Francis Giraud
email: francis.giraud@uca.fr

Béatrice Josselin

Sandrine Ruchaud

Fabrice Anizon
email: fabrice.anizon@uca.fr

Pascale Moreau
email: pascale.moreau@uca.fr

4-yl)(pyridin-4-yl)methanone and derivatives

Keywords: Diheteroarylketone, Pyrido[3, 4-g]quinazoline, (Pyrimidin-4yl)(pyridin-4-yl)methanone, Grignard reagents, molecular shape, protein kinase inhibitors

quinazoline was previously identified as a relevant scaffold for protein kinase inhibition. In order to assess if the planarity of this heterocyclic system was essential to the protein kinase inhibitory potency observed in this series, new compounds were synthesized and evaluated, in which the central cycle was opened to provide (pyridin-4-yl)(pyrimidin-4yl)methane derivatives which were prepared from the corresponding ketone precursor. After preparing (2-aminopyrimidin-4-yl)(pyridin-4-yl)methanone, derivatives were synthesized and evaluated toward a panel of protein kinases. The results demonstrated that the planar pyrido[3,4-g]quinazoline tricyclic system was mandatory to maintain the protein kinase inhibitory potency in this series.

As part of our ongoing studies focused on the design and synthesis of new heteroaromatic compounds with potential protein kinases inhibitory potencies, we recently reported the identification of pyrido [3,4-g]quinazolines as nanomolar Cdclike kinase 1 (CLK1) and Dual-specificity tyrosine-regulated kinase 1A (DYRK1A) inhibitors (Figure 1). 1,2 Figure 1. General formula of pyrido [3,4-g]quinazolines previously identified. New scaffold generated by opening of the central ring.

It was previously demonstrated that aminopyrimidine and pyridine moieties of the pyrido [3,4-g]quinazoline scaffold, as well as substituents at the 10-position, are critical for interactions with the targeted protein kinase. 1,2 However, the pyridoquinazoline system possesses a planar structure which could lead to potential DNA intercalation. To limit the possible side effects due to this off-target activity, we hypothesized that opening the central pyridoquinazoline ring, while conserving aminopyrimidine, pyridine and 10-position moieties, could retain protein kinase inhibition (Figure 1). Thus, this structural modification would ultimately lead to (2-aminopyrimidin-4yl)(pyridin-4-yl)methanamines that could be prepared from the corresponding ketone precursor (Figure 1). Surprisingly, literature review showed not much nitrogen-containing diheteroarylketone syntheses whereas we found many reports on the preparation of these simple molecules in the carbocyclic series. Therefore, the preparation of these targeted molecules is of high interest not only for the medicinal chemistry purpose but also to enlarge chemical space diversity.

Template for SYNTHESIS Thieme

Starting from commercially available compounds I and II, the first strategy for (pyrimidin-4-yl)(pyridin-4-yl)methanone preparation was based on the condensation of a nucleophilic organometallic species with electrophilic aldehyde or carboxylic acid derivatives (Scheme 1).

Scheme 1. Initial retrosynthetic pathway used to prepare the desired diheteroarylketone.

The preparation of starting materials 1-3 using reaction conditions adapted from the literature (see experimental section) were successful (Scheme 2). However, neither the Negishi cross-coupling (organozinc species + acyl chloride) in various conditions [3][4][5][6] nor the addition of a Grignard reagent on a Weinreb amide 7 afforded the desired compound. The only encouraging result was obtained from Weinreb amide 3 9,10 and organolithium derivative prepared from Boc-protected aminopyrimidine 2 8 , leading to ketone 4 in 25% yield (Scheme 2). Protecting group cleavage in TFA provided 5 in 76% yield.

Scheme 2. Synthesis of (2-aminopyrimidin-4-yl)(pyridin-4-yl)methanone 5.

Numerous trials have been set up to optimize the preparation of 4. From compounds 2 and 3, changes in reaction conditions (temperature, stoichiometry, reagent addition order, use of t-BuLi) did not improve the yield. Thus, a second strategy based on the inversion of reactivity between both reaction partners was worked out. Nitrile intermediate 6 was prepared in 95% yield from 1 using CuCN in pyridine. 11 Compound 6 was then reacted with pyridin-4-ylmagnesium bromide prepared by iodinemagnesium exchange from 4-iodopyridine. 12 In these conditions, bis-heteroarylketone 5 was obtained in 30% yield, however without the need of the Boc-protection-deprotection sequence of the aminopyrimidine moiety (Scheme 2).

With compound 5 in hands, we planned to prepare (2aminopyrimidin-4-yl)(pyridin-4-yl)methanamine derivatives by reductive amination reaction. Treatment by ammonia 13 , ammonium acetate 14 or benzylamines 15,16 before reduction with NaBH4 did not afford expected amine derivatives. In these conditions, only the reduction product 7 (which could be prepared in 83% yield directly from 5 17 ) was isolated (Scheme 3), indicating difficulties to generate the intermediate imine.

Next, we expected to synthesize the primary amine via the oxime. Thus, oxime 8 was prepared from 5 and hydroxylamine hydrochloride in 56% yield as a Z/E 6:4 mixture. 18 However, attempts to reduce 8 (Mg/HCOONH4 19 , Zn/HCl 2M 20 ) only resulted in recovered starting material or degradation. The attribution of 1 H and 13 C NMR signals for each oxime isomer was performed using standard NMR experiments ( 1 H-1 H COSY, 1 H- 13 C HSQC, 1 H-13 C HMBC). 1 H-1 H NOESY experiment was also recorded in order to assign signals to the correct isomer. For the two isomers, weak NOE correlations were observed between hydroxyl group and both heterocycles. Strongest correlation was observed with the 3-pyridinyl proton for the minor isomer, and with the 5-pyrimidinyl proton for the major isomer, suggesting E and Z configuration, respectively (Figure 2). In addition, it was previously shown that α-syn-13 C of oximes are upshield shifted compared to α-anti-13 C. 21 Comparison of α-13 C chemical shifts for both isomers showed a similar behavior confirming the assignment. Indeed, pyridinyl α-13 C showed a chemical shift at 141.8 ppm (α-anti) for the major Z-isomer, and 139,2 ppm (αsyn) for the minor E-isomer. Similarly, pyrimidinyl α- 13 C was observed at 159.8 ppm (α-syn) for the major Z-isomer and 161.7 ppm (α-anti) for the E-minor isomer. Additional assignment confirmation was provided by the calculation of 1 H and 13 C NMR chemical shifts using Gaussian software, after geometry optimization calculations to produce the lowest-energy conformation for each isomer (Figure 2). As we found that (2-aminopyrimidin-4-yl)(pyridin-4yl)methanamines could not be obtained, we sought to extend our chemical library. Thus, hydrazone 9 was first prepared from 5 with hydrazine hydrochloride in similar condition than those used for the synthesis of oxime 8. 18 A crude mixture of Z/E isomers was obtained, leading to Z-9 in 57% yield after column chromatography, as well as trace amount of E-9. Assignments were performed by NMR from a mixture of both isomers. In the case of the E-isomer, 1 H-1 H-NOESY showed a correlation between hydrazone NH2, and a 3-pyridinyl proton. For the Z-isomer, weak NOE correlation was observed between hydrazone NH2 and both 3-pyridinyl and 5-pyrimidinyl protons moieties, the effect being weaker on the side of the pyridine moiety. However, a strong effect was observed between 3-pyridinyl and 5-pyrimidinyl protons, which is consistent with the structure obtained by molecular modeling (Figure 2C). As for oximes 8, the upshield shift of α-syn chemical shifts was observed 22,23 (Figures 2C,2D), confirming the assignment, also supported by NMR chemical shift calculations.

Finally, a Wittig reaction was performed in the presence of potassium carbonate using 5 and a phosphorus ylide prepared quantitatively from methyl 2-bromoacetate. 24 Again, the two Z and E diastereoisomers were obtained and isolated by column chromatography leading to 45% and 47% yields of isolated Z-10 and E-10, respectively (Scheme 3). As above, 1 H-1 H NOESY experiments were performed to determine the alkene configuration. Distinct NOE effect was observed between the alkene proton and the 3-pyridinyl position for the Z isomer, and the 5-pyrimidinyl proton for the E isomer (Figure 2E,2F). In the latter case, this proximity between protons was not confirmed by the structure obtained by molecular modeling, indicating a different conformation. However, the assignment was confirmed by chemical shift calculations.

To extend our chemical library, compound 9 was mixed with LiAlH4 in THF 25 and compound 10 with magnesium turnings in MeOH. 26 Nevertheless, no reduction of the C=C double bonds was observed, starting materials were recovered. This might be explained by steric hindrance induced by both heteroaryl rings. * uncertain assignment: 2D NMR experiments did not allow to attribute this carbon; assignment was done with the help of NMR calculation. The images were produced using UCSF Chimera software 27 .

Lastly, compound 5 was engaged in a Johnson-Corey-Chaykovsky epoxidation reaction in the presence of trimethylsulfoxonium iodide (TMSOI) to give 11 in 66% yield (Scheme 3). 28 Epoxide opening with sodium azide led to compound 12 in 81% yield. 29 Then, 12 was converted to the corresponding primary amine 13 under catalytic hydrogenation conditions (Scheme 3). 30 Finally, in order to measure the importance of the planar tricyclic system in the inhibition of protein kinases, compounds 5 and 7-13 were tested, as previously reported, 31 toward 8 kinases (Haspin, CDK9/Cyclin T, GSK3, CK1, CDK5/p25, Pim, CLK1 and DYRK1A). The percentage of residual kinase activity was evaluated in the presence of 10 M and 1 M compound concentrations. The results obtained showed that none of the tested compounds exhibited potent inhibitory potency toward the kinases tested.

In conclusion, we studied the synthetic route to (2aminopyrimidin-4-yl)(pyridin-4-yl)methanone and derivatives. Despite of the simplicity of their structure, the preparation of these compounds is poorly documented in the literature compared to the one of their analogues from the carbocyclic series. The preparation and biological evaluation of bisheteroaryl ketone 5 and analogues 7-13 highlighted the need of a planar polycyclic moiety in the pyridoquinazoline series to inhibit the tested kinases.

The experimental section has no title; please leave this line here.

Procedures

Starting materials were obtained from commercial suppliers and used without further purification. IR spectra were recorded on a PerkinElmer Spectrum 65 FT-IR spectrometer ( in cm -1 ). NMR spectra, performed on a Bruker AVANCE 400 III HD ( 1 H: 400 MHz, 13 C: 101 MHz) or a Bruker AVANCE 500 ( 1 H: 500 MHz, 13 C: 125 MHz) are reported in ppm using the solvent residual peak as an internal standard; the following abbreviations are used: singlet (s), doublet (d), broad signal (br s). Coupling constants are expressed in Hertz. High resolution mass spectra were determined on a high-resolution Waters Micro Q-Tof or Thermo Scientific Q Exactive Q-Orbitrap apparatus (UCA START, Universite Clermont Auvergne, Clermont-Ferrand, France). Chromatographic purifications were performed by column chromatography using 40-63 m silica gel. Reactions were monitored by TLC using fluorescent silica gel plates (60 F254 from Macherey Nagel). Melting points were measured on a Stuart SMP3 apparatus and are uncorrected. The purity of compounds 5 and 7-13 was established by HPLC analysis using a VWR Hitachi chromatograph with DAD detector and a Macherey Nagel Nucleodur gravity column (4.6 mm x 250 mm, 5 mm). Flow rate was 0.5 mL/min and the analysis was performed at 25 °C at 240 nm as detection wavelength for each compound. Solvents were (A) water/0.1% trifluoroacetic acid, (B) acetonitrile. Two methods were designed: method A was a gradient 5:95 A/B for 5 min to 95:5 A/B in 45 min whereas method B was an isocratic mode using 90/10 water/acetonitrile. Derivatives 1-3 were prepared following literature procedures without any modifications. Their structures were proved by 1 H NMR spectral analysis and were in accordance with reported 9,10,[32][33][34][35]

Di-tert-butyl [4-(pyridine-4-carbonyl)pyrimidin-2yl]imidodicarbonate (4)

To a solution of compounds 2 (0.100 g, 0.239 mmol) and 3 (0.033 g, 0.196 mmol) in 3 mL of THF cooled to -78°C, was added n-BuLi (0.265 mL, 1.6 M solution in hexane). The reaction mixture was stirred for 4 h at -78° C before a slow warm up to room temperature overnight. Water was added HPLC method B: purity > 96%,  = 240 nm, tR = 5.33 min.

2-Aminopyrimidine-4-carbonitrile (6)

To a solution of compound 1 (1.228 g, 5.556 mmol) in pyridine (14 mL) at room temperature under argon was added copper cyanide (0.547 g, 6.112 mmol). The mixture was heated at 115 °C overnight before cooling and filtration over Celite pad. Washings with EtOAc (400 mL) and acetone (200 mL) afforded an organic phase which was concentrated to 200 mL. The residue was washed with water and brine, dried over MgSO4 and evaporated to dryness. First a purification by flash column chromatography using Cyclohexane/acetone 1:1 to 1:2 was performed but the product was contaminated with pyridinium salts. It was taken up with EtOAc and washed with NaHCO3 solution and large amount of water. After drying over MgSO4 and evaporation compound 6 (0.640 g, 5.328 mmol) was obtained in 95% yield as a yellow powder.

Experimental data were in accordance with literature. HPLC method B: purity > 99%,  = 240 nm, tR = 6.55 min.

(E/Z)-(2-Aminopyrimidin-4-yl)(pyridin-4-yl)methanone hydrazone (9)

To a solution of compound 5 (74.5 mg, 0.372 mmol) in 7 mL EtOH/H2O 4:1 at room temperature were successively added hydrazine monohydrochloride (40.8 mg, 0.595 mmol) and sodium acetate (61.0 mg, 0.744 mmol). The mixture was stirred at 100 °C overnight, cooled and evaporated to dryness. Ethanol was added in 5 portions (10 mL each) and evaporated to remove water. Purification by flash column chromatography using CH2Cl2 to CH2Cl2/MeOH 88:12 as eluent afforded compound Z-9 (46.1 mg, 0.215 mmol) in 57% yield as a white powder. HPLC method B: purity > 95%,  = 240 nm, tR = 6.74 min.

Rf = 0.

(E)-and (Z)-Methyl 3-(2-aminopyrimidin-4-yl)-3-(pyridin-4yl)prop-2-enoate (10)

To a solution of [(methoxycarbonyl)methylene]triphenylphosphorane prepared following the same procedure as described 24 HPLC method B: purity > 95%,  = 240 nm, tR = 5.58 min.

Molecular Modeling

Structures were optimized using Gaussian 16 software 36 using DFT (density functional theory) method using B3LYP hybrid functional. Optimized geometries were obtained by relaxed potential energy surface scans with 6-31g basis set. Lower-energy structures were optimized in gas phase (B3LYP/6-31g+(d,p)) and vibrational frequencies were computed to check the absence of imaginary frequency. Gauge-Independent Atomic Orbital (GIAO) method was used for the calculation of NMR shielding tensors using the Gaussian 16 default solvation model (IEFPCM, DMSO), using mPW1PW91 functional and 6-311+G(d,p) or 6-311+G(2d,p) basis sets. Isotropic shield tensors were converted into δ chemical shifts using scaling factors reported by G.K. Pierens (with 6-311+G(2d,p) basis set). 37 Agreement between experimental and calculated NMR data was determined with the aid of DP4+ probability (using 6-311+G(d,p) basis set). [38][39][40]

Funding Information

This research was financed by the French government IDEX-ISITE initiative 16-IDEX-0001 (CAP 20-25) (Projet Emergence). We are also grateful to the Mésocentre Clermont Auvergne University for providing computing resources. The authors (FG, FA, PM) also thank WeylChem InnoTec (Frankfurt, Germany) for financial support.

Scheme 3 .

 3 Scheme 3. Synthesis of new compounds 7-13.

Figure 2 .

 2 Figure 2. Modeling of Z-(left) and E-isomer (right) of oxime 8 (A, B), hydrazone 9 (C, D) and methyl ester 10 (E, F). Relevant distances (dashed lines) and experimental NMR chemical shifts of α-syn and α-anti 13 C are indicated. * uncertain assignment: 2D NMR experiments did not allow to attribute this

  Template for SYNTHESIS Thiemebefore extraction with ethyl acetate. The combined organic phases were washed with a brine solution before drying over MgSO4 and evaporation under reduced pressure. The residue was purified by flash column chromatography using EtOAc/Pentane (3:7) to afford compound 4 (Rf = 0.25) in 25% yield (0.02 g, 0.050 mmol) as a green oil.IR (ATR): 1753, 1722, 1535, 1412, 1245 cm -1 .

	1 H NMR (400 MHz, DMSO-d6):  = 9.21 (d, J = 5.2 Hz, 2 H), 8.83 (d, J = 6.0,
	Hz, 1 H), 8.00 (d, J = 4.8 Hz, 2 H), 7.83 (d, J = 6.0 Hz, 1 H), 1.41 (s, 18 H).
	13 C NMR (101 MHz, DMSO-d6):  = 190.8 (CO), 150.3 (2 CO), 160.7 (C),
	157.0 (C), 140.9 (C), 161.9 (CH), 150.2 (2CH), 123.4 (2CH), 118.1 (CH),
	83.6 (2C), 27.9 (6 CH3).
	HRMS (ESI+): m/z [M+H] + calcd for C20H25N4O5: 401.1819; found:
	401.1812. m/z [M+CH3OH+H] + calcd for C21H29N4O6: 433.2087; found:
	433.2075.
	(2-Aminopyrimidin-4-yl)(pyridin-4-yl)methanone (5)
	Procedure A: TFA (0.2 mL) was added to a solution of 4 (0.026 g, 0.065
	mmol) in CH2Cl2 (0.8 mL) under argon. The reaction mixture was stirred
	for 30 min before evaporation and purification by flash column
	chromatography using EtOAc/Pentane (8:2) to give 5 (Rf = 0.2) in 76%
	yield (0.010 g, 0.05 mmol) as a light green powder.
	over
	MgSO4 and concentrated. Purification by flash chromatography using
	EtOAc/cyclohexane 2:1 then pure and finally a gradient from 1 to
	2% of MeOH in EtOAc afforded pure compound 5 in 30% yield (75.7 mg,
	0.378 mmol) as a yellow powder.
	Rf = 0.35 (EtOAc).
	IR (ATR): 3446-2512, 1689, 1634, 1557, 1402, 1233 cm -1 .
	Mp: 162-163 °C (decomposition)

Procedure B: To a solution of 4-iodopyridine (1.014 g, 4.948 mmol) in 3 mL THF under argon was added slowly ethylmagnesium bromide (6.2 mL of a 0.8 mol/L solution in THF, 4.948 mmol). The suspension was stirred for 1 h at room temperature. In a second flask, a solution of compound 6 (148.6 mg, 1.237 mmol) in 3 mL THF under argon was prepared. To this solution was slowly transferred the Grignard reagent and the reaction mixture was heated at 50 °C overnight. After cooling, HCl 1N solution (10 mL) was added and stirring was continued for 30 min at room temperature. The reaction was poured on a saturated NaHCO3 aqueous solution to adjust the pH value to 8. Product was extracted with EtOAc, combined organic phases were washed with water and brine, dried

1

H NMR (400 MHz, DMSO-d6):  = 8.81 (d, J = 6.0 Hz, 2 H), 8.56 (d, J = 4.8 Hz, 1 H), 7.83 (d, J = 6.0 Hz, 2 H), 7.04 (br s, 2 H), 7.02 (d, J = 4.8 Hz, 1 H).

13

C NMR (101 MHz, DMSO-d6):  = 193.0 (CO), 163.1 (C), 160.5 (C), 141.8 (C), 160.7 (CH), 150.3 (2CH), 123.3 (2CH), 107.9 (CH). HRMS (ESI+): m/z [M+H] + calcd for C10H9N4O: 201.0771; found: 201.0770.

  34

  To a solution of compound 5 (30.9 mg, 0.154 mmol) in 1 mL absolute EtOH at 0 °C under argon was added sodium borohydride (8.7 mg, 0.231 mmol). The reaction mixture was warmed at room temperature and stirred for 75 min. Water and NaHCO3 solution were added and the product was extracted with EtOAc. Combined organic phases were dried over MgSO4, evaporated and purified by flash column chromatography using EtOAc/MeOH gradient from 1% to 10%. Pure compound 7 (26.2 mg, 0.129 mmol) was isolated in 83% yield as a beige powder.

	Rf = 0.10 (EtOAc).
	IR (ATR): 3363-2674, 1739, 1652, 1567, 1216 cm -1 .
	Mp: 125-126 °C
	1 H NMR (400 MHz, DMSO-d6):  = 8.50 (d, J = 6.0 Hz, 2 H), 8.21 (d, J = 4.8
	Hz, 1 H), 7.38 (d, J = 6.0 Hz, 2 H), 6.74 (d, J = 4.8 Hz, 1 H), 6.58 (br s, 2 H),
	6.27 (d, J = 4.4 Hz, 1 H), 5.38 (d, J = 4.4 Hz, 1 H).
	13 C NMR (101 MHz, DMSO-d6):  = 171.7 (C), 163.0 (C), 151.6 (C), 158.8
	(CH), 149.4 (2CH), 121.6 (2CH), 105.9 (CH), 73.7 (CH-OH).
	HRMS (ESI+): m/z [M+H] + calcd for C10H11N4O: 203.0927; found:
	203.0923.
	HPLC method B: purity > 95%,  = 240 nm, tR = 5.24 min.
	Rf = 0.15 (EtOAc).
	IR (ATR): 3471-2993, 1562, 1451, 1226 cm -1 .
	Mp > 250 °C
	Z-8 isomer. 1 H NMR (400 MHz, DMSO-d6):  = 12.11 (br s, 1 H), 8.57 (d, J =
	5.2 Hz, 2 H), 8.35 (d, J = 5.2 Hz, 1 H), 7.36 (d, J = 6.0 Hz, 2 H), 6.81 (br s, 2
	H), 6.68 (d, J = 5.2 Hz, 1 H). 13 C NMR (101 MHz, DMSO-d6):  = 163.8 (C),
	159.8 (C), 151.7 (C), 141.8 (C), 158.7 (CH), 149.9 (2CH), 120.7 (2CH),
	110.5 (CH).
	E-8 isomer. 1 H NMR (400 MHz, DMSO-d6):  = 12.20 (br s, 1 H), 8.63 (d, J =
	4.4 Hz, 2 H), 8.27 (d, J = 5.2 Hz, 1 H), 7.31 (d, J = 5.6 Hz, 2 H), 6.98 (d, J = 5.2
	Hz, 1 H), 6.60 (br s, 2 H). 13 C NMR (101 MHz, DMSO-d6):  = 163.3 (C),
	161.7 (C), 152.9 (C), 139.7 (C), 158.5 (CH), 149.2 (2CH), 124.1 (2CH),
	106.3 (CH).
	HRMS (ESI+): m/z [M+H] + calcd for C10H10N5O: 216.0880; found:
	216.0878.

(±)-(2-Aminopyrimidin-4-yl)(pyridin-4-yl)methanol (

7

)

(E/Z)-(2-Aminopyrimidin-4-yl)(pyridin-4-yl)methanone oxime (8)

To a solution of compound 5 (42.7 mg, 0.213 mmol) in 3 mL EtOH/H2O 4:1 at room temperature were successively added hydroxylamine hydrochloride (23.7 mg, 0.341 mmol) and sodium acetate (34.9 mg, 0.426 mmol). The mixture was stirred at 100 °C overnight, cooled and evaporated to dryness. Ethanol was added in 5 portions (10 mL each) and evaporated to remove water. Purification by flash column chromatography using EtOAc/MeOH 98:2 as eluent afforded compound 8 (26.0 mg, 0.121 mmol) in 56% yield as a white powder. 6:4 Z/E mixture was observed by

1

H NMR.

  isomer.1 H NMR (400 MHz, DMSO-d6):  = 9.21 (br s, 2 H), 8.49 (d, J = 6.0 Hz, 2 H), 8.31 (d, J = 5.2 Hz, 1 H), 7.34 (d, J = 4.8 Hz, 2 H), 6.88 (br s, 2 H), 6.34 (d, J = 5.2 Hz, 1 H).13 C NMR (101 MHz, DMSO-d6):  = 163.1 (C), 159.1 (C), 145.8 (C), 133.1 (C), 159.5 (CH), 149.4 (2CH), 121.6 (2CH), 108.3 (CH).

	E-9 isomer (spectra measured from a ~75:25 Z/E mixture). 1 H NMR (400
	MHz, DMSO-d6):  = 8.67 (d, J = 6.0 Hz, 2 H), 8.10 (d, J = 5.6 Hz, 1 H), 7.29
	(br s, 2 H), 7.22 (d, J = 4.4 Hz, 2 H), 6.99 (d, J = 5.6 Hz, 1 H), 6.27 (br s, 2 H).
	13 C NMR (101 MHz, DMSO-d6): 163.8 (C), 163.1 (C), 157.0 (CH), 150.1
	(2CH), 140.2 (C), 138.4 (C), 124.6 (2CH), 104.4 (CH).
	HRMS (ESI+): m/z [M+H] + calcd for C10H11N6: 215.1039; found: 215.1040.

20 and Rf = 0.32 (EtOAc/MeOH 9:1). IR (ATR): 3400-2615, 1641, 1575, 1547, 1448, 1411 cm -1 . Mp: 206-208 °C (decomposition) Template for SYNTHESIS Thieme Z-9

  corresponding to the aminomethyl group was not visible on the spectrum.13 C NMR (101 MHz, DMSO-d6):  = 172.6 (C), 163.0 (C), 153.4 (C), 158.9 (CH), 149.4 (2CH), 121.2 (2CH), 107.1 (CH), 77.9 (C-OH), 50.8 (CH2).HRMS (ESI+): m/z [M+H]+ calcd for C11H14N5O: 232.1193; found: 232.1189.

		2.507 mmol of solid NaOH dissolved in 0.5 mL of water). The reaction
		mixture was refluxed overnight. Brine was added and the product was
		extracted with CH2Cl2, combined organic layers were dried over MgSO4
		and evaporated. Purification by flash column chromatography using
		EtOAc/MeOH 98:2 to 96:4 gradient as eluent afforded compound 11 (35.6
		mg, 0.166 mmol) in 66% yield as a beige powder.
		Rf = 0.10 (EtOAc).
		IR (ATR): 3421-2831, 1636, 1566, 1477, 1408 cm -1 .
		Mp: 146-147 °C (decomposition)
		1 H NMR (400 MHz, DMSO-d6):  = 8.58 (d, J = 4.4 Hz, 2 H), 8.28 (d, J = 5.2
		Hz, 1 H), 7.43 (d, J = 4.4 Hz, 2 H), 6.77 (br s, 2 H), 6.58 (d, J = 5.2 Hz, 1 H),
		3.40 (d, J = 5.6 Hz, 1 H), 3.26 (d, J = 5.2 Hz, 1 H).
		13 C NMR (101 MHz, DMSO-d6):  = 165.7 (C), 163.2 (C), 146.2 (C), 159.5
		(CH), 149.6 (2CH), 122.2 (2CH), 106.8 (CH), 59.7 (C-O), 55.2 (CH2).
		HRMS (ESI+): m/z [M+H] + calcd for C11H11N4O: 215.0927; found:
	(41.8 mg, 0.125	215.0923.
	mmol) in 1 mL CH2Cl2 at room temperature was added potassium	HPLC method B: purity > 97%,  = 240 nm, tR = 6.39 min.
	carbonate (34.6 mg, 0.250 mmol). The suspension was stirred for 10 min
	at this temperature and compound 5 (30 mg, 0.150 mmol) was finally	(±)-1-(2-Aminopyrimidin-4-yl)-2-azido-1-(pyridin-4-yl)ethan-1-ol
	added in one portion as a solid. The reaction mixture was stirred for 4 h,	(12)
	diluted with NaHCO3 solution. The product was extracted with CH2Cl2 and combined organic layers were dried over MgSO4 and evaporated. Purification by flash column chromatography using EtOAc/MeOH 98:2 to	To a solution of compound 11 (49 mg, 0.229 mmol) in 2 mL MeOH at room temperature were added sodium azide (44.6 mg, 0.686 mmol),
	95:5 gradient as eluent afforded 1 st isomer of compound E-10 (15.1 mg,
	0.059 mmol) in 47% yield as a beige powder and later the 2 nd isomer of
	compound Z-10 (14.5 mg, 0.056 mmol) in 45% yield as a colorless oil.
	Characterization data for E-10:
	Rf = 0.12 (EtOAc).
	IR (ATR): 3437-2790, 1709, 1639, 1545, 1431, 1413, 1269 cm -1 .	Rf = 0.70 (EtOAc/MeOH 90:10).
	Mp: 121-122 °C (decomposition)	IR (ATR): 3493, 3397-2550, 2099, 1630, 1571, 1468, 1223 cm -1 .
	1 H NMR (400 MHz, DMSO-d6):  = 8.61 (d, J = 6.0 Hz, 2 H), 8.27 (d, J = 5.2	Mp: 149-150 °C
	Hz, 1 H), 7.23 (d, J = 6.0 Hz, 2 H), 7.15 (s, 1 H), 6.82 (br s, 2 H), 6.16 (d, J = 4.8 Hz, 1 H), 3.55 (s, 3 H).	1 H NMR (400 MHz, DMSO-d6):  = 8.52 (d, J = 4.8 Hz, 2 H), 8.22 (d, J = 5.2 Hz, 1 H), 7.52 (d, J = 4.8 Hz, 2 H), 6.82 (d, J = 5.2 Hz, 1 H), 6.68 (br s, 2 H),
	13 C NMR (101 MHz, DMSO-d6):  = 165.0 (C), 163.3 (C), 161.7 (C), 149.7	6.59 (s, 1 H), 4.05 (d, J = 12.4 Hz, 1 H), 3.89 (d, J = 12.4 Hz, 1 H).
	(C), 144.4 (C), 160.1 (CH), 149.3 (2CH), 123.7 (2CH), 121.4 (CH), 108.8 (CH), 51.5 (CH3).	13 C NMR (101 MHz, DMSO-d6):  = 171.0 (C), 162.8 (C), 151.7 (C), 159.1 (CH), 151.7 (2CH), 120.8 (2CH), 106.2 (CH), 77.6 (C-OH), 57.9 (CH2).
	HRMS (ESI+): m/z [M+H] + calcd for C13H13N4O2: 257.1033; found: 257.1031.	HRMS (ESI+): m/z [M+H] + calcd for C11H12N7O: 258.1098; found: 258.1094.
	HPLC method A: purity > 95%,  = 240 nm, tR = 16.99 min.	HPLC method B: purity > 95%,  = 240 nm, tR = 6.55 min.
	Characterization data for Z-10:
	Rf = 0.08 (EtOAc).	(±)-2-Amino-1-(2-aminopyrimidin-4-yl)-1-(pyridin-4-yl)ethan-1-ol
	IR (ATR): 3529-2831, 1721, 1623, 1549, 1456, 1278 cm -1 .	(13)
	1 H NMR (400 MHz, DMSO-d6):  = 8.60 (d, J = 6.4 Hz, 2 H), 8.28 (d, J = 4.8	To a solution of compound 12 (23.8 mg, 0.092 mmol) in 1 mL EtOH at
	Hz, 1 H), 7.35 (d, J = 6.4 Hz, 2 H), 6.74 (s, 1 H), 6.73 (br s, 2 H), 6.50 (d, J = 4.8 Hz, 1 H), 3.60 (s, 3 H).	room temperature was added palladium on charcoal (2.5 mg). The reaction mixture was placed under 1 atm H2 and stirred at room
	13 C NMR (101 MHz, DMSO-d6):  = 165.0 (C), 164.4 (C), 163.5 (C), 149.2 (C), 144.5 (C), 158.4 (CH), 150.2 (2CH), 121.8 (2CH), 121.4 (CH), 109.7 (CH), 51.5 (CH3).	temperature for 4 h. It was filtered through a short pad of Celite (Pasteur pipette) and washed with EtOH. The filtrate was evaporated and CH2Cl2 was added in three portions to remove solvent residues. Purification by
	HRMS (ESI+): m/z [M+H] + calcd for C13H13N4O2: 257.1033; found: 257.1034.	reverse phase C18 column chromatography using H2O/MeOH gradient from 98:2 to 5:95 as eluent afforded compound 13 (11.8 mg, 0.051 mmol)
	HPLC method A: purity > 95%,  = 240 nm, tR = 16.71 min.	in 55% yield as a white foam.
	(±)-4-[2-(Pyridin-4-yl)oxiran-2-yl]pyrimidin-2-amine (11)
	To a solution of compound 5 (50.2 mg, 0.251 mmol) in 1.5 mL CH2Cl2 at
	room temperature were added trimethylsulfoxonium iodide (110.4 mg,
	0.501 mmol) and a freshly prepared sodium hydroxide solution (100 mg,

ammonium chloride

(27.

6 mg, 0.517 mmol). The reaction mixture was refluxed overnight. Water was added and the product was extracted with CH2Cl2, combined organic layers were washed with brine, dried over MgSO4 and evaporated. Purification by flash column chromatography using EtOAc/MeOH 90:10 as eluent afforded compound 12 (47.9 mg, 0.186 mmol) in 81% yield as a beige powder.

Rf = 0.

20 (EtOAc/MeOH 1:1). IR (ATR): 3467-2840, 1633, 1557, 1456, 1261 cm -1 . Mp: 76-77 °C Template for SYNTHESIS Thieme signal

H NMR (400 MHz, DMSO-d6):  = 8.48 (d, J = 6.0 Hz,

H), 8.17 (d, J = 5.2 Hz, 1 H), 7.51 (d, J = 4.8 Hz, 2 H), 6.80 (d, J = 5.2 Hz, 1 H), 6.62 (br s, 2 H), 5.96 (br s, 1 H),

3.35 (d, J = 13.6 Hz, 1 H), 3.02 (d, J = 13.6 Hz, 1 H). The NH2

Acknowledgment

The authors thank to Aurélie Job for HPLC analysis.

Primary Data

NO.

Supporting Information

YES (this text will be updated with links prior to publication)

Conflict of Interest

The authors declare no conflict of interest.


Richard Michalet
email: richard.michalet@u-bordeaux.fr

| Florian Delerue

Pierre Liancourt

Francisco I Pugnaire

Ragan Callaway

Richard Michale

Florian Delerue

Are complementarity effects of species richness on productivity the strongest in species-rich communities?

Keywords: biomass, competition, diversity, facilitation, natural patterns, niche complementarity, productivity

1. How the relationship between species richness and productivity changes along environmental gradients remains poorly understood.

 humped-back model. We considered several scenarios of variation in competition and facilitation along environmental gradients, either monotonic with the most common or intense facilitation at the most abiotically severe end of gradients or nonlinear with the strongest facilitation at intermediate positions along gradients.

3. How competition shifts to facilitation along environmental gradients is a key for determining where the effect of species richness on productivity occurs. Based on the literature, the original Stress Gradient Hypothesis would likely predict that complementarity effects should be the greatest, or the most important, in the most abiotically stressful environments. Alternatively, both the 'collapse of facilitation' and the 'shift back to competition' scenarios predict that the highest overall complementary effects on productivity, not biomass, would most likely occur at intermediate positions along environmental stress gradients, but this might vary depending on the source of stress. This latter prediction is consistent with a great deal of literature on natural gradients of productivity and species richness. 4. Synthesis. Our predictions illustrate the importance of better understanding the context dependency of complementarity processes and the key role of facilitation along environmental gradients to better focus conservation efforts where ecosystem functioning is more likely to be negatively affected by species loss, in particular in species-rich communities.

F I G U R E 1 Left panels (a) and (b) show the role of plant interactions for explaining natural patterns of diversity along environmental severity and biomass gradients and right panels (c and d), and the effects on productivity of complementarity effects induced by increasing species richness at different positions along natural environmental gradients shown in left panels. Following [START_REF] Grime | Competitive exclusion in herbaceous vegetation[END_REF], upper panels (a and c) focus on stress gradients in undisturbed conditions and lower panels (b and d) on biotic disturbance gradients in conditions of low stress, while 3D diagrams in the left panels show how the two direct gradients interact along complex gradients of environmental severity. Note that the two 3D diagrams are different views of the same diagram that has been rotated from (a) to (b), in order to highlight the stress or disturbance effects, respectively. 2D diagrams in (a) and (b) show the trade-offs between competitive (blue) and stress-tolerant species (red) and ruderals (yellow) along stress and disturbance gradients, respectively, following [START_REF] Grime | Competitive exclusion in herbaceous vegetation[END_REF], while curves below them show variation in competition and direct and indirect facilitation along stress and biotic disturbance gradients following [START_REF] Grime | Competitive exclusion in herbaceous vegetation[END_REF] for mild environmental conditions and [START_REF] Bertness | Positive interactions in communities[END_REF], [START_REF] Maestre | Do positive interactions increase with abiotic stress? A test from a semi-arid steppe[END_REF], [START_REF] Michalet | Do biotic interactions shape both sides of the humped-back model of species richness in plant communities?[END_REF] and Le [START_REF] Bagousse-Pinguet | Facilitation displaces hot-spots of diversity and allows communities to persist in heavily stressed and disturbed environments[END_REF] for severe environmental conditions. Effects of species richness on productivity through complementarity effects in right panels are represented with different curves depending on the scenario of variation in plant interactions proposed in the literature and shown in left panels, and our knowledge from BEF experiments [START_REF] Barry | The future of complementarity: Disentangling causes from consequences[END_REF]. Numbers in black letters in the right panels are positions along environmental gradients, also represented on the top of 2D diagrams and with white letters in the 3D diagrams is thought to increase productivity primarily through niche partitioning and thus, reduced competition (see [START_REF] Hector | Plant diversity and productivity experiments in European grasslands[END_REF][START_REF] Naeem | Declining biodiversity can alter the performance of ecosystems[END_REF]. This niche partitioning was thought to operate at similar intensities at all levels of productivity (see fig. 4b in Loreau et al., 2001). However, this is not consistent with [START_REF] Grime | Competitive exclusion in herbaceous vegetation[END_REF][START_REF] Grime | Global shifts towards positive species interactions with increasing environmental stress[END_REF] ideas about competition being more important and intense in productive communities (also see [START_REF] Brooker | The importance of importance[END_REF][START_REF] Pennings | Salt marsh plant zonation: The relative importance of plant zonation and physical factors[END_REF]. In other words, if niche partitioning reduces competitive exclusion, and competitive exclusion is more likely in productive communities, then the effects of niche partitioning (one component of complementarity) should be more important in highly productive communities. If the processes affecting coexistence change in importance along environmental gradients, then the processes involved in BEF should change as well. If competition is weak in harsh, unproductive environments, competition can only diminish so much with increasing species richness.

The second inconsistency with other literature derived from the assumption that in large-scale biomass-diversity relationships, the abiotic environment drives both diversity and productivity (see fig. 4a, Loreau et al., 2001). This conflicts with [START_REF] Grime | Competitive exclusion in herbaceous vegetation[END_REF], who argued that decreases in species richness at high productivity are due to increased competition (see [START_REF] Brooker | The importance of importance[END_REF]. This conceptual conflict may be due, in part, to some ecologists emphasizing the importance of stochastic environmental processes related to dispersal, and others emphasizing stress induced by climate and soil [START_REF] Brooker | Don't Diss integration: A comment on Ricklefs' disintegrating communities[END_REF][START_REF] Lortie | Rethinking plant community theory[END_REF][START_REF] Michalet | Competition, facilitation and environmental severity shape the relationship between local and regional species richness in plant communities[END_REF]. [START_REF] Michalet | Biotic interactions, biodiversity and community productivity[END_REF] proposed three arguments that might reconcile these conceptual inconsistencies. First, they argued that both points of view should integrate contextdependent effects of biodiversity on productivity while separating increases in productivity with diversity due to resource partitioning (i.e. a decrease in competition) and those due to facilitation (see [START_REF] Michalet | Do biotic interactions shape both sides of the humped-back model of species richness in plant communities?[END_REF][START_REF] Mulder | Physical stress and diversity-productivity relationships: The role of positive interactions[END_REF][START_REF] Wright | The overlooked role of facilitation in biodiversity experiments[END_REF][START_REF] Wright | Biodiversity and ecosystem functioning: Have our experiments and indices been underestimating the role of facilitation[END_REF]. These arguments were further developed by [START_REF] Wright | The overlooked role of facilitation in biodiversity experiments[END_REF] and [START_REF] Barry | The future of complementarity: Disentangling causes from consequences[END_REF], who proposed that complementarity should not only separate resource partitioning and direct facilitative mechanisms (e.g. ambient VPD, this Special Feature, [START_REF] Aguirre | The experimental manipulation of atmospheric drought: Teasing out the role of microclimate in biodiversity experiments[END_REF] but also indirect facilitative mechanisms (e.g. [START_REF] Pugnaire | Climate change effects on plant-soil feedbacks and consequences for biodiversity and functioning of terrestrial ecosystems[END_REF][START_REF] Van Der Putten | Plant-soil feedbacks: The past, the present and future challenges[END_REF][START_REF] Zuppinger-Dingley | Are complementarity effects of species richness on productivity the strongest in species-rich communities?[END_REF]. Second, [START_REF] Michalet | Biotic interactions, biodiversity and community productivity[END_REF] stressed that the refinement of the stress gradient hypothesis (SGH) of [START_REF] Bertness | Positive interactions in communities[END_REF], proposing that facilitation increases with decreasing stress from conditions of high to intermediate environmental severity [START_REF] Michalet | Do biotic interactions shape both sides of the humped-back model of species richness in plant communities?[END_REF], could reconcile BEF experiments with natural patterns, at least in stressful conditions. Indeed, in the [START_REF] Michalet | Do biotic interactions shape both sides of the humped-back model of species richness in plant communities?[END_REF] model, species richness, facilitation, biomass and productivity are positively correlated, consistent with BEF experiments conducted in abiotically stressful conditions (e.g. [START_REF] Mulder | Physical stress and diversity-productivity relationships: The role of positive interactions[END_REF]. However, no BEF experiment has been conducted in exceptionally stressful conditions such as found at the ends of many stress gradients (e.g. [START_REF] Armas | A field test of the Stress-Gradient Hypothesis along an aridity gradient[END_REF][START_REF] Callaway | Facilitative and interfering effects of Arthro cnemum Subterminale on winter annuals[END_REF][START_REF] Callaway | Interdependence among alpine plants increases with stress: A global experiment[END_REF][START_REF] Cavieres | Facilitative plant interactions and climate simultaneously drive alpine plant diversity[END_REF][START_REF] Dohn | Tree effects of grass growth in savannas: Competition, facilitation and the stress-gradient hypothesis[END_REF]He et al., 2013;[START_REF] Molina-Montenegro | Positive interactions between the lichen Usnea antarc tica (Parmeliaceae) and the native flora in Maritime Antarctica[END_REF][START_REF] Pugnaire | No evidence of facilitation collapse in the Tibetan plateau[END_REF]. [START_REF] Michalet | Do biotic interactions shape both sides of the humped-back model of species richness in plant communities?[END_REF] also noted that explicitly distinguishing between productivity and biomass might help to resolve conceptual conflicts in the literature. Loreau et al. (2001) proposed that theoretical models (e.g. [START_REF] Grime | Competitive exclusion in herbaceous vegetation[END_REF][START_REF] Huston | A general hypothesis of species diversity[END_REF] predicted a negative correlation between diversity and productivity under low abiotic stress conditions (also see Adler et al., 2012;[START_REF] Grace | Integrative modelling reveals mechanisms linking productivity and plant species richness[END_REF]. However, most observational, or correlative, studies used final biomass as surrogate for productivity [START_REF] Al-Mufti | A quantitative analysis of shoot phenology and dominance in herbaceous vegetation[END_REF][START_REF] Fraser | Applying plant facilitation to forest restoration: A meta-analysis of the use of shrubs as nurse plants[END_REF]. Furthermore, in the original humped-back model (HBM, [START_REF] Grime | Competitive exclusion in herbaceous vegetation[END_REF], the x-axis is a gradient of 'standing crop', that is, biomass, and not productivity as in Loreau et al. (2001;also, see Schmid, 2002). Productivity is the rate at which living biomass increases over time (g g -1 year -1 ), whereas biomass is a static metric of the accumulation of biomass over undefined periods of time (g/m 2 ). Biomass and productivity can correlate in mesic grasslands dominated by herbaceous species that decay before the beginning of the next growing season, but the assumption of equivalence is problematic in communities that retain biomass over many growing seasons-those with larger proportions of woody plants or recalcitrant standing litter. [START_REF] Grace | Integrative modelling reveals mechanisms linking productivity and plant species richness[END_REF] analysed global grassland data (Nutnet;Adler et al., 2012) and explicitly distinguished between biomass and productivity. Doing so they identified a negative correlation between biomass and species richness, but a positive relationship between species richness and productivity. However, they used current year's biomass increment as proxy of productivity, since the sampling (Adler et al., 2012) was only conducted once, at the peak of biomass during the growing season. The sampling of only current-year leaves and green plant parts (Adler et al., 2012) can underestimate productivity since the yearly accumulated biomass can also be stored in woody stems and roots. Thus, our knowledge of the variation in productivity along natural environmental gradients driving community biomass and species richness is still approximate in grasslands and limited in most woodlands. The distinction between biomass and productivity is crucial, because if biomass, but not productivity, is high when competition decreases species richness, then the results of BEF experiments are not inconsistent with natural patterns of diversity (e.g. [START_REF] Grime | Competitive exclusion in herbaceous vegetation[END_REF], as suggested by [START_REF] Michalet | Biotic interactions, biodiversity and community productivity[END_REF].

In light of advances over the last 20 years [START_REF] Eisenhauer | Aboveground-belowground interactions as a source of complementarity effects in biodiversity experiments[END_REF][START_REF] Isbell | Linking the influence and dependence of people on biodiversity across scales[END_REF][START_REF] Reich | Impacts of biodiversity loss escalate through time as redundancy fades[END_REF][START_REF] Zuppinger-Dingley | Are complementarity effects of species richness on productivity the strongest in species-rich communities?[END_REF] and the clarification of different processes in complementarity [START_REF] Barry | The future of complementarity: Disentangling causes from consequences[END_REF][START_REF] Wright | The overlooked role of facilitation in biodiversity experiments[END_REF], we now elaborate on predictions for the effects of species richness on the productivity of natural plant communities. We integrate the results of BEF experiments with different scenarios for variation in competition and facilitation along environmental gradients.

| A FR AME WORK FOR PRED I C TING THE INFLUEN CE OF S PECIE S RI CHNE SS ON PRODUC TIVIT Y ALONG ENVIRONMENTAL G R AD IENTS

To predict the effect of species richness on productivity along environmental gradients, we build on a framework inspired by fig. 4 in Loreau et al. (2001; Figure 1). In Figure 1, the left two panels show relationships among community biomass, environmental conditions and biotic interactions and species richness following [START_REF] Grime | Competitive exclusion in herbaceous vegetation[END_REF].

The two panels on the right show how complementarity effects might increase productivity with increasing diversity along environmental gradients following the results of BEF experiments. Many textbooks and authors, including Loreau et al. (2001), have simplified [START_REF] Grime | Competitive exclusion in herbaceous vegetation[END_REF] model to a single figure with productivity on the x-axis and species richness on the y-axis. However, Grime originally proposed two conceptual diagrams-reproduced in Figure 1a,b. One diagram (Figure 1a) represented changes in species richness along gradients of stress in undisturbed communities. The other (Figure 1b) showed changes in species richness along disturbance gradients in productive environments with no variation in abiotic stress. These two diagrams allowed Grime to illustrate the switch from competitive to stress-tolerant species, or ruderals, along gradients of abiotic stress or disturbance, respectively. In both diagrams, standing crop (i.e. biomass, not productivity) decreased with increasing abiotic stress or disturbance, and there were no direct measurements of productivity on the x-axis (e.g. [START_REF] Al-Mufti | A quantitative analysis of shoot phenology and dominance in herbaceous vegetation[END_REF]. Likely, because of the similarity between the two curves, many authors (including [START_REF] Grime | Global shifts towards positive species interactions with increasing environmental stress[END_REF] Using this framework, we include the different scenarios of variation in competition and facilitation along stress and disturbance gradients (Figure 1) proposed in the literature [START_REF] Bertness | Positive interactions in communities[END_REF][START_REF] Grime | Competitive exclusion in herbaceous vegetation[END_REF][START_REF] Bagousse-Pinguet | Facilitation displaces hot-spots of diversity and allows communities to persist in heavily stressed and disturbed environments[END_REF][START_REF] Maestre | Do positive interactions increase with abiotic stress? A test from a semi-arid steppe[END_REF][START_REF] Michalet | Do biotic interactions shape both sides of the humped-back model of species richness in plant communities?[END_REF][START_REF] Pugnaire | Changes in plant interactions along a gradient of environmental stress[END_REF]. From these, it is evident that, in conditions of low stress and disturbance (i.e. from positions 1 to 2 or 4 in Figure 1a and b, respectively), competition should decrease-and species richness increase-when biomass decreases. These changes have empirical support (e.g. [START_REF] Michalet | Plant community composition and biomass on calcareous and siliceous substrates in the northern French Alps: Comparative effects of soil chemistry and water status[END_REF][START_REF] Fraser | Applying plant facilitation to forest restoration: A meta-analysis of the use of shrubs as nurse plants[END_REF] see Adler et al., 2012). The stress gradient hypothesis (SGH) further proposed that, at mid-point along the two gradients (i.e. at position 2 or 4), the frequency of competition should gradually shift to facilitation through habitat amelioration (Figure 1a) or through herbivore release (Figure 1b), respectively [START_REF] Bertness | Positive interactions in communities[END_REF].

The SGH model suggested that direct and indirect facilitation should increase monotonically with increasing stress or herbivory towards the severe end of each gradient (i.e. until position 3 or 5, respectively, Figure 1a,b). This model has been supported by the results of a number of experiments, in particular along gradients of cold stress (e.g. [START_REF] Callaway | Interdependence among alpine plants increases with stress: A global experiment[END_REF][START_REF] Cavieres | Facilitative plant interactions and climate simultaneously drive alpine plant diversity[END_REF][START_REF] Michalet | Partitioning net interactions among plants along altitudinal gradients to study community responses to climate change[END_REF]. However, more complex results have been found along drought gradients [START_REF] Maestre | Is the change of plant-plant interactions with abiotic stress predictable? A metaanalysis of field results in arid environments[END_REF], either supporting the SGH (e.g. [START_REF] Armas | A field test of the Stress-Gradient Hypothesis along an aridity gradient[END_REF][START_REF] Callaway | Facilitative and interfering effects of Arthro cnemum Subterminale on winter annuals[END_REF][START_REF] Dohn | Tree effects of grass growth in savannas: Competition, facilitation and the stress-gradient hypothesis[END_REF][START_REF] Fraser | Applying plant facilitation to forest restoration: A meta-analysis of the use of shrubs as nurse plants[END_REF][START_REF] Greenlee | Abiotic stress and the relative importance of interference and facilitation in montane bunchgrass communities in western Montana[END_REF][START_REF] Holzapfel | Annual plant-shrub interactions along an aridity gradient[END_REF][START_REF] Liancourt | Stress tolerance abilities and competitive responses in a watering and fertilization field experiment[END_REF][START_REF] Pugnaire | Changes in plant interactions along a gradient of environmental stress[END_REF] see reviews by [START_REF] Blaser | Facilitative or competitive effects of woody plants on understorey vegetation depend on N-fixation, canopy shape and rainfall[END_REF]He et al., 2013), a few reporting an increase in competition with increasing drought (e.g. [START_REF] Davis | Competition between tree seedlings and herbaceous vegetation: Support for a theory of resource supply and demand[END_REF][START_REF] Maestre | Do positive interactions increase with abiotic stress? A test from a semi-arid steppe[END_REF][START_REF] Tielbörger | Temporal environmental variation tips the balance between facilitation and interference in desert plants[END_REF]but see fig. 4.25, Callaway, 2007) and other finding an absence of interaction at the most stressed end of the gradient (e.g. [START_REF] Chaieb | A regional assessment of changes in plant-plant interactions along topography gradients in Tunisian sebkhas[END_REF][START_REF] Kitzberger | Effects of climatic variability on facilitation of tree establishment in Northern Patagonia[END_REF][START_REF] Bagousse-Pinguet | Importance, but not intensity of plant interactions relates to species diversity under the interplay of stress and disturbance[END_REF]. Facilitation has also been shown to wane in conditions of extreme biotic or physical disturbance [START_REF] Brooker | Transient facilitative effects of heather on Scots pine along a grazing disturbance gradient in Scottish moorland[END_REF][START_REF] Forey | Does disturbance drive the collapse of biotic interactions at the severe end of a diversity-biomass gradient?[END_REF][START_REF] Smit | Nurse plants, tree saplings and grazing pressure: Changes in facilitation along a biotic environmental gradient[END_REF]. Thus, [START_REF] Michalet | Do biotic interactions shape both sides of the humped-back model of species richness in plant communities?[END_REF], [START_REF] Maestre | Refining the stress-gradient hypothesis for competition and facilitation in plant communities[END_REF], [START_REF] Holmgren | Strong facilitation in mild environments: The stress gradient hypothesis revisited[END_REF], [START_REF] Malkinson | What does the stress-gradient hypothesis predict? Resolving the discrepancies[END_REF] and [START_REF] Verwijmeren | Interspecific facilitation and critical transitions in arid ecosystems[END_REF] have proposed several alternatives to monotonic changes in competition and facilitation with increase abiotic stress, but always with a peak of facilitation at intermediate position along environmental severity gradients. [START_REF] Michalet | Two alternatives to the stress-gradient hypothesis at the edge of life: The collapse of facilitation and the switch from facilitation to competition[END_REF] distinguished between the collapse of facilitation described by [START_REF] Michalet | Do biotic interactions shape both sides of the humped-back model of species richness in plant communities?[END_REF] from a shift back to competition. This was because the former is generally due to a decrease in the positive effect of the nurse, more likely to occur on a non-resource gradient (e.g. disturbance or temperature), whereas the latter may be due to increasing competition for resources. The left panels in Figure 1 show different scenarios of variation in competition and facilitation along stress and disturbance gradients. The first scenario corresponds to the original SGH, describing a monotonic increase in facilitation, the second considers that facilitation wanes at high level of stress ('collapse' hereafter), and the third considers that facilitation could shift back to competition at high level of stress ('shift back' hereafter). We examine the influence of species richness on productivity for these three scenarios along a stress gradient, and for the two first only along a disturbance gradient since disturbance is not a resource and, thus, unlikely to induce a shift back to competition.

| THE CONTE X T DEPENDEN C Y OF COMPLEMENTARY EFFEC TS AND IMPLIC ATIONS FOR PRODUC TIVIT Y

We selected five key environmental positions for predicting the effects of species richness on productivity in natural environments (Figure 1c,d). Position 1 shows the least constrained environmental conditions with high biomass and low diversity (due to both low stress and disturbance) but with high competition. Positions 2 and 4 are intermediate positions along the stress and disturbance gradients where species richness is highest due to intermediate levels of stress and disturbance, low competition and either weak facilitation (see [START_REF] Bertness | Positive interactions in communities[END_REF] or high direct or indirect facilitation following other SGH-ideas (Le Bagousse-Pinguet, Xiao, et al., 2014;[START_REF] Maestre | Do positive interactions increase with abiotic stress? A test from a semi-arid steppe[END_REF][START_REF] Michalet | Do biotic interactions shape both sides of the humped-back model of species richness in plant communities?[END_REF]. Finally, positions 3 and 5 point to the severe end of a gradient where species richness is low, with either high or low facilitation, or even competition, depending on the three models of potential interactions described in Figure 1a,b (left panels). [START_REF] Barry | The future of complementarity: Disentangling causes from consequences[END_REF] noted that the relative contribution of complementarity processes to increases in productivity with increasing species richness is highly context dependent. Negative biotic feedbacks from other trophic levels should dominate in monocultures, whereas resource partitioning and indirect facilitation dominate in mixtures under low stress, and direct facilitation under high stress [START_REF] Barry | The future of complementarity: Disentangling causes from consequences[END_REF]. Direct facilitation through microclimate amelioration has been shown to be crucial in driving complementarity effects in BEF experiments conducted under moderately stressful conditions [START_REF] Caldeira | Mecha nisms of positive biodiversity-production relationships: Insights provided by delta C-13 analysis in experimental Mediterranean grassland plots[END_REF][START_REF] Klaus | Plant diversity moderates drought stress in grasslands: Implications from a large real-world study on C-13 natural abundances[END_REF][START_REF] Mulder | Physical stress and diversity-productivity relationships: The role of positive interactions[END_REF][START_REF] Steudel | Biodiversity effects on ecosystem functioning change along environmental stress gradients[END_REF][START_REF] Wright | The overlooked role of facilitation in biodiversity experiments[END_REF]this Special Feature, Aguirre et al., 2021). For example, [START_REF] Mulder | Physical stress and diversity-productivity relationships: The role of positive interactions[END_REF] experimented with bryophyte communities at two levels of water availability, and diversity increased productivity only under dry conditions. This result was due to increasing survival of drought-intolerant species below the canopy of more tolerant species. Thus, following [START_REF] Wright | The overlooked role of facilitation in biodiversity experiments[END_REF] and [START_REF] Barry | The future of complementarity: Disentangling causes from consequences[END_REF], we argue that facilitation is most likely the dominant process explaining complementarity effects in stressful conditions versus resource partitioning or negative biotic feedbacks in milder environmental conditions.

The relative contribution of each complementarity process in natural environments has been minimally documented (but see this Special Feature). Negative biotic feedbacks from other trophic levels are likely to operate on the high-biomass side of Grime's HBM (Figure 1a,b), corresponding with plant communities dominated by a few competitive species, due to low stress and low disturbance. Indeed, since its early formulation [START_REF] Connell | On the role of natural enemies in preventing competitive exclusion in some marine animals and in rain forest trees[END_REF][START_REF] Janzen | Herbivores and the number of tree species in tropical forests[END_REF], negative density-dependent effects have been suggested to be more important in tropical systems with low environmental constraints for plant growth that lead to high species diversity. Thus, negative biotic feedbacks from higher trophic levels should be important complementarity effects driving communities from positions 1 to 2 or 1 to 4 in HBM models, as proposed in Figure 1c,d.

The influence of the abiotic environment on the importance of resource partitioning is certainly an old and open question, for which [START_REF] Braun-Blanquet | Plant sociology: The study of plant communities: Authorized English translation of Pflanzensoziologie[END_REF] proposed a solution. He, like Grime later (1979), argued that competition among species is low in unfavourable environments and increases as environmental conditions improve (but see [START_REF] Tilman | Resource competition and community structure[END_REF]. If competition is less intense and less important in environments supporting less standing biomass [START_REF] Grime | Global shifts towards positive species interactions with increasing environmental stress[END_REF], Figure 1), resource partitioning is less likely to explain species richness. With improving environmental conditions and increasing species richness (and therefore community complexity), we could expect resource partitioning to become more important (see [START_REF] Braun-Blanquet | Plant sociology: The study of plant communities: Authorized English translation of Pflanzensoziologie[END_REF][START_REF] Naeem | Declining biodiversity can alter the performance of ecosystems[END_REF] because the benefits of avoiding competitive exclusion increase. Considering covariation between standing biomass and species richness [START_REF] Fraser | Applying plant facilitation to forest restoration: A meta-analysis of the use of shrubs as nurse plants[END_REF], and if resource partitioning is higher in 'highly organized communities' (i.e. more diverse sensu [START_REF] Braun-Blanquet | Plant sociology: The study of plant communities: Authorized English translation of Pflanzensoziologie[END_REF], resource partitioning should peak at a middle point along severity gradients (i.e. positions 2 and 4 in Figure 1) and should be present but less important at position 1. Following the original SGH model [START_REF] Bertness | Positive interactions in communities[END_REF], resource partitioning would be less important at positions 3 and 5, because competition is assumed to be less frequent.

In contrast, resource partitioning would be still present at position 3 if there is a shift back to competition [START_REF] Maestre | Do positive interactions increase with abiotic stress? A test from a semi-arid steppe[END_REF].

Finally, the relative contribution of direct facilitation to increasing productivity with increasing species richness should be highest at position 3, following the SGH model, but at position 2 following the other two scenarios (Figure 1c). Additionally, consistent with [START_REF] Aschehoug | Diversity increases indirect interactions, attenuates the intensity of competition, and promotes coexistence[END_REF] and [START_REF] Wright | The overlooked role of facilitation in biodiversity experiments[END_REF], indirect facilitation due to competitive release should promote complementarity effects at position 2 because of the higher number of species there (Figure 1c). Release from consumer pressure should be most important with increasing productivity concomitant with increasing species richness at position 5 with SGH [START_REF] Bertness | Positive interactions in communities[END_REF], at position 4 with the 'collapse' scenario (Le Bagousse-Pinguet, Xiao, et al., 2014, Figure 1d).

The overall complementarity effect on productivity is dependent on both the importance of each complementarity process and the species richness at each position on a particular abiotic stress gradient. This overall complementarity effect on productivity is represented in Figure 1c,d. In the absence of facilitation, and with strong competition (position 1), important negative feedbacks and weak resource partitioning, the effect of increasing species richness on productivity should be low. An experimental test of this effect would be hard to carry out, since variation in species richness should ideally represent both the environmental and community characteristics, that is, low disturbance and low number of species. Alternatively, an experimental test might involve species removals in natural communities (e.g. [START_REF] Diaz | Functional diversity reveals by removal experiments[END_REF].

At positions 2 and 4, the overall complementarity effect on productivity should largely depend on the scenario of variation in competition and facilitation along stress and disturbance gradients. It should be the highest in the case of a collapse of facilitation or a shift back to a relatively high frequency of competition [START_REF] Bagousse-Pinguet | Facilitation displaces hot-spots of diversity and allows communities to persist in heavily stressed and disturbed environments[END_REF][START_REF] Maestre | Do positive interactions increase with abiotic stress? A test from a semi-arid steppe[END_REF][START_REF] Michalet | Do biotic interactions shape both sides of the humped-back model of species richness in plant communities?[END_REF], both predicting a maximum of facilitation at these positions (Figure 1c,d), and because all complementarity processes are predicted to operate. The response of dependent species to dominant neighbours at intermediate environmental positions is highly variable and depends on their functional strategies [START_REF] Bai | Contrasting responses of different functional groups stabilise community responses to a dominant shrub under global change[END_REF][START_REF] Michalet | Communities: Are they groups of hidden interactions[END_REF][START_REF] Wang | Stature of dependent forbs is more related to the direct and indirect above-and below-ground effects of a subalpine shrub than are foliage traits[END_REF], which is consistent with [START_REF] Choler | Facilitation and competition on gradients in alpine plant communities[END_REF][START_REF] Liancourt | Stress tolerance and competitive-response ability determine the outcome of biotic interactions[END_REF] and [START_REF] Liancourt | SGH: Stress or strain gradient hypothesis? Insights from an elevation gradient on the roof of the world[END_REF]. Thus, species of different functional strategies (C, S and R; [START_REF] Grime | Competitive exclusion in herbaceous vegetation[END_REF] may contribute specific kinds of complementarity to increasing productivity. At positions 3 and 5, predictions also depend on the scenario, with higher effects expected in the original SGH scenario than in the other two scenarios,

| CON CLUS IONS

Improving our knowledge of the context dependency of complementarity processes in general, and plant-plant interactions in particular, is needed to better understand the effect of biodiversity on ecosystem functions and the possible discrepancy between BEF experiments and natural environmental gradients. Following the predictions of the original SGH [START_REF] Bertness | Positive interactions in communities[END_REF], we predict that the overall complementarity effect should be the greatest in the most abiotically stressful environments. Alternatively, both the 'collapse of facilitation' and the 'shift back to competition' scenarios would predict the highest overall complementary effect for productivity to occur at an intermediate position along environmental gradients, where species richness is highest and where facilitation should peak. More precise measurements of productivity of herbaceous and shrubland communities are needed to test these alternative hypotheses. A better characterization of the context dependency of complementarity processes should help focusing conservation efforts where ecosystem functioning is most likely to be negatively affected by the current diversity loss associated to global change.

  simplified the figure into a single conceptual diagram. Additionally, standing crop (i.e. biomass) was (wrongly) replaced by productivity, as in Loreau et al. (2001) and several textbooks. If we put the two original diagrams into a 3D diagram, we see how stress and disturbance interact along complex environmental gradients to shape biomass and species richness (left panel of Figure 1a,b with two views of the same 3D diagram).

  13652745, 2021, 5, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.13658 by Cochrane France, Wiley Online Library on [27/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License in particular the collapse of facilitation, since the absence of competition precludes resource partitioning.

13652745, 2021, 5, Downloaded from https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2745.13658 by Cochrane France, Wiley Online Library on [27/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

ACK N OWLED G EM ENTS

We thank the editors of this special issue for inviting us to contribute and for their insightful comments. We also thank two anonymous reviewers for constructive criticism on an earlier draft of this manuscript. F.I.P. was supported by the Spanish Research Agency (AEI grant CGL2017-84515-R).

AUTH O R S ' CO NTR I B UTI O N S

R.M. conceived the idea and wrote the first draft of the manuscript; F.D., P.L. and F.I.P. contributed critically to the writing.

DATA AVA I L A B I L I T Y S TAT E M E N T

There are no data to be archived.

O RCI D


The New England Journal Medicine, 1988, 318 (18) 1206-1207 Author's Version May 5, 1988 -Denis E. Corpet

ANTIBIOTIC RESISTANCE FROM FOOD

To the Editor: Resistance to antibiotics is common in the intestinal bacteria of healthy people. Resistant strains of Escherichia coli, a reservoir for transmissible R plasmid, are found in 50 to 80 percent of fecal specimens in the normal population. [START_REF] Hawkey | Resistant bacteria in the normal human flora[END_REF] In the same person, periods when no resistant E. coli isolates can be detected in feces alternate with periods when the resistant bacteria outnumber susceptible bacteria. [START_REF] Corpet | Antibiotic residues and drug resistance in human intestinal flora[END_REF] Because of the absence of person-toperson transmission of E. coli, [START_REF] Levine | Lack of person-to-person transmission of enterotoxigenic Escecherichia coli despite close contact[END_REF] and because some foods carry numerous gram-negative bacilli, [START_REF] Shooter | Isolation of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella front food in hospitals, canteens, and schools[END_REF][START_REF] Levy | Drug resistant bacteria in food for humans and animals[END_REF] we investigated the effect of a sterile diet on the excretion of resistant enterobacteria.

Six healthy volunteers ate a nearly sterile diet for a mean period of 17 days, after a control period of 21 days. They were on their usual diet during the control period, and their foods were heated at 105°C for one hour during the sterile-diet period. This procedure sufficed to destroy an inoculum of 10 8 viable cells of E. coli that was injected into the food. Volunteers washed their hands with ethanol before meals. Fecal samples were collected and analyzed daily. Comparative counting procedures were used to determine the incidence of resistance to ampicillin, tetracycline, and streptomycin in lactose-fermenting enteric bacilli. [START_REF] Corpet | Antibiotic residues and drug resistance in human intestinal flora[END_REF] During the control period, the populations of fecal resistant lac+ enteric bacilli varied with time, periodically reaching a high level of 10 8 per gram. The day after the start of the sterile diet, the fecal concentration of resistant bacteria dropped, and fell to a minimum in a mean time of three days. No resistant strain could be detected in the feces of three volunteers, whereas in those of the other three, some could be detected sporadically. The sterile diet reduced the number of tetracycline-resistant bacilli in all volunteers (Table 1). It also reduced the number of bacilli resistant to ampicillin and streptomycin (data not shown). • Data for the control diet and the sterile diet are means ±SD for 21 and 17 daily counts, respectively. Tet R denotes tetracycline resistant. .  Counts below the detection limit (25 cells per gram) were converted to 1.4 for calculation of mean values. # P<0.01 for the difference between the control and the experimental periods in the same person, by the Mann-Whitney test.

. § P<0.001 for the difference between the control and the experimental periods in the same person, by the Mann-Whitney test.  P<0.001 for the difference between the control and the experimental periods, by paired Student's t-test.

Thus, most fecal resistant lac+ enteric bacilli come from contaminated food: transient strains enter the intestines with food and are excreted in feces. [START_REF] Hartley | Escherichia coli in the faecal flora of man[END_REF][START_REF] Cooke | Fate of ingested Escherichia coli in normal persons[END_REF] Although resistant lac+ enteric bacilli present no direct threat to public health, they may transfer their resistance to pathogens, destroy antibiotics in the gut, or cause urinary tract infections, posing clinical problems. Hence, it may be desirable to reduce the prevalence of resistant bacteria in human intestines and, on the basis of the data reported here, in foods. The foods most likely to carry large numbers of resistant bacteria are raw vegetables and salads. [START_REF] Levy | Drug resistant bacteria in food for humans and animals[END_REF] Thus, the flow of resistant genes from the environment to humans that is caused by the contamination of vegetables by sewage and manure should be re-duced. [START_REF] Lafont | Antibiotic-resistant bacteria in animal wastes: A human health hazard?[END_REF][START_REF] Linton | Flow of resistance genes in the environment and from animals to man[END_REF] In patients with severe granulocytopenia, systemic infections have often been shown to be caused by resistant bacteria from the gut. Our results show that such patients should be given a nearly sterile diet. [START_REF] Remington | Added post-publication: Full data from this study (table, figure) have[END_REF] Denis E. CORPET, Dipl.Bacteriol. Institut Pasteur Institut National de la Recherche Agronomique 31931 Toulouse, France

Table 1 .

 1 Log Number of Total and Tetracycline-Resistant Lac+ Enteric Bacilli per Gram of Feces, in Volunteers on a Sterite Diet.•

	Volunteer	Control Diet	Sterile Diet 
		Total	Tet R	Total	Tet R
	1	6.5±1.4	4.1±1.8	5.5±1.1 #	1.4±0.1 §
	2	7.1±0.7	4.8±1.3	7.1±1.0	1.7±0.7 §
	3	7.4±0.9	4.9±2.5	7.1±0.7	1.7±0.8 §
	4	7.9±1.0	5.7±1.5	6.3±1.0 §	2.3±1.7 §
	5	7.0±0.8	4.0±1.3	7.4±0.9	2.7±1.2 #
	6	8.4±0.6	7.5±1.0	8.4±0.5	5.3±1.0 §
	Total	7.4±0.7	5.2±1.3	6.9±1.0	2.5±1.4 