoindoline-1,3-dione (0.1 g, 0.33 mmol) in EtOAc (10 mL) was then added tin(ll} chloride dihydrate (0.3 g, 4 equiv.). The solution was then maintained at reflux for 1.5 hours, after which LC/MS showed full conversion to the aniline. The reaction mixture was cooled to room temperature, and acetic anhydride {0.3 mL, 10 equiv.) was carefully added. The mixture was then heated and maintained at reflux for another 1.5 hours, after which the reaction mixture was cooled to room temperature, diluted with EtOAc and quenched by the addition of a saturated aqueuous solution of NaHCO:3. Layers were separated and the aqueous was extracted once from EtOAc. The combined organics were washed with brine and dried over MgSOa. The crude product was purified by column chromatography, eluting with a gradient of 0-3% MeOH in dichloromethane affording the title compound as a light yellow solid. Yield: 84% (0.07 g, 0.22 mmol). !H NMR (400 MHz, CDCl) ò 9.43 (s, 1H), 8.75 (d, J=8.3 Hz, 1H), 7.66 (dd, J=8.4, 7.5 Hz, 1H), 7.48 (dd, J = 7.3, 0.7 Hz, 1H), 5.03 (it, J = 8.7,6.2Hz, 1H), 4.72 — 4.64 (m, 1H), 4.48 (dd, J=9.7, 6.4 Hz, 1H), 4.41 (t, J=8.6 Hz, 1H), 4.31 (t, J= 9.4 Hz, 1H), 2.25 (s, 3H), 1.92 (s, 3H). 13C NMR (101 MHz, CDCl3) § 170.61, 169.69, 189.33, 167.02, 137.75, 136.43, 130.97, 125.17, 118.30, 115.08, 54.77, 52.78, 38.36, 25.03, 19.06. LC-MS (ESI) m/z calcd for CisH1sN3Oa4 [M + H]* 301.1, found 302.1. HPLC (method B): 9.97 min, purity 95.1%. 5-chloro-2-methyl-3-nitrobenzoic acid: 9 on

Pg

NU, 5-chloro-2-methylbenzoic acid (5.0 g, 29.3 mmol) was dissolved portionwise in sulfuric acid (34 mL) at -10 °C. After dissolving the material a 2:1 mixture (8.5 mL) of sulfuric acid and nitric acid (65%) was added dropwise, while maintaining a temperature between -10 °C and 0 °C. After addition the reaction mixture was stirred for a further couple of minutes before it was poured onto ice and subsequently extracted from

EtOAc. The organics were dried over MgSO:4, solvents were evaporated in vacuo and the crude product was recrystallized from hot methanol affording the title compound as a light yellow solid. Yield: 37% (2.30 9, 10.7 mmol). !H NMR (400 MHz, CDCI3} 6 11.44 (s, 1H), 8.19 (d, J=2.3 Hz, 1H), 7.90 (d, J = 2.4 Hz, 1H), 2.67 (s, 3H).

P352801NL -49- 5-chloro-3-nitrophthalic acid: 0 rr en

Sy OH

NO, © 5-chloro-2-methyl-3-nitrobenzoic acid (2.16 g, 10 mmol) was suspended in H20 (40 mL) to which was then added sodium hydroxide (0.8 g, 2 equiv.}. The solution was then stirred at 85 °C and to it was added portionwise over 3 hours potassium permanganate (3.16 g, 2 equiv.). After completion of the addition the reaction mixture was maintained at 85 °C one hour. The reaction mixture was cooled tc room temperature, filtered and the solids washed with hot H20 (2x 30 mL). The filtrate was acidified to pH = 1-2 using 2N HCI (aq.), EtOAc added, and the crude product was extracted (3x 100 mL). The combined organics were dried over MgSQ4 and concentrated in vacuo, after which the product was purified by column chromatography eluting first with 99:1:1 DCM/MeOH/AcOH and then 48:48:4 DCM/MeOH/AcOH, affording the title phthalic acid as a yellow solid. Yield: 46% (1.13 g, 4.60 mmol). 2-(1-acetylazetidin-3-yl}-6-chloro-4-nitroisoindoline-1,3-dione: 9 id eed Ea 3 fm Ng, ©

Step one: 5-chloro-3-nitrophthalic acid (0.15 g, 0.61 mmol) and sodium acetate trihydrate (0.08 g, 1 equiv.) were taken up in acetic anhydride (10 mL) and stirred at 80 °C for 1 hour. The solvents were subsequently removed in vacuo and any residual acetic anhydride was removed by co-evaporation with toluene (3x). The crude phthalic anhydride was suspended in THF (4 mL) to which were then added 1- acetylazetidin-3-aminium chloride (0.1 g, 1.1 equiv.), and triethylamine (0.17 mL, 2 equiv.). The resulting suspension was stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo, and carried further without any purification. Step two: The crude 2-((1-acetylazetidin-3-yl)carbamoyl)-4- chloro-6-nitrobenzoic acid was taken up in acetic anhydride (5 mL) and to it was added sodium acetate trihydrate (0.08 g, 1 equiv.). This solution was heated to 80 °C at which it was maintained for 1 hour.

LC/MS showed full conversion of the benzoic acid to the phthalimide, so the reaction mixture was concentrated in vacuo, co-evaporated with toluene (2x) and finally purified by flash column chromatography, eluting using a gradient of 0-10% MeOH in DCM affording the title compound as a light yellow oil. Yield: 27% over 2 steps (0.05 g, 0.15 mmol}. *H NMR (400 MHz, CDCls) 6 8.14 (d, J = 1.7 Hz, 1H), 8.12 (d, J=1.7 Hz, 1H), 5.13 (it, J = 8.7, 6.1 Hz, 1H), 4.74 (dd, J = 8.8, 6.2 Hz, 1H}, 4.55 - 4.41 (m, 2H), 4.36 (dd, J= 10.2, 8.7 Hz, 1H), 1.95 (s, 3H).

P352801NL -50- 2-(1-acetylazetidin-3-yl}-4-amino-6-chloreisoindoline-1,3-dione (10): 2

Ch on PE

Nh, 0

The compound was synthesized according to general procedure C using the following conditions: 2-(1- acetylazetidin-3-yl)-6-chloro-4-nitroisoindoeline-1,3-dione (54 mg, 0.17 mmol) was dissolved in ethyl acetate (10 mL}, to which was then added tin(ll} chloride dihydrate (0.35 g, 9 equiv.). The resulting solution was then stirred at a reflux for 8 hours. The title compound was purified by flash chomatography eluting with a gradient of 0-5% MeOH in chloroform, yielded a bright yellow solid. Yield: 92% (0.05 g, 0.15 mmol). 'H NMR (400 MHz, CDCI:) & 7.07 (s, 1H), 6.90 (s, 1H), 5.50 (br s, 2H), 5.07 — 4.95 (m, 1H), 4.69 t, J=7.4 Hz 1H), 4.57 —4.45 (m, 1H), 4.40 (t, J= 8.6 Hz, 1H), 4.30 (t, J = 9.4 Hz, 1H), 1.93 (s, 3H). °C

NMR (101 MHz, CDCls} ò 170.80, 168.94, 166.94, 146.39, 141.98, 133.71, 120.54, 113.50, 109.20, 54.94, 52.91, 38.24, 19.14. LC-MS (ESI) m/z calcd for C1aH12CIN3Oa [M + H]* 293.1, found 294.0. HPLC (method A): 7.88 min, purity 95%. 3-chloro-6-nitrophthalic acid: eo 0

Nog OH

Nix OQ

To a stirring solution of nitric acid in conc. sulfuric acid (1:2, 9 mL) at 0 °C was added portionwise 3- chlorophthalic anydride (2.5 g, 13.7 mmol). After addition the suspension was allowed to warm to room temperature at which it was then maintained for 3 hours. The reaction mixture was then cooled back down to 0 °C after which ice was added to the mixture. The slurry was filtered and the filtrate was extracted from EtOAc (3x). The combined organics were dried over MgSO4 and the solvents were removed in vacuo, and the crude product was further purified by column chromatography, eluting with 5%

MeOH in dichloromethane affording the title phthalic acid as an off-white solid. Yield: 56% (1.87 g, 7.61 mmol). 'H NMR (400 MHz, DMSO-Ds) 6 14.39 (brs, 2H), 8.16 (d, J = 8.7 Hz, 1H), 7.93 (d, J = 8.7 Hz, 1H). 2-(1-acetylazetidin-3-yl}-4-chloro-7-nitroisoindoline-1,3-dione:

Hog

NO, ©

Step one: 3-chloro-6-nitrophthalic acid (0.2 g, 0.81 mmol) and sodium acetate trihydrate (0.11 g, 1 equiv.) were taken up in acetic anhydride (5 mL} and stirred at 80 °C for 1 hour. The resulting suspension was

P352801NL -51- concentrated in vacuo and any residual acetic anhydride was removed by co-evaporation with toluene (3x). The crude phthalic anhydride was then taken up in THF/H20 (1:1, 25 mL), and to it were added 1- acetylazetidin-3-aminium chloride (0.13 g, 1.06 equiv.) and triethylamine (0.23 mL, 2 equiv.). The solution was stirred at room temperature for 2 hours after which the solvents were removed in vacuo, and residual water was removed by co-evaporation with toluene. Step two: The crude 2-((1-acetylazetidin-3- yhycarbamoyl)-3-chlore-6-nitrobenzoic acid was suspended in acetic anhydride (5 mL) and to it was added sodium acetate trihydrate (0.11 g, 1 equiv.). The resulting solution was stirred at 80 °C for approx. 45 minutes. LC/MS showed full conversion to the desired phthalimide, so the reaction mixture was cooled to room temperature, after which it was diluted with water and the residual acetic anhydride was carefully quenched by tritrating with sat. aq. NaHCOs. Ethyl acetate was then added and the product was extracted (3%). The combined organics were dried over MgSOa and concentrated in vacuo. The title compound was finally purified by column chromatography, eluting with 2% MeOH in DCM affording a white solid. Yield: 19% (0.05 g, 0.15 mmol). 'H NMR (400 MHz, CDCl) ò 8.05 (d, J = 8.6 Hz, 1H), 7.87 (d, J = 8.7 Hz, 1H), 512 (qt, J=8.9, 6.2 Hz, 1H), 4.79 — 4.71 (m, 1H), 4.57 — 4.30 (m, 3H), 1.95 (d, J = 1.6 Hz, 3H). 2-(1-acetylazetidin-3-yl)-4-amino-7-chloroisoindoline-1,3-dione (11): “oo gg AS N ni ©

The compound was synthesized according to general procedure C using the following conditions: 2-(1- acetylazetidin-3-yl)-4-chloro-7-nitroisoindoline-1,3-dione (0.05 g, 0.15 mmol) and tin(il) chloride dihydrate (0.20 g, 6 equiv.) taken up in ethyl acetate (10 mL) and stirred at refluxing temperature for 6 hours, after which LC/MS and TLC showed full conversion to the desired aniline. The crude product was purified by column chromatography eluting with 1.5% MeOH in chloroform, affording the title compound as a bright yellow solid. Yield: 75% (0.03 g, 6.11 mmol). "H NMR (400 MHz, CDCls) 6 7.34 (d, J = 8.8 Hz, 1H), 6.86 (d, J=8.8 Hz, 1H), 5.44 (s, 2H), 5.06 (tt, J = 8.7, 6.2 Hz, 1H), 4.74 (dd, J= 8.4, 6.2 Hz, 1H), 4.53 (dd, J = 10.0,6.3 Hz, 1H), 4.41 (id, J = 8.6, 1.0 Hz, 1H), 4.32 (t, J = 9.4 Hz, 1H), 1.94 (s, 3H). 13C NMR (101 MHz,

CDCls) 6 214.52, 170.71, 165.64, 144.71, 137.44, 127.10, 123.08, 119.36, 54.81, 52.89, 38.22, 19.15.

LC-MS (ESI) m/z calcd for C1aH12CIN3O3 [M + H]* 283.1, found 294.0. HPLC (method A): 7.53 min, purity 98.6%.

P352801NL -52- 4-methyl-3-nitrophthalic acid:

NE

RO; 0

To a stirring solution of nitric acid in conc. sulfuric acid (1:2, 8 mL) was added portion wise at 0 °C 4- methylphthalic acid (2.51 g, 13.9 mmol). The suspension was stirred at 0 °C for 15 minutes, and then allowed to reach room temperature over a period of 2 hours. The reaction mixture was then poured onto ice, solides filtered off and washed with cold water. The combined filtrates were extracted with ethyl acetate (2x), and the combined organics were dried over MgSO.. Solvents were removed in vacuo, and the obtained solids were combined with the precipitated solids, affording a 60/40 mixture of 4-methyl-5- nitrophthalic acid and 4-methyl-3-nitrophthalic acid, respectively, as white solids. Yield: 70% (2.20 g, 10.0 mmol). 2-(1-acetylazetidin-3-yl}-5-methyl-4-nitroisoindoline-1,3-dione: 0 io, ©

Step one: The mixture of 4-methyl-5-nitrophthalic acid and 4-methyl-3-nitrophthalic acid (2.2 g, 10.0 mmol) was taken up in acetic anhydride (16 mL) and stirred at 90 °C for 4 hours. The obtained clear solution was then cooled to room temperature and concentrated in vacuo. Residual amounts of acetic anhydride were removed by co-evaporation with toluene (3x). The intermediate anhydride (0.3 g, 1.45 mmol) was then taken up in THF (5 mL), and to it were added 1-acetylazetidin-3-aminium chloride (0.22 g. 1 equiv.), triethylamine (1 mL, 5 equiv.), and H20 (5 mL). The resulted solution was stirred at room temperature for 1 hour, after which the THF was removed in vacuo, and the reaction mixture was then acidified to pH = 1 by titration with HCI (aq, 2.0 M). The product was extracted with EtOAc (2x), and the combined organics dried over MgSO4 and solvents evaporated to dryness in vacuo. Step two: the intermediate was taken up in acetic anydride (10 mL), and stirred at 80 °C for 1 hour, after which LC/MS showed full conversion to the desired phthalimide. The reaction mixture was concentrated onto silica and purified by column chromatography, eluting with a gradient of 1-2% MeOH in DCM. The title compound was isolated as a 1:1 mixture between 2-(1-acetylazetidin-3-yl)-5-methyl-4-nitroisoindoline-1,3-dione and 2-(1-acetylazetidin-3-yl)-5-methyl-8-nitroisoindoline-1,3-dione, as a colourless oil. Yield: 25% (0.11 g, 0.36 mmol). 'H NMR (400 MHz, CDCls) 6 8.33 (s, 1H), 7.92 (d, J=7.7 Hz, 1H), 7.89 (s, 1H), 7.76 (dd, J = 7.7, 0.5 Hz, 1H), 5.31 (s, 1H), 5.11 (dit, J = 15.0, 8.7, 6.2 Hz, 2H), 4.75 (dt, J = 8.7, 6.0 Hz, 2H), 4.63 — 4.30 (m, 6H), 3.95-3.87 (m, 1H), 2.74 (s, 3H), 2.49 (s, 3H), 1.97 (d, J = 8.5 Hz, 6H).

P352801NL -53- 2-(1-acetylazetidin-3-yl}-4-amino-5-methylisoindoline-1,3-dione (12): © oo, 0

The compound was synthesized according to general procedure C using the following conditions: The mixture of the nitro-substituted methylphthalimides (0.11 g, 0.36 mmol, ~1:1 ratio of isomers) and tin(ll) chloride dihydrate (0.33 g, 4 equiv.) taken up in ethyl acetate (15 mL) and stirred at refluxing temperature for 4 hours. The crude product was purified by column chromatography eluting with a gradient of 1-2%

MeOH in chloroform, affording the title compound as a bright yellow solid. Yield: 45% (0.02 g, 0.08 mmol). 'H NMR (400 MHz, CDCl3) § 7.31 (d, J=7.4 Hz, 1H), 7.08 (d, J = 7.3 Hz, 1H), 5.46-4.82 (m, 2H), 5.03 (it,

J=8.7,6.2 Hz, 1H), 4.81 — 4.66 (m, 1H), 4.62 — 4.47 (m, 1H), 4.46 — 4.28 (m, 2H), 2.24 (s, 3H), 1.94 (s, 3H). BC NMR (101 MHz, CDCl) 6 170.74, 170.03, 168.03, 144.45, 135.89, 130.05, 113.01, 110.49, 55.02, 52.96, 38.07, 19.12, 17.18. LC-MS (ESI) m/z calcd for C14H1sN3O3 [M + H]+ 273.1, found 274.0.

HPLC (method A): 7.31 min, purity 96.9%. 2-(1-acetylazetidin-3-yl}-5-amino-6-methylisoindoline-1,3-dione (13): {3

Hal onl eN 3 0

The compound was synthesized according tc general procedure C using the following conditions: The mixture of the nitro-substituted methylphthalimides (0.11 g, 0.36 mmol, ~1:1 ratio of isomers) and tin(ll} chloride dihydrate (0.33 g, 4 equiv.) taken up in ethyl acetate (15 mL) and stirred at refluxing temperature for 4 hours. The crude product was purified by column chromatography eluting with a gradient of 1-2%

MeOH in chloroform, affording the title compound as an orange-yellow solid. Yield: 55% (0.03 g, 0.10 mmol}, "H NMR (400 MHz, CDCls) 6 7.49 (s, 1H), 7.05 (s, 1H), 5.02 (tt, J= 8.7, 6.2 Hz, 1H), 4.76 — 4.68 t, J=7.2Hz, 1H), 4.62 — 4.23 (m, 5H), 2.24 (s, 3H), 1.93 (s, 3H). 3C NMR (101 MHz, CDCl) ò 170.67, 168.44, 168.18, 150.97, 132.35, 126.91, 126.05, 120.16, 108.33, 55.03, 53.05, 38.16, 19.16, 18.08.

LC-MS (ESI) m/z calcd for C414H1sN2O: [M + H]+ 273.1, found 274.0. HPLC (method A): 6.87 min, purity 97.7%. ethyl 1,3-dioxoisoindoline-2-carboxylate: £3

JEN | 0 he LS

Re ~ | h 0 S

To a stirring solution of phthalimide (7.36 g, 50 mmol) and triethylamine (8.0 mL, 1.3 eq.) in DMF (25 mL) was added at 0 °C in dropwise fashion ethyl chloroformate (5.76 mL, 1.2 eq.). After completion of the

P352801NL - 54 - addition (20 minutes) the resulting suspension was stirred at room temperature for 1.5 hours. The reaction mixture was then poured onto ice and the resulting white precipitate was filtered off, washed with water and subsequently dried tc afford the title compound as a white solid. Yield: 87% (9.0 g, 43,6 mmol). 'H NMR (400 MHz, CDCls) 6 7.98 (dd, J= 5.5, 3.1 Hz, 2H), 7.84 (dd, J = 5.6, 3.0 Hz, 2H), 4.50 (q, J = 7.1

Hz,‚2H), 1.45 (t, J= 7.1 Hz, 3H). tert-butyl 3-(1,3-dioxoisoindolin-2-yl}azetidine-1-carboxylate: <

TN ts a. & x

The compound was synthesized according to general procedure B using the following conditions: tert- butyl 3-aminoazetidine-1-carboxylate (1.0 g, 581 mmol), ethyl 1,3-dioxoisoindoline-2-carboxylate (1.27 g, 1 equiv.), and triethylamine (1.62 ml, 2 equiv.) were taken up in THF (20 mL) and the resulting solution was stirred for 16 hours. The solvents were then removed in vacuo, the crude taken up in EtOAc and washed subsequently with HCL (0.5M, aq.) and brine. The organics were dried over MgSO: and concentrated in vacuo after which column purification eluting with 0-1% MeOH in DCM afforded the title compound as a white solid. Yield: 95% (1.67 g, 5.52 mmol). *H NMR (400 MHz, CDCl:) 6 7.91 — 7.82 (m, 2H), 7.80 — 7.70 (m, 2H), 5.09 — 4.97 (m, 1H), 4.53 (dd, J = 8.9, 6.3 Hz, 2H), 4.24 (t, J = 8.9 Hz, 2H), 1.49 (s, SH). 3-(1,3-dioxoisoindolin-2-yljazetidin-1-ium chloride: 3 ee. SNOT

U tert-butyl 3-(1,3-dioxoisoindolin-2-yl)azetidine-1-carboxylate (1.76 g, 5.82 mmol) was stirred in a solution of HCI in MeOH (2.0 M, 30 mL). After 2 hours TLC indicated full conversion so solvents were removed in vacuo and residual HCI was removed by co-evaporation with toluene (3x), affording the title compound as an off-white solid. Yield: 99% (1.40 g, 5.82 mmol). 'H NMR (400 MHz, DMSO-Ds) 6 10.06 — 8.81 (m, 2H), 7.95-7.83 (m, 4H), 5.15 — 5.02 (m, 1H), 4.60 — 4.51 (m, 2H), 4.22 — 4.08 (m, 2H). 2-(1-acetylazetidin-3-yl}isoindoline-1,3-dione (3): 9

Sl i

The compound synthesized according to general procedure E using the following conditions: 3-(1,3- dioxoisoindolin-2-yl)azetidin-1-ium chloride (0.35 g, 1.45 mmol), acetyl chloride (0.12 mL, 1.2 equiv.}, and

P352801NL -55- triethylamine (0.42 mL, 2.1 equiv.) were stirred in DCM (10 mL) for 1 hour. Column purification eluting with 0-2% MeOH in DCM and a subsequent recrystallization from a mixture of hexanes in EtOAc afforded the title compound as a white solid. Yield: 22% (0.08 g, 0.32 mmol. *H NMR (400 MHz, CDCl:)} 6 7.90 — 7.81 (m, 2H), 7.80 — 7.70 (m, 2H), 5.09 (tt, J = 8.7, 6.2 Hz, 1H), 4.81 — 4.62 (m, 1H), 4.62 — 4.47 (m, 1H), 447-424 (m, 2H), 1.94 (s, 3H). 13C NMR (101 MHz, CDCls) 6 170.66, 167.80, 153.51, 134.61, 131.66, 123.71, 119.93, 38.45, 19.10. LC-MS (ESI) m/z calcd for C13H12N2O: [M + H]+ 244.3, found 245.1. HPLC (method B): 9.77 min, purity 99%.

Material and methods

Mouse models

Cdh5(PAC)-CREERT2: Eng mice were provided by Prof. HM. Arthur (Biosciences Institute, Faculty of medical Sciences, Newcastle University, UK). All experiments were maintained on a C57BL6/J genetic background.

C57BL6/JRj mice were provided by Janvier lab.

For Eng-iKO®. Male and female mice at 8-12 weeks of age were given tamoxifen (2.5 mg per day, total volume 50ml per injection) by intraperitoneal injection for 5 consecutive days to deplete endoglin in endothelial cells. Tamoxifen was purchased from Sigma-Aldrich, (cat. number T5648) and dissolved in 1:4 ethanol (Merck, cat. number 107017)/coil oil (Sigma-Aldrich, cat. number C8267) vehicle. Intraperitoneal injections of either vehicle or thalidomide or analogues began on post-endoglin depletion day 6 (day 0 corresponding to the first injection of tamoxifen) for 2 consecutive days and animals were harvested on day 9.

For irradiation of the head including the eyes, animals were immobilized in a containment device without anesthesia. The heads of adult mice were exposed to an X-ray generator Philips/YXYLON type

MG325 (200kV voltage, 21mA intensity). The X-ray beam was collimated by a bloc of lead located at 50 cm from the source. Four mice were irradiated by run. A total dose of 20Gy was delivered in a single fraction at a dose rate of 0.9Gy/min. Mice were intraperitoneally injected of either vehicle (DMSO), thalidomide or lenalidomide (concentration: 200 mmol kg, total volume 50 ml per injection) on days, -2, -1, +1, +2, +5, +6, and +7, from day O where they received a single fraction of X-ray ionizing radiation. Mice were harvested at day +8, day +90 or day +180 after receiving a single dose of radiation therapy on day 0.

Patch-clamp recordings of pericytes

Patch-clamp experiments were performed as described*. Briefly, fresh mouse retinas were dissected, superfused with bicarbonate-buffered solution containing 124 mM NaCl, 26 mM NaHCOs, 1 mM NaHz2PO,4, 2.5 mM KCI, 1 mM MgClz, 2.5 mM CaCl: and 10 mM glucose, bubbled with 85% O2/5% CO: and were maintained in pH 7.3 at room temperature. An electrode was filled with the same bicarbonate-buffered solution and used to stimulate pericytes by pressing the electrode against the soma to apply voltage pulses {pulses 0.02ms, 10Hz for 5 seconds) and to deliver current intensities ranging from 1 to 3.5 mA. Resistance

P352801NL -56- of the pipette was measured before each experiment in order to define the input voltage. Pericytes were stimulated by applying voltage pulses 20 seconds after patch-clamping and the vascular constriction was recorded during 210 seconds with a total of 300 images taken.

Vascular permeability experiments

Vascular permeability experiments were performed as described. Briefly, Lysine-fixable cadaverine conjugated to Alexa Fluor-555 (5mg.ml” in saline) (Invitrogen, cat. number A30677) was injected intravenously into the tail vein in adult C57BL6/JRj mice at 12 weeks of age. Circulation time was 2 hours for cadaverine Alexa Fluor-555. Animals were harvested through perfusion of HEPES buffer saline for fluorescence tracer quantification or through perfusion of 4% PFA for whole-brain pictures. For fluorescence quantification, the quantity of cadaverine Alexa Fluor-555 injected was 120 mg per 20 grams body weight, respectively. Brains were removed and homogenized in 1% tritonX-100 in PBS pH 7.2. Brains were centrifuged at 16,000 rpm for 20 min and the relative fluorescence of the supernatant was measured on fluorometer POLARstar Omega (BMG Labtech) (ex/em 544/590 nm).

For whole-brain pictures, the quantity of Alexa Fluor-555 or BSA Alexa Fluor-555 injected was 500 mg or 1850 mg per 20 grams body weight, respectively. Brains were removed, post-fixed in 4% PFA overnight and images were taken with an Axio Zoom.V16 fluorescence Stereo Microscope (Zeiss). Images were taken with the exposure time where the fluorescence signal deriving from the brain of irradiated (RT) animals was not saturated.

Antibodies and immunofluorescence staining

All images were acquired with a Leica SP5 confocal microscope (Leica Microsystems). Images were adjusted for brightness and contrast using ImageJ and are presented as maximum intensity projections.

For each image, the endothelial cell surface was defined as Glut1 (+) pixels and determined by the total grey value.

To quantify the number of neurons, 25 microns maximal projection z-stacks were reconstructed and the number of NeuN positive neurons per mm2 was determined as previously described? using ImageJ software Cell Counter plugin analysis tool. In each animal, 6 randomly selected fields (388.74 x 388.74 mm) from the primary somatosensory barrel cortex were analyzed in 3 non adjacent sections (~ 250 mm apart).

Statistical analyses

Statistical analyses were performed with Prism 9 software (GraphPad) using 1-way ANOVA. Results are expressed as mean + SEM. For post hoc pairwise comparisons, the Dunnett's test was used. A value of *P< 0.05, P< 0.01, **P< 0.001 or ***P< 0.001 denoted statistical significance.

P352801NL -57-

References 1-Lebrin, F.; Srun, S.; Raymond, K.; Martin, S.; van den Brink, S.; Freitas, C.; Bréant, C.; Mathivet, T.;

Larrivee, B.; Thomas, J. L.; Arthur, H. M.; Westermann, C. J.; Disch, F.; Mager, J. J.; Snijder, R. J.;

Eichmann, A.; Mummery, C. L. Thalidomide stimulates vessel maturation and reduces epistaxis in individuals with hereditary hemorrhagic telangiectasia. Nat Med 2010, 16, 420-8. 2-Lebrin, F. S., C.; Thalgott, J. Use of thalidomide or analogs thereof for preventing neurologic disorders induced by brain irradiation. 2015. 3-Cheng J, Jiang J, He B, Lin WJ, Li Y, Duan J, Li H, Huang X, Cai J, Xie J, Zhang Z, Yang Y, Xu Y, Hu Xx,

Wu M, Zhuo X, Liu Q, Shi Z, Yu P, Rong X, Ye X, Saw PE, Wu LJ, Simone CB 2nd, Chua MLK, Mai HQ,

Tang Y. A phase 2 study of thalidomide for the treatment of radiation-induced-blood-brain barrier injury.

Sci Transl Med. 2023 Feb 22;15(684):eabm6543. doi: 10.1126/scitransimed.abm6543. Epub 2023 Feb 22.PMID: 36812346 Clinical Trial. 4-Mishra, A.; O'Farrell, F. M.; Reynell, C.; Hamilton, N. B.; Hall, C. N.; Attwell, D. Imaging pericytes and capillary diameter in brain slices and isolated retinae. Naf Profoc 2014, 9, 323-36. 5-Armulik, A.; Genové, G.; Mae, M.; Nisancioglu, M. H.; Wallgard, E.; Niaudet, C.; He, L.; Norlin, J;

Lindblom, P.; Strittmatter, K.; Johansson, B. R.; Betsholtz, C. Pericytes regulate the blood-brain barrier.

Nature 2010, 468, 557-61. 6-Montagne, A.; Nikolakopoulou, A. M.; Zhao, Z.; Sagare, A. P.; Si, G.; Lazic, D.; Barnes, S. R.; Daianu,

M.; Ramanathan, A.; Go, A.; Lawson, E. J.; Wang, Y.; Mack, W. J.; Thompson, P. M.; Schneider, J. A;

Varkey, J.; Langen, R.; Mullins, E.; Jacobs, R. E.; Zlokovic, B. V. Pericyte degeneration causes white matter dysfunction in the mouse central nervous system. Naf Med 2018, 24, 326-337.

Claims (22)

Conclusions 1. A pharmaceutical compound, wherein the compound has a structure according to Formula I or Formula II: 0 vg £0 ad ( * wherein A1 A2 A3 and A4 each individually represent CRI or N; AD and AS each individually represent S, CRI or N; wherein R1 represents hydrogen, halogen, OH, C1 -C8 alkyl, NO2, or NR2R5, wherein R2 and R3 each individually are selected from H, C1 -C8 alkyl, C1 -C4 alkylcarbonyl, and C1 -C8 alkoxycarbonyl; wherein RX represents Y or CHR4R5, wherein Y represents a 4-, 5-, or 6-membered heterocycle comprising 1, 2, or 3 nitrogen atoms, wherein Y is optionally substituted with one or more substituents selected from the group consisting of halide, a saturated or unsaturated C1 -C8 hydrocarbon, C1-C8 alkoxycarbonyl, or C;-C8 alkylcarbonyl, preferably acetyl or propionyl; wherein R4 represents H, C1-C8 alkyl, C1-C4 cycloalkyl, C1-C8 cycloalkylalkyl, heteroaryl, C1-C8 heteroaralkyl, C;-C8 heterocycloalkyl, or C;-C8 heterocycloalkylalkyl, optionally substituted on one or more carbon atoms with one or more groups selected from halogen, amino, sulfinyl and sulfonyl, wherein R° represents H, OH, COOH, C;-C8 alkyl optionally substituted on one or more carbon atoms with one or more groups selected from halogen, amino, sulfinyl and sulfonyl, preferably COOH, i-propyl or t-butyl; wherein R+ and R5 are both different from H. A compound according to claim 1, wherein A! or A? represents C-NH2 or C-NHC(O)CH3, preferably wherein A! represents C-NH2, and A? and A? each individually represent CH, C-CH3 or C-Cl, preferably CH, or wherein A? represents C-NH2, and At, A? and At each individually represent CH, C-CH3 or C-Cl, preferably C. 3. A compound according to claim 1, wherein A1 represents C-NH2, and A+, A6 and At each represent CH, CH3 or CCl, preferably CH. A compound according to claim 1 or 2, wherein Rx represents wherein R5 represents C1-C8 alkyl, C+-C8 alkenyl, C3-C8 alkynyl, C1-C8 alkoxyalkyl, C1-C7 alkoxycarbonyl, aryl, or C1-C4 alkylcarbonyl, preferably acetyl or propionyl. 5. A compound according to claim 1 to 3, wherein RX is CHR4R5, wherein R4 is hydrogen, methyl, ethyl, n-propyl, i- propyl, n-butyl, i-butyl, tert-butyl, neopentyl, isopentyl, cyclopropylmethyl, or thienylmethyl. A compound according to any one of claims 1 to 3 or 5, wherein RX is CHR4R5, wherein R5 is COOH, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, tert-butyl, preferably COOH, methyl, i-propyl or t-butyl. A compound according to claim 1, wherein the compound has a structure according to formula I-a: 3 fo \ " rN NN . VET Ne ere IF Na * Ny {a wherein each Z is independently selected from hydrogen, halo, OH, C1 -C8 alkyl, NO2 , and NR2 R5 , wherein R2 and R5 are each independently selected from H, C1 -C8 alkyl, C1 -C8 alkylcarbonyl, and C1 -C8 alkoxycarbonyl, preferably wherein each Z is independently selected from H, halo, NH2 , and NHC(O)CH3 ; wherein m is 1, 2, 3, or 4, preferably 1 or 2; and wherein RC is saturated or unsaturated C1 -C8 hydrocarbons, C1 -C8 alkoxycarbonyl, or C1 -C8 alkylcarbonyl, preferably acetyl or propionyl. 8. A compound according to claim 1, wherein the compound has a structure according to formula I-b or I-c 0 § re ™ A (24-1 NW Opi? Sha No / / * U) Á Ne es 2} == | > \ nN & Su wee 8 ET Nd Nez ES —~— > SIS ACN > {he wherein each Z is independently selected from hydrogen, halo, OH, C:-C4 alkyl, and NO2, preferably wherein each Z is independently selected from H, CH3, and Cl; wherein m is 1, 2, or 3; and wherein R6 is a saturated or unsaturated C1-C8 hydrocarbon, C1-C8 alkoxycarbonyl, or C;-C8 alkylcarbonyl, preferably acetyl or propionyl. 9. A compound according to any one of claims 1 to 8, wherein the compound does not bind to cereblon (CRBN). 10. A formulation comprising a pharmaceutical compound according to any one of claims 1 to 9, wherein the formulation further comprises a pharmaceutically acceptable excipient, a pharmaceutically acceptable adjuvant, a pharmaceutically acceptable diluent, and/or a pharmaceutically acceptable carrier. 11. A pharmaceutical compound, wherein the compound has a structure according to formula I or II: 8 1 a © ~ © 0 AS A tj an wherein A1 A2 A3 and A4 each individually represent CRL or N; AD and AS each individually represent S, CRI or N; wherein R1 represents hydrogen, halogen, OH, C1-C4 alkyl, NO, or NR2R5, wherein R2 and R° each individually are selected from H, C1-C4 alkyl, C1-C8 alkylcarbonyl, and C1-C8 alkoxycarbonyl; wherein RX represents Y or CHR4R5, wherein Y represents a 4-, 5-, or 6-membered heterocycle comprising 1, 2, or 3 heteroatoms, wherein Y is optionally substituted with one or more substituents selected from the group consisting of halide, a saturated or unsaturated C; -C8 hydrocarbon, C; -C8 alkoxycarbonyl, or C; -C8 alkylcarbonyl, preferably acetyl or propionyl; wherein R* represents H, C1-C8 alkyl, C; -C8 cycloalkyl, C1-C8 cycloalkylalkyl, heteroaryl, C1-C8 heteroaralkyl, C; -Cy heterocycloalkyl, or C; -C8 heterocycloalkylalkyl, wherein R° represents H, OH, COOH, C; -Cy alkyl, preferably COOH, i-propyl or t-butyl; or a formulation comprising such a compound, together with a pharmaceutically acceptable excipient, a pharmaceutically acceptable adjuvant, a pharmaceutically acceptable diluent and/or a pharmaceutically acceptable carrier, for use as a medicament in the treatment of a disease or condition characterised by pericyte dysfunction, by pericyte detachment, and/or by pericyte loss. 12. The compound or formulation for use according to claim 11, wherein the disease or condition characterized by pericyte dysfunction, by pericyte detachment, and/or by pericyte loss is selected from complications of diabetes mellitus, chronic kidney disease (CKD), small vessel disease, and central nervous system (CNS) disorders. The compound or formulation for use according to claim 11, wherein the disease or condition characterized by pericyte dysfunction, by pericyte detachment, and/or by pericyte loss is selected from diabetic nephropathy (DN), diabetic retinopathy (DR), diabetic cardiomyopathy (DCM), and diabetic neuropathy. The compound or formulation for use according to claim 11, wherein the disease or condition characterized by pericyte dysfunction, by pericyte detachment, and/or by pericyte loss is selected from hypertensive renal diseases, IgA nephropathy, congenital nephropathy syndrome, lupus nephritis, polycystic kidney disease, and allograft nephropathy. The compound or formulation for use according to claim 11, wherein the disease or condition characterized by pericyte dysfunction, by pericyte detachment, and/or by pericyte loss is selected from cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (Cadasil), cerebral autosomal-recessive arteriopathy with subcortical infarcts and leukoencephalopathy (Cerebral Autosomal Recessive Arteriopathy with Subcortical Infarcts and Leukoencephalopathy - Carasil), cerebral amyloid angiopathy (Cerebral Amyloid Angiopathy - CAA), retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (Retinal Vasculopathy with Cerebral Leukoencephalopathy and systemic manifestations - RVCL-S), hereditary hemorrhagic telangiectasia (Hereditary Hemorrhagic Telangiectasia - HHT), and cerebral cavernous malformations (CCM). A compound or formulation for use according to claim 11, wherein the disease or condition characterized by pericyte dysfunction, by pericyte detachment, and/or by pericyte loss is selected from stroke, epilepsy, spinal cord injury, vascular dementia, Alzheimer's disease, Huntington's disease, Parkinson's disease, traumatic brain injury, multiple sclerosis, amyotrophic lateral sclerosis, and radiation necrosis. 17. A pharmaceutical compound according to any one of claims 1 to 9, or a formulation according to claim 10, for use as a medicament in the treatment of stabilisation of blood vessels, and/or in the treatment or promotion, restoration or maintenance of pericyte adhesion to blood vessels. 18. The compound or formulation for use according to claim 17, wherein the therapeutic treatment of vascular stabilization comprises protecting the integrity of the blood-brain barrier, controlling blood flow, limiting inflammation, and/or reducing tissue damage. 19. A method of promoting or maintaining pericyte adhesion to blood vessels in a patient, the method comprising administering to the patient a compound according to any one of claims 1 to 9, or a formulation according to claim 10. 20. A method for the therapeutic treatment of a vascular associated disease in a patient, the method comprising administering to the patient a compound according to any one of claims 1 to 9, or a formulation according to claim 10. 21. The method of claim 20, wherein the vascular-associated disease is selected from complications of diabetes mellitus, chronic kidney disease (CKD), small vessel disease, and central nervous system (CNS) disorders; preferably hypertensive kidney disease, IgA nephropathy, congenital nephropathy syndrome, lupus nephritis, polycystic kidney disease, and allograft nephropathy; cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (Cadasil), cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (Carasil), cerebral amyloid angiopathy (CAA), retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S), hereditary hemorrhagic telangiectasia (HHT), and cerebral cavernous malformations (CCM); CVA, epilepsy, spinal cord injury, vascular dementia, Alzheimer's disease, Huntington's disease, Parkinson's disease, traumatic brain injury, multiple sclerosis, amyotrophic lateral sclerosis, and radiation necrosis. 22. A method for maturing an organoid and/or an organ on a chip model system, comprising the use of a compound according to any one of claims 1 to 9.