7H‐Pyrrolo[2,3‐d]pyrimidine‐4‐amines as Potential Inhibitors of Plasmodium falciparum Calcium‐Dependent Protein Kinases

Abstract

A series of pyrrolo[2,3‐d]pyrimidines were designed in silico as potential bumped kinase inhibitors targeting P. falciparum calcium dependent protein kinase 4 (PfCDPK4), with the potential to inhibit PfCDPK1 based on earlier studies of the two kinases. A small series of these compounds were prepared and assessed for inhibitory activity against PfCDPK4 and PfCDPK1 in vitro. Four of the compounds displayed promising inhibitory activity against either PfCDPK4 (IC₅₀=0.210–0.530 μM), or PfCDPK1 (IC₅₀=0.589 μM). These data will enable optimisation of the molecular model to better predict inhibitory activity against PfCDPK4.

Hitting malaria where it hurts: Pyrrolo[2,3‐d]pyrimidines have been designed as potential inhibitors targeting Plasmodium falciparum calcium‐dependent protein kinase 4 (PfCDPK4) using molecular modelling. Compounds displaying good binding interactions in silico were synthesised and assessed for inhibitory activity against PfCDPK4 and PfCDPK1, with several compounds displaying promising inhibitory activity against either PfCDPK4 (IC₅₀=0.210–0.530 μM), or PfCDPK1 (IC₅₀=0.589 μM).

Introduction

Malaria is an infectious disease caused by protozoan parasites of the genus Plasmodium. The majority of the 241 million malaria cases reported worldwide in 2020, and 96 % of the 627 000 fatalities reported that same year, were caused by P. falciparum, the species prevalent in sub‐Saharan Africa. ^[1] Malaria prevention tools (such as preventative therapy in pregnancy (IPTp), seasonal malaria chemoprevention in children (SMC), the use of insecticide treated nets (ITNs) and indoor residual spraying (IRS) of insecticides have played a major role in reducing the number of malaria fatalities worldwide since 2000. However, since 2015 this trend has reversed and the rate of both incidence and morbidity has increased significantly. ^[1a] The COVID‐19 pandemic service disruptions have contributed to an increase in both the number of cases and fatalities observed in 2020. ^[1b] In addition, parasite resistance to current treatments has been observed which, together with increasing insecticide resistance, could have a devastating effect on the positive strides made in the fight against malaria in the last 20 years. ^[1] There is therefore a need for in‐depth consideration of other innovative strategies coupled with identification of novel drug targets and classes of drugs if the WHO Global Technical Strategy goals for malaria eradication are to be met. ^[2]

Calcium‐dependent protein kinases (CDPKs), which are unique to plants and apicomplexans, have been identified as potential drug targets in malaria. ^[3] Several CDPKs in particular are known to play a role in the development of parasitic gametocytes, which transmit malaria from humans to the mosquito during a blood meal. P. falciparum CDPK1 (PfCDPK1) is involved in the regulation of parasite motility and zygote development and transmission, ^[4] while PfCDPK4 is required for male gametocyte exflagellation and sexual reproduction of the parasite. ^[5] As gametocytes can remain viable in the human blood stream for several weeks after asexual forms have been eradicated by drug therapy, there is the potential for continued transmission of the disease. PfCDPK1 and PfCDPK4 therefore serve as potential targets for the development of transmission‐blocking drugs.

Previous studies have described the synthesis of bumped kinase inhibitors (BKIs) containing a pyrazolo[3,4‐d]pyrimidine scaffold, such as compound 1294 (1, Figure 1), which selectively and potently inhibits PfCDPK1 and PfCDPK4 without being toxic to mammalian cells. ^[6] Furthermore, they have shown that gametocyte maturation in the mosquito is blocked by these inhibitors through inhibition of PfCDPK4. ^[7]

Figure 1.

Structure of pyrazolo[3,4‐d]pyrimidine 1294 1 and structure of pyrrolo[2,3‐d]pyrimidines 2.

A bulky group at the C‐3 position of the pyrazolopyrimidine nucleus allows for selectivity over other kinases as PfCDPK4 has a smaller gatekeeper residue than most human kinases. ^[6] It has been shown that the sequence similarity between PfCDPK4 and PfCDPK1 could potentially allow inhibitors of either kinase to act as inhibitors of the other. ^[6]

In this work, we have utilised the crystal structure of P. falciparum CDPK4 to design pyrrolo[2,3‐d]pyrimidine analogues of 1 such as 2 (Figure 1) in order to determine whether these related compounds 2 could be utilised as malarial kinase inhibitors. Related compounds bearing the pyrrolo[2,3‐d]pyrimidine core have been shown to be successful inhibitors of Trypanosoma brucei brucei, ^[8] as inhibitors of merozoite invasion by the malaria parasite ^[9] and as promising BKIs of Toxoplasma gondii CDPK1. ^[10] We now report the results of this study, in which a series of pyrrolo[2,3‐d]pyrimidines were synthesised and assessed for inhibitory activity against PfCDPK1 and PfCDPK4.

Results and Discussion

The X‐ray crystal structure of PfCDPK4 (4QOX from the RSCB protein data bank) was used to design inhibitors with a pyrrolo[2,3‐d]pyrimidine core using Schrodinger (2017‐4, Maestro 11.4), available from the Centre for High Performance Computing (CHPC) in South Africa. Flexible docking protocols were developed and pyrrolo[2,3‐d]pyrimidine derivatives bearing a variety of substituents at N‐7 and C‐5 (2, Figure 1) were docked into the ATP‐binding site of PfCDPK4. As PfCDPK4 has a smaller gatekeeper residue than most human kinases, bulky aromatic groups were considered at C‐5 to bind in the hydrophobic pocket adjacent to this gatekeeper residue to impart selectivity for PfCDPK4. ^[7] Furthermore, as there is an additional hydrophobic pocket near the carbohydrate binding region, both hydrophobic and hydrophilic groups were considered for N‐7. Pyrrolo[2,3‐d]pyrimidines bearing a variety of functional groups at N‐7 and C‐5 were docked into the PfCDPK4 active site (using Glide in Schrodinger) and ligands were ranked according to their docking scores. In addition, poses were assessed visually to ensure that the expected hydrogen‐bonding interactions with key amino acid residues in the hinge region (Tyr150 and Asp148) were present.

The results from these modelling studies showed that pyrrolo[2,3‐d]pyrimidines could potentially act as inhibitors of PfCDPK4, with compounds displaying similar docking scores to compound 1294 (1) (see Table 1). Interestingly, pyrrolo[2,3‐d]pyrimidines bearing a cyclopropylmethyl substituent at N‐7 gave rise to poses with docking scores comparable with those bearing 4‐piperidinyl groups, highlighting that both hydrophobic and hydrophilic groups can demonstrate binding owing to the additional hydrophobic pocket near the carbohydrate binding region.

Table 1.

Docking scores of pyrrolo[2,3‐d]pyrimidine derivatives 2 a–q.

Entry	Compound	R	R′	Docking score[a]	Entry	Compound	R	R′	Docking score[a]
1	Compound 1294			−13.420	10	2 i			−11.873
2	2 a			−13.083	11	2 j			−10.227
3	2 b			−12.787	12	2 k			−9.715
4	2 c			−13.617	13	2 l			−10.013
5	2 d			−11.117	14	2 m			−13.976
6	2 e			−10.823	15	2 n			−10.992
7	2 f			−13.634	16	2 o			−9.697
8	2 g			−13.037	17	2 p			−11.271
9	2 h			−9.682	18	2 q			−12.503

[a] All docking scores given in units cal mol⁻¹.

Compounds bearing a piperidine moiety protected by an acyl or Boc‐protecting group at N‐7 allowed an additional H‐bond with Glu154 in the carbohydrate binding region, as seen with compound 1294, in addition to the important H‐bond acceptor/donor interactions with key amino acids residues (Asp148 and Tyr150) in the hinge region (Figure 2a, b). These same H‐bond acceptor/donor interactions were seen between the compounds bearing a 2‐morpholinoethyl side chain at N‐7 and key amino acids residues Asp148 and Tyr150, but the additional H‐bond with Glu154 in the carbohydrate binding region was not observed for these compounds. Compounds with a benzyl group at N‐7 docked as expected in the active site, but generally gave higher docking scores with naphthyl substituents at C‐5 indicating weaker binding.

Figure 2.

Docked images from the molecular modelling study showing a) pyrrolo[2,3‐d]pyrimidine ligand bearing N‐acylpiperidine substituent at N‐7 H‐bonding to Asp148, Tyr150 and Glu154; b) compound 1294 docked into the active site; c) pyrrolo[2,3‐d]pyrimidine ligand bearing 3,4‐dimethoxyphenyl substituent at C‐5 H‐bonding to Asp215 and d) pyrrolo[2,3‐d]pyrimidine ligand bearing a 2‐methoxy substituent at C‐5 showing pi interaction with Lys99.

In general, compounds of the pyrrolo[2,3‐d]pyrimidine class bearing aromatic substituents at C‐5 showed good binding, with naphthyl substituents filling the large hydrophobic pocket in this region better than phenyl substituents, and often showing a pi interaction with Lys99 (as seen in Figure 2). However, a 3,4‐dimethoxyphenyl substituent at C‐5 could form an additional H‐bond with Asp215 in silico (Figure 2c), while pi interaction with Lys99 was observed for compounds bearing a 2‐methoxyphenyl substituent at C‐5 (Figure 2d).

Thus, the results from our modelling studies showed that pyrrolo[2,3‐d]pyrimidines bearing benzyl, cyclopropylmethyl, (N‐acetylpiperidin‐4‐yl)methyl, 2‐(1‐Boc‐piperidin‐4‐yl)ethyl and 2‐(morpholin‐4‐yl)ethyl on N‐7 and aromatic substituents at C‐5 of the scaffold could potentially act as inhibitors of PfCDPK4. Derivatives displaying the best binding interactions were therefore considered for synthesis.

We envisaged synthesising the desired pyrrolo[2,3‐d]pyrimidine derivatives 2 in three steps from the key intermediate 7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 3. Although 3 is commercially available, we chose to prepare it (as shown in Scheme 1), as it was only available in small quantities. To this end, Raney Nickel desulfurisation of 4,6‐diaminopyrimidine‐2‐thiol afforded 4,6‐diaminopyrimidine 4 in good yield. Iodination of 4 to afford 5, followed by Sonogashira coupling gave 6 in moderate yield. Finally, deprotection to afford 7 was followed by base promoted ring closure under microwave irradiation to yield 3.

Scheme 1.

Reagents and conditions: i) Raney Ni, 25 % aq. NH₃, reflux, 2 h, 96 %; ii) I₂, K₂CO₃, DMF/H₂O, 45 °C, 4 h, 76 %; iii) ethynyltrimethylsilane, CuI (3 mol %), Pd(PPh₃)₄ (5 mol %), diisopropylamine, THF, 70 °C, 4 h, 48 %; iv) TBAF, THF 0 °C–rt, 3 h, 85 %; v) Cs₂CO₃, DMSO, microwave 100 W, 180 °C, 15 min, 50 %.

With 7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 3 in hand, the desired N‐7 substituents of the planned pyrrolo[2,3‐d]pyrimidine derivatives 2 could be introduced by a nucleophilic substitution reaction, while the substituents at C‐5 could be introduced by means of a Suzuki‐Miyaura coupling reaction to the iodinated heterocycle (Scheme 2). To this end, iodination at C‐5 of the pyrrolo[2,3‐d]pyrimidine core of 3 was accomplished using N‐iodosuccinimide (NIS) to afford the desired iodinated compound 8 in a yield of 90 %. This was followed by treatment of 8 with appropriately substituted alkyl bromides or tosylates 9 a–e under basic conditions to introduce the desired substituent at N‐7 of the pyrrolo[2,3‐d]pyrimidine core, affording compounds 10 a–e in moderate yields. The alkyl tosylates 9 b–c were prepared from the corresponding alcohols using reported methods. ^[11] In general, lower yields of 10 a–e were obtained when the N‐alkylation reaction was performed first, followed by iodination at C‐5.

Scheme 2.

Reagents and conditions: i) NIS, CHCl₃, 60 °C, 2 h, 90 %; ii) R−X (9 a–e), Cs₂CO₃, DMF, 70 °C, 18 h, 59–79 %; iii) R’‐B(OH)₂ (11 a–e), 10 % Pd(PPh₃)₄, aq Na₂CO₃, DME, reflux, 18 h, 35 % ‐ 76 %.

The final step in the synthesis of the desired pyrrolo[2,3‐d]pyrimidines 2 was the introduction of an aryl substituent at C‐5, which was accomplished by Suzuki‐Miyaura coupling reaction with suitably substituted boronic acids. Boronic acids 11 a–c were not commercially available but were readily prepared from 6‐bromonaphthalen‐2‐ol 12 in two steps (Scheme 3). The Suzuki‐Miyaura coupling of 5‐iodo‐7H‐pyrrolo[2,3‐d]pyrimidines 10 a–e with boronic acids 11 a–e proceeded smoothly to afford pyrrolo[2,3‐d]pyrimidines 2 a–q in moderate yields.

Scheme 3.

Reagents and conditions: i) K₂CO₃, alkyl halide, acetone, rt, 18 h, 62–94 %; ii) n‐BuLi, B(OⁱPr)₃, dry THF, −78 °C, then rt, HCl (aq), 55 %–83 %.

All of the final compounds synthesised were evaluated for antiplasmodial activity in vitro in a whole cell assay, as potent inhibitors of PfCDPK1 (with high sequence similarity to PfCDPK4) have been shown to display moderate antiplasmodial activity in vitro (Table 2). ^[7] The parasite lactate dehydrogenase (pLDH) assay was performed at a single concentration (20 μM) initially, and IC₅₀ values determined for compounds that inhibited parasite growth by more than 75 %. Four of the compounds showed moderate antiplasmodial activity in vitro in the low micromolar range (2 b, 2 g, 2 j and 2 m, Table 2), three of which were substituted with an ethoxynaphthyl group at C‐5. All the compounds were assessed for cytotoxicity in vitro in a HeLa cell assay at a single concentration, with only one of the compounds showing moderate cytotoxicity at a concentration of 20 μM (2 m, Table 2). Selected compounds were also screened for activity against the target enzymes PfCDPK1 and PfCDPK4. Despite the promising results from our docking studies, only three of the compounds screened showed activity against PfCDPK4 (2 b, 2 f and 2 g, IC₅₀=0.210 – 0.530 μM, Table 2), two of which had shown moderate antiplasmodial activity (2 b and 2 g). Furthermore, only one compound was active against PfCDPK1 (2 e, IC₅₀=0.589 μM, Table 2). All of the active compounds contained either a methoxy‐ or ethoxy‐naphthyl group at C‐5, and either a cyclopropylmethyl or (N‐acetylpiperidin‐4‐yl)methyl at N‐7. These data suggest that the pyrrolo[2,3‐d]pyrimidine scaffold does not adequately compare with the pyrazolo[3,4‐d]pyrimidine scaffold in the design of bumped kinase inhibitors. While a gametocyte exflagellation assay of the compounds active against PfCDPK4 would confirm transmission blocking activity, the moderate activity observed did not warrant further investigation at this stage. Rather, the data generated will be utilised to optimise the molecular model to better predict inhibitory activity against PfCDPK4.

Table 2.

Structure and biological activity of pyrrolo[2,3‐d]pyrimidine derivatives 2 a–q.

Entry	Compound	R	R′	Pf 3D7 % cell viability at 20 μM[a]	Pf 3D7 IC50 [μM][b]	Pf CDPK1 IC50 [μM]0[b]	Pf CDPK4 IC50 [μM][b]	Hela % cell viability at 20 μM[a]
1	2 a			103.0±8.0	–	–	–	93.6±7.1
2	2 b			24.1±3.8	18.7±0.8	>2	0.3694±0.1381	79.8±3.3
3	2 c			93.1±1.4	–	–	–	73±4.1
4	2 d			106.6±6.0	–	>2	>2	63.8±1.5
5	2 e			110.2±10.9	–	0.5885±0.2059	>2	88.8±0.7
6	2 f			35.3±2.2	–	>2	0.2104±0.0680	92.1±3.1
7	2 g			9.3±1.6	14.2±1.7	>2	0.5298±0.1847	92.8±5.7
8	2 h			62.2±9.0	–	>2	>2	94.4±9.3
9	2 i			121.4±8.6	–	>2	>2	93.3±3.2
10	2 j			2.8±3.9	12.1±11.4	>2	>2	79.3±0.8
14	2 k			62.0±2.1	–	–	–	78.9±0.1
15	2 l			66.8±3.9	–	>2	>2	82.4±1.4
16	2 m			12.0±2.6	8.2±3.1	–	–	47.0±7.0
17	2 n			95.0±1.0	–	>2	>2	112.0±2.8
18	2 o			57.0±6.6	–	>2	>2	88.0±11.8
19	2 p			72.4±13.3	–	–	–	97.0±12.5
20	2 q			110.0±4.2	–	–	–	95.4±0.5
21	Chloroquine			–	0.015	–	–	–

[a] Data are presented as the mean ±SD of two replicates. [b] Data are presented as the mean ±SD of three replicates.

Conclusion

We have designed a series of pyrrolo[2,3‐d]pyrimidines as potential bumped kinase inhibitors targeting P. falciparum CDPK4 using molecular modelling, and synthesised compounds that displayed good binding interactions in silico. The compounds were assessed for their inhibitory activity against PfCDPK4 and PfCDPK1 in vitro with only three of the compounds 2 b, 2 f, and 2 g displaying promising inhibitory activity against PfCDPK4 (IC₅₀=0.210–0.530 μM), and one against PfCDPK1 (IC₅₀=0.589 μM). This suggests that the additional nitrogen atom in the pyrazolo[3,4‐d]pyrimidine scaffold, on which the compounds in this study are based, plays an important role in binding in the target enzyme, however, the reason for this was not evident in our modelling study. The data generated here will therefore be utilised to optimise the molecular model to better predict inhibitory activity against PfCDPK4.

Experimental Section

Docking

Chemical structures drawn in ChemDraw and saved as an SD file were imported to Maestro 11.4 (Schrödinger) and prepared using the LigPrep tool. The crystal structure of P. falciparum CDPK4 (4QOX from the RSCB protein data bank) was prepared using the Protein Preparation Wizard in Maestro by making adjustments to bond orders and formal charges, adding hydrogen atoms and predicting the protonation states of polar amino acids at neutral pH using PROPKA. A grid was generated at the ATP‐binding site using the receptor grid generation panel of the Glide tool. Molecular docking calculations were carried out using Glide, Extra Precision (XP) mode, and poses were rescored using Molecular Mechanics/Generalized Born Surface Area (MMGBSA) in Prime with default conditions.

Chemistry

Reagents purchased from Merck were of reagent grade and were used without any further purification unless specified. Ethyl acetate (EtOAc) and hexane used for chromatography or extractions were distilled prior to use. Dimethyl formamide (DMF) was distilled and stored over 4 Å molecular sieves. Tetrahydrofuran (THF) was distilled from sodium prior to use. Reactions were monitored by thin‐layer chromatography (TLC) using precoated aluminium‐backed plates (Merck silica gel 60 F₂₅₄) visualised under UV light (λ=254 nm). Intermediates and final compounds were purified by column chromatography on Fluka silica gel 60 (70‐230 mesh). NMR spectra were acquired on a Bruker 300 or 500 MHz spectrometer at room temperature, using the specified deuterated solvent. For those compounds soluble in deuterated chloroform (CDCl₃), the solvent contained tetramethylsilane (TMS, 0.05 % v/v) as internal standard. For others, the residual solvent signal was used for referencing. Data processing was done using MestreNova Software under license from Mestrelab Research, CA, USA. Infra‐red spectra were recorded neat on a Bruker Tensor‐27 Fourier Transform spectrometer. Mass Spectra (High Resolution) were recorded on a Bruker Compact Q‐TOF high resolution mass spectrophotometer. Melting points were determined on a Stuart SMP10 melting point apparatus and are uncorrected.

Pyrimidine‐4,6‐diamine 4. ^[12] To a mixture of 4,6‐diaminopyrimidine‐2‐thiol (5.00 g, 35.2 mmol) in water (500 ml) and 25 % aqueous ammonia (20.0 ml) was added Raney nickel (10.0 g) in portions. The resulting mixture was heated at reflux for 2 hours after which it was cooled to room temperature, and the catalyst was removed by filtration on a Buchner funnel. The filtrate was evaporated to dryness on a rotary evaporator to furnish pyrimidine‐4,6‐diamine 4 (3.70 g, 96 %) as a light yellow solid. R _f (10 % MeOH/dichloromethane) 0.32; m.p. 271–272 °C; IR (v _max/cm⁻¹) 3445, 3303, 3092, 1634, 1475; ¹ H NMR (300 MHz, DMSO‐d₆ ) δ 7.81 (s, 1H), 6.02 (s, 4H), 5.38 (s, 1H); ¹³C NMR (75 MHz, DMSO‐d₆ ) δ 163.5 (2 C), 157.8, 82.6.

5‐Iodopyrimidine‐4,6‐diamine 5. ^[13] To a suspension of pyrimidine‐4,6‐diamine (3.00 g, 27.2 mmol) in water (70.0 ml) and DMF (20.0 ml) was added potassium carbonate (5.65 g, 40.9 mmol, 1.5 eq) and iodine (13.8 g, 54.4 mmol, 2 eq). The resulting reaction mixture was heated at 40–45 °C for 4 hours, after which the reaction mixture was cooled to room temperature and quenched with 2 M aqueous sodium thiosulfate (40.0 ml) to give a clear solution. The product that formed was collected by filtration and washed with water (3×25 ml) to give 5‐iodopyrimidine‐4,6‐diamine (4.91 g, 76 %) as a yellow solid. R _f (10 % MeOH/dichloromethane) 0.36; m.p. 160 °C; IR (v _max/cm⁻¹) 3455, 3277, 3067, 1627, 1459, 626.6; ¹H NMR (300 MHz, DMSO‐d₆ ) δ 7.72 (s, 1H), 6.31 (s, 4H); ¹³C NMR (75 MHz, DMSO‐d₆ ) δ 162.8 (2 C), 156.9, 55.2.

5‐[(Trimethylsilyl)ethynyl]pyrimidine‐4,6‐diamine 6. ^[14] To a degassed mixture of 5‐iodopyrimidine‐4,6‐diamine (500 mg, 2.12 mmol), copper(I)iodide (12.0 mg, 6.36×10⁻² mmol, 3 mole %) and Pd(PPh₃)₄ (122 mg, 0.106 mmol, 5 mole %) was added a degassed solution of ethynyltrimethylsilane (3.02 ml, 21.2 mmol, 10 eq), diisopropylamine (3.00 ml, 21.2 mmol, 10 eq) and THF (20.0 ml). The resulting mixture was heated at 70 °C for 4 hours under a nitrogen atmosphere, then quenched with a saturated aq NH₄Cl solution and THF removed on a rotary evaporator. The remaining aqueous residue was washed with ethyl acetate (3×100 ml) and the organic layers were combined, dried over MgSO₄, filtered through celite and excess solvent removed on a rotary evaporator. The crude product was purified by column chromatography (80 % EtOAc/hexane) to furnish 5‐[(trimethylsilyl)ethynyl]pyrimidine‐4,6‐diamine (0.21 g, 48 %) as a light yellow solid. R _f (80 % EtOAc/hexane) 0.64. m.p. 190–192 °C; IR (v _max/cm⁻¹) 3457, 3291, 3089, 2136, 1625, 1463; ¹H NMR (300 MHz, DMSO‐d₆ ) δ 7.82 (s, 1H), 6.36 (s, 4H), 0.23 (s, 9H); ¹³C NMR (75 MHz, DMSO‐d₆ ) δ 163.4 (2 C), 156.5, 99.4, 97.2, 78.2, 0.00.

5‐Ethynylpyrimidine‐4,6‐diamine 7. ^[11] To a solution of 5‐[(trimethylsilyl)ethynyl]pyrimidine‐4,6‐diamine (200 mg, 0.969 mmol) in THF (20.0 ml) was added tetrabutylammonium fluoride, 1.0 M in THF, (0.561 ml, 1.94 mmol, 2 eq) at 0 °C. The reaction mixture was then stirred at room temperature for 3 hours; quenched with saturated aq NH₄Cl and was extracted with ethyl acetate (3×25 ml). The combined organic extracts were dried with MgSO₄, filtered through celite and excess solvent was removed on a rotary evaporator. The crude product was purified by column chromatography to give 5‐ethynylpyrimidine‐4,6‐diamine (0.11 g, 85 %) as a yellow solid. R _f (80 % EtOAc/hexane) 0.58; m.p. 202–203 °C; IR (v _max/cm⁻¹) 3551, 3252, 3158, 2092, 1628, 1470; ¹H NMR (300 MHz, DMSO‐d₆ ) δ 7.82 (s, 1H), 6.42 (s, 4H), 4.57 (s, 1H); ¹³C NMR (75 MHz, DMSO‐d₆ ) δ 163.9 (2 C), 156.5, 91.1, 77.4, 76.1.

7H‐Pyrrolo[2,3‐d]pyrimidin‐4‐amine 3. ^[14] A mixture of 5‐ethynylpyrimidine‐4,6‐diamine (300 mg, 2.24 mmol), Cs₂CO₃ (1.46 g, 4.47 mmol) and DMSO (4.00 ml) in a 10.00 ml microwave tube was irradiated at 100 W and 180 °C for 15 minutes. After this time, the reaction mixture was cooled to room temperature and poured into a separating funnel containing 100 ml water and 100 ml ethyl acetate. The aqueous layer was extracted with EtOAc (2×100 ml). The combined organic extracts were dried with MgSO₄, filtered through celite and excess solvent was removed in vacuo. Purification by column chromatography (80 % EtOAc/hexane – 10 % MeOH/CH₂Cl₂) gave 7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine (0.15 g, 50 %) as an off‐white solid. R _f (10 % MeOH/dichloromethane) 0.28; m.p. 257–259 °C; IR (v _max/cm⁻¹) 3414, 3291, 3086, 1640, 1475; ¹H NMR (300 MHz, DMSO‐d₆ ) δ 11.42 (s, 1H), 8.01 (s, 1H), 7.05 (dd, J=3.5, 2.1 Hz, 1H), 6.85 (s, 2H), 6.51 (dd, J=3.4, 1.8 Hz, 1H); ¹³C NMR (75 MHz, DMSO‐d₆ ) δ 157.8, 152.0, 151.1, 121.3, 102.7, 99.3.

5‐Iodo‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 8. 7H‐Pyrrolo[2,3‐d]pyrimidin‐4‐amine 3 (539 mg, 4.02 mmol) was dissolved in chloroform (20 ml) and treated with N‐iodosuccinimide (1.08 g, 4.82 mmol, 1.2 eq). The resulting mixture was heated at reflux for 2 h, the solvent was removed in vacuo and the residue was purified by silica gel column chromatography (10 % MeOH/CH₂Cl₂ as eluent) to give the desired product 8 (0.936 g, 90 %) as a yellow solid. Rf (10 % MeOH/dichloromethane) 0.56; IR (v _max/cm^‐1) 3411, 3288, 3085, 1632, 1460; ¹H NMR (300 MHz, DMSO‐d₆ ) δ 11.95 (s, 1H), 8.05 (s, 1H), 7.36 (d, J=1.6, 1H), 6.54 (s, 2H); ¹³C NMR (75 MHz, DMSO‐d₆ ) δ 156.9, 151.7, 150.5, 126.7, 102.6, 50.1; HRMS (ES⁺) found [M+H]⁺ 260.9641, C₆H₆IN₄ ⁺ requires 260.9632.

7‐(Cyclopropylmethyl)‐5‐iodo‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 10 a. A mixture of 5‐iodo‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 8 (420 mg, 0.769 mmol), Cs₂CO₃ (501 mg, 1.54 mmol, 2 eq) and (bromomethyl)cyclopropane 9 a (0.125 ml, 0.923 mmol, 1.2 eq) were dissolved in dry DMF (20.0 ml) and heated at 70 °C for 18 h. The reaction mixture was cooled to room temperature and poured into a separating funnel containing ethyl acetate (100 ml) and washed with brine (5×100 ml). The combined organic extract was dried over MgSO₄, filtered through celite and excess solvent was removed in vacuo. Purification by column chromatography (60 % EtOAc/chloroform) gave 10 a (0.179 g, 74 %) as a light yellow solid. R _f (80 % EtOAc/hexane) 0.21; m.p. 250–252 °C; IR (v _max/cm⁻¹) 3420, 3292, 3080, 1641; ¹H NMR (300 MHz, DMSO‐d ₆) δ 8.09 (s, 1H), 7.52 (s, 1H), 3.95 (d, J=7.2 Hz, 2H), 1.21 (dqd, J=12.3, 7.5, 4.8 Hz, 1H), 0.50–0.43 (m, 2H), 0.41–0.34 (m, 2H); ¹³C NMR (125 MHz, DMSO‐d₆ ) δ 157.1, 151.8, 149.5, 129.3, 102.8, 49.4, 48.3, 11.6, 3.6; HRMS (ES⁺) found [M+H]⁺ 315.0118, C₁₀H₁₂IN₄ ⁺ requires 315.0101.

1‐{4‐[(4‐Amino‐5‐iodo‐7H‐pyrrolo[2,3‐d]pyrimidin‐7‐yl)methyl]piperidin‐1‐yl}ethanone 10 b. Prepared from 8 (70.0 mg, 0.270 mmol), Cs₂CO₃ (175 mg, 0.538 mmol, 2 eq) and (1‐acetylpiperidin‐4‐yl)methyl 4‐methylbenzenesulfonate 9 b ^[11] (109 mg, 0.349 mmol, 1.3 eq) using the procedure described for 10 a. Purification by silica gel column chromatography (10 % MeOH/chloroform) gave 10 b (85 mg, 79 %) as an off‐white solid. R _f (10 % MeOH/chloroform) 0.28; m.p. 210–212 °C; IR (v _max/cm⁻¹) 3427, 3058, 1649, 1440, 1249, 940.9, ¹H NMR (500 MHz, DMSO‐d₆ ) δ 8.09 (s, 1H), 7.44 (s, 1H), 6.59 (s,2H), 4.31 (d, J=13.0, 1H), 4.00 (d, J=7.2, 2H), 3.76 (d, J=13.1, 1H), 2.97–2.87 (m, 1H), 2.44 (td, J=12.7, 2.9 Hz, 1H), 2.11–2.00 (m, 1H), 1.95 (s, 3H), 1.45 (d, J=12.9, 2H), 1.16–0.95 (m, 2H); ¹³C NMR (125 MHz, DMSO‐d₆ ) δ 168.0, 157.1, 151.8, 149.9, 130.0, 102.8, 49.1, 45.4, 36.4, 29.8, 29.0, 21.3; HRMS (ES⁺) found [M+H]⁺ 400.0619, C₁₄H₁₉IN₅O⁺ requires 400.0629.

tert ‐Butyl 4‐[2‐(4‐amino‐5‐iodo‐7H‐pyrrolo[2,3‐d]pyrimidin‐7‐yl)ethyl]piperidine‐1‐carboxylate 10 c. Prepared from 8 (100 mg, 0.385 mmol), Cs₂CO₃ (0.250 g, 0.769 mmol, 2 eq) and 2‐[1‐(tert‐butoxycarbonyl)piperidin‐4‐yl]ethyl 4‐methylbenzene‐sulfonate 9 c ^[11] (0.193 g, 0.499 mmol, 1.3 eq) in dry DMF (7.00 ml) as described for 10 a. Purification by column chromatography (80 % EtOAc/hexane) gave 10 c (0.120 g, 66 %) as a viscous oil. R _f (80 % EtOAc/hexane) 0.48; IR (v _max/cm⁻¹) 3485, 3277, 2930, 1679, 1453; ¹H NMR (500 MHz, DMSO‐d₆ ) δ 8.10 (s, 1H), 7.50 (s, 1H), 6.59 (s, 2H), 4.16–4.13 (m, 2H), 3.88 (d, J=13.0, 2H), 3.22–3.13 (m, 1H), 2.71–2.64 (m, 1H), 1.67 (q, J=6.3, 4H), 1.39 (s, 9H), 1.34–1.21 (m, 1H), 1.04–0.96 (m, 2H); ¹³C NMR (125 MHz, DMSO‐d₆ ) δ 162.3, 157.1, 153.8, 151.7, 149.6, 129.3, 102.8, 78.4, 49.5, 48.6, 41.4, 36.2, 32.6, 28.1; HRMS (ES⁺) found [M+H]⁺ 472.1210, C₁₈H₂₇IN₅O₂ ⁺ requires 472.1204.

5‐Iodo‐7‐(2‐morpholinoethyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 10 d. Prepared from 8 (0.280 g, 1.08 mmol), Cs₂CO₃ (0.701 g, 2.15 mmol, 2 eq) and 4‐(2‐chloroethyl)morpholine hydrochloride 9 d (0.240 g, 1.29 mmol, 1.2 eq) using the procedure described for 10 a. Purification by silica gel column chromatography (10 % MeOH/chloroform) gave 10 d (0.253 g, 63 %) as a yellow solid. R _f (10 % MeOH/chloroform) 0.26; m.p. 215–218 °C; IR (v _max/cm⁻¹) 3330, 3175, 3066, 1560, 1242, 1061, ¹H NMR (500 MHz, MeOD) δ 8.14 (s, 1H), 7.42 (s, 1H), 4.35 (t, J=6.5, 2H), 3.76–3.61 (m, 4H), 2.81 (t, J=6.5, 2H), 2.64–2.47 (m, 4H); ¹³C NMR (126 MHz, MeOD) δ 158.7, 152.4, 150.9, 131.7, 104.9, 67.8, 59.0, 54.6, 42.7, 29.1; HRMS (ES⁺) found [M+H]⁺ 374.0479, C₁₂H₁₇IN₅O⁺ requires 374.0472.

7‐Benzyl‐5‐iodo‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 10 e. Prepared from 8 (100 mg, 0.385 mmol), Cs₂CO₃ (0.250 g, 0.769 mmol, 2 eq) and benzyl bromide 9 e (0.0500 ml, 0.423 mmol, 1.1 eq) in dry DMF (10.0 ml) using the procedure described for 10 a. Purification by column chromatography (80 % EtOAc/hexane) gave 10 e (0.08 g, 59 %) as a light brown solid. R _f (80 % EtOAc/hexane) 0.37; m.p. 220–221 °C; IR (v _max/cm⁻¹) 3452, 3275, 3064, 1630, 1461, 626.8; ¹H NMR (300 MHz, DMSO‐d ₆) δ 8.12 (s, 1H), 7.53 (s, 1H), 7.40–7.14 (m, 5H), 6.63 (s, 2H), 5.31 (s, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 157.0, 152.6, 150.5, 136.7, 129.1, 129.0, 128.2, 127.9, 104.1, 48.3; HRMS (ES⁺) found: [M+H]⁺ 351.0101, C₁₃H₁₂IN₄ ⁺ requires 351.0108.

6‐Methoxynaphthalen‐2‐yl‐2‐boronic acid 11 a. To a suspension of 6‐bromonaphthalen‐2‐ol 12 (6.00 g, 26.9 mmol) in acetone (50 ml) was added potassium carbonate (7.43 g, 53.8 mmol, 2 eq). The resulting mixture was stirred at room temperature for 30 min before dimethyl sulfate (2.81 ml, 29.59 mmol, 1.1 eq.) was added, and then left to stir at room temperature overnight. After completion, the reaction medium was transferred into a separating funnel containing brine (100 ml), and extracted with EtOAc (3×100 ml). The combined organic extracts were dried with MgSO_4, filtered through celite, and concentrated in vacuo. The residue was purified using silica gel chromatography (hexane) to give 2‐bromo‐6‐methoxynaphthalene ^[15] as a white solid (6.01 g, 94 %). R _f (hexane) 0.21; m.p. 105 °C; IR (v _max/cm⁻¹) 2967, 1262, 1030; ¹H NMR (300 MHz, CDCl₃) δ 7.89 (d, J=2.0, 1H), 7.89–7.56 (m, 2H), 7.48 (dd, J=8.7, 2.0, 1H), 7.14 (dd, J=8.9, 2.5, 1H), 7.07 (d, J=2.5, 1H), 3.89 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 158.0, 133.2, 130.2, 129.8, 129.7, 128.6, 128.5, 119.9, 117.2, 105.9, 55.5. 2‐Bromo‐6‐methoxynaphthalene (1.00 g, 4.22 mmol) was then dissolved in freshly distilled, dry THF (30 ml), degassed for 15 min and cooled to −78 °C under a nitrogen atmosphere. n‐Butyllithium (5.28 ml, 1.6 M/THF, 8.44 mmol, 2 eq) was added dropwise to the reaction mixture and stirred at −78 °C for 1 h. Freshly distilled triisopropyl borate (2.92 ml, 12.7 mmol, 3.0 eq) was then added dropwise to the reaction mixture, which was allowed to warm to room temperature from −78 °C over 2 h. The reaction mixture was quenched with 2 M HCl (aq) (100 ml) and the organic product extracted with EtOAc (2×100 ml). The combined organic extracts were dried over MgSO₄, filtered through celite and concentrated in vacuo. The resulting solid was suspended in hexane, subjected to sonication, and then filtered to afford the product as a white solid (0.469 g, 55 %). Due to decomposition on silica gel, the boronic acid 11 a was used in the next step without further purification or characterisation. However, the crude NMR spectra obtained showed the desired product as the major product. ¹H NMR (400 MHz, DMSO‐d ₆) δ 8.29 (s, 1H), 7.82 (dd, J=8.6, 5.4 Hz, 2H), 7.75 (d, J=8.2 Hz, 1H), 7.29 (d, J=2.4 Hz, 1H), 7.15 (dd, J=8.8, 2.4 Hz, 1H), 3.89 (s, 3H). ¹³C NMR (101 MHz, DMSO‐d ₆) δ 158.2, 135.9, 135.2, 131.7, 130.4, 128.4, 125.9, 118.7, 106.2, 55.7.

6‐Ethoxynaphthalen‐2‐yl‐2‐boronic acid 11 b. 2‐Bromo‐6‐ethoxynaphthalene ^[15] was prepared from 12 (8.00 g, 35.9 mmol), potassium carbonate (9.91 g, 71.7 mmol) and ethyl bromide (2.92 ml, 39.5 mmol) using the procedure described for 11 a, and isolated as an off‐white solid (4.57 g, 62 %). R _f (hexane) 0.26; m.p. 96 °C; IR (v _max/cm⁻¹) 2985, 1453, 1115; ¹H NMR (300 MHz, CDCl₃) δ 7.91 (d, J=2.0, 1H), 7.63 (d, J=9.0 Hz, 1H), 7.58 (d, J=8.8 Hz, 1H), 7.49 (dd, J=8.8, 2.0 Hz, 1H), 7.16 (dd, J=8.9, 2.5 Hz, 1H), 7.08 (d, J=2.4 Hz, 1H), 4.13 (q, J=7.0 Hz, 2H), 1.48 (t, J=7.0 Hz, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 157.4, 133.2, 130.1, 129.8, 129.7, 128.6, 128.5, 120.2, 117.1, 106.7, 63.7, 14.9. 6‐Ethoxynaphthalen‐2‐yl‐2‐boronic acid 11 b was then prepared from 2‐bromo‐6‐ethoxynaphthalene (1.00 g, 3.98 mmol), n‐butyllithium (4.98 ml, 7.96 mmol, 2.0 eq) and triisopropyl borate (2.76 ml, 12.0 mmol, 3.0 eq) using the procedure described for 11 a, and isolated as an off‐white solid (0.53 g, 62 %). ¹H NMR (400 MHz, DMSO‐d ₆) δ 8.28 (s, 1H), 8.04 (s, 2H), 7.80 (dd, J=8.7, 5.0 Hz, 2H), 7.72 (d, J=8.2 Hz, 1H), 7.26 (d, J=2.4 Hz, 1H), 7.13 (dd, J=8.9, 2.4 Hz, 1H), 4.15 (q, J=6.9 Hz, 2H), 1.39 (t, J=6.9 Hz, 3H); ¹³C NMR (126 MHz, DMSO‐d₆ ) δ 157.0, 135.4, 134.7, 131.1, 129.9, 127.8, 125.3, 118.5, 106.3, 63.1, 14.6.

6‐(2‐Ethoxyethoxy)naphthalen‐2‐yl‐2‐boronic acid 11 c. 2‐(2‐Ethoxyethoxy)‐6‐bromonaphthalene was prepared from 12 (5.00 g, 22.4 mmol), potassium carbonate (6.20 g, 44.8 mmol) and 1‐bromo‐2‐ethoxyethane (2.78 ml, 24.7 mmol) using the procedure described for 11 a, and isolated as an off‐white solid (5.65 g, 85 %). Rf (20 % EtOAc/hexane) 0.82; m.p. 76 °C; IR (v _max/cm⁻¹) 2875, 1452, 1061; ¹H NMR (300 MHz, CDCl₃) δ 7.90 (d, J=1.9 Hz, 1H), 7.63 (d, J=9.0 Hz, 1H), 7.58 (d, J=8.8 Hz, 1H), 7.49 (dd, J=8.7, 2.0 Hz, 1H), 7.21 (dd, J=9.0, 2.5 Hz, 1H), 7.10 (d, J=2.5 Hz, 1H), 4.28–4.18 (m, 2H), 3.91–3.81 (m, 2H), 3.63 (q, J=7.0 Hz, 2H), 1.27 (t, J=7.0 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 157.2, 133.1, 130.2, 129.8, 129.7, 128.6, 128.5, 120.2, 117.2, 106.8, 69.0, 67.7, 67.0, 15.3; HRMS (ES⁺) found [M+H]⁺ 295.0312, C₁₄H₁₆ ⁷⁹BrO₂ ⁺ requires 295.0328. 6‐(2‐Ethoxyethoxy)naphthalen‐2‐yl‐2‐boronic acid 11 c was then prepared from 2‐(2‐ethoxyethoxy)‐6‐bromonaphthalene (2.00 g, 6.78 mmol), n‐butyllithium (1.30 ml, 13.6 mmol, 2.0 eq) and triisopropyl borate (4.69 ml, 20.3 mmol, 3.0 eq) using the procedure described for 11 a, and isolated as a brown solid (1.46 g, 83 %).

General procedure for Suzuki‐Miyaura cross coupling to afford pyrrolo[2,3‐d]pyrimidine derivatives 2. To each of 7‐alkyl‐5‐iodo‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amines 10 a–e (0.191 mmol) was added tetrakis(triphenylphosphine) palladium(0) (0.1 eq) and the relevant boronic acid (2 eq), followed by a degassed solution of 1,2‐dimethoxyethane (30.0 ml) and 2 M aqueous sodium carbonate (4 eq). The resulting mixture was heated to 80 °C for 18 h under a nitrogen atmosphere. After completion, the reaction mixture was cooled to room temperature, quenched with water (50 ml) and extracted with ethyl acetate (3×100 ml). The organic layers were combined, dried over MgSO₄, filtered through celite and excess solvent removed on a rotary evaporator. Purification by silica gel column chromatography using an ethyl acetate/hexane mixture (50–80 %) gave title compounds 2 a–2 q in good yields. The following products were prepared using this methodology:

7‐(Cyclopropylmethyl)‐5‐(2‐methoxynaphthalen‐6‐yl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 2 a. Prepared from 10 a (60.0 mg, 0.191 mmol), Pd(PPh₃)₄ (22.1 mg, 0.0191 mmol), 11 a (77.0 mg, 0.382 mmol) and a solution of DME (30.0 ml) and 2 M aqueous Na₂CO₃ (0.400 ml, 4 eq) as described in general procedure above. The product 2 a (0.036 g, 55 %) was isolated as a yellow solid after purification by column chromatography (80 % EtOAc/hexane). R _f (10 % MeOH/EtOAc) 0.48; m.p. 215–217 °C; IR (v _max/cm⁻¹) 3462, 3060, 1500, 1454, 1020; ¹H NMR (300 MHz, MeOD) δ 8.16 (s, 1H), 7.93–7.85 (m, 2H), 7.79 (d, J=9.0 Hz, 1H), 7.57 (dd, J=8.3, 1.9 Hz, 1H), 7.34 (s, 1H), 7.28 (d, J=2.5 Hz, 1H), 7.17 (dd, J=9.0, 2.5 Hz, 1H), 4.10 (d, J=7.1 Hz, 2H), 3.93 (s, 3H), 1.42–1.26 (m, 1H), 0.68–0.55 (m, 2H), 0.51–0.38 (m, 1H); ¹³C NMR (126 MHz, MeOD) δ 159.4, 158.6, 151.8, 150.9, 135.2, 131.0, 130.6, 130.4, 128.7, 128.6, 128.2, 124.9, 120.5, 118.1, 106.8, 102.0, 55.8, 50.0, 12.5, 4.3; HRMS (ES⁺) found [M+H]⁺ 345.1694, C₂₁H₂₁N₄O⁺ requires 345.1710.

7‐(Cyclopropylmethyl)‐5‐(2‐ethoxynaphthalen‐6‐yl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 2 b. Prepared from 10 a (80.0 mg, 0.255 mmol), Pd(PPh₃)₄ (30.0 mg, 0.0255 mmol), 11 b (0.110 g, 0.509 mmol) and a solution of DME (30.0 ml) and 2 M aqueous Na₂CO₃ (0.500 ml, 4 eq) using the procedure described for the preparation of 2 a. The product 2 b (0.055 g, 60 %) was isolated as a light yellow solid after purification by column chromatography (80 % EtOAc/hexane). R _f (10 % MeOH/EtOAc) 0.48; m.p. 215–216 °C; IR (v _max/cm⁻¹) 3464, 3062, 1455, 1222, 1020; ¹H NMR (400 MHz, CDCl₃) δ 8.34 (s, 1H), 7.87 (s, 1H), 7.81 (d, J=8.4 Hz, 1H), 7.77 (d, J=8.8 Hz, 1H), 7.58 (dd, J=8.5, 1.7 Hz, 1H), 7.23–7.15 (m, 3H), 5.26 (s, 2H), 4.19 (q, J=7.1 Hz, 2H), 4.12 (d, J=7.1 Hz, 2H), 1.51 (t, J=6.9 Hz, 3H), 1.38–1.27 (m, 1H), 0.68–0.58 (m, 2H), 0.46 (t, J=5.1 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 157.3, 156.9, 151.6, 150.6, 133.7, 129.9, 129.4, 129.1, 127.72, 127.70, 127.3, 122.9, 120.1, 106.6 (2 C), 105.7, 63.7, 49.1, 15.0, 11.6, 4.2; HRMS (ES⁺) found [M+H]⁺ 359.1848, C₂₂H₂₃N₄O⁺ requires 359.1886.

5‐[2‐(2‐Ethoxyethoxy)naphthalen‐6‐yl]‐7‐(cyclopropylmethyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 2 c. Prepared from 10 a (80.0 mg, 0.255 mmol), Pd(PPh₃)₄ (30.0 mg, 0.0255 mmol), 11 c (0.132 g, 0.509 mmol) and a solution of DME (30.0 ml) and 2 M aqueous Na₂CO₃ (0.500 ml, 4 eq) using the procedure described for the preparation of 2 a. The product 2 c (0.036 g, 35 %) was isolated as a light yellow solid after purification by column chromatography (80 % EtOAc/hexane). R _f (10 % MeOH/EtOAc) 0.48; m.p. 216–218 °C; IR (v _max/cm⁻¹) 3443, 3277, 3065, 1631, 1062; ¹H NMR (300 MHz, MeOD) δ 8.17 (s, 1H), 7.88 (dd, J=5.1, 3.3 Hz, 2H), 7.81 (d, J=9.0 Hz, 1H), 7.58 (dd, J=8.5, 1.7 Hz, 1H), 7.36 (s, 1H), 7.31 (d, J=2.5 Hz, 1H), 7.22 (dd, J=8.9, 2.5 Hz, 1H), 4.29–4.22 (m, 2H), 4.11 (d, J=7.1 Hz, 2H), 3.90–3.84 (m, 2H), 3.64 (q, J=7.0 Hz, 2H), 1.32–1.19 (m, 4H), 0.65–0.57 (m, 2H), 0.50–0.45 (m, 2H); ¹³C NMR (126 MHz, MeOD) δ 158.5, 151.7 (2 C), 150.9, 135.1, 131.0, 130.7, 130.5, 128.8, 128.6, 128.2, 125.0, 120.7, 118.1, 107.8, 102.0, 70.2, 68.7, 67.8, 50.0, 15.4, 12.5, 4.3; HRMS (ES⁺) found [M+H]⁺ 403.2116, C₂₄H₂₇N₄O⁺ requires 403.2129.

7‐(Cyclopropylmethyl)‐5‐(2‐methoxyphenyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 2 d. Prepared from 10 a (60.0 mg, 0.191 mmol), Pd(PPh₃)₄ (22.1 mg, 0.0191 mmol), 2‐methoxyphenylboronic acid 11 d (60.0 mg, 0.382 mmol) and a solution of DME (30.0 ml) and 2 M aqueous Na₂CO₃ (0.400 ml, 4 eq) using the procedure described for the preparation of 2 a. The product 2 d (0.031 g, 55 %) was isolated as a light yellow solid after purification by column chromatography (80 % EtOAc/hexane). R _f (10 % MeOH/EtOAc) 0.48; m.p. 221–222 °C; IR (v _max/cm^‐1) 3445, 3225, 3115, 1632, 1060; ¹H NMR (300 MHz, MeOD) δ 8.12 (s, 1H), 7.40 (ddd, J=8.4, 7.3, 1.8 Hz, 1H), 7.32 (dd, J=7.5, 1.8 Hz, 1H), 7.22 (s, 1H), 7.13 (d, J=8.3 Hz, 1H), 7.06 (td, J=7.5, 1.1 Hz, 1H), 4.09 (d, J=7.1 Hz, 2H), 3.82 (s, 3H), 1.48–1.18 (m, 1H), 0.73–0.51 (m, 2H), 0.51–0.31 (m, 2H); ¹³C NMR (126 MHz, MeOD) δ 159.0, 158.4, 151.7, 150.5, 132.9, 130.4, 125.4, 124.6, 122.1, 113.5, 112.6, 103.8, 56.0, 49.9, 12.5, 4.3; HRMS (ES⁺) found [M+H]⁺ 295.1548 C₁₇H₁₉N₄O⁺ requires 295.1553.

7‐(Cyclopropylmethyl)‐5‐(3,4‐dimethoxyphenyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 2 e. Prepared from 10 a (100 mg, 0.318 mmol), Pd(PPh₃)₄ (36.8 mg, 0.0318 mmol), 3,4‐dimethoxyphenylboronic acid 11 e (0.120 g, 0.637 mmol) and a solution of DME (30 ml) and 2 M aqueous Na₂CO₃ (0.700 ml, 4 eq) using the procedure described for the preparation of 2 a. The product 2 e (0.071 g, 69 %) was isolated as a light yellow solid after purification by column chromatography (80 % EtOAc/hexane). R _f (10 % MeOH/EtOAc) 0.48; m.p. 201–202 °C; IR (v _max/cm⁻¹) 3444, 3202, 3108, 1632, 1061; ¹H NMR (500 MHz, DMSO‐d ₆) δ 8.12 (s, 1H), 7.36 (s, 1H), 7.06 (d, J=8.2 Hz, 1H), 7.03 (d, J=2.0 Hz, 1H), 6.97 (dd, J=8.1, 2.0 Hz, 1H), 6.06 (s, 2H), 4.01 (d, J=7.1 Hz, 2H), 3.81 (s, 3H), 3.80 (s, 3H), 1.35–1.18 (m, 1H), 0.58–0.46 (m, 2H), 0.46–0.36 (m, 2H); ¹³C NMR (126 MHz, DMSO) δ 157.7, 152.0, 150.4, 149.4, 148.3, 127.9, 123.3, 120.9, 115.5, 12.9, 112.8, 100.5, 56.1, 55.9, 48.5, 12.1, 4.1; HRMS (ES⁺) found [M+H]⁺ 325.1672, C₁₈H₂₁N₄O₂ ⁺ requires 325.1659.

1‐{4‐[(4‐Amino‐5‐(2‐methoxynaphthalen‐6‐yl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐7‐yl)methyl]piperidin‐1‐yl}ethanone 2 f. Prepared from 10 b (80.0 mg, 0.200 mmol), Pd(PPh₃)₄ (23.2 mg, 0.0200 mmol), 11 a (81.0 mg, 0.401 mmol) and a degassed solution of DME (30 ml) and 2 M aqueous Na₂CO₃ (0.400 ml, 4 eq) using the procedure described for the preparation of 2 a. The product 2 f (0.064 g, 74 %) was isolated as a yellow solid after purification by column chromatography (10 % MeOH/chloroform). R _f (10 % MeOH/chloroform) 0.28; m.p. 214–216 °C; IR (v _max/cm⁻¹) 3300, 2917, 1606, 1440, 1259, 1026; ¹H NMR (500 MHz, MeOD) δ 8.18 (s, 1H), 7.91–7.87 (m, 2H), 7.80 (d, J=9.0 Hz, 1H), 7.58 (dd, J=8.4, 1.8 Hz, 1H), 7.30 (d, J=2.6 Hz, 1H), 7.29 (s, 1H), 7.18 (dd, J=9.0, 2.5 Hz, 1H), 4.54–4.42 (m, 1H), 4.17 (d, J=7.3 Hz, 2H), 3.94 (s, 3H), 3.92–3.84 (m, 1H), 3.07 (td, J=13.6, 2.7 Hz, 1H), 2.60 (td, J=12.9, 2.9 Hz, 1H), 2.32–2.16 (m, 1H), 2.08 (s, 3H), 1.76–1.58 (m, 2H), 1.39–1.28 (m, 2H); ¹³C NMR (126 MHz, MeOD) δ 171.5, 159.5, 158.5, 151.7, 151.2, 135.2, 130.7, 130.6, 130.4, 128.8, 128.5, 128.2, 125.6, 120.5, 118.3, 106.8, 102.0, 55.9, 42.6, 38.3, 31.2, 30.5, 21.2; HRMS (ES⁺) found [M+H]⁺ 430.2214, C₂₅H₂₈N₅O₂ ⁺ requires 430.2238.

1‐{4‐[(4‐Amino‐5‐(2‐ethoxynaphthalen‐6‐yl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐7‐yl)methyl]piperidin‐1‐yl}ethanone 2 g. Prepared from 10 b (100 mg, 0.251 mmol), Pd(PPh₃)₄ (28.9 mg, 0.0251 mmol), 11 b (0.108 g, 0.501 mmol) and a degassed solution of DME (30 ml) and 2 M aqueous Na₂CO₃ (0.500 ml, 4 eq) using the procedure described for the preparation of 2 a. The product 2 g (0.084 g, 76 %), was isolated as a yellow solid after purification by column chromatography (10 % MeOH/chloroform). R _f (10 % MeOH/chloroform) 0.28; m.p. 215–217 °C; IR (v _max/cm⁻¹) 3468, 2923, 1612, 1440, 1231, 1023; ¹H NMR (500 MHz, MeOD) δ 8.17 (s, 1H), 7.88–7.84 (m, 2H), 7.79 (d, J=8.9 Hz, 1H), 7.56 (dd, J=8.5, 1.8 Hz, 1H), 7.26 (d, J=1.9 Hz, 2H), 7.17 (dd, J=8.9, 2.5 Hz, 1H), 4.51 (ddt, J=13.2, 4.4, 2.4 Hz, 1H), 4.20–4.13 (m, 4H), 3.91 (ddt, J=13.8, 4.8, 2.4 Hz, 1H), 3.06 (ddd, J=13.7, 12.3, 2.7 Hz, 1H), 2.59 (td, J=12.9, 2.9 Hz, 1H), 2.27–2.16 (m, 1H), 2.08 (s, 3H), 1.72–1.59 (m, 2H), 1.46 (t, J=7.0 Hz, 3H), 1.36–1.24 (m, 2H); ¹³C NMR (126 MHz, MeOD) δ 171.4, 158.7, 158.6, 151.8, 151.3, 135.3, 130.6, 130.5, 130.4, 128.7, 128.5, 128.2, 125.6, 120.8, 118.3, 107.6, 102.0, 64.6, 42.5, 38.3, 31.2, 30.5, 21.2, 15.1; HRMS (ES⁺) found [M+H]⁺ 444.2375, C₂₆H₃₀N₅O₂ ⁺ requires 444.2394.

1‐{4‐{[5‐(2‐(2‐Ethoxyethoxy)naphthalen‐6‐yl)‐4‐amino‐7H‐pyrrolo[2,3‐d]pyrimidin‐7‐yl]methyl}piperidin‐1‐yl}ethanone 2 h. Prepared from 10 b (70.0 mg, 0.175 mmol), Pd(PPh₃)₄ (20.3 mg, 0.0175 mmol), 11 c (91.2 mg, 0.351 mmol) and a degassed solution of DME (30 ml) 2 M aqueous Na₂CO₃ (0.400 ml, 4 eq) using the procedure described for the preparation of 2 a to afford, after filtration and column chromatography using 10 % MeOH/chloroform as eluent, 2 h (0.046 g, 54 %) as a light brown viscous oil. R _f (10 % MeOH/chloroform) 0.28; m.p. 215–216 °C; IR (v _max/cm⁻¹) 3301, 3108, 1644, 1473, 1272, 1029; ¹H NMR (500 MHz, DMSO‐d ₆) δ 8.17 (s, 1H), 7.92–7.83 (m, 3H), 7.58 (dd, J=8.4, 1.8 Hz, 1H), 7.41 (s, 1H), 7.38 (d, J=2.5 Hz, 1H), 7.21 (dd, J=8.9, 2.5 Hz, 1H), 6.15 (s, 2H), 4.34 (d, J=13.2 Hz, 1H), 4.27–4.19 (m, 2H), 4.10 (dd, J=7.2, 1.5 Hz, 2H), 3.82–3.72 (m, 3H), 3.54 (q, J=7.0 Hz, 2H), 2.95 (td, J=13.0, 2.7 Hz, 1H), 2.46 (dd, J=12.7, 2.9 Hz, 1H), 2.14 (ddp, J=10.9, 7.1, 3.8 Hz, 1H), 1.96 (s, 3H), 1.60–1.48 (m, 4H), 1.15 (t, J=7.0 Hz, 3H); ¹³C NMR (126 MHz, DMSO) δ 167.8, 157.2, 156.4, 151.5, 150.5, 133.0, 129.8, 129.3, 128.7, 127.5, 127.4, 126.4, 124.1, 119.2, 115.2, 106.7, 99.9, 68.3, 67.3, 65.7, 45.4, 36.4, 29.9, 29.1, 21.3, 15.1; HRMS (ES⁺) found [M+H]⁺ 488.2622, C₂₈H₃₄N₅O₃ ⁺ requires 488.2656.

1‐{4‐[(4‐Amino‐5‐(2‐methoxyphenyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐7‐yl)methyl]piperidin‐1‐yl}ethanone 2 i. Prepared from 10 b (70.0 mg, 0.175 mmol), Pd(PPh₃)₄ (20.0 mg, 0.0175 mmol), 2‐methoxyphenylboronic acid 11 d (53.3 mg, 0.351 mmol) and a degassed solution of DME (30 ml) and 2 M aqueous Na₂CO₃ (0.400 ml, 4 eq) using the procedure described for the preparation of 2 a to afford, after filtration and column chromatography using 10 % MeOH/chloroform as eluent, 2 i (0.036 g, 54 %) as a light yellow solid. R _f (10 % MeOH/chloroform) 0.28; m.p. 215–217 °C; IR (v _max/cm^‐1) 3419, 2933, 1633, 1474, 1252, 1027; ¹H NMR (500 MHz, MeOD) δ 8.11 (s, 1H), 7.39 (ddd, J=8.3, 7.4, 1.8 Hz, 1H), 7.31 (dd, J=7.5, 1.8 Hz, 1H), 7.13 (dd, J=8.4, 1.1 Hz, 1H), 7.11 (s, 1H), 7.05 (td, J=7.5, 1.1 Hz, 1H), 4.51 (ddt, J=13.1, 4.4, 2.4 Hz, 1H), 4.12 (d, J=7.3 Hz, 2H), 3.92 (ddt, J=13.7, 4.8, 2.4 Hz, 1H), 3.81 (s, 3H), 3.06 (ddd, J=13.8, 12.4, 2.8 Hz, 1H), 2.59 (td, J=12.9, 2.9 Hz, 1H), 2.19 (ddt, J=11.5, 7.7, 3.8 Hz, 1H), 2.08 (s, 3H), 1.73–1.55 (m, 2H), 1.36–1.23 (m, 2H); ¹³C NMR (126 MHz, MeOD) δ 171.5, 159.1, 158.3, 151.9, 150.8, 132.9, 130.5, 126.1, 124.4, 122.2, 113.5, 112.6, 103.8, 56.1, 42.6, 38.3, 31.2, 30.5, 21.2; HRMS (ES⁺) found [M+H]⁺ 380.2044, C₂₁H₂₆N₅O₂ ⁺ requires 380.2081.

1‐{4‐[(4‐Amino‐5‐(3,4‐dimethoxyphenyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐7‐yl)methyl]piperidin‐1‐yl}ethanone 2 j. Prepared from 10 b (80.0 mg, 0.200 mmol), Pd(PPh₃)₄ (23.2 mg, 0.0200 mmol), 3,4‐dimethoxyphenylboronic acid 11 e (73.0 mg, 0.400 mmol) and a degassed solution of DME (30 ml) and 2 M aqueous Na₂CO₃ (0.400 ml, 4 eq) using the procedure described for the preparation of 2 a to afford, after filtration and column chromatography using 10 % MeOH/chloroform as eluent, 2 j (0.054 g, 66 %) as a yellow solid. R _f (10 % MeOH/chloroform) 0.28; m.p. 215–216 °C; IR (v _max/cm⁻¹) 3468, 2923, 1612, 1440, 1231, 1023; ¹H NMR (500 MHz, MeOD) δ 8.14 (s, 1H), 7.17 (s, 1H), 7.11–6.95 (m, 3H), 4.51 (ddt, J=13.1, 4.4, 2.4 Hz, 1H), 4.12 (d, J=7.3 Hz, 2H), 3.95–3.89 (m, 1H), 3.88 (s, 3H), 3.878 (s, 3H), 3.05 (ddd, J=13.7, 12.3, 2.8 Hz, 1H), 2.58 (td, J=12.9, 2.9 Hz, 1H), 2.19 (ddt, J=11.5, 7.6, 3.8 Hz, 1H), 2.08 (s, 3H), 1.64 (tdd, J=11.8, 4.0, 2.2 Hz, 2H), 1.37–1.14 (m, 2H); ¹³C NMR (126 MHz, MeOD) δ 171.4, 158.8, 152.2, 151.1, 150.9, 150.1, 128.7, 125.0, 122.4, 117.9, 113.9, 113.5, 102.0, 64.3, 56.5, 42.5, 38.3, 31.2, 30.5, 21.2; HRMS (ES⁺) found [M+H]⁺ 410.2153, C₂₂H₂₈N₅O₃ ⁺ requires 410.2187.

tert ‐Butyl 4‐{2‐[4‐amino‐5‐(2‐methoxyphenyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐7‐yl]ethyl}piperidine‐1‐carboxylate 2 k. Prepared from 10 c (80.0 mg, 0.169 mmol), Pd(PPh₃)₄ (19.6 mg, 0.0169 mmol), 2‐methoxyphenylboronic acid 11 d (51.6 mg, 0.339 mmol) and a solution of DME (30 ml) and 2 M aqueous Na₂CO₃ (0.400 ml) using the procedure described for the preparation of 2 a to afford 2 k (0.035 g, 46 %) as a viscous oil after purification by column chromatography (80 % EtOAc/hexane). R _f (80 % EtOAc/hexane) 0.48; IR (v _max/cm⁻¹) 3485, 2930, 1679, 1453, 1161, 760; ¹H NMR (500 MHz, DMSO‐d ₆) δ 8.10 (s, 1H), 7.37 (ddd, J=8.2, 7.4, 1.8 Hz, 1H), 7.25 (dd, J=7.4, 1.8 Hz, 1H), 7.22 (s, 1H), 7.13 (dd, J=8.4, 1.1 Hz, 1H), 7.03 (td, J=7.4, 1.1 Hz, 1H), 5.82 (s, 2H), 4.18 (t, J=7.3 Hz, 2H), 3.94–3.83 (m, 2H), 3.76 (s, 3H), 3.31–3.26 (m, 2H), 1.77–1.62 (m, 5H), 1.38 (s, 9H), 1.09–0.96 (m, 2H); ¹³C NMR (126 MHz, DMSO‐d ₆) δ 157.5, 156.4, 153.8, 151.3, 149.7, 131.5, 128.8, 123.8, 123.4, 120.8, 111.6, 110.7, 101.8, 78.4, 55.3, 41.2, 36.3, 32.8, 31.4, 28.1; HRMS (ES⁺) found [M+H]⁺ 452.2648, C₂₅H₃₄N₅O₃ ⁺ requires 452.2656.

tert ‐Butyl 4‐{2‐[4‐amino‐5‐(3,4‐dimethoxyphenyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐7‐yl]ethyl}piperidine‐1‐carboxylate 2 l. Prepared from 10 c (90.0 mg, 0.191 mmol), Pd(PPh₃)₄ (20.0 mg, 0.0191 mmol), and 3,4‐dimethoxyphenylboronic acid 11 e (69.5 mg, 0.382 mmol) and a degassed solution of DME (30.0 ml) and 2 M aqueous Na₂CO₃ (0.400 ml) using the procedure described for the preparation of 2 a to afford 2 l (0.0440 g, 48 %) as an oil after purification by column chromatography (80 % EtOAc/hexane). R _f (80 % EtOAc/hexane) 0.48; IR (v _max/cm⁻¹) 3485, 2862, 1678, 1453, 1244, 865.3; ¹H NMR (500 MHz, MeOD) δ 8.14 (s, 1H), 7.20 (s, 1H), 7.08–6.98 (m, 3H), 4.27 (t, J=7.4 Hz, 2H), 4.03 (dt, J=14.6, 3.6 Hz, 2H), 3.88 (s, 6H), 2.75–2.64 (m, 2H), 1.85–1.73 (m, 4H), 1.44 (s, 9H), 1.41 (d, J=2.0 Hz, 1H), 1.13 (qd, J=12.5, 4.3 Hz, 2H); ¹³C NMR (126 MHz, MeOD) δ 158.6, 156.5, 151.8, 150.8, 150.8, 150.0, 128.7, 124.4, 122.4, 118.1, 113.9, 113.5, 102.0, 80.9, 56.5, 43.2, 37.9, 34.7, 33.0, 30.7, 28.7; HRMS (ES⁺) found [M+H]⁺ 482.2743, C₂₆H₃₆N₅O₄ ⁺ requires 482.2762.

5‐(2‐Ethoxynaphthalen‐6‐yl)‐7‐(2‐morpholinoethyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 2 m. Prepared from 10 d (0.250 g, 0.670 mmol), Pd(PPh₃)₄ (77.4 mg, 0.0670 mmol) and 11 b (0.291 g, 1.34 mmol) in DME (40.0 ml) using the procedure described for the preparation of 2 a and purified by column chromatography (10 % MeOH/chloroform) to afford 2 m (85.3 mg, 51 %) as a viscous oil. R _f (10 % MeOH/chloroform) 0.26; IR (v _max/cm⁻¹) 3233, 3079, 2990, 1560, 1070; ¹H NMR (500 MHz, CDCl₃) δ 8.34 (s, 1H), 7.85 (d, J=1.7 Hz, 1H), 7.81 (d, J=8.4 Hz, 1H), 7.77 (d, J=8.9 Hz, 1H), 7.57 (dd, J=8.4, 1.7 Hz, 1H), 7.24–7.15 (m, 2H), 7.12 (s, 1H), 5.12 (s, 2H), 4.38 (t, J=6.6 Hz, 2H), 4.19 (q, J=6.9 Hz, 2H), 3.70 (t, J=4.6 Hz, 4H), 2.82 (t, J=6.6 Hz, 2H), 2.55 (t, J=4.6 Hz, 4H), 1.51 (t, J=6.9 Hz, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 157.2, 157.0, 151.9, 150.8, 133.6, 129.9, 129.3, 129.0, 127.6, 127.1, 123.3, 119.9, 116.3, 106.5, 101.3, 67.0, 63.6, 58.3, 53.7, 41.7, 14.8; HRMS (ES⁺) found [M+H]⁺ 418.2240, C₂₄H₂₈N₅O₂ ⁺ requires 418.2238.

5‐(2‐Methoxyphenyl)‐7‐(2‐morpholinoethyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 2 n. Prepared from 10 d (0.210 g, 0.563 mmol), Pd(PPh₃)₄ (65.0 mg, 0.0563 mmol) and 2‐methoxyphenylboronic acid 11 d (0.171 g, 1.13 mmol) in DME (30.0 ml) using the procedure described for the preparation of 2 a, to afford 2 n (73.2 mg, 52 %) as a light yellow solid after purification by silica gel column chromatography (10 % MeOH/chloroform). R _f (10 % MeOH/chloroform) 0.26; m.p. 221–223 °C; IR (v _max/cm⁻¹) 3327, 3056, 1694, 1460, 1436, 1115; ¹H NMR (500 MHz, DMSO‐d ₆) δ 8.09 (s, 1H), 7.40–7.34 (m, 1H), 7.24 (d, J=7.2 Hz, 2H), 7.13 (d, J=8.4 Hz, 1H), 7.03 (t, J=7.4 Hz, 1H), 4.26 (t, J=6.6 Hz, 2H), 3.75 (s, 3H), 2.77–2.59 (m, 3H), 2.48–2.41 (m, 7H); ¹³C NMR (126 MHz, DMSO‐d ₆) δ 157.6, 156.6, 151.3, 149.8, 131.6, 129.0, 124.5, 123.4, 121.0, 111.8, 110.8, 102.0, 66.3, 57.8, 55.5, 53.3, 41.0; HRMS (ES⁺) found [M+H]⁺ 354.1930, C₁₉H₂₄N₅O₂ ⁺ requires 354.1925.

5‐(3,4‐Dimethoxyphenyl)‐7‐(2‐morpholinoethyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 2 o. Prepared from 10 d (100 mg, 0.268 mmol), Pd(PPh₃)₄ (30.9 mg, 0.0268 mmol) and 3,4‐dimethoxyphenylboronic acid 11 e (97.5 mg, 0.536 mmol) in DME (30.0 ml) using the procedure described for the preparation of 2 a, to afford 2 o (68.0 mg, 66 %) as a yellow solid. R _f (10 % MeOH/chloroform) 0.26; m.p. 220–221 °C; IR (v _max/cm⁻¹) 3230, 3082, 3020, 1566, 1234, 1068; ¹H NMR (500 MHz, DMSO‐d ₆) δ 8.13 (s, 1H), 7.32 (s, 1H), 7.06 (d, J=8.2 Hz, 1H), 7.01 (d, J=2.0 Hz, 1H), 6.96 (dd, J=8.1, 2.0 Hz, 1H), 6.07 (br s, 2H), 4.27 (t, J=6.7 Hz, 2H), 3.80 (s, 3H), 3.79 (s, 3H), 3.54 (t, J=4.6 Hz, 4H), 2.70 (t, J=6.7 Hz, 2H), 2.48–2.42 (m, 4H); ¹³C NMR (126 MHz, DMSO‐d₆ ) δ 157.1, 151.4, 150.1, 148.9, 147.8, 127.4, 123.3, 120.3, 115.0, 112.3, 99.9, 99.9, 66.2, 57.7, 55.6, 55.4, 53.2, 40.7; HRMS (ES⁺) found [M+H]⁺ 384.2003, C₂₀H₂₆N₅O₃ ⁺ requires 384.2030.

7‐Benzyl‐5‐(2‐methoxyphenyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 2 p. Prepared from 10 e (40.0 mg, 0.114 mmol), Pd(PPh₃)₄ (13.2 mg, 0.0114 mmol) and 2‐methoxyphenylboronic acid 11 d (34.7 mg, 0.228 mmol) in a solution of DME (30.0 ml) and 2 M aqueous Na₂CO₃ (0.300 ml) using the procedure described for the synthesis of 2 a to afford 2 p (0.018 g, 48 %) as a light yellow solid after purification by column chromatography (80 % EtOAc/hexane). R _f (10 % MeOH/EtOAc) 0.50; m.p. 240–241 °C; IR (v _max/cm⁻¹) 3476, 3107, 1581, 1452, 1026; ¹H NMR (300 MHz, MeOD) δ 8.14 (s, 1H), 7.71–7.57 (m, 1H), 7.44–7.20 (m, 6H), 7.16–7.07 (m, 2H), 7.03 (td, J=7.3, 1.0 Hz, 1H), 5.42 (s, 2H), 3.79 (s, 3H); ¹³C NMR (75 MHz, MeOD) δ 158.3, 157.9, 151.7, 150.8, 138.9, 132.8, 130.5, 129.8, 128.8, 128.5, 125.5, 122.2, 112.6, 101.4, 56.0, 48.8; HRMS (ES⁺) found [M+H]⁺ 331.1534, C₂₀H₁₉N₄O⁺ requires 331.1553.

7‐Benzyl‐5‐(3,4‐dimethoxyphenyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐amine 2 q. Prepared from 10 e (500 mg, 0.143 mmol), Pd(PPh₃)₄ (16.5 g, 0.0143 mmol), 3,4‐dimethoxyphenylboronic acid 11 e (52.0 mg, 0.286 mmol) and 2 M aqueous Na₂CO₃ (0.300 ml, 4 eq) using the procedure described for the synthesis of 2 a to afford, after filtration and column chromatography using 80 % EtOAc/hexane as eluent, 2 q (0.023 g, 45 %) as a yellow solid. R _f (10 % MeOH/EtOAc) 0.50; m.p. 242–244 °C; IR (v _max/cm^‐1) 3425, 3108, 1579, 1029; ¹H NMR (300 MHz, DMSO‐d ₆) δ 8.16 (s, 1H), 7.36 (s, 1H), 7.37–7.22 (m, 5H), 7.04 (d, J=8.2 Hz, 1H), 7.01 (d, J=2.0 Hz, 1H), 6.95 (dd, J=8.1, 2.0 Hz, 1H), 6.14 (s, 2H), 5.37 (s, 2H), 3.79 (s, 3H), 3.78 (s, 3H); ¹³C NMR (126 MHz, DMSO) δ 157.2, 151.7, 150.1, 148.9, 147.9, 138.1, 128.6, 127.5, 127.4, 127.1, 122.9, 120.4, 115.7, 112.3, 112.3, 100.0, 55.6, 55.4, 47.1; HRMS (ES⁺) found [M+H]⁺ 361.1622, C₂₁H₂₁N₄O₂ ⁺ requires 361.1659.

Biology

Recombinant expression and Enzyme activity assay. Recombinant expression of PfCDPK1 and PfCDPK4 protein was carried out in Rosetta Oxford Escherichia coli (Invitrogen, Carlsbad, CA, USA) at 20 °C using Studier auto‐induction protocols as earlier described.[ ⁷ , ⁸ , ¹⁴ ] Soluble recombinant expressions of the two enzymes were purified by immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography as previously described.[ ⁷ , ⁸ , ¹⁵ ] Recombinant PfCDPK1 and PfCDPK4 activity was detected using a non‐radioactive Kinase Glo luciferase assay (Promega) to measure ATP consumption in the presence of Syntide‐2 peptide substrate (peptide sequence: PLARTLSVAGLPGKK). Kinase phosphorylation reactions were performed in 20 mM HEPES pH 7.5 (KOH), 0.1 % BSA, 10 mM MgCl₂, plus or minus 1 mM CaCl₂, 1 mM EGTA (pH 7.2). The phosphorylation reaction mixture contained 40 μM peptide substrate, 10 nM of PfCDPK1 and 180 nM of PfCDPK4 combined with serial dilutions of inhibitor in a total volume of 25 μl. Reaction was initiated by the addition of 10 μM ATP and terminated after 90 minutes incubation at 30 °C. Positive (BKI‐1294) and negative (DMSO) controls were included in each assay plate run. Unused ATP was measured in luminescence‐based readout as counts per second on an EnVision Multimode Plate Reader (PerkinElmer).

pLDH assay. Malaria parasites (Plasmodium falciparum strain 3D7) were maintained in RPMI 1640 medium containing 2 mM L‐glutamine and 25 mM HEPES (Gibco). The medium was further supplemented with 5 % Albumax II, 20 mM glucose, 0.65 mM hypoxanthine, 60 μg/mL gentamycin and 2–4 % hematocrit human red blood cells. The parasites were cultured at 37 °C under an atmosphere of 5 % CO₂, 5 % O₂, 90 % N₂ in sealed T25 or T75 culture flasks. Compounds at 20 μM were added to parasite cultures (2 % parasitaemia, 1 % haematocrit) in 96‐well plates. Incubations were initiated when parasites were predominantly in the trophozoite stage and continued for 48 h at 37 °C in sealed containers filled with an atmosphere of 5 % CO₂, 5 % O₂, 90 % N₂. After 48 h, the plates were removed from the incubator and a colourimetric assay for parasite lactate dehydrogenase (pLDH) activity in individual wells was carried out. ^[16] Twenty μL of culture was removed from each well and mixed with 125 μL of a mixture of Malstat solution and NBT/PES solution [0.18 M lactate, 0.13 mM acetylpyridine adenine dinucleotide, 0.39 mM nitrotetrazolium blue chloride, 0.048 mM phenazineethosulfate and 0.16 % (v/v) Triton X‐100 in 44 mM Tris (pH 9)] in a fresh 96‐well plate and incubated at ambient temperature for 10–30 minutes, after which Abs₆₂₀ was measured in a Spectramax M3 multiwell plate reader. For each compound concentration, absorbance values were used to calculate percentage parasite viability relative to control wells containing untreated parasite cultures. Compounds were tested in duplicate wells, and a standard deviation (SD) was derived. IC₅₀ values were only obtained for compounds that reduced pLDH activity to less than 25 % parasite viability after dosing for 48 hours. IC₅₀ values were calculated from plots of % viability vs. log(concentration) by non‐linear regression using GraphPad Prism®.

Cytotoxicity assay. HeLa cells were plated in 96 well plates at a density of 2×10⁴ cells per well in DMEM medium containing 10 % fetal bovine serum, 2 mM L‐glutamine and antibiotics (penicillin, streptomycin, amphotericin B) and incubated overnight at 37 °C in a 5 % CO₂ incubator. Test compounds were added to a final concentration of 20 μM and incubation was continued for a further 48 h. Remaining viable cells in the wells were detected by adding resazurin to a final concentration of 50 μM, continuing incubation for 4 hours and measuring resorufin fluorescence (Ex₅₆₀/Em₅₉₀) in a Spectramax M3 plate reader. Fluorescence readings were converted to % cell viability relative to untreated control wells, after subtracting background fluorescence readings obtained from wells without cells.

Conflict of interest

The authors declare no conflict of interest.

Supporting information

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Supporting Information

T. D. Seanego, H. E. Chavalala, H. H. Henning, C. B. de Koning, H. C. Hoppe, K. K. Ojo, A. L. Rousseau, ChemMedChem 2022, 17, e202200421.

Data Availability Statement

The data that support the findings of this study are available in the article supplementary material.