4-(3-Aminoazetidin-1-yl)pyrimidin-2-amines as High-Affinity Non-imidazole Histamine H3 Receptor Agonists with in Vivo Central Nervous System Activity.

Despite the high diversity of histamine H3 receptor (H3R) antagonist/inverse agonist structures, partial or full H3R agonists have typically been imidazole derivatives. An in-house screening campaign intriguingly afforded the non-imidazole 4-(3-azetidin-1-yl)pyrimidin-2-amine 11b as a partial H3R agonist. Here, the design, synthesis, and structure-activity relationships of 11b analogues are described. This series yields several non-imidazole full agonists with potencies varying with the alkyl substitution pattern on the basic amine following the in vitro evaluation of H3R agonism using a cyclic adenosine monophosphate response element-luciferase reporter gene assay. The key compound VUF16839 (14d) combines nanomolar on-target activity (pKi = 8.5, pEC50 = 9.5) with weak activity on cytochrome P450 enzymes and good metabolic stability. The proposed H3R binding mode of 14d indicates key interactions similar to those attained by histamine. In vivo evaluation of 14d in a social recognition test in mice revealed an amnesic effect at 5 mg/kg intraperitoneally. The excellent in vitro and in vivo pharmacological profiles and the non-imidazole structure of 14d make it a promising tool compound in H3R research.

Introduction

The four histamine receptors (n class="Gene">H1R, H2R, H3R, H4R) belong to class A of the G protein-coupled receptor (GPCR) family.[1] The histamine H3 receptor (H3R) was discovered in 1983 by Arrang et al.[2] and regulates the release of several neurotransmitters such as histamine (1), acetylcholine, serotonin, noradrenaline, and dopamine, as both auto- and heteroreceptor.[1] Due to its expression in the cortex, striatum, and hippocampus, H3R regulates several physiological processes such as sleep–wake regulation, cognition, and food intake.[1,3]

During the early years of discovery of H3R ligands, the natural ligand n class="Chemical">histamine served as an initial structure for drug design, leading to a plethora of imidazole-containing ligands. However, imidazole-containing ligands are associated with drug–drug interactions due to the propensity for cytochrome P450 (CYP) inhibition and having poor brain penetration.[4,5] Therefore, research toward therapeutically relevant H3R antagonists focused on druglike non-imidazole structures,[6,7] and these efforts have led to numerous clinical candidates for different indications connected to central nervous system diseases, for example, Alzheimer′s disease, attention deficit hyperactivity disorders, sleep disorders, schizophrenia, obesity, epilepsy, and neuropathic pain and narcolepsy.[8,9] For the latter condition, the H3R ligand pitolisant (Wakix) was approved by the European Medicines Agency in 2016[10] and, most recently, by the Food and Drug Administration in 2019.[11]

In sharp contrast, developing non-imidazole n class="Gene">H3R agonists has not been very successful. The best agonists contain an imidazole ring[12−23] and, compared to the endogenous ligand histamine (1), these derivatives show similar [e.g., imbutamine (2)] or significantly higher affinity (pKi) and/or functional activity (pEC50) on H3R [e.g., imetit (3), methimepip (4), and (1S,2S)-2-(2-aminoethyl)-1-(1H-imidazol-4-yl)cyclopropane (5)] (Figure A). Imidazole-containing agonists have shown some potential application in different therapeutic areas, such as mechanical nociception,[23] obesity, and diabetes mellitus (diet-induced obesity mice test)[24] and stress (rodent-intruder mice test).[21] Some data also support the hypothesis of cardioprotective effect of H3R receptor activation.[25,26] However, it is fair to say that imidazole-containing agonists may suffer from the same imidazole-related drawbacks that were associated with the first generation of imidazole-containing H3R antagonists (vide supra). Future studies on the pharmacological and therapeutic roles of H3R agonists can therefore be helped by having non-imidazole H3R agonists available.

Figure 1

(A) Representative imidazole H3R agonists. Activities are extracted from Igel et al.,[33] Govoni et al.,[18] and Kazuta et al.[12] (B) Published non-imidazole H3R (partial) agonists.[27−32] Unless mentioned otherwise, compounds were tested on the human receptor. α, intrinsic activity compared to histamine; β-gal, cyclic adenosine monophosphate (cAMP) response element (CRE)-β-galactosidase reporter gene assay; cAMP, forskolin-stimulated cAMP accumulation assay.

(A) Representative imidazole n class="Gene">H3R agonists. Activities are extracted from Igel et al.,[33] Govoni et al.,[18] and Kazuta et al.[12] (B) Published non-imidazole H3R (partial) agonists.[27−32] Unless mentioned otherwise, compounds were tested on the human receptor. α, intrinsic activity compared to histamine; β-gal, cyclic adenosine monophosphate (cAMP) response element (CRE)-β-galactosidase reporter gene assay; cAMP, forskolin-stimulated cAMP accumulation assay.

A very limited number of non-imidazole agonists have been published to date (Figure B). n class="Chemical">VUF8430 (6) was designed as an H4R agonist based on the H2R agonist dimaprit and shows micromolar affinity and full H3R agonism as well.[27] The histamine analogues amthamine (7a) and amselamine (7b) were identified as H2R agonists, but both show weak H3R agonist activity in an electrically stimulated guinea-pig jejunum model.[28,29] Three pentacyclic spiroindolinone derivatives were isolated from Penicillium waksmanii, of which PF1270A (8) shows the best affinity for H3R and moderate functional H3R activity in a guanosine 5′-O-[γ-thio]triphosphate (GTPγS) accumulation assay.[30] ZEL-H16 (9) has been reported to have nanomolar binding affinity to the H3R, partial H3R agonism in forskolin-stimulated cAMP accumulation and ERK1/2 signaling assays, and full H3R agonism in a guinea-pig ileum contraction assay.[31] Finally, a compound set with 94 examples in four compound families with either a β-lactam or a pyrrolidinone central core without basic amino moiety was published recently and surprisingly included compounds with nanomolar functional H3R agonist activities (e.g., compound 10).[32] The fungal isolates and the multicomponent reaction product 10 are large and complex molecules, which are difficult to align with known H3R pharmacophores or H3R binding modes for agonists and antagonists. We therefore started a search for novel high-affinity non-imidazole H3R full agonists with simpler structures to generate fundamental knowledge on ligand recognition and signaling of the H3R.

Results

Design

During an in-house compound screen aimed at identifying agonist activities in a set of ligands using a H3R-driven reporter gene assay in n class="CellLine">HEK293T cells, diaminopyrimidine 11b emerged as a H3R partial agonist hit (α = 0.7), while its close derivative 11i showed only weak agonist activity (α = 0.4) (Figure A). Interestingly, a set of four diaminopyrimidine compounds has been tested before by others on H3R en route to imbutamine (2) analogues, but the majority of these diaminopyrimidines were rather inactive and, where applicable, all were shown to be antagonists/inverse agonists.[34] Intrigued by the agonist activity of the diaminopyrimidine 11b and recognizing its core as a thoroughly explored heterocycle in the H4R area,[35] we decided to perform an in-depth patent search on this scaffold in an effort to capture the full array of industrial contributions. This resulted in the identification of 14a (Figure B) as the closest derivative with data associated with H3R (Figure B).[36] Remarkably, 14a was claimed as a H3R agonist by Abbott, although its actual synthesis was not included and only the potential synthetic route was described.[36] In the same patent, 25 related examples have been prepared, and partial agonism at the human H3R is reported.[36]

Figure 2

(A) Initial functional data of compounds 11b and 11i compared to histamine, as obtained by ligand-induced activation of hH3R expressed on HEK293T cells measured by the CRE-luc reporter gene assay. Shown is a representative graph of at least three experiments performed in triplicate. Data are mean ± standard deviation (S.D.). (B) Structures of 11b and 11i, the closest relevant structure (14a) resulting from a subsequent extensive patent search and the compound set designed for the current study.

(A) Initial functional data of compounds 11b and n class="Chemical">11i compared to histamine, as obtained by ligand-induced activation of hH3R expressed on HEK293T cells measured by the CRE-luc reporter gene assay. Shown is a representative graph of at least three experiments performed in triplicate. Data are mean ± standard deviation (S.D.). (B) Structures of 11b and 11i, the closest relevant structure (14a) resulting from a subsequent extensive patent search and the compound set designed for the current study.

Based on n class="Chemical">11b and 14a, we designed a focused series of compounds to explore the H3R affinity and activity in the chemical space between 14a and 11b (Figure B). The design strategy targets four series with iPr, Et, Me, or H as the R1 group at position 6 (11–14). These R1 groups were combined with different substituents on the basic amino groups (R2 and R3). Beyond the evident H (a) and Me (b) substituents, dimethyl derivatives (i) were synthesized due to the potential functional switch that appears to reside in the cases of 11b and 11i. Based on the initial results of the designed compound set (vide infra), the R1 = H series was extended with elongated (d and f), branched (e, g, h), and disubstituted (j, k) amino derivatives. This second design iteration was also inspired by previous work from our labs on the imidazole-containing agonist imbutamine (2), which harbors a functional switch on the basic amine.[18] Beyond 14a, some exact compounds from this designed set are known but none in a context of H3R. That is, 11a, 11b, and 12b have been claimed as H4R ligands,[37,38] while 12i, 13i, 14i, 14j, and 14k were offered in chemical catalogs (April 2019) without any synthesis description and analytical or pharmacological data.

Chemistry

The designed compound set shown in Figure B was synthesized as outlined in Scheme . The key step of the synthetic route was a nucleophilic aromatic substitution on the appropriate aromatic cores with n class="Chemical">aminoazetidine moieties (step ii). The 4-chloro-2-aminopyrimidines were commercially available (19 and 20) or were synthesized (17 and 18) from the appropriate pyrimidin-4(3H)-one derivatives (15 and 16) with POCl3. The R2 = H or Me derivatives of the Boc-protected 3-aminoazetidines (28a and 28b) were commercially available, while the R2 = Et, nPr, and nBu derivatives were synthesized. These intermediates were built from benzhydryl-protected 3-aminoazetidine (25) with Boc protection of the primary amino group to give 26, followed by alkylation with the corresponding iodoalkyl reagents resulting in the orthogonally protected intermediates (27c, 27d, 27f) and the removal of the benzhydryl group with hydrogenation. The resulting mixtures of unprotected azetidine intermediate and diphenylmethane were used directly for the ensuing nucleophilic substitution with 17–20. The key nucleophilic aromatic substitution of the appropriate Boc-protected intermediates was performed in a microwave at 120–150 °C to give 21–24. This was followed by the deprotection under acidic condition to afford the majority of monosubstituted (R2 = H or linear alkyl, R3 = H) products 11–14. Although the Boc protection was necessary at the precursor stage to avoid overalkylation in the case of linear alkyl derivatives, overalkylation was not a problem in the case of the branched-alkyl derivatives as a result of steric hindrance. Therefore, the branched-alkyl derivatives 14e, g, and h as well as the dialkylated derivatives 12i, 13i, 14i, and 14j were synthesized from 14a with a reductive amination. Last, 11i was synthesized directly from 17 and N,N-dimethylazetidin-3-amine with a nucleophilic substitution, while the pyrrolidine ring of 14k was obtained from 1,4-diiodobutane and 14a.

Scheme 1

Syntheses Routes for Final Compounds

Reagents and conditions: (i) POCl3, 110 °C, 3 h, 26–52%; (ii) N,N-diisopropylethylamine (DIPEA), dioxane or N-methyl-2-pyrrolidone (NMP), microwave (μW), 120–150 °C, 0.5–2 h, 27–50% (a, b) or 35–71% (c, d, f, two steps from benzhydryl deprotection); (iii) HCl, dichloromethane (DCM), MeOH, room temperature (rt) to 50 °C, 3 h to overnight, 10% to quant.; (iv) aldehyde/ketone, AcOH, NaHB(OAc)3, DCM, MeOH, rt, 3 h to overnight, 16–44%; or 1,4-diiodobutane, K2CO3, MeCN, reflux, 16 h, 9%; (v) N,N-dimethylazetidin-3-amine dihydrochloride, DIPEA, dioxane, μW, 150 °C, 30 min, 65%; (vi) di-tert-butyl dicarbonate, triethylamine (TEA), tetrahydrofuran (THF), rt, overnight, 63%; (vii) NaH, R2I, THF, 0 °C to rt, overnight, 28–58%; (viii) H2, Pd/C, MeOH, EtOH, rt to 60 °C, 1 h to overnight, not purified and used crude.

Reagents and conditions: (i) POCl3, 110 °C, 3 h, 26–52%; (ii) n class="Chemical">N,N-diisopropylethylamine (DIPEA), dioxane or N-methyl-2-pyrrolidone (NMP), microwave (μW), 120–150 °C, 0.5–2 h, 27–50% (a, b) or 35–71% (c, d, f, two steps from benzhydryl deprotection); (iii) HCl, dichloromethane (DCM), MeOH, room temperature (rt) to 50 °C, 3 h to overnight, 10% to quant.; (iv) aldehyde/ketone, AcOH, NaHB(OAc)3, DCM, MeOH, rt, 3 h to overnight, 16–44%; or 1,4-diiodobutane, K2CO3, MeCN, reflux, 16 h, 9%; (v) N,N-dimethylazetidin-3-amine dihydrochloride, DIPEA, dioxane, μW, 150 °C, 30 min, 65%; (vi) di-tert-butyl dicarbonate, triethylamine (TEA), tetrahydrofuran (THF), rt, overnight, 63%; (vii) NaH, R2I, THF, 0 °C to rt, overnight, 28–58%; (viii) H2, Pd/C, MeOH, EtOH, rt to 60 °C, 1 h to overnight, not purified and used crude.

Pharmacological Evaluation

The synthesized compound set was tested for its activity at the human n class="Gene">H3R transiently expressed in HEK293T cells. Binding affinity (Ki) was evaluated using a [3H]NAMH displacement assay, while potency (EC50) and intrinsic activity (α) were determined as the H3R-mediated inhibition of forskolin-induced CRE-driven luciferase reporter gene activity with histamine as the control (Table and Figure ). During the first iteration, the isopropyl group of 11b was gradually decreased in size to give 11–14, which were all combined with small-size R2/R3 amino substituents (a, b, c, i). The affinities of the unsubstituted derivatives (14) stand out especially within the monomethylated (b) and monoethylated series (c), with both 14b and 14c exceeding the affinity of histamine. Although monomethylated (b) derivatives generally show the highest affinities in each R1 subseries 11–14, the most notable variation was observed in the case of the monoethylated series c, with affinities of Me/Et/iPr derivatives 11c, 12c, and 13c being considerably reduced compared with that of 14c (Figure A). A more indicative trend was observed in the case of the functional results of this c series. Compound 14c has a higher potency than 14b and produces the same intrinsic activity (α) as histamine but with higher potency (EC50), while 12c and 13c show >1 log unit weaker EC50 albeit with maintained full agonism (α ∼ 1.0). Interestingly, isopropyl substitution on the R1 position (11c) turns this full agonism to partial agonism (α = 0.3) (Figure B). A similar trend was observed in the other three series, with the pyrimidine derivatives bearing R1 = H (14a–c and 14i) reaching or exceeding the affinity (Ki) and potency (EC50) of histamine, while any alkyl substituent at position 6 (R1) on the pyrimidine leads to inferior results. The intrinsic activity (α) indicates full or almost full agonism (α ≥ 0.8) in the methyl (12a, 12b, 12i) and ethyl (13a, 13b, 13i) series, while it drops to partial agonism in the isopropyl series (11a, 11b, 11i) (Table ).

Table 1

Pharmacological Evaluation of Designed Compound Set

compound	R1	R2	R3	pKic	pEC50d	α
histamine				7.9 ± 0.2	8.6 ± 0.0	1.0 ± 0.0
11a	iPr	H	H	6.7 ± 0.0	7.1 ± 0.1	0.7 ± 0.0
11b	iPr	Me	H	7.0 ± 0.0	7.9 ± 0.4	0.7 ± 0.1
11c	iPr	Et	H	7.0 ± 0.1	6.8 ± 0.3	0.3 ± 0.1
11i	iPr	Me	Me	6.9 ± 0.0	7.0 ± 0.2	0.4 ± 0.0
12a	Et	H	H	7.1 ± 0.0	7.5 ± 0.0	0.9 ± 0.0
12b	Et	Me	H	7.3 ± 0.1	7.6 ± 0.0	0.8 ± 0.0
12c	Et	Et	H	6.9 ± 0.1	7.5 ± 0.1	0.9 ± 0.0
12i	Et	Me	Me	6.6 ± 0.2	7.2 ± 0.1	0.8 ± 0.0
13a	Me	H	H	7.3 ± 0.1	7.8 ± 0.0	0.9 ± 0.0
13bb	Me	Me	H	7.5 ± 0.1	8.0 ± 0.0	0.8 ± 0.1
13c	Me	Et	H	7.1 ± 0.1	7.9 ± 0.0	1.0 ± 0.0
13i	Me	Me	Me	6.5 ± 0.2	7.4 ± 0.0	0.9 ± 0.1
14aa	H	H	H	7.8 ± 0.1	8.3 ± 0.0	1.1 ± 0.0
14ba	H	Me	H	8.2 ± 0.1	8.9 ± 0.1	1.1 ± 0.1
14ca	H	Et	H	8.0 ± 0.1	9.2 ± 0.0	1.0 ± 0.1
14da	H	nPr	H	8.5 ± 0.1	9.5 ± 0.1	1.2 ± 0.1
14ea	H	iPr	H	7.4 ± 0.1	8.5 ± 0.1	1.2 ± 0.1
14fa	H	nBu	H	7.8 ± 0.1	9.1 ± 0.3	1.2 ± 0.0
14ga	H	rac-sBu	H	7.9 ± 0.2	8.7 ± 0.1	1.1 ± 0.0
14ha	H	iBu	H	7.4 ± 0.2	8.3 ± 0.0	1.1 ± 0.0
14ia	H	Me	Me	7.3 ± 0.1	8.4 ± 0.1	1.0 ± 0.1
14ja	H	Et	Et	7.2 ± 0.1	8.1 ± 0.1	1.2 ± 0.1
14ka	H	-(CH2)4-		7.4 ± 0.1	8.1 ± 0.0	1.3 ± 0.2

Measured as fumarate salt.

Measured as dihydrochloride salt.

Affinity values (pKi) were determined by [3H]NAMH displacement assay on hH3R expressed on HEK293T cell homogenates.

Potency (pEC50) and intrinsic activity (α) were determined by ligand induced activation of hH3R expressed on HEK293T cells as measured by a CRE-luciferase reporter gene assay. Data are mean ± standard error of the mean (S.E.M.) of at least three experiments performed in triplicate.

Figure 3

Representative structure–affinity relationship (A, C) and structure–function relationship (SFR) (B, D) effects selected from Table . (A, B) Different R1 substituents with R2 = Et and R3 = H (11c, 12c, 13c, 14c), (C, D) Different R2 and R3 substituents with R1 = H (14b, 14d, 14e, 14f, 14i). Shown is a representative graph of at least three experiments performed in triplicate. Data are mean ± S.D.

Representative structure–affinity relationship (A, C) and structure–function relationship (SFR) (B, D) effects selected from Table . (A, B) Different R1 substituents with R2 = Et and R3 = H (11c, n class="Chemical">12c, 13c, 14c), (C, D) Different R2 and R3 substituents with R1 = H (14b, 14d, 14e, 14f, 14i). Shown is a representative graph of at least three experiments performed in triplicate. Data are mean ± S.D.

Affinity values (pKi) were determined by [3H]NAMH displacement assay on n class="Gene">hH3R expressed on HEK293T cell homogenates.

Potency (pEC50) and intrinsic activity (α) were determined by ligand induced activation of hH3R expressed on n class="CellLine">HEK293T cells as measured by a CRE-luciferase reporter gene assay. Data are mean ± standard error of the mean (S.E.M.) of at least three experiments performed in triplicate.

Due to the better results of the pyrimidines lacking an R1 substituent (14), in a subsequent iten class="Species">ration the amine NHR2 substituent was replaced with longer linear groups (14d, 14f), branched groups (14e, 14g, 14h), or dialkylamino (14j, 14k) moieties. Representative curves illustrate the structure–activity relationship (SAR) (Figure C) and SFR (Figure D) of this series. The nPr derivative 14d shows the highest affinity (pKi = 8.5) from all linear monoalkyl substituents. Although both shorter and longer R2 moieties resulted in lower binding affinities (e.g., 14c: pKi = 8.0 or 14f: pKi = 7.8), all derivatives remained in the same affinity range (Figure C). The branched-alkyl moieties as well as the dialkylated derivatives display loss of affinity (compare, e.g., 14d vs 14e, or 14b vs 14i). The potencies (EC50) show almost the same trends as observed for the affinities (Figure D). Highly noteworthy, the potency of 14d (pEC50 = 9.5) is almost a log unit higher than that of histamine (pEC50 = 8.6), while chain shortening (e.g., 14b, 14c), chain lengthening (14f), chain branching (e.g., 14e), or dialkylation (e.g., 14i) results in lower potencies. In contrast to the observed differences in affinity and potency, the intrinsic activities indicate that all derivatives of 14 remain full agonists (α ≥ 1.0) (Table ). A combination of highest affinity (pKi = 8.5), highest potency (pEC50 = 9.5), and full agonism resides in 14d. The potential aggregation of GPCR ligands might cause nonspecific effect on the receptor activity,[39] but nephelometry revealed no microprecipitation of 14d up to 100 mM concentration (Figure S1) and underscores the high aqueous (aq) solubility of 14d (soluble up to at least 100 mM in 50 mM Tris–HCl, pH 7.4). All this led to identification of 14d as a key compound (VUF16839) in this study.

Computational Studies on 14d

A combination of molecular docking and molecular dynamics (MD) simulations was used to evaluate the potential binding mode of the key compound 14d and to compare it to the binding mode of the endogenous ligand n class="Chemical">histamine. A homology model of H3R based on the available crystal structure of H1R was used (see the Experimental section). This model was validated by its ability to retrospectively discriminate between known H3R fragmentlike ligands and true inactives.[40] Histamine was docked in the receptor model using PLANTS 1.1 (Figure A). The best-scored docking pose showing interactions with both D1143.32 and E2065.461 (residues known to be involved in H3R ligand binding[40−45]) was selected. During 100 ns of molecular dynamics (MD) simulations, histamine is able to maintain stable interactions with residues D1143.32 and E2065.461 as well as with Y3746.51 (Figure B,E). Using similar procedures, compound 14d was also docked into the same homology model using PLANTS 1.1 (Figure A), and the best-scored docking results show similar interactions of 14d with D1143.32 and E2065.461. The basic amine of 14d forms an ionic interaction with the negatively charged side chain of D1143.32, and the amino group in the pyrimidine ring makes a hydrogen bond with E2065.461. Different docking poses maintain these key interactions but show a different positioning of the linear nPr moiety at the R2 position: toward the extracellular surface of the receptor or toward the intracellular side (Figure S2A,B, respectively). MD simulations of the two alternative models were performed (Movies 1 and 2, Supporting Information). The model in which the nPr group of 14d is directed toward the intracellular half of the receptor was not stable along 100 ns of MD simulations (Figure S2D, Movie 2, Supporting Information), while the model where the nPr group of 14d is pointing toward the extracellular vestibule remained stable throughout the entire simulation time (Figure S2C and Movie 1, Supporting Information). This binding mode is shown in Figure D, and the interactions that remained stable during the simulations are depicted in Figure C and the interaction fingerprints (IFPs) in Figure E. It can be concluded that the non-imidazole H3R ligand 14d exerts its unusual agonist H3R activity by showing a similar pharmacophore as the endogenous H3R ligand. That is, it may achieve its agonist activity by forming similar interactions with the same residues as histamine.

Figure 4

Predicted binding mode of 14d. (A) Overview of the H3R homology model based on the H1R crystal structure (PDB ID: 3RZE).[46] The experimentally validated binding mode of histamine (magenta) is shown in more detail in (B), and the predicted binding mode of 14d (green) is schematically represented in (C) and shown in more detail in (D). Interaction fingerprint representations of histamine and compound 14d are shown in (E), where a one represents the presence of an interaction according to the color coding: blue for apolar, olive green for face-to-face aromatic, green for edge-to-face aromatic, red for protein hydrogen bond donor, purple for ligand hydrogen bond donor, gray for ligand-negative charge, and orange for protein-negative charge.

Predicted binding mode of 14d. (A) Overview of the n class="Gene">H3R homology model based on the H1R crystal structure (PDB ID: 3RZE).[46] The experimentally validated binding mode of histamine (magenta) is shown in more detail in (B), and the predicted binding mode of 14d (green) is schematically represented in (C) and shown in more detail in (D). Interaction fingerprint representations of histamine and compound 14d are shown in (E), where a one represents the presence of an interaction according to the color coding: blue for apolar, olive green for face-to-face aromatic, green for edge-to-face aromatic, red for protein hydrogen bond donor, purple for ligand hydrogen bond donor, gray for ligand-negative charge, and orange for protein-negative charge.

Pharmacological and Pharmacokinetic Characterization of 14d

Functional characterization of key compound 14d in a direct G protein activation assay, that is, the [n class="Chemical">35S]-GTPγS accumulation assay on hH3R expressing cell homogenates (Figure A), results in potent but partial agonism (pEC50 = 8.4 ± 0.3, α = 0.5 ± 0.05) compared with histamine (pEC50 = 7.2 ± 0.3, α = 1.0 ± 0.0).

Figure 5

(A) Dose-dependent Gαi activation by 14d and histamine as measured by [35S]GTPγS accumulation on HEK293T cell homogenates expressing the hH3R. (B, C) Dose–response curves of 14d and histamine for ligand-induced activation of mH3R (B) and mH4R (C) expressed on HEK293T cells as measured by the CRE-luciferase reporter gene assay. Representative graphs of at least three experiments performed in triplicate are shown. Data are mean ± S.D.

(A) Dose-dependent Gαi activation by 14d and n class="Chemical">histamine as measured by [35S]GTPγS accumulation on HEK293T cell homogenates expressing the hH3R. (B, C) Dose–response curves of 14d and histamine for ligand-induced activation of mH3R (B) and mH4R (C) expressed on HEK293T cells as measured by the CRE-luciferase reporter gene assay. Representative graphs of at least three experiments performed in triplicate are shown. Data are mean ± S.D.

Due to the high homology of the hH3R with hHR (43% full sequence identity, 58% predicted transmembrane region identity),[47] several, mainly n class="Chemical">imidazole-containing, hH3R ligands are known to possess high affinity to hH4R as well,[33,44] although there are examples of imidazole-containing H3R agonists with high H3R/H4R selectivity as well (e.g., 4 and 5).[12,33] Therefore, 14d was tested for its H3R/H4R selectivity. The pyrimidine shows only marginal selectivity with respect to binding hH3R or hH4R (hH3R pKi = 8.5 ± 0.1 vs hH4R pKi = 8.1 ± 0.0), but encouragingly a 10-fold selectivity in potency is observed in favor of the hH3R (hH3R pEC50 = 9.5 ± 0.1 vs hH4R pEC50 = 8.5 ± 0.2) with full agonism on hH4R (α = 1.1 ± 0.1) in a CRE-luc reporter gene assay. Moreover, 14d does not activate the H1R and H2R up to 10 μM (Figure S3).

Equally encouragingly, the binding affinity of 14d is increased for n class="Gene">mH3R (pKi = 9.0 ± 0.1) compared with hH3R, while for mH4R, the pKi value is decreased to 7.8 ± 0.0, thus yielding a substantial H3R/H4R binding selectivity for mouse receptors. Compound 14d was also functionally evaluated as an agonist on the mH3R and mH4R using the CRE-luciferase reporter gene assay. In these experiments, 14d displays a 10-fold selectivity in potency (mH3R pEC50 = 10.0 ± 0.1 vs mH4R pEC50 = 9.0 ± 0.1), while it acts as a full agonist on both murine receptors (mH3R α = 1.2 ± 0.1 and mH4R α = 1.1 ± 0.1) (Figure B,C).

The metabolic stability of 14d was determined in vitro by incubation with rodent liver microsomes (Table ). The pharmacokinetic properties for n class="Species">mouse [t1/2 = 130.8 min; Clint = 20.7 mL/(min*kg)] indicate more than 2 times slower elimination compared with the reference control verapamil.[48] For rats, this difference between 14d and verapamil is even more pronounced.

Table 2

Pharmacokinetic Properties of 14d and the Reference Drug Verapamil

	rat liver microsomes		mouse liver microsomes
compound	t1/2 (min)	Clint [mL/(min*kg)]	t1/2 (min)	Clint [mL/(min*kg)]
14d	239.0	7.9	130.8	20.7
verapamil	50.9	37.3	57.3	47.3

The imidazole ring is known to generally be able to interact with n class="Gene">CYP enzymes via coordination of the imidazole with the prosthetic heme iron, which can cause unwanted drug–drug interaction.[49] Since the 2-aminopyrimidine core contains a pattern of adjacent nitrogen atoms, we measured its propensity for CYP inhibition. Compound 14d shows only weak activity on three key CYP enzymes (Figure S4) with IC50 values for binding to CYP3A4, CYP2C9, and CYP2D6 all being larger than 25 μM.

Effect of 14d on Social Recognition in Mice

Given the notion that CNS penetn class="Species">ration of H3R agonists,[1,5] including 14d, is not evident, we evaluated the in vivo CNS effects of 14d in a standard paradigm for H3R action. It is well known that histamine, acting in different brain sites, is an important regulator of memory consolidation and retrieval in various learning paradigms, including the social recognition test.[50,51] We used this behavioral paradigm to investigate the H3R-related CNS activity of compound 14d in vivo. The social recognition memory investigates the ability to remember the identity of a conspecific, which is crucial to the building of social relationships and survival. Twenty-four hours after animals’ habituation to the apparatus, the subject mouse was placed in an open-field arena with an empty cage and another one containing a juvenile mouse. Mice tend to spend more time in the proximity of the cage containing the juvenile mouse than the empty one, offering an indication of sociability. One hour later, the experimental mouse was placed again in the same arena, but this time one cage contained the familiar mouse and the second one a novel juvenile mouse. The exploration times of the familiar and the novel mouse were recorded separately. Compound 14d at a dose of 5 mg/kg or vehicle was given intraperitoneally (i.p.) 30 min before the training session (Figure A). Compound 14d did not affect animals’ sociability as revealed by the longer time that they spent exploring the cage containing the social stimulus compared with the empty cage (Figure S5). In this respect, mice treated with compound 14d behaved like controls. During the test session, control mice recognized the familiar juvenile, since they spent more time exploring the novel one. Conversely, mice treated with 14d did not discriminate between the novel and the familiar mouse (Figure B). This result clearly suggests a social memory impairment, further confirmed by the negative discrimination index (DI) calculated for the group of animals receiving injections of 14d (Figure C).

Figure 6

Compound 14d impairs social recognition in mice. (A) Schematic drawings showing the sequence of procedures and treatment administrations. (B) Results are calculated as means of individual percentage of time spent exploring familiar (white columns) and novel (black columns) social stimuli. ***P < 0.001 vs the respective familiar subject [two-way analysis of variance (ANOVA) and Bonferroni’s MCT]. (C) Discrimination index calculated according to the formula tN – tF/tN + tF. **P < 0.01 vs vehicle (unpaired t-test). Shown are means ± S.E.M. of 10–11 animals per experimental group.

Compound 14d impairs social recognition in n class="Species">mice. (A) Schematic drawings showing the sequence of procedures and treatment administrations. (B) Results are calculated as means of individual percentage of time spent exploring familiar (white columns) and novel (black columns) social stimuli. ***P < 0.001 vs the respective familiar subject [two-way analysis of variance (ANOVA) and Bonferroni’s MCT]. (C) Discrimination index calculated according to the formula tN – tF/tN + tF. **P < 0.01 vs vehicle (unpaired t-test). Shown are means ± S.E.M. of 10–11 animals per experimental group.

Discussion and Conclusions

We present 2-aminopyrimidine derivatives with an n class="Chemical">alkylated 3-amino-azetidine moiety showing low nanomolar affinities for the H3R. Based on the non-imidazole partial agonist 11b identified as an in-house hit, a 23-membered compound set was synthesized and tested on H3R. The reduction of the substituent size at position 6 of the pyrimidine ring (R1) improved both affinity (pKi) and potency (pEC50) on hH3R. Each member of the extended R1 = H series (14) shows full agonism in a CRE-luciferase reporter gene assay, with three derivatives (14b–d) improving upon the endogenous ligand histamine, combining full H3R agonism with high (sub)nanomolar potencies. Most notably, the key non-imidazole H3R agonist 14d (VUF16839) combines high affinity to the H3R (pKi = 8.5) with full agonism at the H3R and a subnanomolar potency (pEC50 = 9.5) in a CRE-luciferase reporter gene assay. However, 14d acts as a partial agonist in an [35S]GTPγS accumulation assay with a 13-fold lower potency as compared with the more downstream CRE-luciferase reporter gene assay, which is most likely the consequence of signal amplification as suggested by a 25-fold higher potency of the full agonist histamine in the CRE-luciferase reporter gene assay as compared with an [35S]GTPγS accumulation assay.

From the recent development of numerous series of H4R antagonists, it is known that n class="Chemical">2-aminopyrimidine is a privileged scaffold for H4R antagonism.[35] As such, it is not surprising that 14d also binds with relatively high affinity to the H4R (pKi = 8.1). It acts as an agonist at H4R, but it is 10-fold less potent than at H3R, while 14d is not active at H1R and H2R.

The subnanomolar potency of the non-imidazole compound n class="Chemical">14d as an agonist at H3R is remarkable, as so far only a few low-potency and/or complex non-imidazole ligands have been reported as agonists at H3R.[27−32] Indeed, replacements of the imidazole ring while maintaining agonism have so far yielded little success. To illustrate, several imbutamine (2) analogues in which the imidazole moiety was replaced with an aminopyrimidine, aminopyridine or aminotriazole ring studied were not effective as H3R agonists.[34] Comparing the activities of the aminopyrimidine analog of 2 with 14d, the effective activation of H3R by 14d suggests a very important role of its azetidine side chain. Molecular docking combined with MD studies affords a predicted binding mode (Figure ) in which 14d interacts with the same key amino acids (D1143.32, E2065.461, Y3746.51) as histamine (1) (Figure E), suggesting that the 2-aminopyrimidine moiety mimics the imidazole ring. The computational studies also suggest that the azetidine side chain makes an ionic interaction with the same amino acid (D1143.32) as the amine group in the ethylamine side chain of histamine. Clearly, both 14d and histamine are able to interact with the same key amino acids of H3R, despite the fact that the binding modes of 14d and histamine do not substantially overlap.

The alkyl substitution of the basic n class="Chemical">amino moiety of 2 was studied previously in our group,[18] and subtle differences in alkyl substituents on the basic amine strongly influence the functional activity of such imbutamine analogs. Based on this SAR, we hypothesize that the basic amine of the 2-aminopyrimidine compound series 11–14 might be a functional “hot spot” as well. In the case of the R1 = iPr series (11), this appears to partially pan out as the chain elongation and dialkylation show moderate drops in intrinsic activity (α) (compare 11b vs 11c and 11i in Table ). However, such a trend was not observed within the other series 12–14 (Table and Figure D).

The analysis of additional properties for key compound 14d underscores its suitability for in vivo characterization in n class="Species">mice. That is, mouse potency data (mH3R: pEC50 = 10.0, mH4R: pEC50 = 9.0) and in vitro elimination parameters in mouse [t1/2 = 130.8 min; Clint = 20.7 mL/(min*kg)] all bode well. The inhibition of CYP enzymes is a general issue of imidazole-containing ligands.[49] Although the diaminopyrimidine core might conceivably also be prone to CYP inhibition, 14d only weakly (IC50 > 25 μM) interacts with selected key CYP enzymes (CYP3A4, CYP2D6, CYP2C9).

Poor brain penetration is a known problem of some n class="Chemical">imidazole-containing ligands,[5] limiting their potential administration routes in CNS-related experiments. As indicated by the effects in the in vivo social memory test, the 2-aminopyrimidine 14d (5 mg/kg i.p.) is clearly penetrating the CNS. Histamine is a known modulator of different types of memory.[3,52] H3R antagonists, such as thioperamide, improve short-term memory, while H3R agonists such as immepip (4) cause amnesia in the social recognition test.[51] The results shown here confirm and expand these observations, since a memory impairment was observed also for systemic treatment with compound 14d in the same paradigm (Figure ). It should be noted that H3R activation affects the circadian rhythm by increasing slow wave sleep and dose-dependently attenuates ciproxifan-induced waking effects.[53] Also, H3R agonists reduce stress-induced behavior in preclinical models.[54] These observations suggest that H3R activation may affect exploratory activity, which could negatively impact arousal and cognition. We did not specifically measure 14d-induced alterations of the sleep–wake cycle; however, 14d-associated social memory impairment does not seem to be related to sedative effects, as there were no statistically significant differences between the control group and 14d-treated mice in exploration time of the cage in the presence or absence of the social stimulus (during training: vehicle = 144.0 ± 60.0 s; 14d = 136.2 ± 44.1 s; during test: vehicle = 150.5 ± 42.4 s; 14d = 148.3 ± 69.4 s). Moreover, based on the mH3R/mH4R selectivity profile of 14d (vide supra), available literature on H4R expression, H4R agonist actions in the CNS, and the behavioral profile of H4R-deficient mice excluding a relevant role of this receptor on the histaminergic modulation of memory processing, it is highly unlikely that the in vivo amnesic effects of 14d are confounded by its H4R activity.[55,56] Indeed, the amnesic effects of 14d are consistent with the memory impairments observed following treatment with different H3R agonists [imetit (3) and (R)-α-methylhistamine] in the object recognition and passive avoidance tests in rats.[57]

To conclude, in this study, 2-aminopyrimidine derivatives with an n class="Chemical">alkylated 3-amino-azetidine side chain are presented as highly potent, non-imidazole agonists for the H3R. The key n-propyl derivative (14d, VUF16839) shows attractive in vitro pharmacological properties on human H3R (pKi = 8.5, pEC50 = 9.5, α ≥ 1.0 in a CRE-luciferase reporter gene assay with a 10-fold lower potency at H4R) and mouse H3R (pKi = 9.0, pEC50 = 10.0, with a >10-fold lower potency at mH4R). It exerts reasonable metabolic stability in rodent liver microsomes and weak activity on CYP enzymes. Moreover, 14d causes amnesic effects in social recognition tests in mice at 5 mg/kg, which is in line with the reported memory loss after administration of other H3R agonists.[51,57] The observed in vivo H3R effects also indicate appreciable brain penetration of 14d. Compound 14d can serve as a useful tool compound for fundamental studies concerning H3R, given its excellent affinity and potency, H3R agonism, and effective brain penetration.

Experimental Section

Pharmacology and ADME

Materials

[3H]NAMH (specific activity: 79.7 Ci/mmol) and [n class="Chemical">3H]histamine (specific activity: 17.5 Ci/mmol) were purchased from PerkinElmer (Groningen, the Netherlands). Human embryonic kidney 293T cells (HEK293T cells) were obtained from ATCC. Ketoconazole, quinidine, sulfaphenazole, and verapamil were obtained from Sigma-Aldrich (St. Louis, MO).

Cell Culture and Transfection

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (n class="Chemical">DMEM) supplemented with 10% fetal bovine serum and 1% penicillin and 1% streptomycin. Two million cells per 10 cm2 dishes were plated 24 h prior to transfection. Cells were transfected using the polyethylenimine (PEI) method.[58] For radioligand displacement assays, HEK293T cells were transfected with 2500 ng of complementary DNA (cDNA) encoding the hH3R (genbank: AF140538), mH3R (genbank: NM_133849.3), hH4R (genbank: AY136745), or mH4R (genbank: NM_153087.2) and 2500 ng of empty plasmid pcDEF3. The DNA/PEI mixture (ratio 1:4) was incubated for 20 min at 22 °C before addition to the cells.

Preparation of Cell Homogenates

Cell homogenates expressing the hH3R were harvested 48 h after transfection as reported previously.[59]

Radioligand Displacement Assays

[3H]NAMH and [n class="Chemical">3H]histamine displacement assays were performed in a binding buffer (50 mM Tris–HCl pH 7.4, 25 °C) by coincubation of 2 nM [3H]NAMH or 10 nM [3H]histamine, increasing concentrations of unlabeled ligand and cell homogenates expressing the hH3R/mH3R or hH4R, respectively. For mH4R displacement, similar studies were performed but with 30 nM [3H]histamine. The assay mixture was incubated for 2 h at 25 °C before rapid filtration over a 0.5% PEI-coated GF/C filter with a PerkinElmer filtermate harvester. The filter plate was dried, and 300 min after 25 μL of Microsint O was added, filter-bound radioactivity was measured with a Microbeta scintillation counter (PerkinElmer).

[35S]GTPγS Accumulation Assay

[35S]GTPγS accumulation experiments on n class="Gene">hH3R were performed as described previously.[60]

Reporter Gene Assay

HEK293T cells were transfected in suspension with cDn class="Chemical">NA encoding hH3R, mH3R, hH4R, mH4R, H1R (1000 ng) or H2R (2500 ng), CRE-luciferase (2500 ng) or NFAT-luciferase (2000 ng) for H1R, and empty pcDEF3 plasmid, and 50.000 cells per well were plated on a poly-l-lysine-coated white 96-well plate and grown for an additional 24 h. Cells were stimulated with increasing ligand concentrations for H3R and H4R in the presence of 1 μM forskolin at 37 °C and 5% CO2. After 6 h, the medium was aspirated and 25 μL of luciferase assay reagent [0.83 mM adenosine 5′-triphosphate, 0.83 mM d-luciferin, 18.7 mM MgCl2, 0.78 μM Na2PO4, 38.9 mM Tris–HCl (pH 7.8), 0.39% glycerol, 0.03% Triton-X-100, and 2.6 μM dithiothreitol] was added to each well. After 30 min of incubation at 37 °C, luminescence was measured with a Mithras plate reader (Berthold, Germany).

Data Analysis

Data were analyzed using GraphPad prism 7.02 (GraphPad Software Inc., San Diego). Shown data are mean ± S.E.M. of three individual experiments performed in triplicate unless stated otherwise. Competition binding curves were fitted to a one-site binding model. Obtained IC50 values were converted into pKi values using the Cheng–Prusoff equation.[61] Dose–response curves were fitted using nonlinear regression.

Metabolic Stability

The pharmacokinetic parameters of 14d and the reference drug n class="Chemical">verapamil were estimated by using rat liver microsomes (RLMs) or mouse liver microsomes (MLMs) obtained from Sigma-Aldrich (St. Louis, MO). The tested compounds (50 μM) were incubated in the presence of microsomes (1 mg/mL) for 5, 15, 30, and 45 min in 10 mM Tris–HCl buffer (pH = 7.4) at 37 °C. Cold methanol with an internal standard (IS) was added to terminate each reaction. Next, the reaction mixtures were centrifuged at 14 500 rpm. The disappearance of the tested compounds in time was calculated by the UPLC/MS Waters ACQUITY TQD system with a TQ Detector (Waters, Milford). The course of the reaction was followed by using the analyte/IS peak height ratio values. For the determination of the t1/2 value, the slope of linear regression from log concentration remaining versus time relationships (−k) was used according to Obach[48] (eq )

Conversion of t1/2 to intrinsic clearance Clint [in units of mL/(min*kg)] was done by using eq where 45 mg of microsomal protein per gram of liver tissue (g liver) and 87 g of liver per kilogram of body weight (kg b.w.) were applied to calculate Clint in mice, whereas 61 mg of microsomal protein per g liver and 45 g of liver per kg b.w. were applied to calculate Clint in n class="Species">rats, according to Huang et al.[62] and Smith et al.[63]

Effect on CYP

Luminescent CYP3A4, n class="Gene">CYP2D6, and CYP2C9 P450-Glo assays and protocols were obtained from Promega (Madison, WI). Compound 14d was tested in triplicate at the final concentrations in range from 0.01 to 25 μM. The luminescent signal was measured by using a microplate reader EnSpire PerkinElmer (Waltham, MA).

Social Recognition Test

Male C57Bl6 mice (8–9 weeks old) behavior was assessed in a test appan class="Species">ratus comprising an open-field plexiglass arena (45 × 25 cm2 and 20 cm high) placed in a sound-attenuated room. The assay paradigm comprises three sessions. In the first session, mice were placed in the arena containing two empty pencil-wire cups placed on opposing sides and left free to explore for 10 min. Twenty-four hours after this session, a juvenile mouse (stimulus, 4–5 weeks old), which had no prior contact with the subject mice, was placed under one of the wire cups while the other cup remained empty. The subject mouse was then placed in the arena and was left free to explore for 10 min. During the third session, performed 1 h later, the same stimulus animal was again placed under the wire cup and a novel unfamiliar juvenile mouse was placed under the opposing cup. Subject mice were then placed again in the arena and tested for discrimination between novel and familiar mice in a 10 min session. Each mouse was subjected to the procedure separately, and care was taken to remove any olfactory/taste cues by cleaning carefully the arena and wire cups between trials. The positions of the social stimuli (empty × social; familiar × novel) were counterbalanced across subjects and trials to prevent bias from place preference. Stimulus mice were habituated to remain under the wire cups several days before behavioral testing. Vehicle or 14d (5 mg/kg) was injected systemically (i.p.) 30 min before the second session. The animal’s behavior during all sessions was videotaped, and the time spent actively exploring the stimuli was analyzed by experienced observers unaware of the experimental groups. Exploration was defined as direct snout-to-cup contact, and the time spent climbing on the cups was not considered. Data are expressed as a percentage of time spent exploring each cup (social × nonsocial during the second session or familiar × novel during the third session), and statistical significance was determined by the two-way ANOVA followed by Bonferroni’s test. We also determined a sociability index, calculated according to the formula [time exploring social cup (tS) – time exploring nonsocial cup (tNS)]/[total exploration time (tS + tNS)], and a discrimination index (DI), which was calculated according to the formula [time exploring the novel mouse (tN) – time exploring the familiar mouse (tS)]/[total exploration time (tN + tF)], both analyzed using unpaired t-tests.

Computational Studies

Residue Numbering

Residue numbering is displayed throughout the manuscript as absolute sequence numbers and with generic numbering from GPCRdb[64] also in superscript, in which the first number denotes the helix, 1–8, and the second number denotes the residue position relative to the most-conserved residue, defined as number 50, in a gapped sequence alignment.

Homology Modeling

A three-dimensional model of the H3R was constructed on Modeller v.9.15[65] n class="Chemical">based on the crystal structure of H1R (PDB ID: 3RZE).[46] The sequence of H3R was obtained from UniProt[66] and aligned to the crystal structure sequence based on the structure-based alignment of GPCRdb. An optimal structure was selected based on its ability to retrospectively discriminate between known H3R fragmentlike ligands and true inactives as described elsewhere.[40]

Docking

A conformational library of all of the compounds was obtained with Corina v3.49[67] and protonated in ChemAxon Calculator.[68] The most energetically favorable conformations were docked using PLANTS v1.1.[69] Hundred docking poses were genen class="Species">rated per conformation and postprocessed with interaction fingerprints (IFPs) inferred from OpenEye’s OChem 1.3 library.[70,71] IFPs are bit vectors that are switched off (0) or on (1) depending on the occurrence of predefined intermolecular interactions [apolar, face-to-face, and face-to-edge aromatic interactions, hydrogen bonds (acceptor or donor), and ionic interactions (cationic or anionic)].

Molecular Dynamics Simulations and Analysis

Ligands were parametrized using the AM1-BCC charges in n class="Chemical">Antechamber.[72] The selected models were energy minimized to optimize protein–ligand interactions and used to run membrane-embedded MD simulations in GROMACS.[73] Each system was simulated for 100 ns after an equilibration of 5 ns, with the parameters and conditions described elsewhere.[74] Potential energy, root-mean-square deviation, root-mean-square fluctuation, and dihedrals of the simulations were analyzed with GROMACS tools, and residue interactions were analyzed with IFPs.

Nephelometry

In transparent flat-bottom 96-well plates, 14d was placed at different concentn class="Species">rations in triplicate (10–1, 10–1.5, 10–2, 10–2.5, 10–3, 10–3.5, 10–4, and 10–4.5 M) in Tris–HCl binding buffer (50 mM Tris–HCl, pH 7.4) at least 1 h before the measurement. A Kaolin dispersion was used as a positive control[75] in each plate at different concentrations (10–2.5, 10–3, 10–3.5, 10–4, 10–4.5, 10–5, and 10–5.5 M) under the same conditions as with compound 14d. Nephelometry measurements were performed with a NEPHELO star Plus (BMG Labtech, Germany) with the following settings: one cycle, measurement start time 0.1 s, measurement interval time 0.1 s, laser intensity 80%, beam focus 2.0 mm, orbital shaking mode at 200 rpm with an additional shaking time of 10 s before each cycle. Results were analyzed using Matlab R2014A (8.3.0.532) software, plotting all available data points and plotting mean and standard deviation values in a line chart compared to the Kaolin control. The linear fit (R2) of the Kaolin control was above 0.985 in all cases.

General Information

Chemicals and solvents were obtained from commercial suppliers and were used without further purification. THF was dried by passing through the PureSolv solvent purification system by Inert. All reactions were carried out under an inert n class="Chemical">N2 atmosphere. Hydrogenation experiments were performed with routine batch technology or the H-cube Mini Plus flow reactor. Microwave reactions were performed with the Biotage Initiator microwave system. Thin-layer chromatography analyses were performed with Merck F254 alumina silica plates using UV visualization or staining. Column purifications were carried out automatically using Biotage Isolera or Teledyne Isco CombiFlash equipment using Silicycle Ultra Pure silica gel. Melting point (mp) for final compounds was determined using a Büchi M-565 melting point apparatus at a rate of 1 °C/min. NMR spectra were recorded on a Bruker 250, 300, 500, or 600 MHz spectrometer. Chemical shifts are reported in ppm (δ), and the residual solvent was used as the internal standard (δ 1H NMR: CDCl3 7.26; CD3OD 4.87; D2O 4.79; 13C NMR: CDCl3 77.16; CD3OD 49.00). Data are reported as follows: chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; sext, sextet; hept, heptet; br, broad signal; m, multiplet; app, apparent), coupling constant(s) (Hz), and integration. High-resolution mass spectra (HRMS) were recorded on a Bruker microTOF mass spectrometer using electrospray ionization in the positive ion mode. Analytical high-performance liquid chromatography (HPLC)-mass spectrometry (MS) analyses were conducted using a Shimadzu LC-20AD liquid chromatograph pump system connected to a Shimadzu SPDM20A diode array detector with MS detection using a Shimadzu HPLC-MS 2010EV mass spectrometer. The column used is an Xbridge C18 5 mm column (50 mm × 4.6 mm). Acidic mode: solvent B (MeCN/0.1% formic acid) and solvent A (water/0.1% formic acid), flow rate of 1.0 mL/min with a run time of 8 min. For compounds whose retention time (tR) was less than 1.5 min with the acidic solvent system, a basic solvent system was used. Basic mode: solvent B (MeCN/10% buffer), solvent A (water/10% buffer). The buffer is a 0.4% (w/v) NH4HCO3 solution in water, adjusted to pH 8.0 with NH4OH. The analysis was conducted using a flow rate of 1.0 mL/min with a total run time of 8 min. Gradient settings (basic and acidic system): start 5% B, linear gradient to 90% B in 4.5 min, then isocratic for 1.5 min at 90% B, then linear gradient to 5% B in 0.5 min, then isocratic for 1.5 min at 5% B. All compounds (except 14c and 14f) have a purity of ≥95% calculated as the percentage peak area of the target compound by UV detection at 254 nm using the analytical HPLC-MS method listed above. Yields reported are not optimized. The compounds described in Table were checked for the presence of pan assay interference compounds (PAINS) substructures as described by Baell and Holloway,[76] and no PAINS substructures were identified.

4-(3-Aminoazetidin-1-yl)-6-isopropylpyrimidin-2-amine (11a)

To a solution of carbamate 21a (351 mg, 1.14 mmol) in n class="Chemical">MeOH (20 mL) was added aq HCl (37%, 0.35 mL, 4.23 mmol). The reaction mixture was stirred at rt overnight. The solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (10:1, 10 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The suspension was filtered. The solvents were removed under reduced pressure. The crude product was dissolved in hot EtOH (5 mL), and after addition of EtOAc (5 mL), a precipitate formed. The formed solid was collected by filtration, washed, and dried in vacuo. Purification by flash chromatography (DCM/MeOH/TEA 100:0:0–90:9:1) gave the title compound as a white solid (53 mg, 22%). mp: 157.0–157.6 °C. 1H NMR (600 MHz, CD3OD) δ 5.58 (s, 1H), 4.25 (t, J = 8.1 Hz, 2H), 3.95–3.85 (m, 1H), 3.72 (dd, J = 9.0, 5.2 Hz, 2H), 2.64 (hept, J = 7.0 Hz, 1H), 1.21 (d, J = 6.9 Hz, 6H). 13C NMR (151 MHz, CD3OD) δ 175.4, 165.7, 164.0, 89.8, 59.9, 44.1, 36.6, 22.0. HPLC-MS (basic mode): tR = 2.6 min, purity: >99%, [M + H]+: 208. HR-MS [M + H]+ calcd for C10H18N5+: 208.1557, found 208.1564.

4-Isopropyl-6-(3-(methylamino)azetidin-1-yl)pyrimidin-2-amine (11b)

To a solution of carbamate 21b (96 mg, 0.30 mmol) in n class="Chemical">DCM (5 mL) was added HCl in dioxane (4 N, 1.0 mL, 4.0 mmol). The reaction mixture was stirred for 3 h at rt. The reaction mixture was diluted with satd. aq Na2CO3 (10 mL) and extracted with DCM (3 × 5 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH/TEA 100:0:0–90:9:1) gave the title compound as a white solid (45 mg, 68%). mp: 81.8–82.4 °C. 1H NMR (500 MHz, CDCl3) δ 5.47 (s, 1H), 5.05 (br, 2H), 4.23–4.15 (m, 2H), 3.76–3.63 (m, 3H), 2.65 (hept, J = 6.9 Hz, 1H), 2.42 (s, 3H), 1.19 (d, J = 6.9 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 173.8, 164.4, 162.0, 89.1, 56.7, 50.5, 35.4, 33.3, 21.6. HPLC-MS (basic mode): tR = 3.1 min, purity: 97.3%, [M + H]+: 222. HR-MS [M + H]+ calcd for C11H20N5+: 222.1713, found 222.1718.

4-(3-(Ethylamino)azetidin-1-yl)-6-isopropylpyrimidin-2-amine (11c)

To a solution of carbamate 21c (79 mg, 0.24 mmol) in n class="Chemical">MeOH (4 mL) was added aq HCl (37%, 0.19 mL, 2.29 mmol). The reaction mixture was stirred at rt overnight. The solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (4:1, 8 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The inorganic salts were filtered off and washed with DCM/MeOH (4:1, 12 mL). The filtrate was concentrated under reduced pressure. The crude product was dissolved in hot EtOH (1 mL), and after addition of EtOAc (5 mL), a precipitate formed. The formed solid was collected by filtration, washed, and dried in vacuo. The title compound was obtained as a white solid (25 mg, 45%). mp: 226.2–227.3 °C. 1H NMR (600 MHz, CD3OD) δ 5.97 (s, 1H), 4.63 (br, 1H), 4.57 (br, 1H), 4.46 (br, 1H), 4.37 (br, 1H), 4.35–4.27 (m, 1H), 3.13 (q, J = 7.2 Hz, 2H), 2.87 (hept, J = 6.8 Hz, 1H), 1.38 (t, J = 7.2 Hz, 3H), 1.32 (d, J = 6.9 Hz, 6H). 13C NMR (151 MHz, CD3OD) δ 164.2, 163.6, 157.0, 91.5, 54.8, 54.4, 48.2, 42.4, 33.3, 20.9, 11.7. HPLC-MS (basic mode): tR = 3.2 min, purity: >99%, [M + H]+: 236. HR-MS [M + H]+ calcd for C12H22N5+: 236.1870, found 236.1868.

4-(3-(Dimethylamino)azetidin-1-yl)-6-isopropylpyrimidin-2-amine (11i)

A microwave vial charged with amine 17 (248 mg, 1.44 mmol), n class="Chemical">N,N-dimethylazetidin-3-amine dihydrochloride (250 mg, 1.44 mmol), DIPEA (0.76 mL, 4.33 mmol), and dioxane (10 mL) was heated for 30 min at 150 °C under microwave irradiation. The reaction mixture was diluted with water (20 mL) and extracted with DCM (3 × 20 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH/TEA 100:0:0–90:9:1) gave the title compound as a white solid (220 mg, 65%). mp: 123.6–123.8 °C. 1H NMR (500 MHz, CDCl3) δ 5.49 (s, 1H), 4.87 (br, 2H), 4.04 (t, J = 7.8 Hz, 2H), 3.94–3.81 (m, 2H), 3.21 (p, J = 5.6 Hz, 1H), 2.65 (hept, J = 6.8 Hz, 1H), 2.20 (s, 6H), 1.20 (d, J = 6.8 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 174.0, 164.6, 162.1, 89.1, 56.2, 53.9, 41.8, 35.5, 21.7. HPLC-MS (basic mode): tR = 3.4 min, purity: 97.8%, [M + H]+: 236. HR-MS [M + H]+ calcd for C12H22N5+: 236.1870, found 236.1878.

4-(3-Aminoazetidin-1-yl)-6-ethylpyrimidin-2-amine (12a)

To a solution of carbamate 22a (250 mg, 0.85 mmol) in n class="Chemical">MeOH (20 mL) was added aq HCl (37%, 0.21 mL, 2.54 mmol). The reaction mixture was stirred at rt overnight. The solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (10:1, 40 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The suspension was filtered. The solvents were removed under reduced pressure. The crude product was dissolved in hot EtOH (5 mL), and after addition of EtOAc (15 mL), a precipitate formed. The formed solid was collected by filtration, washed, and dried in vacuo. Dissolving in H2O (5 mL) and freeze-drying gave the title compound as a white fluffy solid (95 mg, 58%). mp: 224.7–225.9 °C. 1H NMR (600 MHz, CD3OD) δ 5.83 (s, 1H), 4.51–4.37 (m, 2H), 4.16–4.02 (m, 3H), 2.50 (q, J = 7.6 Hz, 2H), 1.18 (t, J = 7.6 Hz, 3H). 13C NMR (151 MHz, CD3OD) δ 164.1, 160.2, 157.4, 92.7, 56.5, 42.6, 27.1, 12.2. HPLC-MS (basic mode): tR = 2.3 min, purity: >99%, [M + H]+: 194. HR-MS [M + H]+ calcd for C9H16N5+: 194.1400, found 194.1410.

4-Ethyl-6-(3-(methylamino)azetidin-1-yl)pyrimidin-2-amine (12b)

To a solution of carbamate 22b (450 mg, 1.46 mmol) in n class="Chemical">MeOH (20 mL) was added aq HCl (37%, 0.36 mL, 4.35 mmol). The reaction mixture was stirred at rt overnight. The solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (10:1, 20 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The suspension was filtered. The solvents were removed under reduced pressure. The crude product was dissolved in hot EtOH (5 mL), and after addition of EtOAc (15 mL), a precipitate formed. The formed solid was collected by filtration, washed, and dried in vacuo. Dissolving in H2O (5 mL) and freeze-drying gave the title compound as a white fluffy solid (150 mg, 50%). mp: 202.8–203.3 °C. 1H NMR (600 MHz, CD3OD) δ 5.89 (s, 1H), 4.51–4.39 (m, 2H), 4.11 (dd, J = 10.1, 3.5 Hz, 2H), 3.96 (br, 1H), 2.58 (q, J = 7.9 Hz, 2H), 2.55 (s, 3H), 1.27 (t, J = 7.6 Hz, 3H). 13C NMR (151 MHz, CD3OD) δ 164.1, 160.4, 157.6, 92.6, 55.9, 50.4, 32.3, 27.2, 12.2. HPLC-MS (basic mode): tR = 2.6 min, purity: >99%, [M + H]+: 208. HR-MS [M + H]+ calcd for C10H18N5+: 208.1557, found 208.1566.

4-Ethyl-6-(3-(ethylamino)azetidin-1-yl)pyrimidin-2-amine (12c)

To a solution of carbamate 22c (117 mg, 0.36 mmol) in n class="Chemical">MeOH (4 mL) was added aq HCl (37%, 0.30 mL, 3.62 mmol). The reaction mixture was stirred at rt overnight. The solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (9:1, 3 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The inorganic salts were filtered off and washed with DCM/MeOH (9:1, 8 mL). The filtrate was concentrated under reduced pressure. The crude product was dissolved in hot EtOH (1.5 mL), and after addition of EtOAc (7 mL), a precipitate formed. The formed solid was collected by filtration, washed, and dried in vacuo. The title compound was obtained as a white solid (8 mg, 10%). mp: 236.6–236.9 °C. 1H NMR (600 MHz, CD3OD) δ 5.99 (s, 1H), 4.60 (br, 2H), 4.48–4.26 (m, 3H), 3.13 (q, J = 7.3 Hz, 2H), 2.64 (q, J = 7.6 Hz, 2H), 1.38 (t, J = 7.2 Hz, 3H), 1.31 (t, J = 7.6 Hz, 3H). 13C NMR (151 MHz, CD3OD) δ 164.2, 159.8, 157.0, 92.8, 54.8, 54.4, 48.2, 42.4, 26.8, 12.0, 11.7. HPLC-MS (basic mode): tR = 2.9 min, purity: >99%, [M + H]+: 222. HR-MS [M + H]+ calcd for C11H20N5+: 222.1713, found 222.1703.

4-(3-(Dimethylamino)azetidin-1-yl-6-ethyl)pyrimidin-2-amine (12i)

To a solution of amine 12b (110 mg, 0.53 mmol) in n class="Chemical">MeOH (5 mL) were added formaline (37%, 47 μL, 0.64 mmol) and AcOH (30 μL, 0.53 mmol). After 10 min of stirring at rt, NaBH(OAc)3 (169 mg, 0.80 mmol) was added, and the resulting mixture was stirred for 3 h at rt. The reaction mixture was quenched with 5 M aq NaOH (two drops). The solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (4:1, 8 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The inorganic salts were filtered and washed with DCM/MeOH (4:1, 12 mL). The filtrate was concentrated under reduced pressure. The crude product was purified by flash chromatography (DCM/MeOH/TEA 100:0:0–90:9:1). The selected fractions were collected, and the solvents were removed under reduced pressure. The residue was dissolved in DCM (5 mL) and washed with satd. aq Na2CO3 (10 mL). The aqueous phase was extracted with DCM (2 × 5 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was dissolved in MeOH (10 mL) and washed with c-hexane (20 mL). The c-hexane layer was extracted with MeOH (2 × 5 mL). The combined MeOH layers were concentrated in vacuo to give the title compound as a white solid (34 mg, 29%). mp: 128.7–128.9 °C. 1H NMR (600 MHz, CD3OD) δ 5.63 (s, 1H), 4.15–4.05 (m, 2H), 3.85 (dd, J = 9.0, 5.1 Hz, 2H), 3.30–3.23 (m, 1H), 2.43 (q, J = 7.6 Hz, 2H), 2.23 (s, 6H), 1.20 (t, J = 7.6 Hz, 3H). 13C NMR (151 MHz, CD3OD) δ 171.6, 165.6, 164.0, 91.3, 57.4, 54.7, 41.9, 31.2, 13.4. HPLC-MS (basic mode): tR = 2.9 min, purity: >99%, [M + H]+: 222. HR-MS [M + H]+ calcd for C11H20N5+: 222.1713, found 222.1705.

4-(3-Aminoazetidin-1-yl)-6-methylpyrimidin-2-amine (13a)

To a solution of carbamate 23a (723 mg, 2.59 mmol) in n class="Chemical">MeOH (25 mL) was added aq HCl (37%, 3.26 mL, 39.4 mmol). The reaction mixture was stirred at rt overnight. The solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (10:1, 40 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The suspension was filtered. The solvents were removed under reduced pressure. The crude product was recrystallized from EtOH. The crystals were collected by filtration, washed, and dried in vacuo. Dissolving in H2O (5 mL) and freeze-drying gave the title compound as a white fluffy solid (189 mg, 43%). mp: 204.7–206.5 °C. 1H NMR (600 MHz, CD3OD) δ 5.89 (s, 1H), 4.50–4.43 (m, 2H), 4.17–4.11 (m, 1H), 4.06 (dd, J = 10.6, 4.8 Hz, 2H), 2.28 (s, 3H). 13C NMR (151 MHz, CD3OD) δ 164.0, 157.8, 155.9, 94.0, 57.4, 43.0, 19.3. HPLC-MS (basic mode): tR = 2.0 min, purity: >99%, [M + H]+: 180. HR-MS [M + H]+ calcd for C8H14N5+: 180.1244, found 180.1244.

4-Methyl-6-(3-(methylamino)azetidin-1-yl)pyrimidin-2-amine Dihydrochloride (13b)

To a solution of carbamate 23b (193 mg, 0.66 mmol) in n class="Chemical">dioxane (2 mL) was added HCl in dioxane (4 N, 2.0 mL, 8.0 mmol). The reaction mixture was stirred at rt overnight. The solvents were removed under reduced pressure. The title compound was obtained as an off-white solid (176 mg, quant.). mp: 220.1–220.5 °C. 1H NMR (600 MHz, CD3OD/D2O) δ 6.01 (s, 1H), 4.69–4.56 (m, 2H), 4.46–4.29 (m, 3H), 2.82 (s, 3H), 2.36 (s, 3H). 13C NMR (151 MHz, CD3OD/D2O) δ 163.4, 156.2, 154.8, 94.3, 54.1, 53.7, 49.3, 31.6, 18.8. HPLC-MS (basic mode): tR = 2.3 min, purity: >99%, [M + H]+: 194. HR-MS [M + H]+ calcd for C9H16N5+: 194.1400, found 194.1401.

4-(3-(Ethylamino)azetidin-1-yl)-6-methylpyrimidin-2-amine (13c)

To a solution of carbamate 23c (124 mg, 0.40 mmol) in n class="Chemical">MeOH (4 mL) was added aq HCl (37%, 0.33 mL, 3.99 mmol). The reaction mixture was stirred for 3 h at rt. The solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (9:1, 8 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The inorganic salts were filtered off and washed with DCM/MeOH (4:1, 12 mL). The filtrate was concentrated under reduced pressure. The title compound was obtained as a white solid (70 mg, 84%). mp: 218.1–219.2 °C. 1H NMR (600 MHz, CD3OD) δ 5.85 (s, 1H), 4.44–4.34 (m, 2H), 4.03 (dd, J = 10.6, 4.8 Hz, 2H), 3.98–3.91 (m, 1H), 2.78 (q, J = 7.2 Hz, 2H), 2.26 (s, 3H), 1.20 (t, J = 7.2 Hz, 3H). 13C NMR (151 MHz, CD3OD) δ 164.0, 158.2, 156.4, 93.9, 56.6, 48.9, 42.2, 19.6, 13.8. HPLC-MS (basic mode): tR = 2.7 min, purity: 96.4%, [M + H]+: 208. HR-MS [M + H]+ calcd for C10H18N5+: 208.1557, found 208.1549.

4-(3-(Dimethylamino)azetidin-1-yl-6-methyl)pyrimidin-2-amine (13i)

Free base n class="Chemical">4-methyl-6-(3-(methylamino)azetidin-1-yl)pyrimidin-2-amine (160 mg, 0.83 mmol) was obtained from 13b by neutralization with NH3 solution (7 N) in MeOH and filtration of the inorganic salt, followed by evaporation of the solvent. The residue was dissolved in MeOH (5 mL). To this were added formaline (37%, 74 μL, 0.99 mmol) and AcOH (47 μL, 0.83 mmol). After 10 min of stirring at rt, NaBH(OAc)3 (263 mg, 1.24 mmol) was added, and the resulting mixture was stirred for 3 h at rt. The reaction mixture was quenched with 5 M aq NaOH (two drops). The solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (4:1, 8 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The inorganic salts were filtered off and washed with DCM/MeOH (4:1, 12 mL). The filtrate was concentrated under reduced pressure. The crude product was purified by flash chromatography (DCM/MeOH/TEA 100:0:0–90:9:1). The selected fractions were collected, and the solvents were removed under reduced pressure. The residue was dissolved in DCM (5 mL) and washed with satd. aq Na2CO3 (10 mL). The aqueous phase was extracted with DCM (2 × 5 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was dissolved in MeOH (10 mL) and washed with c-hexane (20 mL). The c-hexane layer was extracted with MeOH (2 × 5 mL). The combined MeOH layers were concentrated in vacuo to give the title compound as a white solid (65 mg, 38%). mp: 171.5–172.7 °C. 1H NMR (600 MHz, CD3OD) δ 5.63 (app d, J = 3.2 Hz, 1H), 4.15–4.04 (m, 2H), 3.91–3.80 (m, 2H), 3.30–3.23 (m, 1H), 2.23 (d, J = 2.5 Hz, 6H), 2.16 (s, 3H). 13C NMR (151 MHz, CD3OD) δ 166.2, 165.5, 163.9, 92.6, 57.4, 54.7, 41.9, 23.3. HPLC-MS (basic mode): tR = 2.6 min, purity: >99%, [M + H]+: 208. HR-MS [M + H]+ calcd for C10H18N5+: 208.1557, found 208.1550.

4-(3-Aminoazetidin-1-yl)pyrimidin-2-amine Fumarate (14a)

To a solution of carbamate 24a (433 mg, 1.63 mmol) in n class="Chemical">MeOH (25 mL) was added aq HCl (37%, 0.74 mL, 8.94 mmol). The reaction mixture was stirred at rt overnight. The solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (4:1, 25 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The suspension was filtered. The solvents were removed under reduced pressure. The free base (248 mg, 1.50 mmol) was dissolved in MeOH (4 mL), and fumaric acid (88 mg, 0.75 mmol) in MeOH (1 mL) was added. The clear solution was concentrated until a suspension formed. The suspension was cooled overnight at 5 °C. The formed solid was collected by filtration, washed, and dried in vacuo. Dissolving in H2O (5 mL) and freeze-drying gave the title compound as a white fluffy solid (134 mg, 37%). The base-to-fumaric-acid ratio was 1:0.5 based on the 1H NMR peak integration. mp: 222.7–224.1 °C. 1H NMR (600 MHz, CD3OD/D2O) δ 7.62 (d, J = 7.3 Hz, 1H), 6.45 (s, 1H), 6.01 (d, J = 7.3 Hz, 1H), 4.57 (br, 2H), 4.38–4.15 (m, 3H). 13C NMR (151 MHz, CD3OD/D2O) δ 174.7, 162.9, 155.8, 142.4, 136.3, 95.8, 55.4, 54.9, 41.7. HPLC-MS (basic mode): tR = 1.7 min, purity: 96.1%, [M + H]+: 166. HR-MS [M + H]+ calcd for C7H12N5+: 166.1087, found 166.1082.

4-(3-(Methylamino)azetidin-1-yl)pyrimidin-2-amine Fumarate (14b)

To a solution of carbamate 24b (501 mg, 1.79 mmol) in n class="Chemical">MeOH (8 mL) was added aq HCl (37%, 0.54 mL, 6.52 mmol). The reaction mixture was stirred at rt overnight. The solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (9:1, 25 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The suspension was filtered. The solvents were removed under reduced pressure. The crude free base (205 mg, 1.13 mmol) was dissolved in MeOH (25 mL), and fumaric acid (131 mg, 1.13 mmol) in MeOH (10 mL) was added. The clear solution was concentrated until a suspension formed. The suspension was cooled for 1 h at 5 °C. The formed solid was collected by filtration, washed, and dried in vacuo. Dissolving in H2O (5 mL) and freeze-drying gave the title compound as a white fluffy solid (250 mg, 53%). The base-to-fumaric-acid ratio was 1:0.7 based on the 1H NMR peak integration. mp: 230.1–230.5 °C. 1H NMR (600 MHz, CD3OD/D2O) δ 7.64 (d, J = 7.0 Hz, 1H), 6.43 (s, 1H), 5.96 (d, J = 7.1 Hz, 1H), 4.46 (dd, J = 11.1, 7.6 Hz, 2H), 4.18 (dd, J = 11.2, 4.5 Hz, 2H), 4.13–4.06 (m, 1H), 2.62 (s, 3H). 13C NMR (151 MHz, CD3OD/D2O) δ 175.2, 163.3, 157.7, 146.0, 136.5, 95.5, 54.5, 49.5, 31.8. HPLC-MS (basic mode): tR = 2.1 min, purity: 95.6%, [M + H]+: 180. HR-MS [M + H]+ calcd for C8H14N5+: 180.1244, found 180.1238.

4-(3-(Ethylamino)azetidin-1-yl)pyrimidin-2-amine Fumarate (14c)

To a solution of carbamate n class="Chemical">24c (117 mg, 0.40 mmol) in MeOH (4 mL) was added aq HCl (37%, 0.30 mL, 3.62 mmol). The reaction mixture was stirred at rt overnight. The solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (9:1, 4 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The suspension was filtered. The solvents were removed under reduced pressure. The crude free base (54 mg, 0.28 mmol) was dissolved in MeOH (8 mL), and fumaric acid (32 mg, 0.28 mmol) in MeOH (3 mL) was added. After partial evaporation of MeOH and addition of EtOAc (3 mL), a precipitate was formed. The suspension was cooled for 3 h at 5 °C. The formed solid was collected by filtration, washed, and dried in vacuo. Dissolving in H2O (5 mL) and freeze-drying gave the title compound as a white fluffy solid (18 mg, 17%). The base-to-fumaric-acid ratio was 1:0.6 based on the 1H NMR peak integration. mp: 206.8–208.5 °C. 1H NMR (600 MHz, CD3OD/D2O) δ 7.76 (d, J = 7.3 Hz, 1H), 6.70 (s, 1H), 6.16 (d, J = 7.3 Hz, 1H), 4.68 (br, 2H), 4.55–4.31 (m, 3H), 3.19 (q, J = 7.3 Hz, 2H), 1.41 (t, J = 7.3 Hz, 3H). 13C NMR (151 MHz, CD3OD/D2O) δ 171.9, 163.2, 156.0, 142.7, 136.0, 95.7, 54.5, 54.1, 47.7, 42.3, 11.6. HPLC-MS (basic mode): tR = 2.2 min, purity: 91.8%, [M + H]+: 194. HR-MS [M + H]+ calcd for C9H16N5+: 194.1400, found 194.1393.

4-(3-(Propylamino)azetidin-1-yl)pyrimidin-2-amine Fumarate Hydrate (14d, VUF16839)

To a solution of carbamate 24d (1.19 g, 3.87 mmol) in n class="Chemical">MeOH (20 mL) was added aq HCl (37%, 3.2 mL, 38.6 mmol). The reaction mixture was stirred at rt overnight and subsequently at 50 °C for 30 min. The solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (4:1, 20 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The suspension was filtered. The solvents were removed under reduced pressure. The crude free base (871 mg, 3.87 mmol) was dissolved in MeOH (10 mL), and fumaric acid (453 mg, 3.90 mmol) in MeOH (10 mL) was added. The solvent was removed under reduced pressure, and the residue was recrystallized from EtOH. The suspension was cooled for 1 h at 5 °C. The crystals were filtered, washed with EtOH, and dried in vacuo. Dissolving in H2O (20 mL) and freeze-drying gave the title compound as a white fluffy solid (803 mg, 58%). The base-to-fumaric-acid ratio was 1:1 based on the 1H NMR peak integration. mp: 213.8–214.2 °C. 1H NMR (500 MHz, D2O) δ 7.61 (d, J = 7.3 Hz, 1H), 6.43 (s, 2H), 5.99 (d, J = 7.3 Hz, 1H), 4.56 (br, 2H), 4.40–4.19 (m, 3H), 3.06–2.92 (m, 2H), 1.68 (sext, J = 7.5 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H). 13C NMR (151 MHz, CD3OD/D2O) δ 175.2, 162.9, 155.8, 142.7, 136.3, 95.7, 54.4 53.9, 48.4, 47.9, 20.2, 11.1. HPLC-MS (basic mode): tR = 2.7 min, purity: 97.9%, [M + H]+: 208. HR-MS [M + H]+ calcd for C10H18N5+: 208.1557, found 208.1566. Anal. calcd for C10H17N5·C4H4O4·1.75H2O: C, 47.38; H, 6.96; N, 19.73; O, 25.92; Cl, 0.00. Found: C, 47.07; H, 6.97; N, 19.43; O, 25.60; Cl, <0.1.

4-(3-(Isopropylamino)azetidin-1-yl)pyrimidin-2-amine Fumarate (14e)

To a solution of free base n class="Chemical">14a (100 mg, 0.60 mmol) in DCM (4 mL) and MeOH (1 mL) were added acetone (48 μL, 0.66 mmol) and AcOH (34 μL, 0.60 mmol). After 10 min of stirring at rt, NaBH(OAc)3 (201 mg, 0.90 mmol) was added, and the resulting mixture was stirred at rt overnight. The reaction mixture was quenched with 5 M aq NaOH (two drops). The solvents were removed under reduced pressure. The residue was purified by column chromatography (DCM/MeOH/TEA 100:0:0–80:18:2). The selected fractions were collected, and the solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (9:1, 10 mL) and washed with satd. aq Na2CO3 solution (10 mL). The aqueous layer was extracted with DCM/MeOH (9:1, 2 × 5 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. The free base (32 mg, 0.154 mmol) was dissolved in MeOH (1 mL), and fumaric acid (18 mg, 0.154 mmol) in MeOH (1 mL) was added. After partial evaporation of MeOH and addition of EtOAc (5 mL), a precipitate was formed. The suspension was cooled for 1 h at 5 °C. The formed solid was collected by filtration, washed, and dried in vacuo. Dissolving in H2O (5 mL) and freeze-drying gave the title compound as a white fluffy solid (42 mg, 22%). The base-to-fumaric-acid ratio was 1:1 based on the 1H NMR peak integration. mp: 219.0–219.3 °C. 1H NMR (500 MHz, CD3OD/D2O) δ 7.76 (d, J = 7.1 Hz, 1H), 6.68–6.55 (m, 2H), 6.14–6.03 (m, 1H), 4.67–4.56 (m, 2H), 4.43–4.29 (m, 3H), 3.51–3.39 (m, 1H), 1.43–1.34 (m, 6H). 13C NMR (151 MHz, CD3OD/D2O) δ 174.2, 174.0, 163.4, 157.4, 157.1, 144.9, 144.5, 136.7, 95.5, 95.4, 55.3, 55.1, 50.9, 50.6, 46.2, 46.1, 20.0, 19.8 (multiple sets observed). HPLC-MS (basic mode): tR = 2.7 min, purity: >99%, [M + H]+: 208. HR-MS [M + H]+ calcd for C10H18N5+: 208.1557, found 208.1566.

4-(3-(Butylamino)azetidin-1-yl)pyrimidin-2-amine Fumarate (14f)

To a solution of carbamate 24f (32 mg, 0.19 mmol) in n class="Chemical">MeOH (3 mL) was added aq HCl (37%, 0.158 mL, 1.19 mmol). The reaction mixture was stirred at rt overnight. The solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (4:1, 5 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The suspension was filtered. The solvents were removed under reduced pressure. The free base (48 mg, 0.19 mmol) was dissolved in MeOH (1 mL), and fumaric acid (22 mg, 0.19 mmol) in MeOH (1 mL) was added. After partial evaporation of MeOH and addition of EtOAc (5 mL), a precipitate formed. The suspension was cooled for 1 h at 5 °C. The formed solid was collected by filtration, washed, and dried in vacuo. The title compound was obtained as a white solid (34 mg, 58%). The base-to-fumaric-acid ratio was 1:0.75 based on the 1H NMR peak integration. mp: 169.6–169.9 °C. 1H NMR (600 MHz, CD3OD/D2O) δ 7.72–7.66 (m, 1H), 6.66 (s, 1H), 6.06 (d, J = 7.2 Hz, 1H), 4.54 (br, 2H), 4.34 (br, 2H), 4.27–4.21 (m, 1H), 3.02–2.95 (m, 2H), 1.72–1.64 (m, 2H), 1.43 (sext, J = 7.4 Hz, 2H), 0.97 (t, J = 7.4 Hz, 3H). 13C NMR (151 MHz, CD3OD/D2O) δ 170.0, 163.6, 156.7, 143.1, 135.8, 95.5, 54.7, 48.6, 47.0, 29.6, 20.9, 13.9. HPLC-MS (basic mode): tR = 3.1 min, purity: 94.6%, [M + H]+: 222. HR-MS [M + H]+ calcd for C11H20N5+: 222.1713, found 222.1705.

(rac)-4-(3-(sec-Butylamino)azetidin-1-yl)pyrimidin-2-amine Fumarate (14g)

To a solution of free base n class="Chemical">14a (100 mg, 0.60 mmol) in DCM (4 mL) and MeOH (1 mL) were added butane-2-one (64 μL, 0.66 mmol) and AcOH (34 μL, 0.60 mmol). After 10 min of stirring at rt, NaBH(OAc)3 (201 mg, 0.90 mmol) was added, and the resulting mixture was stirred at rt overnight. The reaction mixture was quenched with 5 M aq NaOH (two drops). The solvents were removed under reduced pressure. The residue was purified by column chromatography (DCM/MeOH/TEA 100:0:0–80:18:2). The selected fractions were collected, and the solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (9:1, 10 mL) and washed with satd. aq Na2CO3 solution (10 mL). The aqueous layer was extracted with DCM/MeOH (9:1, 2 × 5 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. The free base (25 mg, 0.113 mmol) was dissolved in MeOH (1 mL), and fumaric acid (13 mg, 0.113 mmol) in MeOH (1 mL) was added. After partial evaporation of MeOH and addition of EtOAc (5 mL), a precipitate formed. The suspension was cooled for 1 h at 5 °C. The formed solid was collected by filtration, washed, and dried in vacuo. Dissolving in H2O (5 mL) and freeze-drying gave the title compound as a white fluffy solid (32 mg, 16%). The base-to-fumaric-acid ratio was 1:1 based on the 1H NMR peak integration. mp: 214.6–215.4 °C. 1H NMR (500 MHz, CD3OD/D2O) δ 7.74 (d, J = 7.1 Hz, 1H), 6.64–6.53 (m, 2H), 6.12–6.05 (m, 1H), 4.62 (br, 2H), 4.47–4.30 (m, 3H), 3.28 (br, 1H), 1.90–1.80 (m, 1H), 1.69–1.57 (m, 1H), 1.38–1.30 (m, 3H), 1.05 (t, J = 7.5 Hz, 3H). 13C NMR (151 MHz, CD3OD/D2O) δ 174.5, 174.1, 163.2, 156.9, 156.8, 144.3, 143.8, 136.7, 95.6, 95.5, 56.1, 56.0, 55.0, 46.1, 46.0, 27.4, 27.2, 16.3, 16.2, 9.8 (multiple sets observed). HPLC-MS (basic mode): tR = 3.0 min, purity: 95.6%, [M + H]+: 222. HR-MS [M + H]+ calcd for C11H20N5+: 222.1713, found 222.1715.

4-(3-(Isobutylamino)azetidin-1-yl)pyrimidin-2-amine Fumarate (14h)

To a solution of free base n class="Chemical">14a (100 mg, 0.60 mmol) in DCM (4 mL) and MeOH (1 mL) were added isobutyraldehyde (66 μL, 0.66 mmol) and AcOH (34 μL, 0.60 mmol). After 10 min of stirring at rt, NaBH(OAc)3 (201 mg, 0.90 mmol) was added, and the resulting mixture was stirred at rt overnight. The reaction mixture was quenched with 5 M aq NaOH (two drops). The solvents were removed under reduced pressure. The residue was purified by column chromatography (DCM/MeOH/TEA 100:0:0–80:18:2). The selected fractions were collected, and the solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (9:1, 10 mL) and washed with satd. aq Na2CO3 solution (10 mL). The aqueous layer was extracted with DCM/MeOH (9:1, 2 × 5 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. The free base (59 mg, 0.267 mmol) was dissolved in MeOH (1 mL), and fumaric acid (31 mg, 0.267 mmol) in MeOH (1 mL) was added. After partial evaporation of MeOH and addition of EtOAc (5 mL), a precipitate formed. The suspension was cooled for 1 h at 5 °C. The formed solid was collected by filtration, washed, and dried in vacuo. Dissolving in H2O (5 mL) and freeze-drying gave the title compound as a white fluffy solid (78 mg, 38%). The base-to-fumaric-acid ratio was 1:1 based on the 1H NMR peak integration. mp: 230.9–231.3 °C. 1H NMR (500 MHz, CD3OD/D2O) δ 7.73 (d, J = 7.2 Hz, 1H), 6.59–6.55 (m, 2H), 6.09 (d, J = 7.2 Hz, 1H), 4.59 (t, J = 9.5 Hz, 2H), 4.37 (br app d, 2H), 4.30–4.23 (m, 1H), 2.88 (dd, J = 7.2, 3.6 Hz, 2H), 2.11–2.03 (m, 1H), 1.08 (d, J = 6.7 Hz, 6H). 13C NMR (151 MHz, CD3ODD2O) δ 174.3, 174.2, 163.2, 156.7, 156.7, 143.7, 143.7, 136.6, 95.6, 95.5, 54.6, 54.0, 48.6, 27.3, 27.2, 20.3 (multiple sets observed). HPLC-MS (basic mode): tR = 3.1 min, purity: >99%, [M + H]+: 222. HR-MS [M + H]+ calcd for C11H20N5+: 222.1713, found 222.1716.

4-(3-(Dimethylamino)azetidin-1-yl)pyrimidin-2-amine Fumarate (14i)

To a solution of amine fumarate 14b (100 mg, 0.34 mmol) in n class="Chemical">MeOH (25 mL) were added formaline (37%, 70 μL, 0.94 mmol) and AcOH (20 μL, 0.34 mmol). After 10 min of stirring at rt, NaBH(OAc)3 (108 mg, 0.68 mmol) was added, and the resulting mixture was stirred at rt overnight. The reaction mixture was quenched with 5 M aq NaOH (two drops). The solvents were removed under reduced pressure. The residue was purified by column chromatography (DCM/MeOH/TEA 100:0:0–90:9:1). The selected fractions were collected, and the solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (9:1, 20 mL). The pH was adjusted to above 10 with NH3 solution (7 N) in MeOH. The suspension was filtered. The solvents were removed under reduced pressure. The crude free base (25 mg, 0.130 mmol) was dissolved in MeOH (5 mL), and fumaric acid (7.5 mg, 0.065 mmol) in MeOH (2.5 mL) was added. The solvent was evaporated until a suspension formed. The suspension was cooled overnight at 5 °C. The formed solid was collected by filtration, washed, and dried in vacuo. Dissolving in H2O (5 mL) and freeze-drying gave the title compound as a white fluffy solid (31 mg, 36%). The base-to-fumaric-acid ratio was 1:0.5 based on the 1H NMR peak integration. mp: 224.0–224.4 °C. 1H NMR (500 MHz, CD3OD/D2O) δ 7.66 (d, J = 7.0 Hz, 1H), 6.50 (s, 1H), 5.98 (d, J = 7.0 Hz, 1H), 4.35–4.27 (m, 2H), 4.08 (dd, J = 10.8, 4.9 Hz, 2H), 3.62–3.53 (m, 1H), 2.36 (s, 6H). 13C NMR (151 MHz, CD3OD/D2O) δ 175.0, 163.2, 157.8, 145.6, 136.7, 95.4, 56.4, 54.4, 41.5. HPLC-MS (basic mode): tR = 2.4 min, purity: >99%, [M + H]+: 194. HR-MS [M + H]+ calcd for C9H16N5+: 194.1400, found 194.1395.

4-(3-(Diethylamino)azetidin-1-yl)pyrimidin-2-amine Fumarate (14j)

To a solution of free base n class="Chemical">14a (100 mg, 0.60 mmol) in DCM (4 mL) and MeOH (1 mL) were added acetaldehyde (0.34 mL, 6 mmol) and AcOH (34 μL, 0.60 mmol). After 10 min of stirring at rt, NaBH(OAc)3 (402 mg, 1.80 mmol) was added, and the resulting mixture was stirred at rt overnight. The reaction mixture was quenched with 5 M aq NaOH (two drops). The solvents were removed under reduced pressure. The residue was purified by column chromatography (DCM/MeOH/TEA 100:0:0–80:18:2). The selected fractions were collected, and the solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (9:1, 10 mL) and washed with satd. aq Na2CO3 solution (10 mL). The aqueous layer was extracted with DCM/MeOH (9:1, 2 × 5 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. The free base (82 mg, 0.37 mmol) was dissolved in MeOH (1 mL), and fumaric acid (43 mg, 0.37 mmol) in MeOH (1 mL) was added. After partial evaporation of MeOH and addition of EtOAc (5 mL), a precipitate formed. The suspension was cooled for 1 h at 5 °C. The formed solid was collected by filtration, washed, and dried in vacuo. Dissolving in H2O (5 mL) and freeze-drying gave the title compound as a white fluffy solid (90 mg, 44%). The base-to-fumaric-acid ratio was 1:1 based on the 1H NMR peak integration. mp: 229.6–229.8 °C. 1H NMR (600 MHz, CD3OD/D2O) δ 7.74 (d, J = 7.1 Hz, 1H), 6.64–6.55 (m, 2H), 6.10 (dd, J = 7.2, 4.0 Hz, 1H), 4.62–4.51 (m, 2H), 4.50–4.41 (m, 2H), 4.41–4.29 (m, 1H), 3.28–3.13 (m, 4H), 1.40–1.27 (m, 6H). 13C NMR (151 MHz, CD3OD/D2O) δ 174.1, 173.8, 163.3, 157.0, 156.9, 144.1, 144.0, 136.6, 95.6, 95.5, 53.9, 52.7, 45.6, 45.4, 9.3, 9.2 (multiple sets observed). HPLC-MS (basic mode): tR = 3.0 min, purity: 97.3%, [M + H]+: 222. HR-MS [M + H]+ calcd for C11H20N5+: 222.1713, found 222.1722.

4-(3-(Pyrrolidin-1-yl)azetidin-1-yl)pyrimidin-2-amine Fumarate (14k)

To a solution of free base n class="Chemical">14a (100 mg, 0.60 mmol) in MeCN (20 mL) were added 1,4-diiodobutane (0.095 mL, 0.72 mmol) and K2CO3 (166 mg, 1.20 mmol). The resulting mixture was heated at reflux for 16 h. The solvent was removed under reduced pressure. The residue was purified by column chromatography (DCM/MeOH/TEA 100:0:0–80:18:2). The selected fractions were collected, and the solvents were removed under reduced pressure. The residue was dissolved in DCM/MeOH (9:1, 10 mL) and washed with satd. aq Na2CO3 solution (10 mL). The aqueous layer was extracted with DCM/MeOH (9:1, 2 × 5 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. The free base (32 mg, 0.146 mmol) was dissolved in MeOH (1 mL), and fumaric acid (17 mg, 0.146 mmol) in MeOH (1 mL) was added. After partial evaporation of MeOH and addition of EtOAc (5 mL), a precipitate formed. The suspension was cooled for 1 h at 5 °C. The formed solid was collected by filtration, washed, and dried in vacuo. Dissolving in H2O (5 mL) and freeze-drying gave the title compound as a white fluffy solid (20 mg, 9%). The base-to-fumaric-acid ratio was 1:1.15 based on the 1H NMR peak integration. mp: 228.3–228.6 °C. 1H NMR (600 MHz, CD3OD/D2O) δ 7.74 (d, J = 7.2 Hz, 1H), 6.58 (s, 2H), 6.12 (d, J = 7.2 Hz, 1H), 4.63 (br, 2H), 4.46 (br, 2H), 4.39–4.31 (m, 1H), 3.42 (br, 4H), 2.24–2.11 (m, 4H). 13C NMR (151 MHz, CD3OD/D2O) δ 173.8, 163.2, 156.3, 143.2, 136.4, 95.6, 54.6, 54.1, 53.3, 24.2. HPLC-MS (basic mode): tR = 2.8 min, purity: >99%, [M + H]+: 220. HR-MS [M + H]+ calcd for C11H18N5+: 220.1557, found 220.1563.

4-Chloro-6-isopropylpyrimidin-2-amine (17)

Pyrimidin-4(3H)-one 15 (4.87 g, 31.8 mmol) was dissolved in n class="Chemical">POCl3 (40 mL, 0.43 mol). The mixture was heated to reflux for 3 h. The solvent was removed under reduced pressure. Ice (150 g) was carefully added to the residue. The pH of the mixture was adjusted to 9–10 with aq NaOH (2.5 M). The mixture was extracted with DCM (3 × 100 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH 10:0–9:1) gave the title compound as an off-white solid (1.41 g, 26%). 1H NMR (500 MHz, CDCl3) δ 6.53 (s, 1H), 5.33 (br, 2H), 2.77 (hept, J = 6.9 Hz, 1H), 1.22 (d, J = 6.9 Hz, 6H). HPLC-MS (acidic mode): tR = 3.5 min, purity: 96.8%, [M + H]+: 172.

4-Chloro-6-ethylpyrimidin-2-amine (18)

Pyrimidin-4(3H)-one 16 (6.13 g, 44.1 mmol) was dissolved in n class="Chemical">POCl3 (50 mL, 0.54 mol). The mixture was heated to reflux for 3 h. The solvent was removed under reduced pressure. Ice (150 g) was carefully added to the residue. The pH of the mixture was adjusted to 9–10 with aq NaOH (2.5 M). The mixture was extracted with DCM (3 × 10 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH 10:0–9:1) gave the title compound as an off-white solid (3.63 g, 52%). 1H (500 MHz, CDCl3) δ 6.53 (s, 1H), 5.43 (br, 2H), 2.57 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). HPLC-MS (acidic mode): tR = 3.0 min, purity: >99%, [M + H]+: 158.

tert-Butyl (1-(2-Amino-6-isopropylpyrimidin-4-yl)azetidin-3-yl)carbamate (21a)

A microwave vial charged with chloride 17 (400 g, 2.33 mmol), n class="Chemical">carbamate 28a (402 mg, 2.33 mmol), DIPEA (0.41 mL, 2.33 mmol), and dioxane (10 mL) was heated for 120 min at 150 °C under microwave irradiation. The reaction mixture was diluted with water (20 mL) and extracted with DCM (3 × 20 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH/TEA 100:0:0–90:9:1) gave the title compound as a colorless oil (351 mg, 49%). 1H NMR (300 MHz, CDCl3) δ 5.48 (s, 1H), 5.01 (br, 1H), 4.80 (br, 2H), 4.57 (br, 1H), 4.31 (t, J = 8.3 Hz, 2H), 3.89–3.74 (m, 2H), 2.65 (hept, J = 6.9 Hz, 1H), 1.45 (s, 9H), 1.19 (d, J = 6.9 Hz, 6H). HPLC-MS (acidic mode): tR = 3.1 min, purity: >99%, [M + H]+: 308.

tert-Butyl (1-(2-Amino-6-isopropylpyrimidin-4-yl)azetidin-3-yl)(methyl)carbamate (21b)

A microwave vial charged with chloride 17 (300 mg, 1.75 mmol), n class="Chemical">carbamate hydrochloride 28b (389 mg, 1.75 mmol), DIPEA (0.61 mL, 3.50 mmol), and dioxane (10 mL) was heated for 30 min at 150 °C under microwave irradiation. The reaction mixture was diluted with water (20 mL) and extracted with DCM (3 × 15 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH 10:0–9:1) gave the title compound as a yellow oil (280 mg, 50%). 1H NMR (250 MHz, CDCl3) δ 5.50 (s, 1H), 5.03 (br, 1H), 4.79 (br, 2H), 4.20 (t, J = 8.6 Hz, 2H), 4.07–3.95 (m, 2H), 2.93 (s, 3H), 2.65 (hept, J = 6.9 Hz, 1H), 1.46 (s, 9H), 1.20 (d, J = 6.9 Hz, 6H). HPLC-MS (acidic mode): tR = 3.4 min, purity: >99%, [M + H]+: 322.

tert-Butyl (1-(2-Amino-6-isopropylpyrimidin-4-yl)azetidin-3-yl)(ethyl)carbamate (21c)

Carbamate 27c (1.02 g, 2.78 mmol) was dissolved in n class="Chemical">MeOH/EtOH (10:10 mL) and reacted with H2 gas under atmospheric pressure using Pd/C (5%, 0.60 g) overnight at rt. The mixture was filtered over Celite, and the filtrate was concentrated in vacuo. The resulting yellowish oil (900 mg, a mixture of intermediate and diphenylmethane) was used in the next step without further purification. A microwave vial charged with chloride 17 (103 mg, 0.60 mmol), crude intermediate (221 mg), DIPEA (0.105 mL, 0.60 mmol), and dioxane (5 mL) was heated for 90 min at 150 °C under microwave irradiation. The reaction mixture was diluted with water (10 mL) and extracted with DCM (3 × 10 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH 10:0–9:1) gave the title compound as a yellowish oil (79 mg, 35% over two steps, extrapolated). 1H NMR (300 MHz, CDCl3) δ 6.03 (br, 2H), 5.46 (s, 1H), 5.06 (br, 1H), 4.73 (br, 1H), 4.25 (t, J = 8.7 Hz, 2H), 4.17–4.05 (m, 2H), 3.33 (q, J = 7.0 Hz, 2H), 2.76 (hept, J = 6.9 Hz, 1H), 1.46 (s, 9H), 1.23 (d, J = 6.9 Hz, 6H), 1.14 (t, J = 7.0 Hz, 3H). HPLC-MS (acidic mode): tR = 3.6 min, purity: >99%, [M + H]+: 336.

tert-Butyl (1-(2-Amino-6-ethylpyrimidin-4-yl)azetidin-3-yl)carbamate (22a)

A microwave vial charged with chloride 18 (500 mg, 3.17 mmol), n class="Chemical">carbamate 28a (546 mg, 3.17 mmol), DIPEA (0.55 mL, 3.17 mmol), and dioxane (11 mL) was heated for 45 min at 150 °C under microwave irradiation. The reaction mixture was diluted with water (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH 10:0–9:1) gave the title compound as a colorless oil (250 mg, 27%). 1H NMR (300 MHz, CDCl3) δ 5.49 (s, 1H), 4.99 (br, 1H), 4.78 (br, 2H), 4.58 (br, 1H), 4.31 (t, J = 8.3 Hz, 2H), 3.85–3.73 (m, 2H), 2.46 (q, J = 7.6 Hz, 2H), 1.45 (s, 9H), 1.20 (t, J = 7.6 Hz, 3H). HPLC-MS (acidic mode): tR = 3.0 min, purity: 94.5%, [M + H]+: 294.

tert-Butyl (1-(2-Amino-6-ethylpyrimidin-4-yl)azetidin-3-yl)(methyl)carbamate (22b)

A microwave vial charged with chloride 18 (500 mg, 3.17 mmol), n class="Chemical">carbamate hydrochloride 28b (707 mg, 3.17 mmol), DIPEA (1.11 mL, 6.35 mmol), and dioxane (11 mL) was heated for 45 min at 150 °C under microwave irradiation. The reaction mixture was diluted with water (15 mL) and extracted with DCM (3 × 10 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH 10:0–9:1) gave the title compound as a colorless oil (450 mg, 46%). 1H NMR (300 MHz, CDCl3) δ 5.52 (s, 1H), 5.00 (br, 1H), 4.77 (br, 2H), 4.20 (t, J = 8.6 Hz, 2H), 4.07–3.94 (m, 2H), 2.92 (s, 3H), 2.47 (q, J = 7.6 Hz, 2H), 1.46 (s, 9H), 1.20 (t, J = 7.6 Hz, 3H). HPLC-MS (acidic mode): tR = 3.1 min, purity: >99%, [M + H]+: 308.

tert-Butyl (1-(2-Amino-6-ethylpyrimidin-4-yl)azetidin-3-yl)(ethyl)carbamate (22c)

Carbamate 27c (1.02 g, 2.78 mmol) was dissolved in n class="Chemical">MeOH/EtOH (10:10 mL) and reacted with H2 gas under atmospheric pressure using Pd/C (5%, 0.60 g) overnight at rt. The mixture was filtered over Celite, and the filtrate was concentrated in vacuo. The resulting yellowish oil (900 mg, a mixture of intermediate and diphenylmethane) was used in the next step without further purification. A microwave vial charged with chloride 18 (95 mg, 0.60 mmol), crude intermediate (221 mg), DIPEA (0.105 mL, 0.60 mmol), and dioxane (5 mL) was heated for 90 min at 150 °C under microwave irradiation. The reaction mixture was diluted with water (10 mL) and extracted with DCM (3 × 10 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH 10:0–9:1) gave the title compound as a yellowish oil (92 mg, 42% over two steps, extrapolated). 1H NMR (300 MHz, CDCl3) δ 5.54 (s, 1H), 4.83 (br, 3H), 4.24 (t, J = 8.5 Hz, 2H), 4.12–3.96 (m, 2H), 3.36 (q, J = 7.1 Hz, 2H), 2.49 (q, J = 7.6 Hz, 2H), 1.48 (s, 9H), 1.29–1.11 (m, 6H). HPLC-MS (acidic mode): tR = 3.3 min, purity: >99% %, [M + H]+: 322.

tert-Butyl (1-(2-Amino-6-methylpyrimidin-4-yl)azetidin-3-yl)carbamate (23a)

A microwave vial charged with chloride 19 (1.00 g, 6.97 mmol), n class="Chemical">carbamate 28a (1.20 g, 6.97 mmol), DIPEA (1.22 mL, 6.99 mmol), and dioxane (20 mL) was heated for 60 min at 150 °C under microwave irradiation. The reaction mixture was diluted with water (40 mL) and extracted with DCM (3 × 40 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH 10:0–9:1) gave the title compound as a yellowish solid (726 mg, 36%). 1H NMR (300 MHz, CDCl3) δ 5.50 (s, 1H), 4.99 (br, 1H), 4.78 (br, 2H), 4.58 (br, 1H), 4.30 (t, J = 8.3 Hz, 2H), 3.83–3.73 (m, 2H), 2.20 (s, 3H), 1.45 (s, 9H). HPLC-MS (acidic mode): tR = 2.7 min, purity: 95.5%, [M + H]+: 280.

tert-Butyl (1-(2-Amino-6-methylpyrimidin-4-yl)azetidin-3-yl)(methyl)carbamate (23b)

A microwave vial charged with chloride 19 (287 mg, 2.00 mmol), n class="Chemical">carbamate hydrochloride 28b (445 mg, 2.00 mmol), DIPEA (0.70 mL, 4.00 mmol), and dioxane (4 mL) was heated for 60 min at 150 °C under microwave irradiation. The reaction mixture was diluted with water (15 mL) and extracted with DCM (3 × 10 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH 100:0–92:8) gave the title compound as a colorless oil (501 mg, 46%). 1H NMR (300 MHz, CDCl3) δ 5.51 (s, 1H), 4.99 (br, 1H), 4.81 (br, 2H), 4.19 (t, J = 8.6 Hz, 2H), 4.06–3.93 (m, 2H), 2.91 (s, 3H), 2.19 (s, 3H), 1.46 (s, 9H). HPLC-MS (acidic mode): tR = 2.9 min, purity: 95.9%, [M + H]+: 294.

tert-Butyl (1-(2-Amino-6-methylpyrimidin-4-yl)azetidin-3-yl)(ethyl)carbamate (23c)

Carbamate 27c (1.02 g, 2.78 mmol) was dissolved in n class="Chemical">MeOH/EtOH (10:10 mL) and reacted with H2 gas under atmospheric pressure using Pd/C (5%, 0.60 g) overnight at rt. The mixture was filtered over Celite, and the filtrate was concentrated in vacuo. The resulting yellowish oil (900 mg, a mixture of intermediate and diphenylmethane) was used in the next step without further purification. A microwave vial charged with chloride 19 (101 mg, 0.71 mmol), crude intermediate (260 mg), DIPEA (0.123 mL, 0.71 mmol), and dioxane (3 mL) was heated for 90 min at 150 °C under microwave irradiation. The reaction mixture was diluted with water (10 mL) and extracted with DCM (3 × 10 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH 10:0–9:1) gave the title compound as a colorless oil (125 mg, 56% over two steps, extrapolated). 1H NMR (300 MHz, CDCl3) δ 5.52 (s, 1H), 4.75 (br, 3H), 4.20 (t, J = 8.5 Hz, 2H), 4.07–3.95 (m, 2H), 3.33 (q, J = 7.0 Hz, 2H), 2.20 (s, 3H), 1.46 (s, 9H), 1.15 (t, J = 7.0 Hz, 3H). HPLC-MS (acidic mode): tR = 3.2 min, purity: >99% %, [M + H]+: 308.

tert-Butyl (1-(2-Aminopyrimidin-4-yl)azetidin-3-yl)carbamate (24a)

A microwave vial charged with chloride 20 (500 mg, 3.86 mmol), n class="Chemical">carbamate 28a (665 mg, 3.86 mmol), DIPEA (0.67 mL, 3.86 mmol), and dioxane (10 mL) was heated for 60 min at 150 °C under microwave irradiation. The reaction mixture was diluted with water (40 mL) and extracted with DCM (3 × 40 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH 100:0–88:12) gave the title compound as a colorless oil (434 mg, 39%). 1H NMR (300 MHz, CDCl3) δ 7.83 (d, J = 5.8 Hz, 1H), 5.61 (d, J = 5.8 Hz, 1H), 5.10 (br, 1H), 4.82 (s, 2H), 4.58 (br, 1H), 4.31 (t, J = 8.4 Hz, 2H), 3.86–3.74 (m, 2H), 1.45 (s, 9H). HPLC-MS (acidic mode): tR = 2.6 min, purity: 91.6%, [M + H]+: 266.

tert-Butyl (1-(2-Aminopyrimidin-4-yl)azetidin-3-yl)(methyl)carbamate (24b)

A microwave vial charged with chloride 20 (500 mg, 3.86 mmol), n class="Chemical">carbamate hydrochloride 28b (723 mg, 3.86 mmol), DIPEA (1.35 mL, 7.72 mmol), and dioxane (10 mL) was heated for 30 min at 150 °C under microwave irradiation. The reaction mixture was diluted with water (40 mL) and extracted with DCM (3 × 40 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH 100:0–88:12) gave the title compound as a colorless oil (501 mg, 46%). 1H NMR (300 MHz, CDCl3) δ 7.84 (d, J = 5.9 Hz, 1H), 5.65 (d, J = 5.9 Hz, 1H), 5.21–4.59 (m, 3H), 4.22 (t, J = 8.7 Hz, 2H), 4.09–3.98 (m, 2H), 2.93 (s, 3H), 1.47 (s, 9H). HPLC-MS (acidic mode): tR = 2.8 min, purity: >99%, [M + H]+: 280.

tert-Butyl (1-(2-Aminopyrimidin-4-yl)azetidin-3-yl)(ethyl)carbamate (24c)

Carbamate 27c (1.02 g, 2.78 mmol) was dissolved in n class="Chemical">MeOH/EtOH (10:10 mL) and reacted with H2 gas under atmospheric pressure using Pd/C (5%, 0.60 g) overnight at rt. The mixture was filtered through Celite. The Celite cake was washed with MeOH (2 × 5 mL), and the combined filtrates were concentrated in vacuo. The resulting yellowish oil (900 mg, a mixture of intermediate and diphenylmethane) was used in the next step without further purification. A microwave vial charged with chloride 20 (78 mg, 0.60 mmol), crude intermediate (221 mg), DIPEA (0.105 mL, 0.60 mmol), and dioxane (5 mL) was heated for 90 min at 150 °C under microwave irradiation. The reaction mixture was diluted with water (10 mL) and extracted with DCM (3 × 10 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (DCM/MeOH 10:0–9:1) gave the title compound as a colorless oil (117 mg, 50% over two steps, extrapolated). 1H NMR (300 MHz, CDCl3) δ 7.84 (d, J = 5.8 Hz, 1H), 5.64 (d, J = 5.8 Hz, 1H), 4.77 (br, 3H), 4.22 (t, J = 8.5 Hz, 2H), 4.10–3.97 (m, 2H), 3.33 (q, J = 7.0 Hz, 2H), 1.45 (s, 9H), 1.15 (t, J = 7.0 Hz, 3H). HPLC-MS (acidic mode): tR = 3.0 min, purity: 95.5%, [M + H]+: 294.

tert-Butyl (1-(2-Aminopyrimidin-4-yl)azetidin-3-yl)(propyl)carbamate (24d)

Carbamate 27d (2.09 g, 5.49 mmol) in n class="Chemical">MeOH (100 mL) was passed through an H-cube fitted with a Pd/C (10%) catalyst cartridge at a flow rate of 1 mL/min at 60 °C and at 10 atm H2 pressure. The solvent was removed in vacuo. The resulting colorless oil (2.03 g, a mixture of intermediate and diphenylmethane) was used in the next step without further purification. A microwave vial charged with chloride 20 (687 mg, 5.31 mmol), crude intermediate (2.03 g), DIPEA (0.93 mL, 5.27 mmol), and NMP (5 mL) was heated for 30 min at 120 °C under microwave irradiation. The mixture was purified by flash chromatography (DCM/MeOH/TEA 100:0:0–90:9:1). The selected fractions were collected, and the solvents were removed under reduced pressure. The residue was dissolved in DCM (50 mL) and washed with satd. aq Na2CO3 (80 mL). The aqueous phase was extracted with DCM (2 × 50 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. The title compound was obtained as a colorless oil (1.19 g, 71% over two steps). 1H NMR (500 MHz, CDCl3) δ 7.83 (d, J = 5.8 Hz, 1H), 5.64 (d, J = 5.8 Hz, 1H), 5.06–4.38 (m, 3H), 4.20 (t, J = 8.5 Hz, 2H), 4.07 (t, J = 7.6 Hz, 2H), 3.22 (t, J = 7.6 Hz, 2H), 1.54 (sext, J = 7.4 Hz, 2H), 1.44 (s, 9H), 0.88 (t, J = 7.4 Hz, 3H). HPLC-MS (acidic mode): tR = 3.3 min, purity: >99%, [M + H]+: 308.

tert-Butyl (1-(2-Aminopyrimidin-4-yl)azetidin-3-yl)(butyl)carbamate (24f)

A solution of carbamate 27f (178 mg, 0.45 mmol) in n class="Chemical">MeOH (16 mL) was passed through an H-cube fitted with a Pd/C (10%) catalyst cartridge at a flow rate of 1 mL/min at 60 °C and at 10 atm H2 pressure. The solvent was removed in vacuo. The resulting colorless oil (136 mg, a mixture of intermediate and diphenylmethane) was used in the next step without further purification. A microwave vial charged with chloride 20 (44 mg, 0.34 mmol), crude intermediate (136 mg), DIPEA (0.060 mL, 0.34 mmol), and NMP (0.5 mL) was heated for 30 min at 120 °C under microwave irradiation. The mixture was purified by flash chromatography (DCM/MeOH/TEA 100:0:0–80:18:2). The title compound was obtained as an off-white solid (62 mg, 46% over two steps). 1H NMR (300 MHz, CD3OD) δ 7.71 (d, J = 6.2 Hz, 1H), 5.77 (d, J = 6.0 Hz, 1H), 4.54 (s, 1H), 4.32–4.14 (m, 4H), 3.35–3.25 (m, 2H), 1.62–1.47 (m, 2H), 1.44 (s, 9H), 1.38–1.24 (m, 2H), 0.98–0.90 (m, 3H). HPLC-MS (acidic mode): tR = 3.4 min, purity: 97.5%, [M + H]+: 322.

tert-Butyl (1-Benzhydrylazetidin-3-yl)carbamate (26)

To a solution of amine 25 (5.00 g, 21.0 mmol) in n class="Chemical">THF (40 mL) at 0 °C was added a solution of di-tert-butyl dicarbonate (5.40, 25.2 mmol) in THF (40 mL) and TEA (3.51 mL, 25.2 mmol). The reaction mixture was warmed to rt and stirred for 2 h. The solvent was evaporated. Purification by flash chromatography (c-hexane/EtOAc/TEA 100:0:0–0:95:5) gave the title compound as a white solid (4.45 g, 63%). 1H NMR (300 MHz, CDCl3) δ 7.39 (d, J = 7.6 Hz, 4H), 7.32–7.22 (m, 4H), 7.22–7.12 (m, 2H), 4.87 (br, 1H), 4.30 (br, 2H), 3.53 (t, J = 6.3 Hz, 2H), 2.82 (br, 2H), 1.42 (s, 9H). HPLC-MS (acidic mode): tR = 3.5 min, purity: 97.5%, [M + H]+: 339.

tert-Butyl (1-Benzhydrylazetidin-3-yl)(ethyl)carbamate (27c)

To a solution of carbamate 26 (3.10 g, 9.2 mmol) in n class="Chemical">THF (30 mL) at 0 °C, NaH (60%, 0.44 g, 11.0 mmol) was added. When the evolution of H2 gas subsided, iodoethane (0.81 mL, 10.1 mmol) was added dropwise to the reaction mixture. The resulting mixture was stirred overnight at rt. The reaction mixture was diluted with water (60 mL) and extracted with DCM (3 × 40 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (c-hexane/EtOAc 10:0–7:3) gave the title compound as a white solid (1.95 g, 58%). 1H NMR (300 MHz, CDCl3) δ 7.48–7.35 (m, 4H), 7.32–7.23 (m, 4H), 7.23–7.12 (m, 2H), 4.34 (br, 2H), 3.51 (br, 2H), 3.27 (q, J = 7.1 Hz, 2H), 2.95 (br, 2H), 1.42 (s, 9H), 1.05 (t, J = 7.0 Hz, 3H). HPLC-MS (acidic mode): tR = 3.9 min, purity: 99.6%, [M + H]+: 367.

tert-Butyl (1-Benzhydrylazetidin-3-yl)(propyl)carbamate (27d)

To a solution of carbamate 26 (4.55 g, 13.2 mmol) in n class="Chemical">THF (30 mL) at 0 °C, NaH (95%, 0.40 g, 10.5 mmol) was added. When the evolution of H2 gas subsided, iodopropane (1.44 mL, 14.7 mmol) was added dropwise to the reaction mixture. The resulting mixture was stirred overnight at rt. The reaction mixture was diluted with water (200 mL) and extracted with DCM (3 × 120 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (c-hexane/EtOAc/TEA 100:0:0–70:28.5:1.5) gave the title compound as a white solid (2.09 g, 42%). 1H NMR (300 MHz, CDCl3) δ 7.46–7.35 (m, 4H), 7.33–7.23 (m, 4H), 7.23–7.14 (m, 2H), 4.33 (s, 2H), 3.50 (t, J = 7.2 Hz, 2H), 3.22–3.10 (m, 2H), 2.93 (t, J = 6.9 Hz, 2H), 1.51–1.36 (m, 11H), 0.85 (t, J = 7.4 Hz, 3H). HPLC-MS (acidic mode): tR = 3.9 min, purity: 99.3%, [M + H]+: 381.

tert-Butyl (1-Benzhydrylazetidin-3-yl)(butyl)carbamate (27f)

To a solution of carbamate 26 (552 mg, 1.60 mmol) in n class="Chemical">THF (50 mL) at 0 °C, NaH (60%, 77 mg, 1.92 mmol) was added. When the evolution of H2 gas subsided, iodobutane (0.20 mL, 1.76 mmol) was added dropwise to the reaction mixture. The resulting mixture was stirred overnight at rt. The reaction mixture was diluted with water (20 mL) and extracted with DCM (3 × 15 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (c-hexane/EtOAc/TEA 100:0:0–0:95:5) gave the title compound as a white solid (178 mg, 28%). 1H NMR (300 MHz, CDCl3) δ 7.46–7.36 (m, 4H), 7.32–7.23 (m, 4H), 7.23–7.14 (m, 2H), 4.33 (br, 2H), 3.59–3.35 (m, 2H), 3.26–3.12 (m, 2H), 2.93 (br, 2H), 1.50–1.34 (m, 11H, overlaps with residual c-hexane), 1.33–1.17 (m, 2H), 0.90 (t, J = 7.2 Hz, 3H). HPLC-MS (acidic mode): tR = 4.4 min, purity: 97.8%, [M + H]+: 395.